cAMP-Response Element-Binding 3-Like Protein 1 (CREB3L1) is Required for Decidualization and its Expression is Decreased in Women with Endometriosis

Abstract

Endometriosis is a major cause of infertility and pelvic pain, affecting more than 10% of reproductive-aged women. Progesterone resistance has been observed in the endometrium of women with this disease, as evidenced by alterations in progesterone-responsive gene and protein expression. cAMPResponse Element-Binding 3-like protein 1 (Creb3l1) has previously been identified as a progesterone receptor (PR) target gene in mouse uterus via high density DNA microarray analysis. However, CREB3L1 function has not been studied in the context of endometriosis and uterine biology. In this study, we validated progesterone (P4) regulation of Creb3l1 in the uteri of wild-type and progesterone receptor knockout (PRKO) mice. Furthermore, we observed that CREB3L1 expression was significantly higher in secretory phase human endometrium compared to proliferative phase and that CREB3L1 expression was significantly decreased in the endometrium of women with endometriosis. Lastly, by transfecting CREB3L1 siRNA into cultured human endometrial stromal cells (hESCs) prior to hormonal induction of in vitro decidualization, we showed that CREB3L1 is required for the decidualization process. Interestingly, phosphorylation of ERK1/2, critical factor for decidualization, was also significantly reduced in CREB3L1-silenced hESCs. It is known that hESCs from patients with endometriosis show impaired decidualization and that dysregulation of the P4-PR signaling axis is linked to a variety of endometrial diseases including infertility and endometriosis. Therefore, these results suggest that CREB3L1 is required for decidualization in mice and humans and may be linked to the pathogenesis of endometriosis in a P4-dependent manner.

Figure 1

The expression of CREB3L1 in wild-type (WT) or progesterone receptor knock-out (PRKO) mouse. (A) The expression level of Creb3l1 from Progesterone (P4) injected WT or PRKO uteri by real-time RT-PCR assay. The samples were prepared from wild-type or PRKO uterus that was injected vehicle (Sesami oil) or P4 after 6 hours. The results represent the mean ± SEM. **p<0.01, ***p<0.001. (B) The localization pattern of CREB3L1 by immunohistochemistry in the vehicle or P4-treated uterus. Uterine sections were collected from P4 or vehicle treated WT and PRKO mice for 6 hours. Nuclei were counterstained with hematoxylin.

Figure 2

The expression of CREB3L1 during early pregnancy. The localization pattern of CREB3L1 during natural pregnancy by immunohistochemistry were determined at 0.5 dpc (a and b), 2.5 dpc (c and d), 3.5 dpc (e and f), 4.5 dpc (g and h), 5.5 dpc (i and j), and 7.5 dpc (k and l).

Figure 3

The expression profile of CREB3L1 in human endometrium during estrus cycle. (A) The expression level and localization of CREB3L1 in proliferative (a and b) and early (c and d), mid (e and f), and late (g and h) secretory phase of human endometrium. CREB3L1 expression was high expression in secretary phase endometrial epithelium. Black arrow head indicates a decidualized cell. (B) was H-Score by measuring expression intensity of endometrial cells. CREB3L1 expression intensity was significantly high level in secretary phase endometrium. The results represent the mean ± SEM. *p<0.05, ***p<0.001.

Figure 4

The expression profile of CREB3L1 in secretory endometrium from women with or without endometriosis (A) The expression level and localization of CREB3L1 in human endometrium with or without endometriosis. CREB3L1 expression was low expression in human endometrium with endometriosis. Nuclei were counterstained with hematoxylin. (B) H-Score of CREB3L1 expression in human endometrium with or without endometriosis. CREB3L1 expression intensity was significantly low level in human endometrium with endometriosis. The results represent the mean ± SEM. **p<0.01.

Figure 5

The nuclear translocalization of CREB3L1 during in vitro decidualization of human endometrial stromal cells (hESCs). Nuclear localization of CREB3L1 was detected at day 3 and day 6 in vitro decidualization hESCs with EPC. Neclei were conterstained with DAPI staining.

Figure 6

The effect of inhibition of CREB3L1 in human endometrial stromal cells (hESCs) during in vitro decidualization. CREB3L1 expression was inhibited by treatment with CREB3L1 siRNA during in vitro decidualization. (A) The expression of CREB3L1 gene was examined during in vitro decidualization. CREB3L1 were significantly decreased in hESCs treated with CREB3L1 siRNA during in vitro decidualization. The results represent the mean ± SEM. ***p<0.001. (B) Morphological change of hESCs was observed during in vitro decidualization on day 3 and day 6. The expression of decidualization marker gene IGFBP1(C) and PRL (D) were examined during in vitro decidualization. PRL and IGFBP1 were significantly decreased in transfected CREB3L1 siRNA hESC during in vitro decidualization. The results represent the mean ± SEM. *p<0.05, ***p<0.001.

Figure 7

The regulation of phospho-ERK1/2 by CREB3L1 during hESCs decidualization. (A) The expression level of ERK1/2 phosphorylation during CREB3L1 inhibited hESCs decidualization. ERK1/2 phosphorylation was decreased at day 3 and day 6 in CREB3L1 siRNA treatment. (B) Quantification of phospho-ERK1/2 protein level during in vitro decidualization with or without CREB3L1 target siRNA treatment. The results represent the mean ± SEM. ***p<0.001. (C) Nuclear phospho-ERK1/2 was reduced at day 3 and day 6 in vitro decidualization hESCs with CREB3L1 siRNA compared to transfected with non targeting pool siRNA. Neclei were conterstained with DAPI staining.