DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling
DHCR24-knockout embryonic fibroblasts are susceptible to serum withdrawal-induced apoptosis because of dysfunction of caveolae and insulin-Akt-Bad signaling
复制标题
DOI：
10.1210/en.2005-1426
复制
发表时间：
2006-06-01
期刊：
影响因子：
4.8
通讯作者：
Seo, Hisao
中科院分区：
文献类型：
--
作者：
Lu, Xiuli;Kambe, Fukushi;Seo, Hisao
DHCR 24基因编码催化胆固醇生物合成的最后一步的酶，即链甾醇转化为胆固醇。为了阐明哺乳动物细胞中胆固醇生物合成的生理意义，我们研究了从DHCR 24（-/-）小鼠制备的小鼠胚胎成纤维细胞（MEFs）的增殖。DHCR 24（-/-）和野生型MEF在培养基中血清存在下增殖。然而，通过血清戒断抑制外部胆固醇供应诱导DHCR 24（-/-）MEFs凋亡，这与细胞内和质膜胆固醇水平显著降低、Akt失活和Bad去磷酸化相关。胰岛素是一种能够刺激Akt-Bad级联反应的抗凋亡因子，其受体（IR）富集在质膜的小窝、富含胆固醇的微区中。因此，我们分析了IR和小窝在胆固醇耗尽MEFs的关联。亚细胞分级和免疫细胞化学分析表明，在MEFs的小窝组分中，IR和小窝蛋白-1的含量明显降低，表明小窝的破坏，并且除了小窝蛋白-1外，在质膜上还存在大量IR，表明IR与小窝的解偶联。与这些发现一致，胰岛素受体底物-1，Akt和Bad的胰岛素依赖性磷酸化在胆固醇耗尽的MEFs中受损。然而，这种损害是部分的，因为用胰岛素治疗MEFs恢复了Akt活化并防止了细胞凋亡。胆固醇供应也阻止了细胞凋亡。这些结果表明，细胞胆固醇生物合成对于响应于生长因子如胰岛素的Akt-Bad细胞存活级联的激活和维持是关键的。
The DHCR24 gene encodes an enzyme catalyzing the last step of cholesterol biosynthesis, the conversion of desmosterol to cholesterol. To elucidate the physiological significance of cholesterol biosynthesis in mammalian cells, we investigated proliferation of mouse embryonic fibroblasts (MEFs) prepared from DHCR24(-/-) mice. Both DHCR24(-/-) and wild-type MEFs proliferated in the presence of serum in culture media. However, the inhibition of external cholesterol supply by serum withdrawal induced apoptosis of DHCR24(-/-) MEFs, which was associated with a marked decrease in the intracellular and plasma membrane cholesterol levels, Akt inactivation, and Bad dephosphorylation. Insulin is an antiapoptotic factor capable of stimulating the Akt-Bad cascade, and its receptor (IR) is enriched in caveolae, cholesterol-rich microdomains of plasma membrane. We thus analyzed the association of IR and caveolae in the cholesterol-depleted MEFs. Subcellular fractionation and immunocytochemical analyses revealed that the IR and caveolin-1 contents were markedly reduced in the caveolae fraction of the MEFs, suggesting the disruption of caveolae, and that large amounts of IR were present apart from caveolin-1 on plasma membrane, indicating the uncoupling of IR with caveolae. Consistent with these findings, insulin-dependent phosphorylations of insulin receptor substrate-1, Akt, and Bad were impaired in the cholesterol-depleted MEFs. However, this impairment was partial because treatment of the MEFs with insulin restored Akt activation and prevented apoptosis. Cholesterol supply also prevented apoptosis. These results demonstrate that the cellular cholesterol biosynthesis is critical for the activation and maintenance of the Akt-Bad cell survival cascade in response to growth factors such as insulin.