No abstract

Phenotypic switching of vascular smooth muscle cells (SMCs), such as increased proliferation, enhanced migration, and downregulation of SMC differentiation marker genes, is known to play a key role in the development of atherosclerosis. However, the factors and mechanisms controlling this process are not fully understood. We recently showed that oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), which accumulate in atherosclerotic lesions, are potent repressors of expression of SMC differentiation marker genes in cultured SMCs as well as in rat carotid arteries in vivo. Here, we examined the molecular mechanisms whereby POVPC induces suppression of SMC differentiation marker genes in cultured SMCs. Results showed that POVPC induced phosphorylation of ERK1/2 and Elk-1. The MEK inhibitors U-0126 and PD-98059 attenuated POVPC-induced suppression of smooth muscle (SM) alpha-actin and SM-myosin heavy chain. POVPC also induced expression of Krüppel-like factor 4 (Klf4). Chromatin immunoprecipitation assays revealed that POVPC caused simultaneous binding of Elk-1 and Klf4 to the promoter region of the SM alpha-actin gene. Moreover, coimmunoprecipitation assays showed a physical interaction between Elk-1 and Klf4. Results in Klf4-null SMCs showed that blockade of both Klf4 induction and Elk-1 phosphorylation completely abolished POVPC-induced suppression of SMC differentiation marker genes. POVPC-induced suppression of SMC differentiation marker genes was also accompanied by hypoacetylation of histone H4 at the SM alpha-actin promoter, which was mediated by the recruitment of histone deacetylases (HDACs), HDAC2 and HDAC5. Coimmunoprecipitation assays showed that Klf4 interacted with HDAC5. Results provide evidence that Klf4, Elk-1, and HDACs coordinately mediate POVPC-induced suppression of SMC differentiation marker genes.

A hallmark of smooth muscle cell (SMC) phenotypic switching is suppression of SMC marker gene expression. Although myocardin has been shown to be a key regulator of this process, the role of its related factors, MKL1 and MKL2, in SMC phenotypic switching remains unknown. The present studies were aimed at determining if: 1) MKL factors contribute to the expression of SMC marker genes in cultured SMCs; and 2) platelet-derived growth factor-BB (PDGF-BB)-induced repression of SMC marker genes is mediated by suppression of MKL factors. Results of gain- and loss-of-function experiments showed that MKL factors regulated the expression of single and multiple CArG [CC(AT-rich)(6)GG]-containing SMC marker genes, such as smooth muscle (SM) alpha-actin and telokin, but not CArG-independent SMC marker genes such as smoothelin-B. Treatment with PDGF-BB reduced the expression of CArG-containing SMC marker genes, as well as myocardin expression in cultured SMCs, while it had no effect on expression of MKL1 and MKL2. However, of interest, PDGF-BB induced the dissociation of MKL factors from the CArG-containing region of SMC marker genes, as determined by chromatin immunoprecipitation assays. This dissociation was caused by the competition between MKL factors and phosphorylated Elk-1 at early time points, but subsequently by the reduction in acetylated histone H4 levels at these promoter regions mediated by histone deacetylases, HDAC2, HDAC4, and HDAC5. Results provide novel evidence that PDGF-BB-induced repression of SMC marker genes is mediated through combinatorial mechanisms, including downregulation of myocardin expression and inhibition of the association of myocardin/MKL factors with CArG-containing SMC marker gene promoters.

There is clear evidence that the phenotypic modulation of smooth muscle cells (SMCs) contributes to the pathophysiology of vascular disease. Phenotypic modulation refers to the unique ability of SMCs to alter their phenotype in response to extracellular stimuli and is hallmarked by the loss of SMC marker gene expression. The transcription factor Krüppel-like factor 4 (KLF4) is a known powerful negative regulator of SMC marker gene expression that works, in part, by decreasing the expression of the serum response factor (SRF) myocardin. KLF4 is not expressed in healthy adult SMCs but is increased in SMCs in response to vascular injury in vivo or PDGF-BB treatment in vitro. The aim of the present study was to determine the molecular mechanisms that regulate the expression of KLF4 in phenotypically modulated SMCs. The results demonstrated that the transcription factor stimulating protein-1 (Sp1) regulated the expression of KLF4 in SMCs. The KLF4 promoter contains three consensus Sp1 binding sites. Using a series of truncated KLF4 promoters, we showed that only fragments containing these Sp1 sites could be activated by PDGF-BB. In addition, overexpression of Sp1 alone was sufficient to increase the activity of the KLF4 promoter. Moreover, inhibiting Sp1 expression with small-interfering RNA attenuated the effects of PDGF-BB on KLF4 expression. Mutation of the three Sp1 sites within the KLF4 promoter abolished both baseline and PDGF-BB-induced activity. Finally, the results demonstrated enhanced Sp1 binding to the KLF4 promoter in SMCs treated with PDGF-BB in vitro and following vascular injury in vivo. Taken together, the results suggest a novel role for Sp1 in increasing the expression of KLF4 in phenotypically modulated SMCs.

Epigenetic mechanisms, such as histone modifications and DNA methylation, have been shown to play a key role in the regulation of gene transcription. Results of recent studies indicate that a novel "bivalent" chromatin structure marks key developmental genes in embryonic stem cells (ESCs), wherein a number of untranscribed lineagecontrol genes, such as Sox1, Nkx2-2, Msx1, Irx3, and Pax3, are epigenetically modified with a unique combination of activating and repressive histone modifications that prime them for potential activation (or repression) upon cell lineage induction and differentiation. However, results of these studies also showed that a subset of lineagecontrol genes, such as Myf5 and Mash1, were not marked by these histone modifications, suggesting that distinct epigenetic mechanisms might exist for lineage-control genes in ESCs. In this review article, we summarize evidence regarding possible mechanisms that control these unique histone modifications at lineage-control gene loci in ESCs and consider their possible contribution to ESC pluripotency. In addition, we propose a novel "histone modification pulsing" model wherein individual pluripotent stem cells within the inner cell mass of blastocysts undergo transient asynchronous histone modifications at these developmental gene loci, thereby conferring differential responsiveness to environmental cues and morphogenic gradients important for cell lineage determination. Finally, we consider how these rapid histone modification exchanges become progressively more stable as ESCs undergo differentiation and maturation into specialized cell lineages. STEM CELLS 2007;25:2-9