In vivo BİP expression in human endometrial endothelial cells and in vitro modulation of BİP by the inflammatory cytokines TNF-alpha and Interleukin 1 (IL-1) beta

Purpose:In this thesis the effects of TNF-? and IL-1ß on unfolding protein response (UPR) related with endoplasmic reticulum (ER) stress in human endometrial endothelial cells (HEECs) was investigated.Materials and Methods:In vivo 28 endometrial tissues from women with different menstrual cycle stages and 7 decidual tissues from early pregnancy were examined for BIP expression using immunohistochemistry. In in vitro: HEECs were treated for 24 and 48 hours with TNF-? and IL-1 ? and evaluated for BIP, total (total-) and phosphorylation (phospho-) eIF-2? expressions by using immunocytochemistry and for apoptosis using TUNEL technique. HEECs also were treated with TUDCA and tunicamycin to analyze for cell proliferation by MTS assay and IL-8 secretion by ELISA. Furthermore, expression of BIP, phospho- and total- eIF-2? in HEECs stimulated by TNF-?, IL-1 ? and sex hormones estrogen and progesterone were also evaluated by Western blot analysis.Results:In vivo: BIP immunostaining was detected at the highest level in HEEC during the late-secretory phase of the menstrual cycle and the lowest in HEEC from the mid-secretory phase and early pregnancy samples. In vitro: Tunicamycin decreased HEECs proliferation compared with control in a dose dependent manner. IL-1ß increased proliferation of HEECs, while TUDCA decreased this proliferative effect of IL-1ß. Furthermore, tunicamycin increased IL-8 secretion in HEECs in a dose-dependent manner which was blunted by TUDCA. Similarly, TNF-? increased IL-8 secretion in HEECs and combination of TUDCA eliminated this effect of TNF-?. TUNEL analysis demonstrated that TNF-? increased the apoptotic cell number in HEECs. Western blot results revealed that BIP protein level was increased by TNF- ? and IL-1 ? while ovarian hormones unchanged the BIP protein level compared with control. Phospho-eIF-2? protein expressions were increased by TNF- ? and IL-1 ? , but decreased by progesterone while estrogen had no effect. According to immunoctyochemical analysis BIP and phospho-eIF-2? expression in cytokine-stimulated HEECs were increased compared to control.Conclusion:Our results suggest that UPR signaling pathway is involved in molecular control of endometrial endothelial cell proliferation and apoptosis, and that process is controlled in a menstrual cycle-dependent manner. Furthermore, our findings reveal that the inflammatory cytokines TNF- ? and IL-1 ? are one of the factors responsible from activation of UPR signaling in HEEC.