Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17beta-estradiol at estrus

Abstract

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17beta-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17beta-estradiol treatment. Expression of NF-kappaB and IkappaB proteins, and IkappaB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.

Figure 1

Cleaved caspase-3, -6, and -7 expression in rat endometrium during estrous cycle. Polyoestrus rats were sacrificed at each stage of the estrous cycle (diestrus, proestrus, estrus and metestrus) and total endometrial proteins were collected. A) Western blots of cleaved caspase-3, -6 and -7 (one blot presented out of 4). Graphics shows Western blots densitometric analysis. Data represent the mean ± SEM of four independent experiments (four different rats). B) Immunohistochemistry of cleaved caspase-3. IHC shown are from one representative experiment (a total of four different uterine sections from four different rats per day of the estrous cycle have been tested). *Significantly different from diestrus and proestrus (p < 0.05).

Figure 2

Smac/DIABLO and XIAP expression in rat endometrium during estrous cycle. Total endometrial proteins were collected from polyoestrus cycling rats at each stage of the estrous cycle (diestrus, proestrus, estrus and metestrus). A) Western blots analyses of Smac/DIABLO and XIAP (one blot presented out of 6). Graphics shows Western blots densitometric analysis. Data represent the mean ± SEM of four independent experiments (four different rats). *Significantly different from all other days of estrus cycle (p < 0.05).

Figure 3

Immunohistochemistry of Smac/DIABLO and XIAP in rat endometrium during estrous cycle. IHC shown are from one representative experiment carried out on four different uterine sections (a total of four different uterine sections from four different rats per day of the estrous cycle have been tested). Negative control: primary antibody absent.

Figure 4

Expression of cleaved caspase-3, -6, -7, Smac/DIABLO and XIAP in response to 17β-estradiol (E₂) in ovariectomized rats. Ovariectomized rats received daily subcutanous injections of E₂ (40 μg/Kg/day) or vehicle for control group for 3 days. Graphics shows Western blots (one blot presented out of 4) densitometric analysis. Data represent the mean ± SEM of four independent experiments (4 rats per group). *Significantly different from control (p < 0.05).

Figure 5

Immunohistochemistry of Smac/DIABLO and XIAP in rat endometrium of treated ovariectomized rats. Ovariectomized rats received daily subcutanous injections of E₂ (40 μg/Kg/day) or vehicle for control group for 3 days. IHC shown are from one representative experiment per group (a total of four different uterine sections from four different treated ovariectomized rats have been tested).

Figure 6

NF-κB, IκB and phospho-IκB expression in rat endometrium during estrous cycle. Total endometrial proteins were collected from polyoestrus cycling rats at each stage of the estrous cycle (diestrus, proestrus, estrus and metestrus). A) Western blots analyses of NF-κB, IκB and phospho-IκB (one blot presented out of 6). Graphics shows Western blots densitometric analysis. Data represent the mean ± SEM of six independent experiments (six different rats)