Activation of the JAK/STAT pathway by ceramide in cultured human fibroblasts.
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.
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Laboratoire de Biochimie, Université de Picardie Jules Verne, CHRU Amiens, Hôpital Nord, 80054 Amiens Cedex 01, France. cmaziere@nnx.com
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D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002518:Ceramides / Q000096:biosynthesis*; D003505:Cyclodextrins / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D053614:Janus Kinase 2; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D047392:beta-Cyclodextrins
D002518:Ceramides; D003505:Cyclodextrins; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D013108:Sphingomyelin Phosphodiesterase