Objective: To investigate the effect of protein kinase C (PKC)-nuclear factor-kappa B (NF-kappaB) signal transduction pathway on proliferation and expression of vascular endothelial growth factor (VEGF) in human pulmonary artery smooth muscle cells (HPASMCs).
Methods: Cultured HPASMCs in normoxia or hypoxia conditions were divided into three groups and stimulated with or without phorbol 12-myristate 13-acetate (PMA) and pyrrolidine dithiocarbamate (PDTC) in vitro. The three groups were the control group, the PMA group and the PMA + PDTC group. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect VEGF mRNA expression, and the expression of VEGF protein and the inhibitor protein IkappaBalpha were observed by Western blot, while the location and expression of NF-kappaB p65 were observed by immunocytochemical staining, and cell cycle phases were analyzed by flow cytometry.
Results: (1) As for the positive rate of nucleolar staining for NF-kappaB p65, the relative expression of IkappaBalpha protein, and the percentage of G(2)/M phases of cell cycle, there were significant differences between the PMA group and the control group or PMA + PDTC group, both in normoxia and hypoxia conditions (P < 0.05, respectively), and there was also a significant difference between the normoxia and hypoxia PMA groups (P < 0.05). (2) There were no significant differences in VEGF mRNA and protein expression among the three groups (P > 0.05, respectively) in normoxia, but the expression was higher in hypoxia PMA group than in hypoxia control and hypoxia PMA + PDTC or normoxia PMA group (P < 0.05, respectively). (3) There was a positive correlation between the positive rate of nucleolar staining for NF-kappaB p65, the relative expression of VEGF protein and the percentage of G(2)/M phases of cell cycle in hypoxia PMA group (r = 0.587 - 0.710, P < 0.05, respectively).
Conclusions: There is a signal transduction pathway of PKC-NF-kappaB in HPASMCs. The activity of PKC can be enhanced in hypoxia, concomitant NF-kappaB activation or VEGF overexpression to be involved in the proliferation. These results suggest that the activation of NF-kappaB can be considered as a downstream of PKC signal transduction pathway, and the activation of PKC-NF-kappaB signal transduction pathway and VEGF overexpression may contribute to the process of hypoxic pulmonary hypertension.