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-alpha-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-alpha-actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM-alpha-actin promoter CArG (CC(AT)(6)GG) 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.
Highlights
Vascular smooth muscle cells (VSMCs)4 can reversibly change their phenotype from a differentiated, “contractile” phenotype with high levels of smooth muscle (SM)-specific gene expression to a de-differentiated, “synthetic” phenotype with reduced levels of SM-specific gene expression [1, 2]
Dedifferentiated cells show increased expression of growth factor receptors, extracellular matrix, and inflammatory adhesion proteins and have increased proliferative and migratory potential [1, 2]. This phenotypic switching plays an important role in the development of vascular diseases: in acutely injured blood vessels, e.g. after balloon angioplasty, VSMCs proliferate and migrate from the medial layer of the vessel wall contributing to a “neo-intimal” layer, and the majority of SM-like cells found in atherosclerotic plaques appear to represent de-differentiated VSMCs originating from the medial layer [1, 3, 4]
To determine whether CRP4 interacted with PKG I in intact VSMCs, we performed co-immunoprecipitation experiments using a CRP4 antibody, which we showed did not cross-react with CRP2 (Fig. 3A is a Western blot from transfected cells expressing epitope-tagged CRP2 and CRP4)
Summary
DNA Constructs, Antibodies, and Reagents—Expression vectors encoding PKG I␣ and I were described previously [34]. For PKG-CRP4 interaction studies, cell extracts were subjected to immunoprecipitation using the indicated antibodies on protein G-agarose beads, or were incubated with glutathione Sepharose beads for GST pulldown experiments. ChIP—Approximately 107 PAC1 cells were incubated in situ with 1% formaldehyde for 10 min at room temperature to crosslink DNA and proteins; cells were washed and scraped into 1 ml of lysis buffer containing 10 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% Nonidet P-40, and protease inhibitor mixture. The supernatant was shaken gently for 16 h at 4 °C with 5 g of either control immunoglobulin G or antibodies specific for SRF or CRP4; after centrifugation, 25 l of blocked protein A/G-agarose was added for an additional 2 h. The Student t test was employed for pair-wise comparisons, and a one-way analysis of variance with Dunnett’s post-test analysis for multiple comparisons to the control group; a p value of Ͻ0.05 was considered to indicate statistical significance
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