Inhibition of vascular smooth muscle growth via signaling crosstalk between AMP-activated protein kinase and cAMP-dependent protein kinase

Abstract

Abnormal vascular smooth muscle (VSM) growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP)-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKA). Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remain unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells (VSMC), the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSMC migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashion. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.

AMP-activated protein kinase inhibits transforming growth factor-β-mediated vascular smooth muscle cell growth: implications for a Smad-3-dependent mechanism

“…VSMCs were seeded in 96-well plates and, once confluent, were treated with specified agents. Cells were then formalin fixed, and protein expression was determined by In-Cell Western analysis as described (1,11,24). Briefly, fixed cells were permeabilized with 0.1% Triton X, blocked with IRDye blocking buffer (Invitrogen), and treated with rabbit antirat primary antibodies (1 h; room temperature) in 96-well plates.…”

“…Cells were plated in 12-well plates at 80,000 cells/well in complete media until ϳ50% confluent. Cells were quiesced in 0.5% FBS for 24 h followed by treatment in complete growth media (DMEM, 10% FBS; Primocin) containing select pharmacological agents for 24 h. Cells were trypsinized, fixed, and stained with propidium iodide or Draq5 for flow cytometry or DAPI for iCys per the manufacturer's recommendations (11,24). The fraction of cells in each phase of the cell cycle was assessed by flow cytometry or iCys LSC.…”

“…Primary VSMCs were obtained from thoracic aorta of male Sprague Dawley rats (ϳ125 g; Charles River Labs) by collagenase and elastase digestion and characterized morphologically as described (5). VSMCs were cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with fetal bovine serum (FBS, 0.5-10%), 2 mM L-glutamine, and 1:500 dilution of 50 g/ml Primocin at 37°C in 95% air-5% CO 2, and were serially split and used through passage 10 (commercial) or passage 6 (primary) to avoid onset of phenotypic switching (1,11,23,24) unless otherwise stated.…”

“…Based on our previous observations showing growth inhibition by AMPK in VSM (23,24), and considering the potential growthpromoting nature of TGF-␤/Smad3 in VSM, an interesting yet unexplored relationship may exist between these two signaling molecules. The purpose of this investigation was to characterize the capacity of AMPK to modulate growth in response to TGF-␤ in rat VSMCs.…”

“…Activation of AMPK requires AMP binding and catalytic phosphorylation (17,20) and leads to energy-producing pathways being upregulated while energy-consuming pathways are shut off or rendered minimally active (7,8,17,(22)(23)(24). AMPK has many tissue-dependent functional targets leading to phosphorylation of key metabolic enzymes and, specifically in the vasculature, has been implicated in increased endothelial nitric oxide synthase activity and nitric oxide bioavailability, the promotion of angiogenesis, and cytostasis of commercial and primary VSMCs via inhibition of cell cycle progression (10,13,(22)(23)(24). Moreover, AMPK activation has been shown to inhibit neointima formation in rat arteries following injury (13,23).…”