Heparin inhibits pulmonary artery smooth muscle cell proliferation through guanine nucleotide exchange factor-H1/RhoA/Rho kinase/p27.
Ras homolog gene family member A (RhoA) through Rho kinase kinase (ROCK), one of its downstream effectors, regulates a wide range of cell physiological functions, including vascular smooth muscle cell (SMC) proliferation, by degrading cyclin-dependent kinase inhibitor, p27. Our previous studies found that heparin inhibition of pulmonary artery SMC (PASMC) proliferation and pulmonary hypertension was dependent on p27 up-regulation. To investigate whether ROCK, a regulator of p27, is involved in regulation of heparin inhibition of PASMC proliferation, we analyzed ROCK expression in the lungs from mice and from human PASMCs exposed to hypoxia, and investigated the effect of ROCK expression in vitro by RhoA cDNA transfection. We also investigated the effect of guanine nucleotide exchange factor (GEF)-H1, an upstream regulator of RhoA, on heparin inhibition of PASMC proliferation by GEF-H1 cDNA transfection. We found that: (1) hypoxia increased ROCK expression in mice and PASMCs; (2) overexpression of RhoA diminished the inhibitory effect of heparin on PASMC proliferation and down-regulated p27 expression; and (3) overexpression of GEF-H1 negated heparin inhibition of PASMC proliferation, which was accompanied by increased GTP-RhoA and decreased p27. This study demonstrates that the RhoA/ROCK pathway plays an important role in heparin inhibition on PASMC proliferation, and reveals that heparin inhibits PASMC proliferation through GEF-H1/RhoA/ROCK/p27 signaling pathway, by down-regulating GEF-H1, RhoA, and ROCK, and then up-regulating p27.
Yu
Lunyin
L
Pulmonary and Critical Care Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114-2696, USA. lyu3@partners.org
Quinn
Deborah A
DA
Garg
Hari G
HG
Hales
Charles A
CA
D000818:Animals; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D008954:Models, Biological; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D011651:Pulmonary Artery / Q000166:cytology*; D034741:RNA, Small Interfering / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D054460:rho-Associated Kinases / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C577356:ARHGEF2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D050760:Cyclin-Dependent Kinase Inhibitor p27; D006160:Guanosine Triphosphate; D006493:Heparin; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein