Mazière C, Conte M A, Mazière J C
Laboratoire de Biochimie, Université de Picardie Jules Verne, CHRU Amiens, Hôpital Nord, 80054 Amiens Cedex 01, France.
FEBS Lett. 2001 Oct 26;507(2):163-8. doi: 10.1016/s0014-5793(01)02977-5.
Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1-0.3 U/ml SMase induced a dose-dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose-dependent Tyr-phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose-dependent manner. Concomitantly, SMase enhanced the Tyr-phosphorylation of Janus kinase (JAK) 2, a Tyr-kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr-kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr-phosphorylation, together with STAT1 and STAT3 Tyr-phosphorylation and binding activity. The SMase-induced increase in STAT1/3 binding activity was prevented by methyl-beta-cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER-activated intracellular kinases.
通过用蜡样芽孢杆菌的鞘磷脂酶（SMase）处理培养的成纤维细胞来产生内源性神经酰胺（CER）。用0.1 - 0.3 U/ml SMase处理30分钟可诱导CER细胞内水平呈剂量依赖性增加。通过免疫印迹法测定磷酸化状态以及通过电泳迁移率变动分析测定DNA结合活性，研究了SMase对转录因子信号转导和转录激活因子（STAT）1和STAT3的激活作用。SMase处理诱导STAT1/3的酪氨酸磷酸化呈剂量依赖性。SMase还以剂量依赖性方式增强STAT1/3的DNA结合活性。同时，SMase增强了Janus激酶（JAK）2的酪氨酸磷酸化，JAK2是JAK/STAT途径中位于STAT上游的酪氨酸激酶。酪氨酸激酶抑制剂染料木黄酮和JAK抑制剂AG490均能阻止JAK2的酪氨酸磷酸化，以及STAT1和STAT3的酪氨酸磷酸化和结合活性。甲基-β-环糊精是一种胆固醇结合剂，可导致位于小窝中的分子失去区室化，它能阻止SMase诱导的STAT1/3结合活性增加。MEK抑制剂PD98059也能阻止这种增加，从而证明了MEK/ERK途径在该系统中的作用。除了ERK，SMase还激活了其他信号激酶，如JNK和p38。外源性天然CER也激活了STAT1/3结合活性，这表明内源性CER很可能是参与SMase作用的第二信使。这些结果描述了SMase/CER和JAK/STAT信号通路之间的串扰，并将JAK2纳入CER激活的细胞内激酶范围内。
