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

Abstract

Stone JD, Holt AW, Vuncannon JR, Brault JJ, Tulis DA. AMP-activated protein kinase inhibits transforming growth factor-␤-mediated vascular smooth muscle cell growth: implications for a Smad-3-dependent mechanism.

AMP-Activated Protein Kinase Inhibits Arterial Smooth Muscle Cell Proliferation in Vasodilator-Stimulated Phosphoprotein-Dependent Manner

“…Cell culture: Rat A7r5 ASM cells (ATCC, Manassas, VA) were cultured under sterile conditions in Dulbecco's Modified Eagles Medium (DMEM) supplemented with fetal bovine serum (FBS, 0.2-10%), 2 mM L-glutamine, a 1:500 dilution of 50 µg/mL Primocin (Invivogen) at 37°C in 95% air/5% CO 2 and were serially split and used through passage 6 as described [11,13,22].…”

“…Expression analyses: Cells were seeded in 96-well plates until confluent and treated with vehicle (DMSO) or AICAR (1 mM in DMSO), and after specified times total and phosphorylated protein expression was determined by ECL-based Western blotting on cell homogenates and by In-Cell Western blotting on intact, adherent cells as we have described [9][10][11]13,[20][21][22]. In brief, fixed cells were permeabilized with 0.1% Triton-X, blocked with IRDye blocking buffer and treated with rabbit anti-rat primary antibodies for 1 hour at room temperature (RT).…”

“…Fluorescence was detected and analyzed using Li-Cor Odyssey Infrared Imaging System and software. Additionally, ECL Western blots were performed on cell lysates to verify In-Cell Western data for select experiments as previously performed [9][10][11]13,[20][21][22].…”

“…Previous work from our lab established an AICAR dosing regimen that significantly induces AMPK expression and activity in rat primary and A7r5 ASM cells [9][10][11]. Following this approach, in the current study we used AICAR to stimulate AMPK signaling and evaluated downstream targets including VASP in rat A7r5 ASM cell growth.…”

“…In complement, we recently showed that AMPK has capacity to communicate with the cyclic AMP/protein kinase A (PKA) pathway in growth suppression of ASM under both in vitro and in vivo conditions [9][10][11][12][13].…”