Griffin M, Lee H W, Zhao L, Eghbali-Webb M
Department of Anesthesiology, Yale School of Medicine, New Haven, CT 06510, USA.
Mol Cell Biochem. 2000 Dec;215(1-2):21-30. doi: 10.1023/a:1026585420021.
Ischemic heart disease is more prevalent in men than in women. The remodeling of extracellular matrix, is a structural correlate of heart failure of ischemic origin and proliferation of cardiac fibroblasts is a key factor in this remodeling. We asked if proliferative response of male and female cardiac fibroblasts is differentially susceptible to hypoxia. DNA synthesis, using 3H-thymidine incorporation was compared under hypoxia (2% O2) in cardiac fibroblasts obtained from adult, age-matched male and female rat heart. In female cells DNA synthesis remained unchanged under hypoxia and this resistance was dependent on tyrosine kinase activation, as it was abolished in the presence of genistein, a tyrosine kinase inhibitor. Male cells, on the other hand, were susceptible to hypoxia and their DNA synthesis was reduced significantly (70%, (p < 0.0001). This effect was partially reversed by inhibition of tyrosine kinase. Western analysis showed a higher abundance of tyrosine phosphorylated proteins in male cells compared to female cells as well as differences in molecular weight of basal and hypoxia-induced tyrosine-phosphorylated proteins between male and female cells. The presence of estrogen (17-beta estradiol, 10 nM) altered the response of both cells to hypoxia. In female cells the combined effect of hypoxia and estrogen led to inhibition of DNA synthesis, whereas in male cells estrogen partially reversed the hypoxia-induced inhibition of DNA synthesis (37% (p < 0.01) inhibition in the presence of estrogen vs. 70% (p < 0.0001) inhibition in the absence of estrogen). The effects of estrogen in male and female cells were mediated via estrogen receptors as they were reversed by the pure anti-estrogen, ICI 182,780. Western analysis of cell lysate showed hypoxia-induced increase in the level of estrogen receptor beta in both male and female cells. Gel shift analysis showed hypoxia-induced increase in cytoplasmic ERE (estrogen response element)-binding activity and decrease in nuclear ERE-binding in male cells. In female cells cytoplasmic and nuclear ERE-binding activities remained unchanged under hypoxia. Together, these data demonstrate that while female cells are resistant to hypoxia-induced inhibition in DNA synthesis, male cells are susceptible; intracellular pathways involving tyrosine phosphorylation are involved in the response of both cells; and estrogen, via estrogen-receptor-dependent mechanisms, differentially alters the response of male and female cells to hypoxia.
缺血性心脏病在男性中比在女性中更为普遍。细胞外基质的重塑是缺血性心力衰竭的一种结构关联，而心脏成纤维细胞的增殖是这种重塑的关键因素。我们探讨了雄性和雌性心脏成纤维细胞的增殖反应对缺氧的易感性是否存在差异。利用3H-胸腺嘧啶核苷掺入法比较了从成年、年龄匹配的雄性和雌性大鼠心脏获取的心脏成纤维细胞在缺氧（2% O2）条件下的DNA合成情况。在雌性细胞中，缺氧条件下DNA合成保持不变，这种抗性依赖于酪氨酸激酶激活，因为在酪氨酸激酶抑制剂染料木黄酮存在时这种抗性消失。另一方面，雄性细胞对缺氧敏感，其DNA合成显著减少（70%，（p < 0.0001）。酪氨酸激酶抑制可部分逆转这种效应。蛋白质免疫印迹分析显示，与雌性细胞相比，雄性细胞中酪氨酸磷酸化蛋白的丰度更高，并且雄性和雌性细胞中基础及缺氧诱导的酪氨酸磷酸化蛋白的分子量存在差异。雌激素（17-β雌二醇，10 nM）的存在改变了两种细胞对缺氧的反应。在雌性细胞中，缺氧和雌激素的联合作用导致DNA合成受到抑制，而在雄性细胞中，雌激素部分逆转了缺氧诱导的DNA合成抑制（存在雌激素时抑制37%（p < 0.01），而不存在雌激素时抑制70%（p < 0.0001））。雌激素在雄性和雌性细胞中的作用是通过雌激素受体介导的，因为它们可被纯抗雌激素ICI 182,780逆转。对细胞裂解物的蛋白质免疫印迹分析显示，缺氧诱导雄性和雌性细胞中雌激素受体β水平升高。凝胶迁移分析显示，缺氧诱导雄性细胞中细胞质雌激素反应元件（ERE）结合活性增加，细胞核ERE结合减少。在雌性细胞中，缺氧条件下细胞质和细胞核ERE结合活性保持不变。总之，这些数据表明，虽然雌性细胞对缺氧诱导的DNA合成抑制具有抗性，而雄性细胞敏感；涉及酪氨酸磷酸化的细胞内途径参与了两种细胞的反应；并且雌激素通过雌激素受体依赖性机制，不同程度地改变了雄性和雌性细胞对缺氧的反应。
