Abstract
Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A(-/-) cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF.MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.
Highlights
Neovascularization in mechanically challenged smooth muscle-rich organs in particular is promoted by various mechanoresponsive angiogenic factors including CCN1, formerly known as cysteine-rich protein 61 (Cyr61), a functionally multifaceted matricellular protein that appears in the extracellular environment during development and pathological states [3, 4]
Specific Regulation of CCN1 Promoter Activity by myocardin-related transcription factor (MRTF)-A in Response to Cyclic Strain—Mechanical induction of the CCN gene was tested in smooth muscle cells (SMCs) derived from mouse bladder harboring a loss-of-function mutation in MRTF-A, a major transcriptional co-activators of p67SRF
Nuclear Translocation of MRTF-A Is Necessary but Not Sufficient for Mechanical Strain-dependent Activation of the CCN1 Promoter—Because nuclear import of MRTF-A is a key step in strain-induced CCN1 gene expression, we examined the signaling requirement for MRTF-A nuclear import in the absence of export
Summary
Chronically by remodeling their extracellular environment. These compensatory responses are associated with the activation of angiogenesis to meet increased metabolic demands and improve tissue perfusion [1, 2]. ChIP analysis showed that mechanical strain and/or LMA treatment induced ϳ2.5-fold enrichment of the CCN1 promoter region with MRTF-A as compared with nontreated cells (Fig. 4B).
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