title: cAMP-induced actin cytoskeleton remodelling inhibits MKL1-dependent expression of the chemotactic and pro-proliferative factor, CCN1.
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.
authors.affiliation: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, BS2 8HW, UK.
authors.forename: Aparna
authors.initials: A
authors.lastname: Duggirala
authors.affiliation: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, BS2 8HW, UK.
authors.forename: Tomomi E
authors.initials: TE
authors.lastname: Kimura
authors.affiliation: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, BS2 8HW, UK.
authors.forename: Graciela B
authors.initials: GB
authors.lastname: Sala-Newby
authors.affiliation: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, BS2 8HW, UK.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Johnson
authors.affiliation: Department of Medicine, MacKay Medical College, New Taipei, Taiwan; Cardiovascular Division, Department of Internal Medicine, MacKay Memorial Hospital, New Taipei, Taiwan.
authors.forename: Yih-Jer
authors.initials: YJ
authors.lastname: Wu
authors.affiliation: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, BS2 8HW, UK.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Newby
authors.affiliation: Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, BS2 8HW, UK. Electronic address: mark.bond@bris.ac.uk.
authors.forename: Mark
authors.initials: M
authors.lastname: Bond
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects*; D000241:Adenosine / Q000494:pharmacology; D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D002339:Carotid Arteries / Q000187:drug effects / Q000473:pathology; D049109:Cell Proliferation / Q000187:drug effects; D002633:Chemotaxis / Q000187:drug effects*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D055515:Cysteine-Rich Protein 61 / Q000235:genetics* / Q000378:metabolism; D011464:Epoprostenol / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008297:Male; D008934:Mitogens / Q000494:pharmacology; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D026362:Serum Response Factor / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000187:drug effects; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000631:Aminopyridines; C501849:CCN1 protein, rat; D055515:Cysteine-Rich Protein 61; D008934:Mitogens; C000594702:Mrtfa protein, rat; D026362:Serum Response Factor; D015534:Trans-Activators; D014157:Transcription Factors; D005576:Colforsin; D011464:Epoprostenol; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D000241:Adenosine; C043867:cicaprost
keywords: 3′-5′-Cyclic adenosine monophosphate; CCN1; Cyr61; MKL1; VSMC; cAMP