Oliver G. McDonald

Platelet-derived growth factor BB (PDGF-BB) has been shown to be an extremely potent negative regulator of smooth muscle cell (SMC) differentiation. Moreover, previous studies have demonstrated that the Kruppel-like transcription factor (KLF) 4 potently represses the expression of multiple SMC genes. However, the mechanisms whereby KLF4 suppresses SMC gene expression are not known, nor is it clear whether KLF4 contributes to PDGF-BB-induced down-regulation of SMC genes. The goals of the present studies were to determine the molecular mechanisms by which KLF4 represses expression of SMC genes and whether it contributes to PDGF-BB-induced suppression of these genes. Results demonstrated that KLF4 markedly repressed both myocardin-induced activation of SMC genes and expression of myocardin. KLF4 was rapidly up-regulated in PDGF-BB-treated, cultured SMC, and a small interfering RNA to KLF4 partially blocked PDGF-BB-induced SMC gene repression. Both PDGF-BB and KLF4 markedly reduced serum response factor binding to CArG containing regions within intact chromatin. Finally, KLF4, which is normally not expressed in differentiated SMC in vivo, was rapidly up-regulated in vivo in response to vascular injury. Taken together, results indicate that KLF4 represses SMC genes by both down-regulating myocardin expression and preventing serum response factor/myocardin from associating with SMC gene promoters, and suggest that KLF4 may be a key effector of PDGF-BB and injury-induced phenotypic switching of SMC.

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.