P-393: C-Jun N-terminal kinase (JNK) signaling regulates cell proliferation and apoptosis in endometrial cells

Abstract

Mitogen-activated protein kinases (MAPK)s regulate many cellular and molecular activities ranging from cytokine expression, cell proliferation to apoptosis. c-Jun N-terminal kinase (JNK) is one of the main subfamilies of MAPK superfamily. JNK is activated in response to stress and modulate inflammation and apoptosis. Our hypothesis is that temporal and spatial changes in JNK activity regulate cell survival and inflammation throughout menstrual cycle and early pregnancy. Therefore, we studied to determine total- and active- (phosphorylated-) JNK expression in endometrial tissues in vivo and its impact on endometrial cell proliferation and apoptosis, in vitro. A prospective in vitro and in vivo study assessing activity and function of JNK in endometrium. Serial sections from endometrial (n=30) tissues were stained with phosphorylated (p-) and total (t-) JNK and evaluated semi-quantitatively using HSCORE system. Endometrial stromal and glandular cells were isolated from endometrial biopsies obtained from women undergoing surgery for benign gynecologic conditions. Endometrial stromal and epithelial cells were cultured in DMEM containing 10% FBS, and grown to confluence. Experiments were performed in 96-well microplates and 4-well chamber slides after incubating cells in serum-free, phenol red-free DMEM for 24 h. Thereafter, cells were incubated with the JNK inhibitor [SP600125; 10 μM] alone or with estradiol (E2; 10-8 M) for 24-72 h and evaluated with BrdU cell proliferation and TUNEL apoptosis assays. Statistical analysis of the data was performed using ANOVA. Human endometrial tissues were grouped according to the menstrual cycle phase and examined by immunohistochemistry for t-JNK and p-JNK. Both stromal and glandular cells revealed moderate cytoplasmic and nuclear t-JNK immunoreactivity without significant changes throughout the menstrual cycle. On the other hand, mostly nuclear immunoreactivity was detected in endometrial cells for p-JNK. The highest p-JNK immunoreactivity was detected in late secretory endometrial epithelium and stroma (p<0.03) compared to other cycle phases. Interestingly, the lowest p-JNK level was detected in mid-secretory phase endometrial samples. In culture, SP600125-tretead endometrial stromal cells showed a 3-fold higher apoptosis ratio after 72 h of incubation (p<0.05). Moreover, SP600125 treatment significantly lowered the DNA synthesis in both endometrial glandular and stromal cells (60% and 80% less than control cells, respectively) as assessed by BrdU-incorporation assay. When combined with E2, SP600125 significantly reduced the E2-stimulated BrdU-incorporation (p<0.05). Increased expression of p-JNK during the late-secretory phase suggests that JNK signaling may be involved in regulation of inflammatory processes in endometrium. Moreover, our in vitro results also support that JNK signaling may directly regulate endometrial cell survival by affecting proliferation/apoptosis ratio.