Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27(kip1)

BACKGROUND: Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27(kip1), a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27(kip1 )and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood. METHODS: We investigated the role of p27(kip1 )in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27(kip1 )mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27(kip1 )in HPASMC proliferation using p27(kip1 )gene knockdown using small interfering RNA (siRNA) transfection. RESULTS: Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27(kip1 )protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27(kip1 )degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27(kip1). Moderate hypoxia did not affect the stability of p27(kip1 )protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27(kip1 )protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27(kip1 )mRNA degradation. Furthermore, p27(kip1 )gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation. CONCLUSION: Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27(kip1 )down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27(kip1 )mRNA degradation through cAMP pathway.