Heparin inhibits pulmonary artery smooth muscle cell proliferation through guanine nucleotide exchange factor-H1/RhoA/Rho kinase/p27

Abstract

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.

Figure 1.

Effect of heparin on Rho kinase (ROCK) expression in hypoxic mice, and effect of hypoxia on ROCK expression in human pulmonary artery smooth muscle cells (PASMCs). (A) ROCK expression in hypoxic mice: total protein from mouse lungs was isolated and then subjected to Western blot analysis. Representative images of Western blot (left) and quantitative results (right), showing relative ROCK expression, setting normoxic control as 1. *P < 0.05 as compared with other groups. (B). ROCK expression in human PASMCs: representative images of RT-PCR and Western blot (left) and quantitative results (right). *P < 0.05 as compared with previous time-point group. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for equal loading control. The results are representative of three separate experiments.

Figure 2.

Effect of over expressing RhoA on heparin inhibition of PASMC proliferation and on RhoA activity and location, as well as p27 expression. (A) Overexpression of RhoA and heparin inhibition of PASMC proliferation. Cells grown in 10% FBS and 0.1% FBS were used for positive and negative controls. The cells in heparin treatment groups were grown in 10% FBS plus different doses of heparin (HP). The results are representative of three separate experiments (total n = 15). *P < 0.05 as compared with 10% FBS. (B) GTP-RhoA pulldown assay showing relative GTP-RhoA and total RhoA expression. Control was cells growing in 10% FBS (thereafter). (C) RhoA localization and (D) p27 expression. Representative images of Western blot (left) and quantitative results (right), setting control as 1. The results are representative of three separate experiments. *P < 0.05 as compared with control cells.

Figure 3.

Effect of overexpressing guanine nucleotide exchange factor (GEF)–H1 on heparin inhibition of PASMC proliferation. (A) GEF-H1 mRNA and protein expression. Total RNA and protein from the lungs of hypoxic mice treated with or without heparin were isolated for RT-PCR and Western blot analysis. Representative images of RT-PCR and Western blot (left) and quantitative results (right), setting control as 1. *P < 0.05 as compared with control cells. The results are representative of three separate experiments. (B) GEF-H1 overexpression and PASMC proliferation. *P < 0.05 as compared with 10% FBS. The results are representative of three separate experiments (total n = 15). GEF-H1, GEF-H1 cDNA.

Figure 4.

Effect of overexpressing RhoA and GEF-H1 on human PASMC proliferation and cell cycle progression under hypoxia. (A) RhoA and GEF-H1 cDNA transfection and human PASMC proliferation, (B) cell cycle analysis, and (C) RhoA and GEF-H1 small interfering RNA (siRNA) silencing and PASMC proliferation. *P < 0.05 as compared with control. The results are representative of three separate experiments (total n = 15).

Figure 5.

Effect of overexpressing RhoA and GEF-H1 on GEF-H1, GTP-RhoA, and p27 in human PASMCs under hypoxia. Proteins were isolated from human PASMCs under hypoxia treated with GEF-H1 cDNA, RhoA cDNA, and heparin, respectively, for Western blot analysis (total protein for GEF-H1, p27, and GAPDH; GTP pulldown protein for GTP-RhoA). (A) Effect of GEF-H1 on GTP-RhoA and p27 expression. (B) Effect of RhoA on GEF-H1 and p27 expression. (C) Heparin and p27 phosphroylation. Representative images of Western blot (left) and quantitative results (right), setting control as 1. *P < 0.05 as compared with control group. The results are representative of three separate experiments.

Figure 6.

Effect of heparin on the GEF-H1/RhoA/ROCK/p27 signaling pathway in inhibition of PASMC proliferation. Under normal physiopathological condition, the GEF-H1/RhoA/ROCK/p27 signaling pathway regulates a variety of cellular functions, including cell proliferation. Growth stimulus, including hypoxia, can stimulate GEF-H1 activity, which catalyzes the change of GDP for GTP to generate GTP-RhoA. GTP-RhoA consequently activates its downstream effecter, ROCK, and then ROCK degrades p27, which eventually results in PASMC proliferation and development of pulmonary hypertension. Heparin inhibits PASMC proliferation and hypoxic pulmonary hypertension through inhibiting GEF-H1, which results in a decrease in GTP-RhoA and ROCK. The decrease in ROCK attenuates degradation of p27, which then results in an increase in p27. Dashed arrows, growth stimulation; solid arrows, heparin treatment.