The role of inositol requiring enzyme- 1- c- Jun N- terminal kinase signaling pathways in stress- induced apoptosis of myoblasts

Abstract

Objective To explore the role of inositol requiring enzyme- 1(IRE- 1)- c- Jun N-terminal kinase(JNK) signaling pathways in stretch- induced apoptosis of myoblasts. Methods As experimental objects, L6 rat myoblasts were used to construct the culture- mechanical stimulation model in vitro.Then cells were subjected to 15% surface elongation with a frequency of 10 cycles/ min cyclic stretch using multi- channel stress loading system.Cells were harvested after different durations(1, 2, 6, 12, 24 h) of cyclic exposure.After cyclic stretch, Hochest 33258 staining and Annexin V- fluoresceine isothiocyanate(FITC)/propidium iodide(PI) staining was performed to observe the morphology of the apoptotic cells.Reverse transcriptase- polymerase chain reaction(RT - PCR)was used to detect the m RNA expression of endoplasmic reticulum stress related factors[C/ EBP homologous protein(CHOP), tumor necrosis factor receptor associated factor2(TRAF2), apoptosis signal regulating kinase- 1(ASK- 1)and JNK].IRE- 1 specific inhibitor(STF- 083010) was used to detect the TRAF2, ASK- 1 and JNK m RNA expression to investigate the role of IRE-1- JNK signaling pathway in stretch- induced apoptosis. Results The stretch- induced apoptosis rate of myoblasts was increased in a time- dependent manner, peaked at 24 h(14.34±0.77). CHOP, TRAF2 and ASK- 1 mRNA expression levels were increased with the extension of stretch time, and reached the maximum at 24 h[(4.19±1.76),(3.46±0.84), and(3.78±1.38), P< 0.05]; but JNK m RNA expression was decreased followed by an increase range from 0 h to 2 h,and then increased again within 24 h and peaked at 24 h(3.04±0.79, P< 0.05). After treatment with IRE- 1 specific inhibitor(STF- 083010), the real- time PCR results showed that TRAF2 and ASK - 1 m RNA expression was significantly decreased(P< 0.05). Conclusion Cyclic stretch can induce myoblast apoptosis and within 24 h the apoptosis rate of myoblasts was increased in a time- dependent manner.The increase of CHOP, TRAF2, ASK- 1,and JNK mRNA indicates that endoplasmic reticulum stress may involve in stretch- induced apoptosis.After treatment with IRE- 1 specific inhibitor, the significant decrease of CHOP, TRAF2, ASK- 1 and JNK mRNA,and JNK protein indicates that IRE- 1-JNK signaling pathway may involve in stretch- induced apoptosis. Key words: Inositol requiring enzyme- 1; c- Jun N- terminal kinase; Endoplasmic reticulum stress; Cyclic stretch; Apoptosis