Effects of typical PKC subtypes on the proliferation of mouse pulmonary artery smooth muscle cells and the expression of ERK1/2 and Akt induced by hypoxia

Abstract

Objective: To study the effects of specific isoforms of classic protein kinase C (cPKCs) on hypoxia-induced proliferation and the expression of ERK1/2 and Akt using drug intervention or virus transfection in vitro. Methods: Dynal MPC-1 magnetic particle concentrator was used to separate iron-containing pulmonary arterioles fragments, and the pulmonary artery smooth muscle cells (PASMCs) were primary cultured and identified. The cells were intervened by PKC agonist (PMA), PKCα inhibitor (safingol), PKCβⅠ inhibitor (Go6976) and PKCβⅡ inhibitor (LY333531) respectively, and the changes in protein expressions of cPKCs, and the phosphorylation levels of ERK1/2 and Akt were observed by immunoblotting under the condition of normal oxygen or hypoxia. The lentiviral vectors of PKCα and PKCβ were used to specifically knock-down the activity of target genes by virus transfection techniques, and Western blotting was used to observe the protein expressions of cPKCs, and the phosphorylation levels of ERK1/2 and Akt in hypoxia-induced PASMCs in mice. Results: With Brdu method, the proliferation of PASMCs induced by hypoxia was significantly inhibited by safingol, Go6976 and LY333531 by inhibiting cPKCα, βⅠ and βⅡ respectively. Compared with the hypoxic control group, the rates of Brdu positive cells were (7.35±0.26)% vs (11.28±0.43)%, (3.76±0.25)% vs (7.98±0.28)% and (4.12±0.46)% vs (7.78±0.53)%. We also observed that PMA could significantly promote the proliferation of PASMCs under normoxic condition. Compared with the normoxia control group, the Brdu-positive cell rates were (9.65±0.47)% vs (6.34±0.52)%, (9.34±0.38)% vs (5.42±0.21)% and (7.78±0.53)% vs (4.12±0.46)%. In addition, after transfection with PKCα or PKCβ lentiviral vector, the proliferation of PASMCs was significantly lower in hypoxia transfection group than in the control group. The rates of Brdu positive cells were (3.58±0.54)% vs (5.97±0.63)%, respectively. Using Western blotting, we also observed that after being inhibited by safingol, Go6976 and LY333531 respectively, the phosphorylation levels of ERK1/2 and Akt in PASMCs induced by hypoxia was significantly lower than the control group. After using safingol, the phosphorylation levels of ERK1/2 and Akt were (0.56±0.07) vs (1.08±0.13) and (0.49±0.04) vs (0.97±0.08). After using Go6976, the phosphorylation levels of ERK1/2 and Akt were (0.41±0.09) vs (0.79±0.10) and (0.48±0.09) vs (0.82±0.16), after using LY333531, the phosphorylation levels of ERK1/2 and Akt were (0.42±0.03) vs (0.87±0.06) and (0.34±0.07) vs (0.78±0.05). While PMA could promote the phosphorylation levels of ERK1/2 and Akt under normoxic condition, 1.25±0.12 vs 0.41±0.07 and 0.98±0.06 vs 0.37±0.08, respectively. Using transfection technique to specifically knock down the expression of cPKCα and β, we found that under hypoxic conditions, transfection of PASMCs could significantly lower the phosphorylation levels of ERK1/2, its phosphorylation level was 0.29±0.06 vs 0.76±0.05, with no evident change in the phosphorylation levels of Akt. Conclusions: Hypoxia may lead to phosphorylation of ERK1/2 by promoting the protein expression of cPKCα, cPKCβⅠ and cPKCβⅡ respectively, which eventually induces abnormal proliferation of PASMCs from the distal pulmonary arteries, participating in the development of hypoxic pulmonary hypertension (HPH) of the mice. Regulation of the expression of cPKCα, cPKCβⅠ and cPKCβⅡ may help to attenuate the formation of pulmonary vascular remodeling. Target therapy based on cPKCs is expected to be a new direction for HPH therapy in the future.