Adenylyl Cyclase Isoform–Selective Regulation of Vascular Smooth Muscle Proliferation and Cytoskeletal Reorganization

Abstract

Abstract-Compartmentation of cAMP signaling been demonstrated to be attributable to the structural association of protein kinase A (PKA) (via association with A-kinase anchoring proteins [AKAPs]) with phosphodiesterase and AKAP-dependent effector molecules. However, other mechanisms contributing to compartmentalization have not been rigorously explored, including the possibility that different isoforms of adenylyl cyclase (AC) may be functionally "compartmentalized" because of differential association with tethering or signaling molecules. To this end, we examined the effect of adenoviral transduction of representative AC isoforms (AC1, AC2, AC5, and AC6) on cellular cAMP production, PKA activation, extracellular signal-regulated kinase (ERK) activation, cell doubling and proliferation, as well as arborization responses (an index of cAMP-mediated cytoskeletal re-organization) in vascular smooth muscle cells. When isoforms were expressed at levels to achieve comparable forskolin-stimulated AC activity, only gene transfer of AC6 significantly enhanced PKA-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and arborization responses. Treatment of control cells, which express AC6 endogenously, as well as vascular smooth overexpressing the AC6 isoform with small interfering RNA directed against AC6, significantly suppressed both isoproterenol-stimulated cAMP accumulation and arborization. Notably, the selective effects of AC6 expression were abrogated in the presence of phosphodiesterase suppression. In contrast, only the expression of AC1 enhanced forskolin-stimulated association of ERK with AC, demonstrated by coimmuno-isolation of ERK with Flag-tagged AC1, but not with Flag-tagged AC6. To determine whether these isoform-selective effects of AC were unique to differentiated and morphologically compartmentalized vascular smooth muscle cells or were a general property of these isoforms, we examined the consequence of expression of these various isoforms in human embryonic kidney (HEK) cells. Indeed, we observed similar isoform-dependent association of AC1 with ERK, activation of ERK by stimulation of AC1 with forskolin, and AC1-dependent lengthening of doubling time, indicating that these properties of AC1 are cell autologous and likely result from AC1-dependent protein-protein interactions. In aggregate, these findings suggest that isoform-

β2-Adrenergic Receptor Agonists Modulate Human Airway Smooth Muscle Cell Migration via Vasodilator-Stimulated Phosphoprotein

“…Evidence suggests that cAMP-inducing agents may modulate the actin cytoskeleton during cell motility through an activation of PKA, which regulates the activity of VASP by specific phosphorylation at Ser157. The PKA-dependent of VASP was demonstrated in rat aortic vascular smooth muscle cells (20). The stimulation of human neutrophils by a chemoattractant induces the PKA-dependent phosphorylation of VASP at Ser157 (21).…”

Adenylyl Cyclase 2 Selectively Couples to E Prostanoid Type 2 Receptors, Whereas Adenylyl Cyclase 3 Is Not Receptor-Regulated in Airway Smooth Muscle

“…Arborization of smooth muscle cells is a useful response to monitor, because it is mediated by cAMP and can be readily detected in single cells (Gros et al, 2006). We found that arborization rate stimulated by forskolin (which activates all AC isoforms, although not necessarily to the same degree; is enhanced by overexpression of AC3 or AC6 but not AC2.…”

“…One response that can be assayed in single cells is arborization. Arborization is a cell shape change that is stimulated by cAMP signaling in smooth muscle cells (Gros et al, 2006). We measured the rate of arborization in individual cells by analyzing the pixel density change over time (40 min) under a digital microscope.…”

G Protein regulation of MAPK networks

“…Whereas Ga s -mediated stimulation of C-Raf-MEK1/2-ERK1/2 module involves PKA-dependent or -independent activation of Ras, Ga s -mediated inhibition involves PKA-mediated phosphorylation or Rap-1-mediated sequestration of C-Raf. In light of recent findings that adenylyl cyclase as well as PKA are functionally and/or physically compartmentalized through their interactions with proteins such as ERKs (Gros et al, 2006) or AKAPs (Dumaz and Marais, 2005;Houslay, 2006), it is quite possible that the Ga s -mediated differential regulation of ERK modules by Ga s involves different pools of adenylyl cyclase and/or PKA in a single cell.…”