Abstract
Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. However, the mechanisms are incompletely understood. Here we investigated whether cAMP-induced actin-cytoskeleton remodelling modulates VSMC behaviour by inhibiting expression of CCN1. In cultured rat VSMC, CCN1-silencing significantly inhibited BrdU incorporation and migration in a wound healing assay. Recombinant CCN1 enhanced chemotaxis in a Boyden chamber. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Forskolin also inhibited CCN1 expression in balloon injured rat carotid arteries in vivo. Inhibiting RhoA activity, which regulates actin-polymerisation, by cAMP-elevation or pharmacologically with C3-transferase, or inhibiting its downstream kinase, ROCK, with Y27632, significantly inhibited CCN1 expression. Conversely, expression of constitutively active RhoA reversed the inhibitory effects of forskolin on CCN1 mRNA. Furthermore, CCN1 mRNA levels were significantly decreased by inhibiting actin-polymerisation with latrunculin B or increased by stimulating actin-polymerisation with Jasplakinolide. We next tested the role of the actin-dependent SRF co-factor, MKL1, in CCN1 expression. Forskolin inhibited nuclear translocation of MKL1 and binding of MKL1 to the CCN1 promoter. Constitutively-active MKL1 enhanced basal promoter activity of wild-type but not SRE-mutated CCN1; and prevented forskolin inhibition. Furthermore, pharmacological MKL-inhibition with CCG-1423 significantly inhibited CCN1 promoter activity as well as mRNA and protein expression. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel cAMP-dependent mechanism controlling VSMC behaviour.
Highlights
Elevation of intracellular cyclic adenosine monophosphate (cAMP) concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression
We hypothesised that cAMP-mediated repression of Cysteinerich protein 61 (CCN1) might contribute towards reduced vascular smooth muscle cells (VSMC) proliferation and migration
As a first step to investigate the regulation of CCN1 in VSMC in vivo, we studied the effect of peri-adventitial application of forskolin on expression of CCN1 protein in rat carotid arteries after balloon injury
Summary
Elevation of intracellular cAMP concentration has numerous vascular protective effects that are in part mediated via actin cytoskeleton-remodelling and subsequent regulation of gene expression. Adding db-cAMP, or elevating cAMP using forskolin, significantly inhibited CCN1 mRNA and protein expression in vitro; transcriptional regulation was demonstrated by measuring pre-spliced CCN1 mRNA and CCN1-promoter activity. Our data demonstrates that cAMP-induced actin-cytoskeleton remodelling regulates expression of CCN1 through MKL1: it highlights a novel cAMP-dependent mechanism controlling VSMC behaviour. In vascular smooth muscle cells (VSMC), elevated cAMP levels inhibit mitogen stimulated proliferation in vitro [1] and injury induced proliferation in vivo [6]. RhoA or Rac inhibition using pharmacological inhibitors, siRNA or dominant-negative approaches mimics the effects of cAMP on VSMC morphology, proliferation, migration and relaxation in vitro and in vivo [20,23], demonstrating the functional role of Rho GTPase inhibition for cAMP responses. Regulation of transcription factor activity via these actin-dependent mechanisms typically controls cell behaviour by rapidly modulating expression of immediate early genes
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