[Involvement of JNK signal pathway in hypoxia related upregulation of calcium/calmodulin-dependent serine protein kinase in endothelial cells].
OBJECTIVE
To investigate the expression of calcium/calmodulin-dependent serine protein kinase (CASK) induced by short-term hypoxia, and to explore the role of JNK pathway in this signal event.


METHODS
EA. hy926 cells were cultured in normoxic condition for 0, 12, 24, 48, 72 h after being exposed to hypoxic condition for 3 h, then the cellular lysates were extracted. CASK promoter luciferase reporter recombinant was constructed and transfected into EA. hy926 cells for 48h. Cellular lysates were extracted 1, 3, 6, 12 h after hypoxia treatment and were used to detect firefly luciferase activity and rinella luciferase activity with luminometer. EA. hy926 cells were cultured under hypoxic condition for 1, 3, 6, 12 h or under normoxic condition, then the cell lysates were extracted and used to detect phospho-JNK with Western blot. EA. hy926 cells were pretreated with different concentrations of JNK specific inhibitor SP 600125 (0, 10, 100 nmol/L and 1,10 micromol/L) 1h before hypoxic treatment of various duration, and the cell lysates were extracted to detect CASK expression with Western blot.


RESULTS
CASK expression was obviously elevated by hypoxia, and the high expression sustained for 72 h when the hypoxic cells were cultured in normal conditions, and it was significantly higher than that of normal controls. Dual luciferase reporter assay showed that CASK promoter activity was significantly increased after hypoxia (0.010 +/- 0.003, P < 0.01), and it reached the peak 12 hrs after hypoxia (0.192 +/- 0.023, P < 0.01). The phosphorylation of JNK was enhanced with the prolongation of hypoxic time. CASK protein expression was suppressed by JNK specific inhibitor SP600125 in a dose dependent manner, and it decreased to the lowest level with 10 micromol/L SP600125 pretreatment.


CONCLUSIONS
JNK signal pathway is involved in short-term hypoxia related CASK upregulation.
Su
Yong-Yue
YY
Institutie of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, The Third Military Medical University, Chongqing 400038, PR China.
Liang
Guang-Ping
GP
Liu
You-Sheng
YS
Chen
Jian
J
Yang
Zong-Cheng
ZC
Luo
Xiang-Dong
XD
D002118:Calcium / Q000378:metabolism*; D015687:Cell Hypoxia; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology; D051528:Guanylate Kinases / Q000378:metabolism*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction
C106868:CASK kinases; D048031:JNK Mitogen-Activated Protein Kinases; D051528:Guanylate Kinases; D002118:Calcium