Kadowaki Maiko, Mizuno Shiro, Demura Yoshiki, Ameshima Shingo, Miyamori Isamu, Ishizaki Takeshi
Third Department of Internal Medicine, University of Fukui, 23-3 Eiheiji-cho, Matsuoka, Yoshida-gun, Fukui, Japan.
Respir Res. 2007 Nov 1;8(1):77. doi: 10.1186/1465-9921-8-77.
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 p27kip1, 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 p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.
We investigated the role of p27kip1 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 p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.
Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 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 p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.
Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.
缺氧在体内和体外均可诱导肺动脉平滑肌细胞（PASMC）增殖，而前列环素类似物被认为可抑制PASMC生长。既往研究表明，p27kip1作为一种细胞周期蛋白依赖性激酶抑制剂，在平滑肌细胞增殖中发挥重要作用。然而，缺氧机制以及人肺动脉平滑肌细胞（HPASMC）中p27kip1与前列环素类似物之间的亚细胞相互作用尚未完全明确。
我们研究了p27kip1在贝前列素钠（BPS，一种稳定的前列环素类似物）抑制缺氧时HPASMC增殖能力中的作用。为阐明低氧空气暴露和BPS对HPASMC的生物学效应，将细胞在不同氧浓度（0.1% - 21%）的缺氧培养箱中培养。此后，通过溴脱氧尿苷（BrdU）掺入法测定DNA合成，用碘化丙啶染色通过流式细胞术分析细胞周期。用实时逆转录 - 聚合酶链反应（RT - PCR）和蛋白质免疫印迹法检测p27kip1 mRNA和蛋白表达及其稳定性。此外，我们通过小干扰RNA（siRNA）转染敲低p27kip1基因，评估其在HPASMC增殖中的作用。
虽然严重缺氧（0.1%氧气）抑制血清刺激的HPASMC增殖，但中度缺氧（2%氧气）通过增强p27kip1蛋白降解促进增殖，而BPS在缺氧和常氧条件下均通过细胞内cAMP升高抑制p27kip1降解来抑制HPASMC增殖。环磷腺苷类似物8 - 溴环磷酸腺苷（8 - Br - cAMP）在调节p27kip1方面具有与BPS类似的作用。中度缺氧不影响p27kip1蛋白表达的稳定性，但血小板衍生生长因子（PDGF，已知的主要缺氧诱导生长因子）显著降低p27kip1蛋白稳定性。我们还证明，BPS和8 - Br - cAMP在缺氧和常氧条件下均通过阻断p27kip1 mRNA降解来抑制HPASMC增殖。此外，p27kip1基因沉默部分减弱了BPS的作用，并部分恢复了缺氧诱导的增殖。
我们的研究表明，中度缺氧诱导HPASMC增殖，这可能部分依赖于通过诱导如PDGF等生长因子导致的p27kip1下调，而BPS通过cAMP途径抑制细胞增殖和p27kip1 mRNA降解。
