Protein Kinase Cδ Negatively Regulates Hedgehog Signaling by Inhibition of Gli1 Activity*
- ‡Department of Surgery, §Sealy Center for Cancer Cell Biology, and the ¶Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555
- 2 To whom correspondence should be addressed: Dept. of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0536. Tel.: 409-772-5612; Fax: 409-747-4819; E-mail: mevers{at}utmb.edu.
Abstract
Constitutive activation of the hedgehog pathway is implicated in the development of many human malignancies; hedgehog targets, PTCH1 and Gli1, are markers of hedgehog signaling activation and are expressed in most hedgehog-associated tumors. Protein kinase Cδ (PKCδ) generally slows proliferation and induces cell cycle arrest of various cell lines. In this study, we show that activated PKCδ (wild-type PKCδ stimulated by phorbol 12-myristate 13-acetate or constitutively active PKCδ) decreased Gli-luciferase reporter activity in NIH/3T3 cells, as well as the endogenous hedgehog-responsive gene PTCH1. In human hepatoma (i.e. Hep3B) cells, wild-type PKCδ and constitutively active PKCδ decreased the expression levels of endogenous Gli1 and PTCH1. In contrast, PKCδ siRNA increased the expression levels of these target genes. Silencing of PKCδ by siRNA rescued the inhibition of cell growth by KAAD-cyclopamine, an antagonist of hedgehog signaling element Smoothened, suggesting that PKCδ acts downstream of Smoothened. The biological relevance of our study is shown in hepatocellular carcinoma where we found that hepatocellular carcinoma with detectable hedgehog signaling had weak or no detectable expression of PKCδ, whereas PKCδ highly expressing tumors had no detectable hedgehog signaling. Our results demonstrate that PKCδ alters hedgehog signaling by inhibition of Gli protein transcriptional activity. Furthermore, our findings suggest that, in certain cancers, PKCδ plays a role as a negative regulator of tumorigenesis by regulating hedgehog signaling.
Footnotes
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↵3 The abbreviations used are: Hh, hedgehog; HCC, hepatocellular carcinoma; PTCH, patched; Smo, smoothened; MEK1, mitogen-activated protein kinase kinase 1; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; HA, hemagglutinin; siRNA, small interference RNA; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ERK, extracellular signal-regulated kinase; KD, kinase-dead; WT, wild type; RT, reverse transcription.
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↵* This work was supported, in whole or in part, by National Institutes of Health Grants R01 CA104748, R01 DK48489, R01 DK35608, and R01 CA94160. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 Both authors contributed equally to this work.
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- Received April 28, 2008.
- Revision received November 17, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
