Abstract
Vascular smooth muscle cell (VSMC) proliferation has been implicated in the development of restenosis after angioplasty, vein graft intimal thickening and atherogenesis. We investigated the mechanisms underlying positive and negative regulation of VSMC proliferation by the transcription factor cyclic AMP response element binding protein (CREB). Incubation with the cAMP elevating stimuli, adenosine, prostacyclin mimetics or low levels of forksolin activated CREB without changing CREB phosphorylation on serine-133 but induced nuclear translocation of the CREB co-factors CRTC-2 and CRTC-3. Overexpression of CRTC-2 or -3 significantly increased CREB activity and inhibited VSMC proliferation, whereas CRTC-2/3 silencing inhibited CREB activity and reversed the anti-mitogenic effects of adenosine A2B receptor agonists. By contrast, stimulation with serum or PDGFBB significantly increased CREB activity, dependent on increased CREB phosphorylation at serine-133 but not on CRTC-2/3 activation. CREB silencing significantly inhibited basal and PDGF induced proliferation. These data demonstrate that cAMP activation of CREB, which is CRTC2/3 dependent and serine-133 independent, is anti-mitogenic. Growth factor activation of CREB, which is serine-133-dependent and CRTC2/3 independent, is pro-mitogenic. Hence, CREB plays a dual role in the regulation of VSMC proliferation with the mode of activation determining its pro- or anti-mitogenic function.
Highlights
The second messenger 3′-5′ cyclic adenosine monophosphate is synthesized in cells by adenylyl cyclase enzymes in response to vasoactive Gs protein-coupled receptor stimulation
Activation of cyclic AMP response element binding protein (CREB) reporter activity by forskolin was not associated with an increase in CREB phosphorylation at serine-133 (Fig. 1B and C), despite robust Protein Kinase A (PKA) activation detected by increased phosphorylation of VASP (Fig. 1B and D)
We investigated the role played by CREB phosphorylation at serine-133 and the CREB co-factors, CRTC2 and CRTC3, in mediating the pro- or anti-mitogenic effects of elevated cyclic adenosine monophosphate (cAMP) in vascular smooth muscle cells
Summary
The second messenger 3′-5′ cyclic adenosine monophosphate (cAMP) is synthesized in cells by adenylyl cyclase enzymes in response to vasoactive Gs protein-coupled receptor stimulation. VSMC proliferation induced by angiotensin II26, thrombin[27] or the interferon-γ inducible protein-1028 is dependent on CREB activation These observations suggest that CREB activation may play a role in both mitogenic and anti-mitogenic responses in VSMC. Selective PKA activation fails to inhibit VSMC proliferation[2], suggesting that PKA-mediated phosphorylation of CREB at serine-133 may not be sufficient for the anti-mitogenic effects of cAMP in these cells. Several studies have suggested that phosphorylation of CREB at serine-133 may not be essential for CREB-dependent gene expression, implicating alternative mechanisms. T-cell receptor stimulation of Jurkat cells induces high levels of CREB serine-133 phosphorylation without inducing target gene expression[31]. We investigated the relative importance of CREB serine-133 phosphorylation and nuclear translocation of CRTCs in pro- and anti-mitogenic effects on VSMCs
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