Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta in lung epithelial cells.
Regulation of neutral sphingomyelinase-2 (nSMase2) by tumor necrosis factor-alpha involves protein kinase C-delta in lung epithelial cells.
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肿瘤坏死因子-Alpha对中性鞘磷脂酶2（NSMASE2）的调节涉及肺上皮细胞中的蛋白激酶C-二烷。
DOI：
10.1124/mol.108.046250
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发表时间：
2008-10
影响因子：
3.6
通讯作者：
Hannun YA
中科院分区：
文献类型：
--
作者：
Clarke CJ;Guthrie JM;Hannun YA
中性鞘磷脂酶（N-SMases）是应激诱导神经酰胺产生的主要候选酶，但对克隆的N-SMase酶nSMase 2的调控机制仍知之甚少。我们已经报道，在肿瘤坏死因子-α（TNF-α）刺激的A549细胞中，p38丝裂原活化蛋白激酶（MAPK）位于nSMase 2的上游（J Biol Chem 282：1384-1396，2007）。在这里，我们报告了蛋白激酶C（PKC）在介导TNF诱导的nSMase 2从高尔基体到质膜（PM）的易位中的作用。在A549细胞中，PKC的药理学抑制阻止TNF刺激的nSMase 2易位到PM。使用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）作为一种工具，解剖PKC的反应，我们发现，PMA诱导nSMase 2易位到PM在时间和剂量依赖性的方式。药理学抑制剂和特异性siRNA提示新型PKC，特别是PKC-δ参与TNF和PMA刺激的nSMase 2易位。PMA不能增加N-SMase活性，PKC-δ不能调节TNF诱导的N-SMase活性。此外，PKC-δ和nSMase 2没有共免疫沉淀，表明可能涉及其他信号蛋白。PMA刺激的nSMase 2易位不依赖于p38 MAPK，PKC抑制剂和小干扰RNA对TNF刺激的p38 MAPK活化均无显著影响，表明PKC-δ在调节nSMase 2中不通过p38 MAPK起作用。最后，PKC-δ的下调抑制了血管细胞和细胞间粘附分子的诱导，这些分子先前被鉴定为A549细胞中nSMase 2的下游。综上所述，这些数据暗示PKC-δ是nSMase 2的调节剂，并首次将nSMase 2确定为PKC和鞘脂途径之间的串扰点。
Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production, but there is still limited knowledge of the regulatory mechanisms of the cloned N-SMase enzyme—nSMase2. We have reported that p38 mitogen-activated protein kinase (MAPK) was upstream of nSMase2 in tumor necrosis-α (TNF-α)-stimulated A549 cells (J Biol Chem 282:1384–1396, 2007). Here, we report a role for protein kinase C (PKC) in mediating TNF-induced translocation of nSMase2 from the Golgi to the plasma membrane (PM). Pharmacological inhibition of PKCs prevented TNF-stimulated nSMase2 translocation to the PM in A549 cells. Using phorbol 12-myristate 13-acetate (PMA) as a tool to dissect PKC responses, we found that PMA induced nSMase2 translocation to the PM in a time- and dose-dependent manner. Pharmacological inhibitors and specific siRNA implicated the novel PKCs, specifically PKC-δ, in both TNF and PMA-stimulated nSMase2 translocation. However, PMA did not increase in vitro N-SMase activity and PKC-δ did not regulate TNF-induced N-SMase activity. Furthermore, PKC-δ and nSMase2 did not coimmunoprecipitate, suggesting that other signaling proteins may be involved. PMA-stimulated nSMase2 translocation was independent of p38 MAPK, and neither PKC inhibitors nor small interfering RNA had significant effects on TNF-stimulated p38 MAPK activation, indicating that PKC-δ does not act through p38 MAPK in regulating nSMase2. Finally, down-regulation of PKC-δ inhibited induction of vascular cell and intercellular adhesion molecules, previously identified as downstream of nSMase2 in A549 cells. Taken together, these data implicate PKC-δ as a regulator of nSMase2 and, for the first time, identify nSMase2 as a point of cross-talk between the PKC and sphingolipid pathways.