A rapid, PPAR-gamma-dependent effect of pioglitazone on the phosphorylation of MYPT.
Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, thiazolidinediones, have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO; 20 muM) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit of myosin phosphatase (MYPT). PIO decreased the phosphorylation of Thr(697) on MYPT within 15 min, and the inhibition was maintained up to 6 h. The PPAR-gamma antagonist GW-9662 (5 microM) abrogated the inhibition of Thr(697) phosphorylation mediated by PIO. Because longer-term PIO treatment inhibits RhoA/Rho kinase (ROCK) signaling and Thr(697) phosphorylation, we tested the effect of the ROCK inhibitor Y-27632 (10 muM) on the inhibition of Thr(697) phosphorylation by PIO. Y-27632 alone inhibited Thr(697) phosphorylation, and there was an additive effect with PIO. In addition, up to 1 h of PIO treatment did not affect RhoA localization or decrease ROCK-dependent phosphorylation of Thr(855). These results suggest that the effect of PIO is independent of inhibition of RhoA/ROCK. PIO increased the phosphorylation of Ser(696) in the same time course as its effect on Thr(697). Ser(696) has been shown to be phosphorylated by PKA and PKG. PKA inhibitor H-89 (10 microM) and PKG inhibitor KT-5823 (0.5 microM) abrogated the effect of PIO on both Thr(697) and Ser(696) phosphorylation. The constitutive turnover of phosphorylation of Thr(697) is rapid, suggesting that the decreased phosphorylation of Thr(697) by PIO is due to enhanced phosphorylation of Ser(696). This is supported by the finding that PIO blocks ANG II-stimulated phosphorylation of Thr(697) but not ANG II-stimulated RhoA translocation. Therefore, the effect of shorter-term PIO apparently is to increase myosin light chain phosphatase activity, thereby desensitizing the vascular smooth muscle to agonist signaling.
Atkins
Kevin B
KB
University of Michigan, Ann Arbor, MI 48109-0676, USA. katkins@umich.edu
Irey
Brittany
B
Xiang
Nan
N
Brosius
Frank C
FC
D000577:Amides / Q000494:pharmacology; D000813:Anilides / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D047495:PPAR gamma / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D000077205:Pioglitazone; D054645:Protein Phosphatase 1 / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D045162:Thiazolidinediones / Q000494:pharmacology*; D013912:Threonine / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
C457499:2-chloro-5-nitrobenzanilide; D000577:Amides; D000813:Anilides; D004791:Enzyme Inhibitors; D007004:Hypoglycemic Agents; D047495:PPAR gamma; D011725:Pyridines; D045162:Thiazolidinediones; C108830:Y 27632; D013912:Threonine; D054460:rho-Associated Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1; D000077205:Pioglitazone