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

Abstract

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-α-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-α-actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM-α-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.

cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation

“…Knowledge of the cellular pathways that control SMC-specific gene expression and differentiation is important for understanding this phenotypic switch. In this study, we investigated mechanisms relating to the cGMP/PKG pathway in SMCspecific gene expression because studies in our laboratory (3) and others (56,58) have shown that this pathway stimulates vascular SMC differentiation.…”

“…Previous work by our laboratory (3) and that of Zhang et al (56) demonstrated that the expression of PKG-I in passaged SMCs, deficient in endogenous PKG-I, induced SMC-specific gene expression. Because myocardin is a key transcriptional regulator of the expression of SMC-specific gene expression, we tested whether PKG-I expression affected myocardin-induced SMC gene promoter activity in COS-7 cells.…”

A DNA Polymerase-α·Primase Cofactor with Homology to Replication Protein A-32 Regulates DNA Replication in Mammalian Cells

“…Probes were generated by PCR and labeled by random hexamer priming using either [␣- 32 In Vitro Transcription/Translation of Epitope-tagged AAF-44 and AAF-132-5Ј fusion sites were created using PCR and the following sets of primers: 5Ј-GGATCCATGGCTGCTT-GCCGAGCACAAC-3Ј and 5Ј-CTTTGGGTCAAAAGTAGC-CCAAGCC-3Јfor AAF-132 and 5Ј-CTCGAGGAGATGCCA-CAGCCCTGCCTCTTG-3Ј and 5Ј-TCTGCTCGATGGTCT-CCTGC-3Ј for AAF-44. The PCR products were sequenced and spliced into the full-length coding sequences in pcDNA3-Myc, pcDNA3-HA, or pXJ40-FLAG, creating an in-frame fusion with an N-terminal Myc, hemagglutinin (HA), or FLAG epitope tag (16,17). AAF-44 and/or AAF-132 in pcDNA3-Myc was used as a template for a coupled transcription/translation reaction using T7 RNA polymerase, [ 35 S]methionine, and the TNT TM reticulocyte lysate system according to the manufacturer's instructions (Promega).…”

The molecular interaction of heart LIM protein (HLP) with RyR2 and caveolin-3 is essential for Ca 2+ -induced Ca 2+ release in the heart

“…Another study showed that both cGKIα and cGKIβ interact with the third zinc finger motif of HLP in a phosphorylation-dependent manner [15]. Recently, the functional importance of an interaction between HLP and nuclear factor-κB (NF-κB) in cancer development has also been suggested [16].…”

“…HLP is a protein of 208 aa and its expression is abundant in the adult heart, as well as in the brain and lung [9,11,12]. Several binding partners of HLP have been previously identified in various tissues [11,[13][14][15][16]. Ham et al…”