Platelet-derived growth factor-BB represses smooth muscle cell marker genes via changes in binding of MKL factors and histone deacetylases to their promoters

Endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN) is up-regulated in the neointima of remodeling arteries and modulates vascular smooth muscle cell (VSMC) growth. Platelet-derived growth factor (PDGF) is the prototypic growth factor for VSMCs and plays a key role in vascular remodeling. Here, we sought to further define ESDN function in primary human VSMCs. ESDN down-regulation by RNA interference significantly enhanced PDGF-induced VSMC DNA synthesis and migration. This was associated with increased ERK1/2, Src, and PDGF receptor (PDGFR)␤ phosphorylation, without altering total PDGFR␤ expression levels. In binding assays, ESDN down-regulation significantly increased 125 I-PDGF maximum binding (B max ) to PDGF receptors on VSMCs without altering the binding constant (K d ), raising the possibility that ESDN regulates PDGFR processing. ESDN down-regulation significantly reduced ligand-induced PDGFR␤ ubiquitination. This was associated with a significant reduction in the expression level of c-Cbl, an E3 ubiquitin ligase that ubiquitinylates PDGFR␤. Thus, ESDN modulates PDGF signaling in VSMCs via regulation of PDGFR surface levels. The ESDN effect is mediated, at least in part, through effects on PDGFR␤ ubiquitination. ESDN may serve as a target for regulating PDGFR␤ signaling in VSMCs.Vascular injury initiates a cascade of events that ultimately leads to vascular remodeling and often intimal hyperplasia. Vascular smooth muscle cell (VSMC) 2 proliferation and migration are key cellular events in this process. Platelet-derived growth factor (PDGF)-BB is released by platelets, endothelial cells, VSMCs, and inflammatory cells at the sites of vascular injury and is a particularly potent regulator of VSMC proliferation and migration (1). PDGF binding to PDGF receptor (PDGFR)␤ in VSMCs leads to receptor dimerization, autophosphorylation, and activation of downstream signaling pathways, including MAPK. The ligand-bound receptor is internalized through the endocytotic pathway and may either recycle to the membrane or undergo ubiquitination and lysosomal degradation (2). A number of endogenous stimulatory and inhibitory regulators, including the E3 ubiquitin ligase, c-Cbl (3), tightly regulate the mitogenic stimulus by modulating the duration and intensity of the signal.We have identified endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN, also called CLCP1 or DCBLD2) as a marker and regulator of cell proliferation in vascular remodeling (4). ESDN is a transmembrane protein with a domain structure similar to neuropilins (5, 6). ESDN can be induced by PDGF-BB and serum and is highly expressed in the neointima of injured rat (5), mouse (4), and human (4) arteries. ESDN expression parallels cell proliferation in the vessel wall in vivo (4). Furthermore, ESDN is up-regulated in proliferating VSMCs, and ESDN overexpression inhibits VSMC growth (4). Here, we expand the scope of our previous studies to demonstrate that ESDN regulates PDGF-induced VSMC migration and inhibits PDGF signa...

Section: Discussionmentioning

confidence: 82%

Endothelial and Smooth Muscle-derived Neuropilin-like Protein Regulates Platelet-derived Growth Factor Signaling in Human Vascular Smooth Muscle Cells by Modulating Receptor Ubiquitination

Guo

Nie

Esmailzadeh

et al. 2009

“…Real-time RT-PCR, Western Blotting, and Coimmunoprecipitation Assays-Total RNA was prepared from cultured cells for real-time RT-PCR using TRIzol as described previously (25). RNA from Xenopus laevis was prepared by using Lysis Matrix D beads in TRIzol.…”

Section: Methodsmentioning

confidence: 99%

“…RNA from Xenopus laevis was prepared by using Lysis Matrix D beads in TRIzol. Primers for SM ␣-actin, SM22␣, myocardin, MRTF A and MRTF B, and 18 S ribosomal RNA were described previously (25). Western blotting and co-immunoprecipitation assays were performed as previously described (26).…”

Section: Methodsmentioning

confidence: 99%

WD Repeat-containing Protein 5, a Ubiquitously Expressed Histone Methyltransferase Adaptor Protein, Regulates Smooth Muscle Cell-selective Gene Activation through Interaction with Pituitary Homeobox 2

Gan¹,

Thiébaud²,

Thézé³

et al. 2011

Self Cite

WD repeat-containing protein 5 (WDR5) is a common component of mammalian mixed lineage leukemia methyltransferase family members and is important for histone H3 lysine 4 methylation (H3K4me), which has been implicated in control of activation of cell lineage genes during embryogenesis. However, WDR5 has not been considered to play a specific regulatory role in epigenetic programming of cell lineage because it is ubiquitously expressed. Previous work from our laboratory showed the appearance of histone H3K4me within smooth muscle cell (SMC)-marker gene promoters during the early stages of development of SMC from multipotential embryonic cells but did not elucidate the underlying mechanisms that mediate SMC-specific and locus-selective H3K4me. Results presented herein show that knockdown of WDR5 significantly decreased SMCmarker gene expression in cultured SMC differentiation systems and in Xenopus laevis embryos in vivo. In addition, we showed that WDR5 complexes within SMC progenitor cells contained H3K4 methyltransferase enzymatic activity and that knockdown of WDR5 selectively decreased H3K4me1 and H3K4me3 enrichment within SMC-marker gene promoter loci. Moreover, we present evidence that it is recruited to these gene promoter loci through interaction with a SMC-selective pituitary homeobox 2 (Pitx2). Taken together, studies provide evidence for a novel mechanism for epigenetic control of SMCmarker gene expression during development through interaction of WDR5, homeodomain proteins, and chromatin remodeling enzymes.A critical question in developmental biology is how cell lineage-specific gene expression patterns are established and propagated during embryogenesis. Although the exact mechanisms by which complex patterns of gene expression are established during development are unclear, epigenetic modification of chromatin appears to play an important role in the transition of embryonic stem cells (ESCs) 2 into committed cell lineages (1, 2). Among the epigenetic modifications, histone methylation is an important primary determinant of cellular identity. In pluripotent ESCs, many lineage-specific genes are labeled with a unique bivalent histone modification pattern consisting of "activating" histone H3 lysine 4 methylation (H3K4me) and "repressive" H3 lysine 27 methylation (H3K27me) modifications. During the process of lineage determination from ESCs, these "bivalent domains" are resolved into constitutive H3K4me loci within genes that are actively expressed or H3K27me loci within genes that are repressed in specific cell lineages (2-4). In mammalian cells, H3K4me is mainly carried out by the mixed lineage leukemia (MLL) family members and SET1A/SET1B (5-7). These MLL/SET members have H3K4 methyltransferase (HKMT) activity through a conserved SET domain and form a complex with conserved subunits WDR5, Ash2L, and RbBP5 (8, 9). WDR5 is required to keep the MLL/ SET associated with the rest of the complex and is required for its activity (10). Although WDR5 was previously shown to be an H3K4me-binding protein impor...

“…Although HDAC4 has been shown to mediate platelet-derived growth factor-BB pro-proliferation of SMCs (30), the function of HDAC4 on ESC/SMC differentiation is unclear. To further explore the function of HDAC4 on SMC differentiation in this differentiation system, the down-regulated HDAC4 expression was rescued by transient transfection of a HDAC4 expression plasmid in mouse ESCs, and subsequently, transfected cells were induced to differentiate with RA.…”

Section: Inhibition Of Mir-10a Impairs Smc Differentiation Frommentioning

confidence: 99%

miR-10a Contributes to Retinoid Acid-induced Smooth Muscle Cell Differentiation

Huang

Xie

Sun

et al. 2010

119

105

MicroRNAs (miRs) have been reported to play a critical role in muscle differentiation and function. The purpose of this study is to determine the role of miRs during smooth muscle cell (SMC) differentiation from embryonic stem cells (ESCs). MicroRNA profiling showed that miR-10a expression is steadily increased during in vitro differentiation of mouse ESCs into SMCs. Lossof-function approaches using miR-10a inhibitors uncovered that miR-10a is a critical mediator for SMC lineage determination in our retinoic acid-induced ESC/SMC differentiation system. In addition, we have documented for the first time that histone deacetylase 4 is a novel target of miR-10a and mediates miR-10a function during ESC/SMC differentiation. To determine the molecular mechanism through which retinoic acid induced miR-10a expression, a consensus NF-B element was identified in the miR-10a gene promoter by bioinformatics analysis, and chromatin immunoprecipitation assay confirmed that NF-B could bind to this element. Finally, inhibition of NF-B nuclear translocation repressed miR-10a expression and decreased SMC differentiation from ESCs. Our data demonstrate for the first time that miR-10a is a novel regulator in SMC differentiation from ESCs. These studies suggest that miR-10a may play important roles in vascular biology and have implications for the diagnosis and treatment of vascular diseases.