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Chen, Wei

Overview:

My general area of interest relates to how cancer develops and how to identify cancer therapeutic agents. In particular I hope to identify molecular signals that underlie the changes necessary for directing normal tissue to a malignant state in cancer. Therefore, I have been studying how extracellular signals are deciphered by seven trans-membrane receptors and their regulatory proteins to control cell proliferation and differentiation. My major research focuses on studying GPCR, Smoothened, TGF-beta and Frizzled receptor trafficking and signaling as well as their roles in tumor biology. Abnormalities in the receptors or other proteins they interact with either directly or indirectly result in malignancies. Moreover, as a result of our research, we have established a state-of-the-art high throughput, high content screening platform in my laboratory to identify novel small molecules that modulate the activity of these receptors. We have found many new small molecules that block Hedgehog pathway. These chemical compounds may have the potential to become new therapeutic agents to treat many refractory cancers of the pancreas, liver, breast, prostate, and skin.

Positions:

Associate Professor in Medicine

Medicine, Gastroenterology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1999

Ph.D. — University of Toledo

Grants:

Translational Research in Surgical Oncology

Administered By
Surgery, Surgical Sciences
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
January 01, 2002
End Date
August 31, 2021

Inhibition of Wnt/B-Catenin Signaling in Colorectal Cancer Therapy

Administered By
Medicine, Gastroenterology
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
September 19, 2013
End Date
July 31, 2018

Targeting the WNT/beta-catenin Pathway in Triple Negative Breast Cancer

Administered By
Medicine, Medical Oncology
AwardedBy
Department of Defense
Role
Partnering PI
Start Date
September 30, 2013
End Date
September 29, 2017

Targeting the WNT/beta-catenin pathway in triple negative breast cancer

Administered By
Medicine, Gastroenterology
AwardedBy
Department of Defense
Role
Principal Investigator
Start Date
September 30, 2013
End Date
September 29, 2017

Developing a HER3 Vaccine to Prevent Resistance to Endocrine Therapy

Administered By
Surgery, Surgical Sciences
AwardedBy
Department of Defense
Role
Co Investigator
Start Date
September 30, 2012
End Date
September 29, 2017

Duke Training Grant in Digestive Diseases and Nutrition

Administered By
Medicine, Gastroenterology
AwardedBy
National Institutes of Health
Role
Mentor
Start Date
July 01, 1988
End Date
June 30, 2017

Rac1 Promotes Hepatic Stellate Accumulation/Activation in Liver Injury

Administered By
Medicine, Gastroenterology
AwardedBy
National Institutes of Health
Role
Co-Mentor
Start Date
July 22, 2009
End Date
June 30, 2014

Drug Abuse: Discovering Ligands for Pertinent GPCRs

Administered By
Cell Biology
AwardedBy
National Institutes of Health
Role
Collaborator
Start Date
September 30, 2006
End Date
June 30, 2010
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Publications:

Benzimidazole inhibitors from the Niclosamide chemotype inhibit Wnt/β-catenin signaling with selectivity over effects on ATP homeostasis.

The Wnt signaling pathway plays a key role in organ and tissue homeostasis, and when dysregulated, can become a major underlying mechanism of disease, particularly cancer. We reported previously that the anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. To define Niclosamide's mechanism of Wnt/β-catenin inhibition, and to improve its selectivity and pharmacokinetic properties as an anticancer treatment, we designed a novel class of benzimidazole inhibitors of Wnt/β-catenin signaling based on SAR studies of the Niclosamide salicylanilide chemotype. Niclosamide has multiple biological activities. To address selectivity in our design, we interrogated a protonophore SAR model and used the principle of conformational restriction to identify novel Wnt/β-catenin inhibitors with less effect on ATP cellular homeostasis. These studies led to the identification of 4-chloro-2-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) phenol (4) and related derivatives with greater selectivity for Wnt/β-catenin signaling inhibition vs. differential effects on cellular ATP homeostasis. This is the first report that the Wnt signaling inhibitory activity of Niclosamide can be translated into a new chemical class and to show that its effects on ATP homeostasis can be separated from its inhibitory effects on Wnt signaling. These compounds could be useful tools to elucidate the mechanism of Niclosamide's inhibition of Wnt signaling, and aid the discovery of inhibitors with improved pharmacologic properties to treat cancer and diseases in which Niclosamide has important biological activity.

Authors
Mook, RA; Ren, X-R; Wang, J; Piao, H; Barak, LS; Kim Lyerly, H; Chen, W
MLA Citation
Mook, RA, Ren, X-R, Wang, J, Piao, H, Barak, LS, Kim Lyerly, H, and Chen, W. "Benzimidazole inhibitors from the Niclosamide chemotype inhibit Wnt/β-catenin signaling with selectivity over effects on ATP homeostasis." Bioorganic & medicinal chemistry 25.6 (March 2017): 1804-1816.
PMID
28233680
Source
epmc
Published In
Bioorganic & Medicinal Chemistry
Volume
25
Issue
6
Publish Date
2017
Start Page
1804
End Page
1816
DOI
10.1016/j.bmc.2017.01.046

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

INTRODUCTION: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment. METHODS: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform. RESULTS: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. CONCLUSIONS: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Authors
Ren, XR; Wang, J; Osada, T; Mook, RA; Morse, MA; Barak, LS; Lyerly, HK; Chen, W
MLA Citation
Ren, XR, Wang, J, Osada, T, Mook, RA, Morse, MA, Barak, LS, Lyerly, HK, and Chen, W. "Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth." Breast cancer research : BCR 17.1 (December 2015): 528-.
PMID
25778364
Source
epmc
Published In
Breast Cancer Research
Volume
17
Issue
1
Publish Date
2015
Start Page
528

Genetic Deletion of β-Arrestin-2 and the Mitigation of Established Airway Hyperresponsiveness in a Murine Asthma Model.

β-Arrestin-2 (βarr2) is a ubiquitously expressed cytosolic protein that terminates G protein-coupled receptor signaling and transduces G protein-independent signaling. We previously showed that mice lacking βarr2 do not develop an asthma phenotype when sensitized to, and challenged with, allergens. The current study evaluates if an established asthma phenotype can be mitigated by deletion of βarr2 using an inducible Cre recombinase. We sensitized and challenged mice to ovalbumin (OVA) and demonstrated that on Day (d) 24 the allergic asthma phenotype was apparent in uninduced βarr2 and wild-type (WT) mice. In a second group of OVA-treated mice, tamoxifen was injected on d24 to d28 to activate Cre recombinase, and OVA aerosol challenge was continued through d44. The asthma phenotype was assessed using lung mechanics measurements, bronchoalveolar lavage cell analysis, and histological assessment of mucin and airway inflammation. Compared with their respective saline-treated controls, OVA-treated WT mice and mice expressing the inducible Cre recombinase displayed a significant asthma phenotype at d45. Whereas tamoxifen treatment had no significant effect on the asthma phenotype in WT mice, it inhibited βarr2 expression and caused a significant reduction in airway hyper-responsiveness (AHR) in Cre-inducible mice. These findings suggest that βarr2 is actively required for perpetuation of the AHR component of the allergic asthma phenotype. Our finding that βarr2 participates in the perpetuation of AHR in an asthma model means that targeting βarr2 may provide immediate and potentially long-term relief from daily asthma symptoms due to AHR irrespective of inflammation.

Authors
Chen, M; Hegde, A; Choi, YH; Theriot, BS; Premont, RT; Chen, W; Walker, JKL
MLA Citation
Chen, M, Hegde, A, Choi, YH, Theriot, BS, Premont, RT, Chen, W, and Walker, JKL. "Genetic Deletion of β-Arrestin-2 and the Mitigation of Established Airway Hyperresponsiveness in a Murine Asthma Model." American journal of respiratory cell and molecular biology 53.3 (September 2015): 346-354.
PMID
25569510
Source
epmc
Published In
American journal of respiratory cell and molecular biology
Volume
53
Issue
3
Publish Date
2015
Start Page
346
End Page
354
DOI
10.1165/rcmb.2014-0231oc

Structure-activity studies of Wnt/β-catenin inhibition in the Niclosamide chemotype: Identification of derivatives with improved drug exposure.

The Wnt signaling pathway plays a key role in regulation of organ development and tissue homeostasis. Dysregulated Wnt activity is one of the major underlying mechanisms responsible for many diseases including cancer. We previously reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling and suppresses colon cancer cell growth in vitro and in vivo. Niclosamide is a multi-functional drug that possesses important biological activity in addition to inhibition of Wnt/β-catenin signaling. Here, we studied the SAR of Wnt signaling inhibition in the anilide and salicylamide region of Niclosamide. We found that the 4'-nitro substituent can be effectively replaced by trifluoromethyl or chlorine and that the potency of inhibition was dependent on the substitution pattern in the anilide ring. Non-anilide, N-methyl amides and reverse amide derivatives lost significant potency, while acylated salicylamide derivatives inhibited signaling with potency similar to non-acyl derivatives. Niclosamide's low systemic exposure when dosed orally may hinder its use to treat systemic disease. To overcome this limitation we identified an acyl derivative of Niclosamide, DK-520 (compound 32), that significantly increased both the plasma concentration and the duration of exposure of Niclosamide when dosed orally. The studies herein provide a medicinal chemical foundation to improve the pharmacokinetic exposure of Niclosamide and Wnt-signaling inhibitors based on the Niclosamide chemotype. The identification of novel derivatives of Niclosamide that metabolize to Niclosamide and increase its drug exposure may provide important research tools for in vivo studies and provide drug candidates for treating cancers with dysregulated Wnt signaling including drug-resistant cancers. Moreover, since Niclosamide is a multi-functional drug, new research tools such as DK520 could directly result in novel treatments against bacterial and viral infection, lupus, and metabolic diseases such as type II diabetes, NASH and NAFLD.

Authors
Mook, RA; Wang, J; Ren, X-R; Chen, M; Spasojevic, I; Barak, LS; Lyerly, HK; Chen, W
MLA Citation
Mook, RA, Wang, J, Ren, X-R, Chen, M, Spasojevic, I, Barak, LS, Lyerly, HK, and Chen, W. "Structure-activity studies of Wnt/β-catenin inhibition in the Niclosamide chemotype: Identification of derivatives with improved drug exposure." Bioorganic & medicinal chemistry 23.17 (September 2015): 5829-5838.
PMID
26272032
Source
epmc
Published In
Bioorganic & Medicinal Chemistry
Volume
23
Issue
17
Publish Date
2015
Start Page
5829
End Page
5838
DOI
10.1016/j.bmc.2015.07.001

A metabolomic study of the PPARδ agonist GW501516 for enhancing running endurance in Kunming mice

Authors
Chen, W; Gao, R; Xie, X; Zheng, Z; Li, H; Li, S; Dong, F; Wang, L
MLA Citation
Chen, W, Gao, R, Xie, X, Zheng, Z, Li, H, Li, S, Dong, F, and Wang, L. "A metabolomic study of the PPARδ agonist GW501516 for enhancing running endurance in Kunming mice." Scientific Reports 5.1 (September 2015).
Source
crossref
Published In
Scientific Reports
Volume
5
Issue
1
Publish Date
2015
DOI
10.1038/srep09884

Preoperative Single-Fraction Partial Breast Radiation Therapy: A Novel Phase 1, Dose-Escalation Protocol With Radiation Response Biomarkers.

Women with biologically favorable early-stage breast cancer are increasingly treated with accelerated partial breast radiation (PBI). However, treatment-related morbidities have been linked to the large postoperative treatment volumes required for external beam PBI. Relative to external beam delivery, alternative PBI techniques require equipment that is not universally available. To address these issues, we designed a phase 1 trial utilizing widely available technology to 1) evaluate the safety of a single radiation treatment delivered preoperatively to the small-volume, intact breast tumor and 2) identify imaging and genomic markers of radiation response.Women aged ≥55 years with clinically node-negative, estrogen receptor-positive, and/or progesterone receptor-positive HER2-, T1 invasive carcinomas, or low- to intermediate-grade in situ disease ≤2 cm were enrolled (n=32). Intensity modulated radiation therapy was used to deliver 15 Gy (n=8), 18 Gy (n=8), or 21 Gy (n=16) to the tumor with a 1.5-cm margin. Lumpectomy was performed within 10 days. Paired pre- and postradiation magnetic resonance images and patient tumor samples were analyzed.No dose-limiting toxicity was observed. At a median follow-up of 23 months, there have been no recurrences. Physician-rated cosmetic outcomes were good/excellent, and chronic toxicities were grade 1 to 2 (fibrosis, hyperpigmentation) in patients receiving preoperative radiation only. Evidence of dose-dependent changes in vascular permeability, cell density, and expression of genes regulating immunity and cell death were seen in response to radiation.Preoperative single-dose radiation therapy to intact breast tumors is well tolerated. Radiation response is marked by early indicators of cell death in this biologically favorable patient cohort. This study represents a first step toward a novel partial breast radiation approach. Preoperative radiation should be tested in future clinical trials because it has the potential to challenge the current treatment paradigm and provide a path forward to identify radiation response biomarkers.

Authors
Horton, JK; Blitzblau, RC; Yoo, S; Geradts, J; Chang, Z; Baker, JA; Georgiade, GS; Chen, W; Siamakpour-Reihani, S; Wang, C; Broadwater, G; Groth, J; Palta, M; Dewhirst, M; Barry, WT; Duffy, EA; Chi, J-TA; Hwang, ES
MLA Citation
Horton, JK, Blitzblau, RC, Yoo, S, Geradts, J, Chang, Z, Baker, JA, Georgiade, GS, Chen, W, Siamakpour-Reihani, S, Wang, C, Broadwater, G, Groth, J, Palta, M, Dewhirst, M, Barry, WT, Duffy, EA, Chi, J-TA, and Hwang, ES. "Preoperative Single-Fraction Partial Breast Radiation Therapy: A Novel Phase 1, Dose-Escalation Protocol With Radiation Response Biomarkers." International journal of radiation oncology, biology, physics 92.4 (July 2015): 846-855.
PMID
26104938
Source
epmc
Published In
International Journal of Radiation Oncology, Biology, Physics
Volume
92
Issue
4
Publish Date
2015
Start Page
846
End Page
855
DOI
10.1016/j.ijrobp.2015.03.007

Differential Angiogenic Gene Expression in TP53 Wild-Type and Mutant Ovarian Cancer Cell Lines.

Underlying mechanisms regulating angiogenesis in ovarian cancer have not been completely elucidated. Evidence suggests that the TP53 tumor suppressor pathway and tumor microenvironment play integral roles. We utilized microarray technology to study the interaction between TP53 mutational status and hypoxia on angiogenic gene expression.Affymetrix U133A arrays were analyzed for angiogenic gene expression in 19 ovarian cancer cell lines stratified both by TP53 mutation status and A2780 wild-type (wt) TP53 vs. mutated (m) TP53 cell lines after treatment under hypoxic conditions or with ionizing radiation.Twenty-eight differentially expressed angiogenic genes were identified in the mTP53 cell lines compared to wtTP53 lines. Five genes were upregulated in mTP53 cells: 40% involved in extracellular matrix (ECM) degradation [matrix metalloproteinase 10 (MMP10)/15] and 60% in angiogenesis (fibroblast growth factor receptor 3/VEGFA/ephrin receptor-B4). Twenty-three genes were upregulated in wtTP53: nearly 22% were ECM constituents or involved in ECM degradation; over 40% were growth factors or mediators of angiogenesis. Five genes were upregulated in the A2780mTP53 cells: 40% involved in ECM remodeling (MMP10, ADAMTS1), 40% with pro-angiogenic activity (EFNB2, factor 2 receptor), and 20% with anti-angiogenic properties (ADAMTS1). Three genes were upregulated in hypoxia treated cells compared to controls: one with anti-angiogenic activity (angiopoietin-like 4) and two with pro-angiogenic activity (VEGFA, EFNA3). No significant gene fold changes were noted after exposure to radiation. Four genes continued to demonstrate significant differential expression (p ≤ 0.05) after adjusting for multiple comparisons. These genes included endoglin upregulation in wt lines (pro-angiogenesis) and upregulation of FGF20 (growth factor), ADAMTS1 (anti-angiogenesis) and MMP10 (ECM degradation) in mTP53 cell lines.Our exploratory findings indicate that non-overlapping angiogenic pathways may be altered by TP53 mutations and hypoxic conditions in the tumor microenvironment. Further evaluation is needed for confirmation.

Authors
Davidson, BA; Rubatt, JM; Corcoran, DL; Teoh, DK; Bernardini, MQ; Grace, LA; Soper, WJ; Berchuck, A; Siamakpour-Reihani, S; Chen, W; Owzar, K; Murphy, SK; Secord, AA
MLA Citation
Davidson, BA, Rubatt, JM, Corcoran, DL, Teoh, DK, Bernardini, MQ, Grace, LA, Soper, WJ, Berchuck, A, Siamakpour-Reihani, S, Chen, W, Owzar, K, Murphy, SK, and Secord, AA. "Differential Angiogenic Gene Expression in TP53 Wild-Type and Mutant Ovarian Cancer Cell Lines." Frontiers in oncology 4 (January 2014): 163-.
PMID
24999452
Source
epmc
Published In
Frontiers in Oncology
Volume
4
Publish Date
2014
Start Page
163
DOI
10.3389/fonc.2014.00163

Triphenylmethane dye activation of beta-arrestin.

β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy.

Authors
Barak, LS; Bai, Y; Snyder, JC; Wang, J; Chen, W; Caron, MG
MLA Citation
Barak, LS, Bai, Y, Snyder, JC, Wang, J, Chen, W, and Caron, MG. "Triphenylmethane dye activation of beta-arrestin." Biochemistry 52.32 (August 2013): 5403-5414.
PMID
23865508
Source
epmc
Published In
Biochemistry
Volume
52
Issue
32
Publish Date
2013
Start Page
5403
End Page
5414
DOI
10.1021/bi400217r

Small molecule modulators of Wnt/β-catenin signaling.

The Wnt signal transduction pathway is dysregulated in many highly prevalent diseases, including cancer. Unfortunately, drug discovery efforts have been hampered by the paucity of targets and drug-like lead molecules amenable to drug discovery. Recently, we reported the FDA-approved anthelmintic drug Niclosamide inhibits Wnt/β-catenin signaling by a unique mechanism, though the target responsible remains unknown. We interrogated the mechanism and structure-activity relationships to understand drivers of potency and to assist target identification efforts. We found inhibition of Wnt signaling by Niclosamide appears unique among the structurally-related anthelmintic agents tested and found the potency and functional response was dependent on small changes in the chemical structure of Niclosamide. Overall, these findings support efforts to identify the target of Niclosamide inhibition of Wnt/β-catenin signaling and the discovery of potent and selective modulators to treat human disease.

Authors
Mook, RA; Chen, M; Lu, J; Barak, LS; Lyerly, HK; Chen, W
MLA Citation
Mook, RA, Chen, M, Lu, J, Barak, LS, Lyerly, HK, and Chen, W. "Small molecule modulators of Wnt/β-catenin signaling." Bioorg Med Chem Lett 23.7 (April 1, 2013): 2187-2191.
PMID
23453073
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry Letters
Volume
23
Issue
7
Publish Date
2013
Start Page
2187
End Page
2191
DOI
10.1016/j.bmcl.2013.01.101

MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro

Authors
Fang, S; Crews, AL; Chen, W; Park, J; Yin, Q; Ren, X-R; Adler, KB
MLA Citation
Fang, S, Crews, AL, Chen, W, Park, J, Yin, Q, Ren, X-R, and Adler, KB. "MARCKS and HSP70 interactions regulate mucin secretion by human airway epithelial cells in vitro." AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY 304.8 (April 2013): L511-L518.
PMID
23377348
Source
wos-lite
Published In
American journal of physiology. Lung cellular and molecular physiology
Volume
304
Issue
8
Publish Date
2013
Start Page
L511
End Page
L518
DOI
10.1152/ajplung.00337.2012

Correction: phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion.

Authors
Hamilton, E; Blackwell, K; Hobeika, AC; Clay, TM; Broadwater, G; Ren, XR; Chen, W; Castro, H; Lehmann, F; Spector, N; Wei, J; Osada, T; Lyerly, HK; Morse, MA
MLA Citation
Hamilton, E, Blackwell, K, Hobeika, AC, Clay, TM, Broadwater, G, Ren, XR, Chen, W, Castro, H, Lehmann, F, Spector, N, Wei, J, Osada, T, Lyerly, HK, and Morse, MA. "Correction: phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibtion." J Transl Med 11 (2013): 82-.
PMID
23536971
Source
pubmed
Published In
Journal of Translational Medicine
Volume
11
Publish Date
2013
Start Page
82
DOI
10.1186/1479-5876-11-82

Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1.

Smoothened (Smo) mediated Hedgehog (Hh) signaling plays an essential role in regulating embryonic development and postnatal tissue homeostasis. Aberrant activation of the Hh pathway contributes to the formation and progression of various cancers. In vertebrates, however, key regulatory mechanisms responsible for transducing signals from Smo to the nucleus remain to be delineated. Here, we report the identification of Myc-interacting Zinc finger protein 1 (Miz1) as a Smo and Gli2 binding protein that positively regulates Hh signaling. Overexpression of Miz1 increases Gli luciferase reporter activity, whereas knockdown of endogenous Miz1 has the opposite effect. Activation of Smo induces translocation of Miz1 to the primary cilia together with Smo and Gli2. Furthermore, Miz1 is localized to the nucleus upon Hh activation in a Smo-dependent manner, and loss of Miz1 prevents the nuclear translocation of Gli2. More importantly, silencing Miz1 expression inhibits cell proliferation in vitro and the growth of Hh-driven medulloblastoma tumors allografted in SCID mice. Taken together, these results identify Miz1 as a novel regulator in the Hh pathway that plays an important role in mediating Smo-dependent oncogenic signaling.

Authors
Lu, J; Chen, M; Ren, X-R; Wang, J; Lyerly, HK; Barak, L; Chen, W
MLA Citation
Lu, J, Chen, M, Ren, X-R, Wang, J, Lyerly, HK, Barak, L, and Chen, W. "Regulation of hedgehog signaling by Myc-interacting zinc finger protein 1, Miz1. (Published online)" PLoS One 8.5 (2013): e63353-.
PMID
23671675
Source
pubmed
Published In
PloS one
Volume
8
Issue
5
Publish Date
2013
Start Page
e63353
DOI
10.1371/journal.pone.0063353

Cadmium is a potent inhibitor of PPM phosphatases and targets the M1 binding site

The heavy metal cadmium is a non-degradable pollutant. By screening the effects of a panel of metal ions on the phosphatase activity, we unexpectedly identified cadmium as a potent inhibitor of PPM1A and PPM1G. In contrast, low micromolar concentrations of cadmium did not inhibit PP1 or tyrosine phosphatases. Kinetic studies revealed that cadmium inhibits PPM phosphatases through the M1 metal ion binding site. In particular, the negative charged D441 in PPM1G specific recognized cadmium. Our results suggest that cadmium is likely a potent inhibitor of most PPM family members except for PHLPPs. Furthermore, we demonstrated that cadmium inhibits PPM1A-regulated MAPK signaling and PPM1G-regulated AKT signaling potently in vivo. Cadmium reversed PPM1A-induced cell cycle arrest and cadmium insensitive PPM1A mutant rescued cadmium induced cell death. Taken together, these findings provide a better understanding of the effects of the toxicity of cadmium in the contexts of human physiology and pathology.

Authors
Pan, C; Liu, H-D; Gong, Z; Yu, X; Hou, X-B; Xie, D-D; Zhu, X-B; Li, H-W; Tang, J-Y; Xu, Y-F; Yu, J-Q; Zhang, L-Y; Fang, H; Xiao, K-H; Chen, Y-G; Wang, J-Y; Pang, Q; Chen, W; Sun, J-P
MLA Citation
Pan, C, Liu, H-D, Gong, Z, Yu, X, Hou, X-B, Xie, D-D, Zhu, X-B, Li, H-W, Tang, J-Y, Xu, Y-F, Yu, J-Q, Zhang, L-Y, Fang, H, Xiao, K-H, Chen, Y-G, Wang, J-Y, Pang, Q, Chen, W, and Sun, J-P. "Cadmium is a potent inhibitor of PPM phosphatases and targets the M1 binding site." Scientific Reports 3 (2013).
PMID
23903585
Source
scival
Published In
Scientific Reports
Volume
3
Publish Date
2013
DOI
10.1038/srep02333

Identification of a novel Smoothened antagonist that potently suppresses Hedgehog signaling.

The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of β-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat Hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma.

Authors
Wang, J; Mook, RA; Lu, J; Gooden, DM; Ribeiro, A; Guo, A; Barak, LS; Lyerly, HK; Chen, W
MLA Citation
Wang, J, Mook, RA, Lu, J, Gooden, DM, Ribeiro, A, Guo, A, Barak, LS, Lyerly, HK, and Chen, W. "Identification of a novel Smoothened antagonist that potently suppresses Hedgehog signaling." Bioorg Med Chem 20.22 (November 15, 2012): 6751-6757.
PMID
23063522
Source
pubmed
Published In
Bioorganic & Medicinal Chemistry
Volume
20
Issue
22
Publish Date
2012
Start Page
6751
End Page
6757
DOI
10.1016/j.bmc.2012.09.030

Identification of Nedd4 as a novel regulator in Hedgehog signaling

Authors
Qing-feng, L; Wei, C; Shu-tian, Z
MLA Citation
Qing-feng, L, Wei, C, and Shu-tian, Z. "Identification of Nedd4 as a novel regulator in Hedgehog signaling." CHINESE MEDICAL JOURNAL 125.21 (November 5, 2012): 3851-3855.
PMID
23106887
Source
wos-lite
Published In
Chinese medical journal
Volume
125
Issue
21
Publish Date
2012
Start Page
3851
End Page
3855
DOI
10.3760/cma.j.issn.0366-6999.2012.21.020

The insecticide synergist piperonyl butoxide inhibits hedgehog signaling: assessing chemical risks.

The spread of chemicals, including insecticides, into the environment often raises public health concerns, as exemplified by a recent epidemiologic study associating in utero piperonyl butoxide (PBO) exposure with delayed mental development. The insecticide synergist PBO is listed among the top 10 chemicals detected in indoor dust; a systematic assessment of risks from PBO exposure, as for many toxicants unfortunately, may be underdeveloped when important biological targets that can cause toxicity are unknown. Hedgehog/Smoothened signaling is critical in neurological development. This study was designed to use novel high-throughput in vitro drug screening technology to identify modulators of Hedgehog signaling in environmental chemicals to assist the assessment of their potential risks. A directed library of 1408 environmental toxicants was screened for Hedgehog/Smoothened antagonist activity using a high-content assay that evaluated the interaction between Smoothened and βarrestin2 green fluorescent protein. PBO was identified as a Hedgehog/Smoothened antagonist capable of inhibiting Hedgehog signaling. We found that PBO bound Smoothened and blocked Smoothened overexpression-induced Gli-luciferase reporter activity but had no effect on Gli-1 downstream transcriptional factor-induced Gli activity. PBO inhibited Sonic Hedgehog ligand-induced Gli signaling and mouse cerebellar granular precursor cell proliferation. Moreover, PBO disrupted zebrafish development. Our findings demonstrate the value of high-throughput target-based screening strategies that can successfully evaluate large numbers of environmental toxicants and identify key targets and unknown biological activity that is helpful in properly assessing potential risks.

Authors
Wang, J; Lu, J; Mook, RA; Zhang, M; Zhao, S; Barak, LS; Freedman, JH; Lyerly, HK; Chen, W
MLA Citation
Wang, J, Lu, J, Mook, RA, Zhang, M, Zhao, S, Barak, LS, Freedman, JH, Lyerly, HK, and Chen, W. "The insecticide synergist piperonyl butoxide inhibits hedgehog signaling: assessing chemical risks." Toxicol Sci 128.2 (August 2012): 517-523.
PMID
22552772
Source
pubmed
Published In
Toxicological Sciences (Elsevier)
Volume
128
Issue
2
Publish Date
2012
Start Page
517
End Page
523
DOI
10.1093/toxsci/kfs165

Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells.

INTRODUCTION: Sustained HER2 signaling at the cell surface is an oncogenic mechanism in a significant proportion of breast cancers. While clinically effective therapies targeting HER2 such as mAbs and tyrosine kinase inhibitors exist, tumors overexpressing HER2 eventually progress despite treatment. Thus, abrogation of persistent HER2 expression at the plasma membrane to synergize with current approaches may represent a novel therapeutic strategy. METHODS: We generated polyclonal anti-HER2 antibodies (HER2-VIA) by vaccinating mice with an adenovirus expressing human HER2, and assessed their signaling effects in vitro and anti-tumor effects in a xenograft model. In addition, we studied the signaling effects of human HER2-specific antibodies induced by vaccinating breast cancer patients with a HER2 protein vaccine. RESULTS: HER2-VIA bound HER2 at the plasma membrane, initially activating the downstream kinases extracellular signal-regulated protein kinase 1/2 and Akt, but subsequently inducing receptor internalization in clathrin-coated pits in a HER2 kinase-independent manner, followed by ubiquitination and degradation of HER2 into a 130 kDa fragment phosphorylated at tyrosine residues 1,221/1,222 and 1,248. Following vaccination of breast cancer patients with the HER2 protein vaccine, HER2-specific antibodies were detectable and these antibodies bound to cell surface-expressed HER2 and inhibited HER2 signaling through blocking tyrosine 877 phosphorylation of HER2. In contrast to the murine antibodies, human anti-HER2 antibodies induced by protein vaccination did not mediate receptor internalization and degradation. CONCLUSION: These data provide new insight into HER2 trafficking at the plasma membrane and the changes induced by polyclonal HER2-specific antibodies. The reduction of HER2 membrane expression and HER2 signaling by polyclonal antibodies induced by adenoviral HER2 vaccines supports human clinical trials with this strategy for those breast cancer patients with HER2 therapy-resistant disease.

Authors
Ren, X-R; Wei, J; Lei, G; Wang, J; Lu, J; Xia, W; Spector, N; Barak, LS; Clay, TM; Osada, T; Hamilton, E; Blackwell, K; Hobeika, AC; Morse, MA; Lyerly, HK; Chen, W
MLA Citation
Ren, X-R, Wei, J, Lei, G, Wang, J, Lu, J, Xia, W, Spector, N, Barak, LS, Clay, TM, Osada, T, Hamilton, E, Blackwell, K, Hobeika, AC, Morse, MA, Lyerly, HK, and Chen, W. "Polyclonal HER2-specific antibodies induced by vaccination mediate receptor internalization and degradation in tumor cells. (Published online)" Breast Cancer Res 14.3 (June 7, 2012): R89-.
PMID
22676470
Source
pubmed
Published In
Breast Cancer Research
Volume
14
Issue
3
Publish Date
2012
Start Page
R89
DOI
10.1186/bcr3204

Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition [corrected].

BACKGROUND: Patients with HER2-overexpressing metastatic breast cancer, despite initially benefiting from the monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib, will eventually have progressive disease. HER2-based vaccines induce polyclonal antibody responses against HER2 that demonstrate enhanced anti-tumor activity when combined with lapatinib in murine models. We wished to test the clinical safety, immunogenicity, and activity of a HER2-based cancer vaccine, when combined with lapatinib. METHODS: We immunized women (n = 12) with metastatic, trastuzumab-refractory, HER2-overexpressing breast cancer with dHER2, a recombinant protein consisting of extracellular domain (ECD) and a portion of the intracellular domain (ICD) of HER2 combined with the adjuvant AS15, containing MPL, QS21, CpG and liposome. Lapatinib (1250 mg/day) was administered concurrently. Peripheral blood antibody and T cell responses were measured. RESULTS: This regimen was well tolerated, with no cardiotoxicity. Anti-HER2-specific antibody was induced in all patients whereas HER2-specific T cells were detected in one patient. Preliminary analyses of patient serum demonstrated downstream signaling inhibition in HER2 expressing tumor cells. The median time to progression was 55 days, with the majority of patients progressing prior to induction of peak anti-HER2 immune responses; however, 300-day overall survival was 92% (95% CI: 77-100%). CONCLUSIONS: dHER2 combined with lapatinib was safe and immunogenic with promising long term survival in those with HER2-overexpressing breast cancers refractory to trastuzumab. Further studies to define the anticancer activity of the antibodies induced by HER2 vaccines along with lapatinib are underway. TRIAL REGISTRY: ClinicalTrials.gov NCT00952692.

Authors
Hamilton, E; Blackwell, K; Hobeika, AC; Clay, TM; Broadwater, G; Ren, X-R; Chen, W; Castro, H; Lehmann, F; Spector, N; Wei, J; Osada, T; Lyerly, HK; Morse, MA
MLA Citation
Hamilton, E, Blackwell, K, Hobeika, AC, Clay, TM, Broadwater, G, Ren, X-R, Chen, W, Castro, H, Lehmann, F, Spector, N, Wei, J, Osada, T, Lyerly, HK, and Morse, MA. "Phase 1 clinical trial of HER2-specific immunotherapy with concomitant HER2 kinase inhibition [corrected]. (Published online)" J Transl Med 10 (February 10, 2012): 28-.
PMID
22325452
Source
pubmed
Published In
Journal of Translational Medicine
Volume
10
Publish Date
2012
Start Page
28
DOI
10.1186/1479-5876-10-28

Growth Arrest Specific 8 (Gas8) and G protein-coupled receptor kinase 2 (GRK2) cooperate in the control of Smoothened signaling.

The G protein-coupled receptor (GPCR)-like molecule Smoothened (Smo) undergoes dynamic intracellular trafficking modulated by the microtubule associated kinase GRK2 and recruitment of β-arrestin. Of this trafficking, especially the translocation of Smo into primary cilia and back to the cytoplasm is essential for the activation of Hedgehog (Hh) signaling in vertebrates. The complete mechanism of this bidirectional transport, however, is not completely understood. Here we demonstrate that Growth Arrest Specific 8 (Gas8), a microtubule associated subunit of the Dynein Regulatory Complex (DRC), interacts with Smo to modulate this process. Gas8 knockdown in ciliated cells reduces Smo signaling activity and ciliary localization whereas overexpression stimulates Smo activity in a GRK2-dependent manner. The C terminus of Gas8 is important for both Gas8 interaction with Smo and facilitating Smo signaling. In zebrafish, knocking down Gas8 results in attenuated Hh transcriptional responses and impaired early muscle development. These effects can be reversed by the co-injection of Gas8 mRNA or by constitutive activation of the downstream Gli transcription factors. Furthermore, Gas8 and GRK2 display a synergistic effect on zebrafish early muscle development and some effects of GRK2 knockdown can be rescued by Gas8 mRNA. Interestingly, Gas8 does not interfere with cilia assembly, as the primary cilia architecture is unchanged upon Gas8 knock down or heterologous expression. This is in contrast to cells stably expressing both GRK2 and Smo, in which cilia are significantly elongated. These results identify Gas8 as a positive regulator of Hh signaling that cooperates with GRK2 to control Smo signaling.

Authors
Evron, T; Philipp, M; Lu, J; Meloni, AR; Burkhalter, M; Chen, W; Caron, MG
MLA Citation
Evron, T, Philipp, M, Lu, J, Meloni, AR, Burkhalter, M, Chen, W, and Caron, MG. "Growth Arrest Specific 8 (Gas8) and G protein-coupled receptor kinase 2 (GRK2) cooperate in the control of Smoothened signaling." J Biol Chem 286.31 (August 5, 2011): 27676-27686.
PMID
21659505
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
31
Publish Date
2011
Start Page
27676
End Page
27686
DOI
10.1074/jbc.M111.234666

Increased production of sonic hedgehog by ballooned hepatocytes.

Ballooned hepatocytes distinguish non-alcoholic steatohepatitis (NASH) from steatosis. Such cells contain dilated endoplasmic reticulum and ubiquitin aggregates, characteristics of endoplasmic reticulum stress. Hepatocyte ballooning increases the risk for fibrosis in NASH, suggesting that ballooned hepatocytes release pro-fibrogenic factors. Hedgehog ligands function as pro-fibrogenic factors in liver diseases, but mechanisms for hedgehog ligand production remain poorly understood. We evaluated the hypothesis that endoplasmic reticulum stress induces hepatocyte production of hedgehog ligands that provide paracrine pro-fibrogenic signals to neighbouring cells. In livers from NASH patients, keratin 8/18 and ubiquitin staining demonstrated enlarged, keratin 8/18-negative/ubiquitin-positive hepatocytes (ballooned hepatocytes) that were positive for Sonic hedgehog. In order to model endoplasmic reticulum stress in vitro, primary mouse hepatocytes were treated with tunicamycin. Compared to vehicle, tunicamycin significantly increased Sonic hedgehog and Indian hedgehog expression. Furthermore, conditioned medium from tunicamycin-treated hepatocytes increased Gli-luciferase reporter activity 14-fold more than conditioned medium from vehicle-treated hepatocytes. Cyclopamine (hedgehog signalling inhibitor) abrogated the effect of conditioned medium from tunicamycin-treated hepatocytes, verifying that soluble hepatocyte-derived factors activate hedgehog signalling. Ballooned hepatocytes in NASH patients did not express the hedgehog target gene, Gli2, α-smooth muscle actin or vimentin, but were surrounded by Gli2-positive stromal cells expressing these myofibroblast markers. Trichrome staining demonstrated the accumulation of ballooned hepatocytes in areas of matrix deposition, and numbers of Sonic hedgehog-positive hepatocytes correlated with the degree of ballooning and fibrosis stage. Hepatocytes undergoing endoplasmic reticiulum stress generate hedgehog ligands which act as paracrine pro-fibrogenic factors for hedgehog-responsive stromal cells. These results help to explain why fibrosis stage correlates with hepatocyte ballooning in NASH.

Authors
Rangwala, F; Guy, CD; Lu, J; Suzuki, A; Burchette, JL; Abdelmalek, MF; Chen, W; Diehl, AM
MLA Citation
Rangwala, F, Guy, CD, Lu, J, Suzuki, A, Burchette, JL, Abdelmalek, MF, Chen, W, and Diehl, AM. "Increased production of sonic hedgehog by ballooned hepatocytes." J Pathol 224.3 (July 2011): 401-410.
PMID
21547909
Source
pubmed
Published In
The Journal of Pathology
Volume
224
Issue
3
Publish Date
2011
Start Page
401
End Page
410
DOI
10.1002/path.2888

Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations.

Wnt/β-catenin pathway activation caused by adenomatous polyposis coli (APC) mutations occurs in approximately 80% of sporadic colorectal cancers (CRC). The antihelminth compound niclosamide downregulates components of the Wnt pathway, specifically Dishevelled-2 (Dvl2) expression, resulting in diminished downstream β-catenin signaling. In this study, we determined whether niclosamide could inhibit the Wnt/β-catenin pathway in human CRCs and whether its inhibition might elicit antitumor effects in the presence of APC mutations. We found that niclosamide inhibited Wnt/β-catenin pathway activation, downregulated Dvl2, decreased downstream β-catenin signaling, and exerted antiproliferative effects in human colon cancer cell lines and CRC cells isolated by surgical resection of metastatic disease, regardless of mutations in APC. In contrast, inhibition of NF-κB or mTOR did not exert similar antiproliferative effects in these CRC model systems. In mice implanted with human CRC xenografts, orally administered niclosamide was well tolerated, achieved plasma and tumor levels associated with biologic activity, and led to tumor control. Our findings support clinical explorations to reposition niclosamide for the treatment of CRC.

Authors
Osada, T; Chen, M; Yang, XY; Spasojevic, I; Vandeusen, JB; Hsu, D; Clary, BM; Clay, TM; Chen, W; Morse, MA; Lyerly, HK
MLA Citation
Osada, T, Chen, M, Yang, XY, Spasojevic, I, Vandeusen, JB, Hsu, D, Clary, BM, Clay, TM, Chen, W, Morse, MA, and Lyerly, HK. "Antihelminth compound niclosamide downregulates Wnt signaling and elicits antitumor responses in tumors with activating APC mutations." Cancer Res 71.12 (June 15, 2011): 4172-4182.
PMID
21531761
Source
pubmed
Published In
Cancer Research
Volume
71
Issue
12
Publish Date
2011
Start Page
4172
End Page
4182
DOI
10.1158/0008-5472.CAN-10-3978

Paracrine modulation of cholangiocyte serotonin synthesis orchestrates biliary remodeling in adults.

Paracrine signaling between cholangiocytes and stromal cells regulates biliary remodeling. Cholangiocytes have neuroepithelial characteristics and serotonin receptor agonists inhibit their growth, but whether they are capable of serotonin biosynthesis is unknown. We hypothesized that cholangiocytes synthesize serotonin and that cross talk between liver myofibroblasts (MF) and cholangiocytes regulates this process to influence biliary remodeling. Transwell cultures of cholangiocytes ± MF, and tryptophan hydroxylase-2 knockin (TPH2KI) mice with an inactivating mutation of the neuronal tryptophan hydroxylase (TPH) isoform, TPH2, were evaluated. Results in the cell culture models confirm that cholangiocytes have serotonin receptors and demonstrate for the first time that these cells express TPH2 and produce serotonin, which autoinhibits their growth but stimulates MF production of TGF-β(1). Increased TGF-β(1), in turn, counteracts autocrine inhibition of cholangiocyte growth by repressing cholangiocyte TPH2 expression. Studies of TPH2KI mice confirm that TPH2-mediated production of serotonin plays an important role in remodeling damaged bile ducts because mice with decreased TPH2 function have reduced biliary serotonin levels and exhibit excessive cholangiocyte proliferation, accumulation of aberrant ductules and liver progenitors, and increased liver fibrosis after bile duct ligation. This new evidence that cholangiocytes express the so-called neuronal isoform of TPH, synthesize serotonin de novo, and deploy serotonin as an autocrine/paracrine signal to regulate regeneration of the biliary tree complements earlier work that revealed that passive release of serotonin from platelets stimulates hepatocyte proliferation. Given the prevalent use of serotonin-modulating drugs, these findings have potentially important implications for recovery from various types of liver damage.

Authors
Omenetti, A; Yang, L; Gainetdinov, RR; Guy, CD; Choi, SS; Chen, W; Caron, MG; Diehl, AM
MLA Citation
Omenetti, A, Yang, L, Gainetdinov, RR, Guy, CD, Choi, SS, Chen, W, Caron, MG, and Diehl, AM. "Paracrine modulation of cholangiocyte serotonin synthesis orchestrates biliary remodeling in adults." Am J Physiol Gastrointest Liver Physiol 300.2 (February 2011): G303-G315.
PMID
21071507
Source
pubmed
Published In
American journal of physiology. Gastrointestinal and liver physiology
Volume
300
Issue
2
Publish Date
2011
Start Page
G303
End Page
G315
DOI
10.1152/ajpgi.00368.2010

Glucocorticoid hedgehog agonists in neurogenesis.

The process of neurogenesis in mammals, which is prolific and widespread at birth, gradually slows with aging and in humans becomes restricted to areas including the cerebellum and hippocampus. It has been reported that exposure to glucocorticoids can impair neurogenesis in both adults and children. Glucocorticoids are known to bind with high affinity to intracellular receptors. Glucocorticoid blood levels are normally regulated by environmental stresses, but because of their clinical utility, exogenous glucocorticoids are frequently administered in drug formulations. Consequently, concerns have arisen about the consequences of glucocorticoid use on neurogenesis and health, especially in the pediatric population. In this article, we will review recent findings that a select number of related glucocorticoids, halcinonide, fluticasone propionate, clobetasol propionate, and fluocinonide, also bind the hedgehog pathway receptor Smoothened. We will discuss their pharmacology and also a most surprising result; that this select group of compounds, which includes FDA approved drugs, unlike typical glucocorticoids such as dexamethasone, stimulate stem cell growth, and thus enhance neurogenesis.

Authors
Wang, J; Barak, LS; Mook, RA; Chen, W
MLA Citation
Wang, J, Barak, LS, Mook, RA, and Chen, W. "Glucocorticoid hedgehog agonists in neurogenesis." Vitam Horm 87 (2011): 207-215. (Review)
PMID
22127244
Source
pubmed
Published In
Vitamins and hormones
Volume
87
Publish Date
2011
Start Page
207
End Page
215
DOI
10.1016/B978-0-12-386015-6.00030-5

Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress

Many environmental stimuli, including light, biotic and abiotic stress factors, induce changes in cellular Ca 2+ concentrations in plants. Such Ca 2+ signatures are perceived by sensor molecules such as calcineurin B-like (CBL) proteins. AtCBL1, a member of the CBL family which is highly inducible by multiple stress signals, is known to function in the salt stress signal transduction pathway and to positively regulate the plant tolerance to salt. To shed light into the molecular mechanisms of the salt stress response mediated by AtCBL1, a two-dimensional DIGE proteomic approach was applied to identify the differentially expressed proteins in Arabibdopsis wild-type and cbl1 null mutant plants in response to salt stress. Seventy-three spots were found altered in expression by least 1.2-fold and 50 proteins were identified by MALDI-TOF/TOF-MS, including some well-known and novel salt-responsive proteins. These proteins function in various processes, such as signal transduction, ROS scavenging, energy production, carbon fixation, metabolism, mRNA processing, protein processing and structural stability. Receptor for activated C kinase 1C (RACK1C, spot 715), a WD40 repeat protein, was up-regulated in the cbl1 null mutant, and two rack1c mutant lines showed decreased tolerance to salt stress, suggesting that RACK1C plays a role in salt stress resistance. In conclusion, our work demonstrated the advantages of the proteomic approach in studies of plant biology and identified candidate proteins in CBL1-mediated salt stress signaling network. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Authors
Shi, S; Chen, W; Sun, W
MLA Citation
Shi, S, Chen, W, and Sun, W. "Comparative proteomic analysis of the Arabidopsis cbl1 mutant in response to salt stress." Proteomics 11.24 (2011): 4712-4725.
Source
scival
Published In
Proteomics
Volume
11
Issue
24
Publish Date
2011
Start Page
4712
End Page
4725
DOI
10.1002/pmic.201100042

Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of extracellular signal-regulated kinase and β-arrestin2 with antinociceptive activity.

Known agonists of the orphan receptor GPR35 are kynurenic acid, zaprinast, 5-nitro-2-(3-phenylproplyamino) benzoic acid, and lysophosphatidic acids. Their relatively low affinities for GPR35 and prominent off-target effects at other pathways, however, diminish their utility for understanding GPR35 signaling and for identifying potential therapeutic uses of GPR35. In a screen of the Prestwick Library of drugs and drug-like compounds, we have found that pamoic acid is a potent GPR35 agonist. Pamoic acid is considered by the Food and Drug Administration as an inactive compound that enables long-acting formulations of numerous drugs, such as the antihelminthics oxantel pamoate and pyrantel pamoate; the psychoactive compounds hydroxyzine pamoate (Vistaril) and imipramine pamoate (Tofranil-PM); and the peptide hormones triptorelin pamoate (Trelstar) and octreotide pamoate (OncoLar). We have found that pamoic acid induces a G(i/o)-linked, GPR35-mediated increase in the phosphorylation of extracellular signal-regulated kinase 1/2, recruitment of β-arrestin2 to GPR35, and internalization of GPR35. In mice, it attenuates visceral pain perception, indicating an antinociceptive effect, possibly through GPR35 receptors. We have also identified in collaboration with the Sanford-Burnham Institute Molecular Libraries Probe Production Center new classes of GPR35 antagonist compounds, including the nanomolar potency antagonist methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID2745687). Pamoic acid and potent antagonists such as CID2745687 present novel opportunities for expanding the chemical space of GPR35, elucidating GPR35 pharmacology, and stimulating GPR35-associated drug development. Our results indicate that the unexpected biological functions of pamoic acid may yield potential new uses for a common drug constituent.

Authors
Zhao, P; Sharir, H; Kapur, A; Cowan, A; Geller, EB; Adler, MW; Seltzman, HH; Reggio, PH; Heynen-Genel, S; Sauer, M; Chung, TDY; Bai, Y; Chen, W; Caron, MG; Barak, LS; Abood, ME
MLA Citation
Zhao, P, Sharir, H, Kapur, A, Cowan, A, Geller, EB, Adler, MW, Seltzman, HH, Reggio, PH, Heynen-Genel, S, Sauer, M, Chung, TDY, Bai, Y, Chen, W, Caron, MG, Barak, LS, and Abood, ME. "Targeting of the orphan receptor GPR35 by pamoic acid: a potent activator of extracellular signal-regulated kinase and β-arrestin2 with antinociceptive activity." Mol Pharmacol 78.4 (October 2010): 560-568.
PMID
20826425
Source
pubmed
Published In
Molecular pharmacology
Volume
78
Issue
4
Publish Date
2010
Start Page
560
End Page
568
DOI
10.1124/mol.110.066746

Development of small molecules targeting the Wnt pathway for the treatment of colon cancer: a high-throughput screening approach.

Wnt proteins play major roles in development and differentiation, and abnormalities in their regulation are believed to contribute to the formation of many cancers, including colorectal malignancies. As a result, there has been an interest in identifying small molecule inhibitors of Wnt signaling as tool compounds for research or as precursors to new generations of anticancer drugs. Advancements in robotic technology along with reductions in the costs of equipment, chemical libraries, and information handling have made high-throughput drug discovery programs possible in an academic setting. In this minireview we discuss the most plausible protein targets for inhibiting Wnt signaling in colon cancer therapy, list small molecule Wnt inhibitors that have been identified through recent drug discovery efforts, and provide our laboratory's strategy for identifying novel Wnt signaling antagonists using high-throughput screening. In particular, we summarize the results of a screen of over 1,200 drug and druglike compounds we recently completed in which niclosamide was identified as a Wnt pathway antagonist.

Authors
Chen, W; Chen, M; Barak, LS
MLA Citation
Chen, W, Chen, M, and Barak, LS. "Development of small molecules targeting the Wnt pathway for the treatment of colon cancer: a high-throughput screening approach." Am J Physiol Gastrointest Liver Physiol 299.2 (August 2010): G293-G300. (Review)
PMID
20508156
Source
pubmed
Published In
American journal of physiology. Gastrointestinal and liver physiology
Volume
299
Issue
2
Publish Date
2010
Start Page
G293
End Page
G300
DOI
10.1152/ajpgi.00005.2010

Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo.

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.

Authors
Morse, MA; Wei, J; Hartman, Z; Xia, W; Ren, X-R; Lei, G; Barry, WT; Osada, T; Hobeika, AC; Peplinski, S; Jiang, H; Devi, GR; Chen, W; Spector, N; Amalfitano, A; Lyerly, HK; Clay, TM
MLA Citation
Morse, MA, Wei, J, Hartman, Z, Xia, W, Ren, X-R, Lei, G, Barry, WT, Osada, T, Hobeika, AC, Peplinski, S, Jiang, H, Devi, GR, Chen, W, Spector, N, Amalfitano, A, Lyerly, HK, and Clay, TM. "Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo." Int J Cancer 126.12 (June 15, 2010): 2893-2903.
PMID
19856307
Source
pubmed
Published In
International Journal of Cancer
Volume
126
Issue
12
Publish Date
2010
Start Page
2893
End Page
2903
DOI
10.1002/ijc.24995

Identification of select glucocorticoids as Smoothened agonists: potential utility for regenerative medicine.

Regenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process for regulating stem cell proliferation. The development of Hedgehog-related therapies has been impeded by a lack of US Food and Drug Administration (FDA)-approved Smo agonists. Using a high-content screen with cells expressing Smo receptors and a beta-arrestin2-GFP reporter, we identified four FDA-approved drugs, halcinonide, fluticasone, clobetasol, and fluocinonide, as Smo agonists that activate Hedgehog signaling. These drugs demonstrated an ability to bind Smo, promote Smo internalization, activate Gli, and stimulate the proliferation of primary neuronal precursor cells alone and synergistically in the presence of Sonic Hedgehog protein. Halcinonide, fluticasone, clobetasol, and fluocinonide provide an unprecedented opportunity to develop unique clinical strategies to treat Hedgehog-dependent illnesses.

Authors
Wang, J; Lu, J; Bond, MC; Chen, M; Ren, X-R; Lyerly, HK; Barak, LS; Chen, W
MLA Citation
Wang, J, Lu, J, Bond, MC, Chen, M, Ren, X-R, Lyerly, HK, Barak, LS, and Chen, W. "Identification of select glucocorticoids as Smoothened agonists: potential utility for regenerative medicine." Proc Natl Acad Sci U S A 107.20 (May 18, 2010): 9323-9328.
PMID
20439738
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
107
Issue
20
Publish Date
2010
Start Page
9323
End Page
9328
DOI
10.1073/pnas.0910712107

G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway.

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3beta, a cytosolic kinase for phosphorylation of a downstream effector beta-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of beta-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished beta-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Authors
Chen, M; Philipp, M; Wang, J; Premont, RT; Garrison, TR; Caron, MG; Lefkowitz, RJ; Chen, W
MLA Citation
Chen, M, Philipp, M, Wang, J, Premont, RT, Garrison, TR, Caron, MG, Lefkowitz, RJ, and Chen, W. "G Protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway." J Biol Chem 284.50 (December 11, 2009): 35040-35048.
PMID
19801552
Source
pubmed
Published In
The Journal of biological chemistry
Volume
284
Issue
50
Publish Date
2009
Start Page
35040
End Page
35048
DOI
10.1074/jbc.M109.047456

The anti-helminthic niclosamide inhibits Wnt/Frizzled1 signaling.

Wnt proteins bind to seven-transmembrane Frizzled receptors to mediate the important developmental, morphogenetic, and stem cell related tissue-regenerative effects of Wnt signaling. Dysregulated Wnt signaling is associated with many cancers. Currently, there are no drug candidates or even tool compounds that modulate Wnt-mediated receptor trafficking, and subsequent Wnt signaling. We examined libraries of FDA-approved drugs for their utility as Frizzled internalization modulators, employing a primary imaged-based GFP fluorescence assay that uses Frizzled1 endocytosis as the readout. We now report that the anti-helminthic niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, downregulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated beta-catenin stabilization and LEF/TCF reporter activity. Additionally, following niclosamide-mediated internalization, the Frizzled1 receptor colocalizes in vesicles containing transferrin and agonist-activated beta(2)-adrenergic receptor. Therefore, niclosamide may serve as a negative modulator of Wnt/Frizzled1 signaling by depleting upstream signaling molecules (i.e., Frizzled and Dishevelled) and moreover may provide a valuable means of studying the physiological consequences of Wnt signaling.

Authors
Chen, M; Wang, J; Lu, J; Bond, MC; Ren, X-R; Lyerly, HK; Barak, LS; Chen, W
MLA Citation
Chen, M, Wang, J, Lu, J, Bond, MC, Ren, X-R, Lyerly, HK, Barak, LS, and Chen, W. "The anti-helminthic niclosamide inhibits Wnt/Frizzled1 signaling." Biochemistry 48.43 (November 3, 2009): 10267-10274.
PMID
19772353
Source
pubmed
Published In
Biochemistry
Volume
48
Issue
43
Publish Date
2009
Start Page
10267
End Page
10274
DOI
10.1021/bi9009677

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

BACKGROUND & AIMS: Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). METHODS: MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. RESULTS: Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. CONCLUSIONS: Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

Authors
Witek, RP; Yang, L; Liu, R; Jung, Y; Omenetti, A; Syn, W-K; Choi, SS; Cheong, Y; Fearing, CM; Agboola, KM; Chen, W; Diehl, AM
MLA Citation
Witek, RP, Yang, L, Liu, R, Jung, Y, Omenetti, A, Syn, W-K, Choi, SS, Cheong, Y, Fearing, CM, Agboola, KM, Chen, W, and Diehl, AM. "Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells." Gastroenterology 136.1 (January 2009): 320-330.e2.
PMID
19013163
Source
pubmed
Published In
Gastroenterology
Volume
136
Issue
1
Publish Date
2009
Start Page
320
End Page
330.e2
DOI
10.1053/j.gastro.2008.09.066

Smoothened signaling in vertebrates is facilitated by a G protein-coupled receptor kinase.

Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein-coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein-dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development.

Authors
Philipp, M; Fralish, GB; Meloni, AR; Chen, W; MacInnes, AW; Barak, LS; Caron, MG
MLA Citation
Philipp, M, Fralish, GB, Meloni, AR, Chen, W, MacInnes, AW, Barak, LS, and Caron, MG. "Smoothened signaling in vertebrates is facilitated by a G protein-coupled receptor kinase." Mol Biol Cell 19.12 (December 2008): 5478-5489.
PMID
18815277
Source
pubmed
Published In
Molecular Biology of the Cell
Volume
19
Issue
12
Publish Date
2008
Start Page
5478
End Page
5489
DOI
10.1091/mbc.E08-05-0448

Beta-arrestin-mediated localization of smoothened to the primary cilium.

beta-Arrestins have important roles in the regulation of seven-transmembrane receptors (7TMRs). Smoothened (Smo) is a 7TMR that mediates effects of Hedgehog on developmental processes and whose dysregulation may cause tumorigenesis. beta-Arrestins are required for endocytosis of Smo and signaling to Gli transcription factors. In mammalian cells, Smo-dependent signaling requires translocation to primary cilia. We demonstrated that beta-arrestins mediate the activity-dependent interaction of Smo and the kinesin motor protein Kif3A. This multimeric complex localized to primary cilia and was disrupted in cells transfected with beta-arrestin small interfering RNA. beta-Arrestin 1 or beta-arrestin 2 depletion prevented the localization of Smo to primary cilia and the Smo-dependent activation of Gli. These results suggest roles for beta-arrestins in mediating the intracellular transport of a 7TMR to its obligate subcellular location for signaling.

Authors
Kovacs, JJ; Whalen, EJ; Liu, R; Xiao, K; Kim, J; Chen, M; Wang, J; Chen, W; Lefkowitz, RJ
MLA Citation
Kovacs, JJ, Whalen, EJ, Liu, R, Xiao, K, Kim, J, Chen, M, Wang, J, Chen, W, and Lefkowitz, RJ. "Beta-arrestin-mediated localization of smoothened to the primary cilium." Science 320.5884 (June 27, 2008): 1777-1781.
PMID
18497258
Source
pubmed
Published In
Science
Volume
320
Issue
5884
Publish Date
2008
Start Page
1777
End Page
1781
DOI
10.1126/science.1157983

Novel Breast Cancer Biomarkers Identified by Integrative Proteomic and Gene Expression Mapping

Proteomic and transcriptomic platforms both play important roles in cancer research, with differing strengths and limitations. Here, we describe a proteo-transcriptomic integrative strategy for discovering novel cancer biomarkers, combining the direct visualization of differentially expressed proteins with the high-throughput scale of gene expression profiling. Using breast cancer as a case example, we generated comprehensive two-dimensional electrophoresis (2DE)/mass spectrometry (MS) proteomic maps of cancer (MCF-7 and HCC-38) and control (CCD-1059Sk) cell lines, identifying 1724 expressed protein spots representing 484 different protein species. The differentially expressed cell-line proteins were then mapped to mRNA transcript databases of cancer cell lines and primary breast tumors to identify candidate biomarkers that were concordantly expressed at the gene expression level. Of the top nine selected biomarker candidates, we reidentified ANX1, a protein previously reported to be differentially expressed in breast cancers and normal tissues, and validated three other novel candidates, CRAB, 6PGL, and CAZ2, as differentially expressed proteins by immunohistochemistry on breast tissue microarrays. In total, close to half (4/9) of our protein biomarker candidates were successfully validated. Our study thus illustrates how the systematic integration of proteomic and transcriptomic data from both cell line and primary tissue samples can prove advantageous for accelerating cancer biomarker discovery. © 2008 American Chemical Society.

Authors
Ou, K; Yu, K; Kesuma, D; Hooi, M; Huang, N; Chen, W; Lee, SY; Goh, XP; Tan, LK; Liu, J; Soon, SY; Rashid, SBA; Putti, TC; Jikuya, H; Ichikawa, T; Nishimura, O; Salto-Tellez, M; Tan, P
MLA Citation
Ou, K, Yu, K, Kesuma, D, Hooi, M, Huang, N, Chen, W, Lee, SY, Goh, XP, Tan, LK, Liu, J, Soon, SY, Rashid, SBA, Putti, TC, Jikuya, H, Ichikawa, T, Nishimura, O, Salto-Tellez, M, and Tan, P. "Novel Breast Cancer Biomarkers Identified by Integrative Proteomic and Gene Expression Mapping." Journal of Proteome Research 7.4 (2008): 1518-1528.
PMID
18318472
Source
scival
Published In
Journal of Proteome Research
Volume
7
Issue
4
Publish Date
2008
Start Page
1518
End Page
1528
DOI
10.1021/pr700820g

Pharmacologic disruption of polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells

Polycomb-repressive complex 2 (PRC2)-mediated histone methylation plays an important role in aberrant cancer gene silencing and is a potential target for cancer therapy. Here we show that S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) induces efficient apoptotic cell death in cancer cells but not in normal cells. We found that DZNep effectively depleted cellular levels of PRC2 components EZH2, SUZ12, and EED and inhibited associated histone H3 Lys 27 methylation (but not H3 Lys 9 methylation). By integrating RNA interference (RNAi), genome-wide expression analysis, and chromatin immunoprecipitation (ChIP) studies, we have identified a prominent set of genes selectively repressed by PRC2 in breast cancer that can be reactivated by DZNep. We further demonstrate that the preferential reactivation of a set of these genes by DZNep, including a novel apoptosis affector, FBXO32, contributes to DZNep-induced apoptosis in breast cancer cells. Our results demonstrate the unique feature of DZNep as a novel chromatin remodeling compound and suggest that pharmacologic reversal of PRC2-mediated gene repression by DZNep may constitute a novel approach for cancer therapy. © 2007 by Cold Spring Harbor Laboratory Press.

Authors
Tan, J; Yang, X; Zhuang, L; Jiang, X; Chen, W; Puay, LL; Karuturi, RKM; Tan, PBO; Liu, ET; Yu, Q
MLA Citation
Tan, J, Yang, X, Zhuang, L, Jiang, X, Chen, W, Puay, LL, Karuturi, RKM, Tan, PBO, Liu, ET, and Yu, Q. "Pharmacologic disruption of polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells." Genes and Development 21.9 (2007): 1050-1063.
PMID
17437993
Source
scival
Published In
Genes & development
Volume
21
Issue
9
Publish Date
2007
Start Page
1050
End Page
1063
DOI
10.1101/gad.1524107

PPARgamma agonists prevent TGFbeta1/Smad3-signaling in human hepatic stellate cells.

PPARgamma agonists inhibit liver fibrosis, but the mechanisms involved are uncertain. We hypothesized that PPARgamma agonists inhibit transforming growth factor (TGF)beta1-activation of TGFbeta receptor (TGFbetaR)-1 signaling in quiescent stellate cells, thereby abrogating Smad3-dependent induction of extracellular matrix (ECM) genes, such as PAI-1 and collagen-1alphaI. To test this, human HSC were cultured to induce a quiescent phenotype, characterized by lipid accumulation and PPARgamma expression and transcriptional activity. These adipocytic HSC were then treated with TGFbeta1+/-a TGFbetaR-1 kinase inhibitor (SB431542) or a PPARgamma agonist (GW7845). TGFbeta1 caused dose- and time-dependent increases in Smad3 phosphorylation, followed by induction of collagen and PAI-1 expression. Like the TGFbetaR-1 kinase inhibitor, the PPARgamma agonist caused dose-dependent inhibition of all of these responses without effecting HSC proliferation or viability. Thus, the anti-fibrotic actions of PPARgamma agonists reflect their ability to inhibit TGFbeta1-TGFbetaR1 signaling that initiates ECM gene expression in quiescent HSC.

Authors
Zhao, C; Chen, W; Yang, L; Chen, L; Stimpson, SA; Diehl, AM
MLA Citation
Zhao, C, Chen, W, Yang, L, Chen, L, Stimpson, SA, and Diehl, AM. "PPARgamma agonists prevent TGFbeta1/Smad3-signaling in human hepatic stellate cells." Biochem Biophys Res Commun 350.2 (November 17, 2006): 385-391.
PMID
17010940
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
350
Issue
2
Publish Date
2006
Start Page
385
End Page
391
DOI
10.1016/j.bbrc.2006.09.069

Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice.

Rac, a small, GTP-binding protein in the Rho family, regulates several cellular functions, including the activation of NADPH oxidase, a major intracellular producer of reactive oxygen species (ROS). Hepatic stellate cells (HSCs) isolated from mice that are genetically deficient in NADPH oxidase produce less ROS, and their activation during chronic liver injury is abrogated, resulting in decreased liver fibrosis. Therefore, we hypothesized that HSC ROS production and activation would be enhanced, and fibrosis worsened, by increasing Rac expression in HSCs. To achieve this, we used transgenic mice that express constitutively active human Rac1 under the control of the alpha-smooth muscle actin (alpha-sma) promoter, because alpha-sma expression is induced spontaneously during HSC activation. Transgene expression was upregulated progressively during culture of primary Rac-transgenic HSCs, and this increased HSC ROS production as well as expression of activation markers and collagen. Similarly, Rac mice treated with carbon tetrachloride (CCl(4)) accumulated greater numbers of activated HSCs and had more liver damage, hepatocyte apoptosis, and liver fibrosis-as well as higher mortality-than CCl(4)-treated wild-type mice. In conclusion, sustained activation of Rac in HSCs perpetuates their activation and exacerbates toxin-induced liver injury and fibrosis, prompting speculation that Rac may be a therapeutic target in patients with cirrhosis.

Authors
Choi, SS; Sicklick, JK; Ma, Q; Yang, L; Huang, J; Qi, Y; Chen, W; Li, Y-X; Goldschmidt-Clermont, PJ; Diehl, AM
MLA Citation
Choi, SS, Sicklick, JK, Ma, Q, Yang, L, Huang, J, Qi, Y, Chen, W, Li, Y-X, Goldschmidt-Clermont, PJ, and Diehl, AM. "Sustained activation of Rac1 in hepatic stellate cells promotes liver injury and fibrosis in mice." Hepatology 44.5 (November 2006): 1267-1277.
PMID
17058265
Source
pubmed
Published In
Hepatology
Volume
44
Issue
5
Publish Date
2006
Start Page
1267
End Page
1277
DOI
10.1002/hep.21375

Dysregulation of the Hedgehog pathway in human hepatocarcinogenesis.

Hedgehog (Hh) pathway activation promotes tumors in several endodermally derived tissues, but its role in the pathogenesis of hepatocellular carcinoma (HCC) is unknown. Although normal hepatocytes lack Hh signaling, activation of the Hh pathway in endodermal progenitors is required for liver development. Thus, we hypothesized that hepatocarcinogenesis may involve regulation of Hh signaling. This pathway is activated when Hh ligand binds to its receptor, Patched (PTC). In an unoccupied state, PTC normally functions as a tumor suppressor that inhibits Smoothened (SMO), a proto-oncoprotein, from activating downstream components and transcription of target genes. Here we show that in HCCs, overexpression of the Smo proto-oncogene, as well as an increase in the stoichiometric ratio of Smo to Ptc mRNA levels, correlated with tumor size, a prognostic indicator in HCC biology. In one tumor we identified a novel Smo mutation in an evolutionarily conserved residue. We also demonstrated that HCC cell lines (HepG2 and Hep3B) expressed Hh pathway components and activated Hh transcriptional targets. In Hep3B cells, cyclopamine, an inhibitor of wild-type SMO, had no effect, but KAAD-cyclopamine, a blocker of oncogenic SMO, inhibited Hh signaling activity by 50%, decreased expression of the hepatocarcinogenic oncogene, c-myc, by 8-fold, and inhibited the growth rate of Hep3B cells by 94%. These data support our hypothesis that Hh signaling is dysregulated in human hepatocarcinogenesis. We demonstrate that overexpression and/or tumorigenic activation of the Smo proto-oncogene mediates c-myc overexpression which plays a critical role in hepatocarcinogenesis and suggests that Smo is a prognostic factor in HCC tumorigenesis.

Authors
Sicklick, JK; Li, Y-X; Jayaraman, A; Kannangai, R; Qi, Y; Vivekanandan, P; Ludlow, JW; Owzar, K; Chen, W; Torbenson, MS; Diehl, AM
MLA Citation
Sicklick, JK, Li, Y-X, Jayaraman, A, Kannangai, R, Qi, Y, Vivekanandan, P, Ludlow, JW, Owzar, K, Chen, W, Torbenson, MS, and Diehl, AM. "Dysregulation of the Hedgehog pathway in human hepatocarcinogenesis." Carcinogenesis 27.4 (April 2006): 748-757.
PMID
16339184
Source
pubmed
Published In
Carcinogenesis
Volume
27
Issue
4
Publish Date
2006
Start Page
748
End Page
757
DOI
10.1093/carcin/bgi292

Role for hedgehog signaling in hepatic stellate cell activation and viability.

Hepatic stellate cells (HSC) have a complex phenotype that includes both neural and myofibroblastic features. The Hedgehog (Hh) pathway has been shown to direct the fate of neural and myofibroblastic cells during embryogenesis and during tissue remodeling in adults. Therefore, we hypothesized that Hh signaling may regulate the fate of HSC in adults. In this study, we find that freshly isolated stellate cells from adult Patched-lacZ transgenic mice exhibit beta-galactosidase activity, indicating Hh pathway activity. Transcripts of Hh ligands, the Hh pathway receptor, and Hh-regulated transcription factors are expressed by stellate cells from mice, rats, and humans. Transfection experiments in a cell line using a Hh-inducible luciferase reporter demonstrate constitutive Hh pathway activity. Moreover, neutralizing antibodies to Hh increase apoptosis, while viability is restored by treatment with Hh ligand. In vitro treatment of primary stellate cells with cyclopamine (Cyc), a pharmacologic inhibitor of the Hh pathway, inhibits activation and slightly decreases cell survival, while a single injection of Cyc into healthy adult mice reduces activation of HSC by more than 50% without producing obvious liver damage. Our findings reveal a novel mechanism, namely the Hh pathway, that regulates the activation and viability of HSC.

Authors
Sicklick, JK; Li, Y-X; Choi, SS; Qi, Y; Chen, W; Bustamante, M; Huang, J; Zdanowicz, M; Camp, T; Torbenson, MS; Rojkind, M; Diehl, AM
MLA Citation
Sicklick, JK, Li, Y-X, Choi, SS, Qi, Y, Chen, W, Bustamante, M, Huang, J, Zdanowicz, M, Camp, T, Torbenson, MS, Rojkind, M, and Diehl, AM. "Role for hedgehog signaling in hepatic stellate cell activation and viability." Lab Invest 85.11 (November 2005): 1368-1380.
PMID
16170335
Source
pubmed
Published In
Laboratory Investigation
Volume
85
Issue
11
Publish Date
2005
Start Page
1368
End Page
1380
DOI
10.1038/labinvest.3700349

Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor.

Signaling through beta-arrestins is a recently appreciated mechanism used by seven-transmembrane receptors. Because G protein-coupled receptor kinase (GRK) phosphorylation of such receptors is generally a prerequisite for beta-arrestin binding, we studied the roles of different GRKs in promoting beta-arrestin-mediated extracellular signal-regulated kinase (ERK) activation by a typical seven-transmembrane receptor, the Gs-coupled V2 vasopressin receptor. Gs- and beta-arrestin-mediated pathways to ERK activation could be distinguished with H89, an inhibitor of protein kinase A, and beta-arrestin 2 small interfering RNA, respectively. The roles of GRK2, -3, -5, and -6 were assessed by suppressing their expression with specific small interfering RNA sequences. By using this approach, we demonstrated that GRK2 and -3 are responsible for most of the agonist-dependent receptor phosphorylation, desensitization, and recruitment of beta-arrestins. In contrast, GRK5 and -6 mediated much less receptor phosphorylation and beta-arrestin recruitment, but yet appeared exclusively to support beta-arrestin 2-mediated ERK activation. GRK2 suppression actually increased beta-arrestin-stimulated ERK activation. These results suggest that beta-arrestin recruited in response to receptor phosphorylation by different GRKs has distinct functional potentials.

Authors
Ren, X-R; Reiter, E; Ahn, S; Kim, J; Chen, W; Lefkowitz, RJ
MLA Citation
Ren, X-R, Reiter, E, Ahn, S, Kim, J, Chen, W, and Lefkowitz, RJ. "Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor." Proc Natl Acad Sci U S A 102.5 (February 1, 2005): 1448-1453.
PMID
15671180
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
5
Publish Date
2005
Start Page
1448
End Page
1453
DOI
10.1073/pnas.0409534102

Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2.

Binding of Sonic Hedgehog (Shh) to Patched (Ptc) relieves the latter's tonic inhibition of Smoothened (Smo), a receptor that spans the cell membrane seven times. This initiates signaling which, by unknown mechanisms, regulates vertebrate developmental processes. We find that two molecules interact with mammalian Smo in an activation-dependent manner: G protein-coupled receptor kinase 2 (GRK2) leads to phosphorylation of Smo, and beta-arrestin 2 fused to green fluorescent protein interacts with Smo. These two processes promote endocytosis of Smo in clathrin-coated pits. Ptc inhibits association of beta-arrestin 2 with Smo, and this inhibition is relieved in cells treated with Shh. A Smo agonist stimulated and a Smo antagonist (cyclopamine) inhibited both phosphorylation of Smo by GRK2 and interaction of beta-arrestin 2 with Smo. beta-Arrestin 2 and GRK2 are thus potential mediators of signaling by activated Smo.

Authors
Chen, W; Ren, X-R; Nelson, CD; Barak, LS; Chen, JK; Beachy, PA; de Sauvage, F; Lefkowitz, RJ
MLA Citation
Chen, W, Ren, X-R, Nelson, CD, Barak, LS, Chen, JK, Beachy, PA, de Sauvage, F, and Lefkowitz, RJ. "Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2." Science 306.5705 (December 24, 2004): 2257-2260.
PMID
15618519
Source
pubmed
Published In
Science
Volume
306
Issue
5705
Publish Date
2004
Start Page
2257
End Page
2260
DOI
10.1126/science.1104135

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca2+ imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Gα subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.

Authors
Fukuto, HS; Ferkey, DM; Apicella, AJ; Lans, H; Sharmeen, T; Chen, W; Lefkowitz, RJ; Jansen, G; Schafer, WR; Hart, AC
MLA Citation
Fukuto, HS, Ferkey, DM, Apicella, AJ, Lans, H, Sharmeen, T, Chen, W, Lefkowitz, RJ, Jansen, G, Schafer, WR, and Hart, AC. "G protein-coupled receptor kinase function is essential for chemosensation in C. elegans." Neuron 42.4 (2004): 581-593.
PMID
15157420
Source
scival
Published In
Neuron
Volume
42
Issue
4
Publish Date
2004
Start Page
581
End Page
593
DOI
10.1016/S0896-6273(04)00252-1

Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling.

beta-Arrestins bind to activated seven transmembrane-spanning (7TMS) receptors (G protein-coupled receptors) after the receptors are phosphorylated by G protein-coupled receptor kinases (GRKs), thereby regulating their signaling and internalization. Here, we demonstrate an unexpected and analogous role of beta-arrestin 2 (betaarr2) for the single transmembrane-spanning type III transforming growth factor-beta (TGF-beta) receptor (TbetaRIII, also referred to as betaglycan). Binding of betaarr2 to TbetaRIII was also triggered by phosphorylation of the receptor on its cytoplasmic domain (likely at threonine 841). However, such phosphorylation was mediated by the type II TGF-beta receptor (TbetaRII), which is itself a kinase, rather than by a GRK. Association with betaarr2 led to internalization of both receptors and down-regulation of TGF-beta signaling. Thus, the regulatory actions of beta-arrestins are broader than previously appreciated, extending to the TGF-beta receptor family as well.

Authors
Chen, W; Kirkbride, KC; How, T; Nelson, CD; Mo, J; Frederick, JP; Wang, X-F; Lefkowitz, RJ; Blobe, GC
MLA Citation
Chen, W, Kirkbride, KC, How, T, Nelson, CD, Mo, J, Frederick, JP, Wang, X-F, Lefkowitz, RJ, and Blobe, GC. "Beta-arrestin 2 mediates endocytosis of type III TGF-beta receptor and down-regulation of its signaling." Science 301.5638 (September 5, 2003): 1394-1397.
PMID
12958365
Source
pubmed
Published In
Science
Volume
301
Issue
5638
Publish Date
2003
Start Page
1394
End Page
1397
DOI
10.1126/science.1083195

Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.

Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane.Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells was dependent on added Wnt5A protein and was accomplished by the multifunctional adaptor protein beta-arrestin 2 (betaarr2), which was recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of beta-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling.

Authors
Chen, W; ten Berge, D; Brown, J; Ahn, S; Hu, LA; Miller, WE; Caron, MG; Barak, LS; Nusse, R; Lefkowitz, RJ
MLA Citation
Chen, W, ten Berge, D, Brown, J, Ahn, S, Hu, LA, Miller, WE, Caron, MG, Barak, LS, Nusse, R, and Lefkowitz, RJ. "Dishevelled 2 recruits beta-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4." Science 301.5638 (September 5, 2003): 1391-1394.
PMID
12958364
Source
pubmed
Published In
Science
Volume
301
Issue
5638
Publish Date
2003
Start Page
1391
End Page
1394
DOI
10.1126/science.1082808

GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

Authors
Hu, LA; Chen, W; Martin, NP; Whalen, EJ; Premont, RT; Lefkowitz, RJ
MLA Citation
Hu, LA, Chen, W, Martin, NP, Whalen, EJ, Premont, RT, and Lefkowitz, RJ. "GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation." J Biol Chem 278.28 (July 11, 2003): 26295-26301.
PMID
12724327
Source
pubmed
Published In
The Journal of biological chemistry
Volume
278
Issue
28
Publish Date
2003
Start Page
26295
End Page
26301
DOI
10.1074/jbc.M212352200

Regulation of ALK-1 signaling by the nuclear receptor LXRbeta.

The transforming growth factor beta (TGF-beta) receptor, ALK-1, is expressed specifically on endothelial cells and is essential for angiogenesis, as demonstrated by its targeted deletion in mice and its mutation in the human disease hereditary hemorrhagic telangiectasia. Although ALK-1 and another endothelial-specific TGF-beta receptor, endoglin, both bind TGF-beta with identical isoform specificity and form a complex together, neither has been shown to signal in response to TGF-beta, and the mechanism by which these receptors signal in endothelial cells remains unknown. Here we report the identification of the nuclear receptor liver X receptor beta (LXRbeta) as a modulator/mediator of ALK-1 signaling. The cytoplasmic domain of ALK-1 specifically binds to LXRbeta in vitro and in vivo. Expression of activated ALK-1 results in translocation of LXRbeta from the nuclear compartment to the cytoplasmic compartment. The interaction of activated ALK-1 with LXRbeta in the cytoplasmic compartment results in the specific phosphorylation of LXRbeta by ALK-1, primarily on serine residues. LXRbeta subsequently modulates signaling by ALK-1 and the closely related TGF-beta receptor, ALK-2, as demonstrated by specific and potent inhibition of ALK-1- and ALK-2-mediated transcriptional responses, establishing LXRbeta as a potential modulator/mediator of ALK-1/ALK-2 signaling.

Authors
Mo, J; Fang, SJ; Chen, W; Blobe, GC
MLA Citation
Mo, J, Fang, SJ, Chen, W, and Blobe, GC. "Regulation of ALK-1 signaling by the nuclear receptor LXRbeta." J Biol Chem 277.52 (December 27, 2002): 50788-50794.
PMID
12393874
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
52
Publish Date
2002
Start Page
50788
End Page
50794
DOI
10.1074/jbc.M210376200

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors.

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.

Authors
Lin, F-T; Chen, W; Shenoy, S; Cong, M; Exum, ST; Lefkowitz, RJ
MLA Citation
Lin, F-T, Chen, W, Shenoy, S, Cong, M, Exum, ST, and Lefkowitz, RJ. "Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors." Biochemistry 41.34 (August 27, 2002): 10692-10699.
PMID
12186555
Source
pubmed
Published In
Biochemistry
Volume
41
Issue
34
Publish Date
2002
Start Page
10692
End Page
10699

G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95.

We previously reported that the beta(1)-adrenergic receptor (beta(1)AR) associates with PSD-95 through a PDZ domain-mediated interaction, by which PSD-95 modulates beta(1)AR function and facilitates the physical association of beta(1)AR with other synaptic proteins such as N-methyl-d-aspartate receptors. Here we demonstrate that beta(1)AR association with PSD-95 is regulated by G protein-coupled receptor kinase 5 (GRK5). When beta(1)AR and PSD-95 were coexpressed with either GRK2 or GRK5 in COS-7 cells, GRK5 alone dramatically decreased the association of beta(1)AR with PSD-95, although GRK2 and GRK5 both could be co-immunoprecipitated with beta(1)AR and both could enhance receptor phosphorylation in vivo. Increasing expression of GRK5 in the cells led to further decreased beta(1)AR association with PSD-95. Stimulation with the beta(1)AR agonist isoproterenol further decreased PSD-95 binding to beta(1)AR. In addition, GRK5 protein kinase activity was required for this regulatory effect since a kinase-inactive GRK5 mutant had no effect on PSD-95 binding to beta(1)AR. Moreover, the regulatory effect of GRK5 on beta(1)AR association with PSD-95 was observed only when GRK5 was expressed together with the receptor, but not when GRK5 was coexpressed with PSD-95. Thus, we propose that GRK5 regulates beta(1)AR association with PSD-95 through phosphorylation of beta(1)AR. Regulation of protein association through receptor phosphorylation may be a general mechanism used by G protein-coupled receptors that associate via PDZ domain-mediated protein/protein interactions.

Authors
Hu, LA; Chen, W; Premont, RT; Cong, M; Lefkowitz, RJ
MLA Citation
Hu, LA, Chen, W, Premont, RT, Cong, M, and Lefkowitz, RJ. "G protein-coupled receptor kinase 5 regulates beta 1-adrenergic receptor association with PSD-95." J Biol Chem 277.2 (January 11, 2002): 1607-1613.
PMID
11700307
Source
pubmed
Published In
The Journal of biological chemistry
Volume
277
Issue
2
Publish Date
2002
Start Page
1607
End Page
1613
DOI
10.1074/jbc.M107297200

beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.

One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.

Authors
Chen, W; Hu, LA; Semenov, MV; Yanagawa, S; Kikuchi, A; Lefkowitz, RJ; Miller, WE
MLA Citation
Chen, W, Hu, LA, Semenov, MV, Yanagawa, S, Kikuchi, A, Lefkowitz, RJ, and Miller, WE. "beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins." Proc Natl Acad Sci U S A 98.26 (December 18, 2001): 14889-14894.
Website
http://hdl.handle.net/10161/7809
PMID
11742073
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
98
Issue
26
Publish Date
2001
Start Page
14889
End Page
14894
DOI
10.1073/pnas.211572798

beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis.

beta-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which beta-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-ribosylation factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the beta(2)-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange factor, ARNO, to enhance receptor endocytosis. Endogenous beta-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, beta-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including beta-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.

Authors
Claing, A; Chen, W; Miller, WE; Vitale, N; Moss, J; Premont, RT; Lefkowitz, RJ
MLA Citation
Claing, A, Chen, W, Miller, WE, Vitale, N, Moss, J, Premont, RT, and Lefkowitz, RJ. "beta-Arrestin-mediated ADP-ribosylation factor 6 activation and beta 2-adrenergic receptor endocytosis." J Biol Chem 276.45 (November 9, 2001): 42509-42513.
PMID
11533043
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
45
Publish Date
2001
Start Page
42509
End Page
42513
DOI
10.1074/jbc.M108399200

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79.

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.

Authors
Cong, M; Perry, SJ; Lin, FT; Fraser, ID; Hu, LA; Chen, W; Pitcher, JA; Scott, JD; Lefkowitz, RJ
MLA Citation
Cong, M, Perry, SJ, Lin, FT, Fraser, ID, Hu, LA, Chen, W, Pitcher, JA, Scott, JD, and Lefkowitz, RJ. "Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79." J Biol Chem 276.18 (May 4, 2001): 15192-15199.
PMID
11278469
Source
pubmed
Published In
The Journal of biological chemistry
Volume
276
Issue
18
Publish Date
2001
Start Page
15192
End Page
15199
DOI
10.1074/jbc.M009130200

Dual-specific Cdc25B phosphatase: in search of the catalytic acid.

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly structurally homologous to other well-characterized members, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. However, the catalytic acid needed for protonation of the leaving group has yet to be identified. To elucidate the role and identity of this key catalytic residue, we have performed a detailed pH-dependent kinetic analysis of Cdc25B. The pK(a) of the catalytic cysteine was found to be 5.6-6.3 in steady state and one-turnover burst experiments using the small molecule substrates p-nitrophenyl phosphate and 3-O-methylfluorescein phosphate. Interestingly, Cdc25B does not exhibit the typical bell-shaped pH-rate profile with small molecule substrates seen in other cysteine phosphatases and indicative of the catalytic acid because it lacks pH dependence between 6.5 and 9. Reactions of Cdc25B with the natural substrate Cdk2-pTpY/CycA, however, did yield a bell-shaped pH-rate profile with a pK(a) of 6.1 for the catalytic acid residue. Recent structural studies of Cdc25 have suggested that Glu474 [Fauman, E. B., et al. (1998) Cell 93, 617-625] or Glu478 [Reynolds, R. A., et al. (1999) J. Mol. Biol. 293, 559-568] could function as the catalytic acid in Cdc25B. Using site-directed mutagenesis and truncation experiments, however, we found that neither of these residues, nor the unstructured C-terminus, is responsible for the observed pH dependence. These results indicate that the catalytic acid does not appear to lie within the known structure of Cdc25B and may lie on its protein substrate.

Authors
Chen, W; Wilborn, M; Rudolph, J
MLA Citation
Chen, W, Wilborn, M, and Rudolph, J. "Dual-specific Cdc25B phosphatase: in search of the catalytic acid." Biochemistry 39.35 (September 5, 2000): 10781-10789.
PMID
10978163
Source
pubmed
Published In
Biochemistry
Volume
39
Issue
35
Publish Date
2000
Start Page
10781
End Page
10789
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