Sodhi C P, Batlle D, Sahai A
Division of Nephrology and Hypertension, Northwestern University Medical School, Chicago, IL 60611, USA.
Kidney Int. 2000 Aug;58(2):691-700. doi: 10.1046/j.1523-1755.2000.00215.x.
We previously reported that hypoxia induces the proliferation of cultured mesangial cells mediated by the stimulation of intracellular calcium and the activation of protein kinase C (PKC). In the present study, we examined the roles of mesangial cell specific growth factors (platelet-derived growth factor and endothelin-1) and osteopontin (OPN) in hypoxia-induced proliferation of mesangial cells. In addition, we determined the effect of hypoxia on p38 mitogen-activated protein (MAP) kinase activity and the roles of both PKC and p38 MAP kinase in hypoxia-induced alterations in OPN and mesangial cell growth.
Quiescent cultures of mesangial cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and [3H]-thymidine incorporation, OPN protein and mRNA expression, and p38 MAP kinase activity were assessed.
Hypoxic-conditioned medium mimicked the effect of hypoxia on thymidine incorporation, suggesting the release of diffusable growth promoting factor(s) by hypoxia. Neither anti-endothelin-1 nor anti-platelet-derived growth factor-neutralizing antibodies had an effect on increased thymidine incorporation induced by hypoxia. However, blocking the effects of OPN either with anti-OPN antibody or its beta3 integrin receptor antibody completely prevented the hypoxia-induced increase in thymidine incorporation. Hypoxia also stimulated OPN protein and mRNA levels. Hypoxia caused an acute activation of p38 MAP kinase, which was inhibited by both verapamil and an inhibitor of PKC (calph C). PKC inhibitor and an inhibitor of p38 MAP kinase (SB203580) reduced the hypoxia-induced stimulation of both OPN and cell growth.
These studies provide, to our knowledge, the first evidence demonstrating the role of OPN in hypoxia-induced proliferation of mesangial cells. In addition, hypoxia causes an activation of p38 MAP kinase in a calcium- and PKC-dependent manner, and the activation of PKC and p38 MAP kinase appears to be involved in the stimulation of both OPN and mesangial cell proliferation induced by hypoxia.
我们之前报道过，缺氧通过刺激细胞内钙和激活蛋白激酶C（PKC）介导培养的系膜细胞增殖。在本研究中，我们检测了系膜细胞特异性生长因子（血小板衍生生长因子和内皮素-1）和骨桥蛋白（OPN）在缺氧诱导的系膜细胞增殖中的作用。此外，我们确定了缺氧对p38丝裂原活化蛋白（MAP）激酶活性的影响，以及PKC和p38 MAP激酶在缺氧诱导的OPN和系膜细胞生长改变中的作用。
将系膜细胞的静止培养物在无血清培养基中暴露于缺氧（3%氧气）或常氧（18%氧气）环境，评估[3H]-胸腺嘧啶核苷掺入、OPN蛋白和mRNA表达以及p38 MAP激酶活性。
缺氧条件培养基模拟了缺氧对胸腺嘧啶核苷掺入的影响，提示缺氧释放了可扩散的生长促进因子。抗内皮素-1抗体和抗血小板衍生生长因子中和抗体均对缺氧诱导的胸腺嘧啶核苷掺入增加无影响。然而，用抗OPN抗体或其β3整合素受体抗体阻断OPN的作用可完全阻止缺氧诱导的胸腺嘧啶核苷掺入增加。缺氧还刺激了OPN蛋白和mRNA水平。缺氧导致p38 MAP激酶急性激活，维拉帕米和PKC抑制剂（钙磷蛋白C）均可抑制该激活。PKC抑制剂和p38 MAP激酶抑制剂（SB203580）可降低缺氧诱导的OPN和细胞生长刺激。
据我们所知，这些研究首次提供了证据，证明OPN在缺氧诱导的系膜细胞增殖中起作用。此外，缺氧以钙和PKC依赖的方式导致p38 MAP激酶激活，PKC和p38 MAP激酶的激活似乎参与了缺氧诱导的OPN和系膜细胞增殖的刺激。
