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J. Biol. Chem., Vol. 282, Issue 46, 33367-33380, November 16, 2007

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A Cysteine-rich LIM-only Protein Mediates Regulation of Smooth Muscle-specific Gene Expression by cGMP-dependent Protein Kinase^*

Tong Zhang, Shunhui Zhuang, Darren E. Casteel¹, David J. Looney², Gerry R. Boss^¶, and Renate B. Pilz^¶3

From the Department of Medicine and ^¶Cancer Center, University of California, San Diego, California 92093 and the Veterans Administration Medical Center, La Jolla, California 92161

Vascular smooth muscle cells (VSMCs) undergo phenotypic modulation, changing from a differentiated, contractile to a de-differentiated, synthetic phenotype; the change is associated with decreased expression of smooth muscle (SM)-specific genes and loss of cGMP-dependent protein kinase (PKG), but transfection of PKG into de-differentiated VSMCs restores SM-specific gene expression. We show that small interference RNA-mediated down-regulation or pharmacologic inhibition of PKG reduced SM-specific gene expression in differentiated VSMCs and provide a mechanism for cGMP/PKG regulation of SM-specific genes involving the cysteine-rich LIM-only protein CRP4. PKG associated with CRP4 and phosphorylated the protein in intact cells. CRP4 had no intrinsic transcriptional activity, but exhibited adaptor function, because it acted synergistically with serum response factor (SRF) and GATA6 to activate the SM--actin promoter. cGMP stimulation of the promoter required PKG and CRP4 co-expression with SRF and GATA6. A phosphorylation-deficient mutant CRP4 and a CRP4 deletion mutant deficient in PKG binding did not support cGMP/PKG stimulation of the SM--actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM--actin promoter CArG (CC(AT)₆GG) element. CRP4 and SRF associated with CArG elements of endogenous SM-specific genes in intact chromatin. Small interference RNA-mediated down-regulation of CRP4 prevented the positive effects of cGMP/PKG on SM-specific gene expression. In the presence of CRP4, cGMP/PKG increased SRF- and GATA6-dependent expression of endogenous SM-specific genes in pluripotent 10T1/2 cells. Thus, CRP4 mediates cGMP/PKG stimulation of SM-specific gene expression, and PKG plays an important role in regulating the phenotype of VSMCs.

Received for publication, August 27, 2007

^* This work was supported in part by the Tobacco-related Disease Research Program Award 14RT-0020 (to R. B. P.), National Institutes of Health (NIH) Grant R01-AR051300 (to R. B. P.), and American Heart Association Fellowship Award 0525091Y (to T. Z.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1 and S2.

¹ Supported by NIH Training Grant 5T32-AI007469 (principal investigator: D. H. Broide).

² Supported by NIH Grant P30-AI36214 (principal investigator: D. Richman).

³ To whom correspondence should be addressed: Dept. of Medicine, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0652. Tel.: 858-534-8805; Fax: 848-534-1421; E-mail: rpilz{at}ucsd.edu.

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