Dynamic Regulation of CYP19A1 Promoter Region under Control of CREB Family Members in Endometrial Tissues of Women with Endometriosis: A Case-Control Study

Endometriosis is a hormonal and inflammatory disease primarily affecting the pelvis ( 1 ), featuring estrogendependent cell growth and increased local estrogen production. This state is due to aberrations in steroidogenesis enzymes ( 2 ) and increased pro-inflammatory cytokines and chemokines production ( 1 ). Clinical observations show that the biosynthesis of estrogen is essential for the development of endometriosis in endometriotic tissues ( 3 , 4 ). Expression of steroidogenic enzymes capacitates endometriotic tissue to synthesize estradiol (E2), The biologically active estrogen, de novo from cholesterol ( 5 ). Through the biosynthesis of estrogen, the rate-limiting enzyme is cytochrome P450 aromatase (P450arom encoded by CYP19A1 ), which converts the androstenedione to estrone ( 6 ). Kitawaki et al. ( 7 ) showed that the expression of the aromatase gene is significantly higher in endometriosis implants compared to eutopic endometrium in endometriosis women. In contrast, aromatase gene expression was undetectable in the normal endometrium of women without endometriosis. Aromatase gene expression is tissue-specific and under the control of several alternative promoters ( 8 ). In endometriosis tissues, aromatase is expressed in the stromal cells, and its expression is regulated primarily by promoter II ( 9 ). Promoter II of aromatase is largely regulated via cyclic adenosine 3′, 5′-monophosphate (cAMP) dependent mechanisms by cAMP response element (CRE) binding protein (CREB) family transcription factors ( 10 ). It has been reported that CREB family members binds to two distinct CRE-like sequences, namely CRE1 and CRE2 (approximately 80 bp. upstream of CRE1), within the promoter II region of the CYP19A1 gene and regulates this promoter ( 10 ). CREB family is a member of a large family of transcription factors containing basic leucine zipper (bZIP), comprising three members of CREB, cAMP response element modulator (CREM) ( 11 ), and Activating transcription factors (ATF), which stimulate target gene expression at promoters that contain CRE ( 12 ). CRE is known as an 8-base-pair Palindrome, TGACGTCA ( 13 ), and also as a half-site motif (TGACG or CGTCA), which is less active than the full CRE palindrome for cAMP binding and responsiveness ( 14 ). There is a high degree of similarity between CREB and CREM, especially in the bZIP domain, which is consistent with the finding that these agents can form homo and heterodimers and can bind to the same cis-regulatory element ( 15 ). CRE-mediated gene transcription depends on the competitive binding of several dimerized transcription factors, including activators and repressors of gene transcription. CREM gene contains several exons and encodes both transcriptional activator and repressor proteins generated by alternative splicing ( 16 ), thus, Transcriptional activation by CREB/CREM is greatly influenced by the expression of CREM isoforms which include DNA-binding and dimerization domains but lacks transactivation domains ( 17 ). Inducible cAMP early repressor (ICER) encoded from an alternative intronic promoter in the CREM gene. ICER is a potent endogenous repressor of CRE-mediated gene transcription and is highly inducible by a variety of stimuli. ICER represses transcription through its binding to the CRE sites of target gene promoters or by the formation of inactive heterodimers with CREB or other associated transactivators ( 17 ). The transactivation potential of CREB is largely mediated by phosphorylation upon stimulation with cAMP in the kinase inducible domain, at Ser133, by protein kinase A (PKA) ( 18 ), but that is not sufficient to stimulate the activation of all CREB target genes ( 14 ). A family of CREB co-activators, termed CREB-regulated transcription co-activators (CRTCs) has been identified ( 19 , 20 ). The highly conserved N-terminal CREB binding domain of the CRTCs is known to interact with the bZIP domain of CREB ( 19 ). Furthermore, CRTCs are known to act as co-activators of CREB independent of their phosphorylation status at Ser133 ( 19 ). Beyond their role in transcription, CRTCs appear to modulate the alternative splicing of certain CREB target genes through a conserved Pro-rich domain ( 21 , 22 ). In 2019 a group of researchers designed research to investigate the effect of Farnesoid X on disruption of the CREB-CRTC2 complex in endometriosis. A receptor called Farnesoid X can disrupt the CREB-CRTC2 complex and inhibit the transcriptional activity of CREB ( 23 ). They revealed that activation of the Farnesoid X receptor (have a noticeable expression in endometriotic tissue) decreases aromatase expression ( 24 ). Due to the role of CREB, CREM, and CRTC2 genes in estrogen biosynthesis and the role of estrogen in pathogenesis of endometriosis, in this study, we decided to evaluate the gene expression levels of CREB, CREM , and CRTC2 and also the binding of ICER to the promoter II of aromatase gene in eutopic and ectopic tissues of women with endometriosis compared to the control endometrial tissues.

In this study, 12 women were examined in each group: control and endometriosis group. The demographic and clinical characteristics of the studied women, including age, body mass index (BMI), and menstrual phase distribution are summarized in Table 2. There were no statistical differences between two groups in regard to age and BMI. The expression of the genes CREB, CREM, CRTC2 , and GAPDH was analyzed using realtime PCR. Primer sequence and length of PCR product PCR; Polymerase chain reaction. Characteristics of both studied groups Data are presented as mean ± SD. BMI; Body mass index. CREB gene expression was significantly increased in ectopic tissues of women with endometriosis compared to both the control group (P=0.006) and the eutopic tissues of women with endometriosis (P=0.008). In contrast, although there was a slight increase in CREB expression in eutopic tissues of women with endometriosis compared to the control group, this difference was not statistically significant (P=0.992, Fig .1A ). The expression level of the CREM gene was significantly higher in ectopic tissues of women with endometriosis compared to both the control group (P=0.001) and eutopic tissues from the same patients (P=0.007). On the other hand, the comparison of CREM expression between eutopic tissues and the control group showed only a slight increase. However, this increase was not statistically significant (P=0.664, Fig .1B ). The results also showed an increase in CRTC2 gene expression in both ectopic and eutopic tissues of women with endometriosis compared to the control group. There was a significant increase in CRTC2 gene expression in ectopic tissues compared to the control group (P=0.005), but this increase was not significant in the eutopic tissues compared to controls (P=0.211). Additionally, while CRTC2 expression was higher in ectopic tissues compared to eutopic tissues, this difference was not statistically significant (P=0.243, Fig .1C ). Expression profile of genes in eutopic and ectopic endometrial tis- sues of women with endometriosis compared to control endometrial tissues. Real-time polymerase chain reaction (PCR) data of A. CREB , B. CREM , and C. CRTC2 . Different letters indicate a significant difference. The alterations in ICER binding to promoter II of the CYP19A1 gene were assessed using a ChIP assay. Results showed that ICER incorporation was significantly decreased in both ectopic and eutopic tissues of women with endometriosis compared to the control group (P=0.001 and P<0.001, respectively). Although ICER incorporation was lower in eutopic tissues than in ectopic tissues, this difference was not statistically significant (P=0.96, Fig .2 ). Incorporation of ICER into PII promoter of CYP19A1 in eutopic and ectopic tissues vs. normal endometrium using Chromatin immunoprecipi- tation real-time polymerase chain reaction (ChIP-qPCR).

Endometriosis is an estrogen-dependent disease that is defined by the presence of endometrial glands and stroma outside the uterine cavity, mainly in the pelvic cavity ( 26 ). In this case-control study, we evaluated the gene expression of CREB, CREM , and CRTC2 in endometriosis and control endometrial tissues as well as the binding of ICER to the regulatory region of the CYP19A1 gene. According to the results, CREB, CREM , and CRTC2 have almost the same gene expression profile and they increased in the endometriosis tissues compared to non-endometriosis samples. In addition, incorporation of ICER into the PII promoter of CYP19A1 in eutopic and ectopic tissues of women with endometriosis was decreased compared to the controls. Since endometriosis is an estrogen-dependent disease, aromatase, because of its rate-limiting feature in the synthesis of estrogen, is an excellent target for synthesis inhibition of the E2 de novo in the endometriotic tissues. Promoter II aromatase has two CRE-like sequences, namely CRE1 and CRE2 for CRE binding proteins and are largely regulated via cAMP-dependent mechanisms ( 10 ). Bulun et al. ( 2 ) proposed that pro-inflammatory cytokines through activation of cyclooxygenase-2 (COX-2) increase prostaglandin E2 (PGE2) production which is a potent inducer of some key genes involved in the production of E2 such as aromatase. There is a positive feedback process, elevated E2 production results in further induction of COX-2. PGE2 by elevating intracellular levels of cAMP activate PKA signaling pathway ( 9 ), which regulates the steroidogenic enzyme expression through phosphorylation induction of the CREB family transcription factors ( 27 ) and nuclear localization of CRTC2 ( 28 ). These processes lead to increased inflammation due to elevated PGE2 levels and also increased lesion growth because of the potent mitogenic effect of E2. CREB family members, most notably CREM, have an important role in the cAMP-mediated regulation of steroidogenesis. The role of CREM became clear by the observation that ICER inhibited the ability of endogenous CREM proteins to induce steroid hormone biosynthesis in response to cAMP ( 29 ). Also, Morales et al. ( 30 ) have reported that ICER can repress rat ovarian CYP19A1 promoter. cAMP through another pathway regulate aromatase gene expression. In the basal state, CRTCs are sequestered in the cytoplasm through phosphorylation-dependent interactions. Exposure of the cell to cAMP causes CRTCs to be dephosphorylated and transported to the nucleus, which then binds to CREB over relevant promoters. The binding of CRTCs to the bZIP domain of CREB leads to increased CREB occupancy over cognate binding sites ( 31 ). According to the results of this study, increased gene expression of CREB and CREM in ectopic tissues of women with endometriosis through their bindings to the CRE elements and thus expression activation of aromatase could be a reason for the presence of significant expression levels of aromatase. Also, decreased ICER incorporation to the promoter region of aromatase, as an inhibitor of the CREB family, in ectopic and eutopic endometriosis tissue may be a cause for increased aromatase expression in endometriosis tissue. Even though endometriosis is a benign disease, many studies have suggested that endometriosis represents the early stages of neoplastic processes. Endometriosis probably represents a form of transition from benign disease to cancer. Common features of endometriosis and cancer include the ability to escape apoptosis, angiogenesis, growth, adult stem cell-like dysregulation, and implantation in distant locations and to create an environment that functions independently of the immune system. Some studies have suggested that alterations in the eutopic endometrial characteristics of women with endometriosis (for example unusual secretion of different cytokines, growth factors, or angiogenesis factors) may contribute to the development and maintenance of the disease. Another possibility may be a change in the expression of specific cancer-related genes ( 32 ). Overexpression of CREB has been reported in many types of solid tumors such as breast cancer, and estrogen dependent cancers, compared with adjacent normal tissues ( 33 , 34 ) as well as in hematopoietic malignancies ( 35 ). In addition, its overexpression is associated with clinicopathologic parameters including grade, tumor stage, increased recurrence, metastasis, worse prognosis, and decreased survival of tumor patients ( 34 , 36 , 37 ). There is evidence of a causal relationship between CREB activation, tumor initiation and progression ( 38 ). This is due to the involvement of CREB in the upregulation of target gene expression with CRE sequences ( 38 ). Also, many studies have reported that CRTCs in signaling networks regulate cell growth and differentiation, proliferation, survival, DNA damage repair, and apoptosis. Therefore, several links between aberrant activation of CRTC and carcinogenesis have been identified with a growing list of different cancers. Direct links between activated CRTCs and the biological capabilities of malignant cells have been identified ( 39 ). Therefore, increased expression of CRTC2, both as CREB co-activator and as its carcinogenesis role, could be important in pathogenesis of endometriosis.

According to these results, the increased expression of CREB, CREM and CRTC2 transcription factor genes in ectopic and eutopic tissues of women with endometriosis may indicate their role in the pathogenesis of endometriosis through their regulatory effect on the expression of downstream genes such as enzymes involved in estrogen biosynthesis (aromatase), and cancer-related genes. On the other hand, decreased binding of ICER to the promoter II region of aromatase in endometriosis tissues could be a reason for increased expression of aromatase in this disease.

In this case-control study, 24 women with and without endometriosis were enrolled (12 women in each group). This study was approved by the Institutional Ethics Committee of Royan Institute (IR.ACECR.ROYAN.REC.1398.006). All women signed the informed consent form. The inclusion criteria were age between 20-45 years, not receiving any hormonal treatments during the last three months, regular menstrual cycle, and not having any other endometrial disease. Control endometrial samples collected from women who had no evidence of endometriosis during diagnostic laparoscopy surgery. Ectopic samples were obtained during laparoscopy surgery from endometriosis women while eutopic endometrial samples of these women were obtained by pipelle. To study the gene expression, 12 eutopic, 12 ectopic, and 12 control endometrial samples were examined. All endometriosis women were in stage III and IV of the disease, as classified by the American Society for Reproductive Medicine (ASRM, 1997). After endometrial tissue collection, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. The purity and concentration of RNA samples were determined using NanoDrop (NanoDrop™ 2000/2000c Spectrophotometers). Extracted RNA was treated using DNase I endonuclease (Thermo Fisher Scientific, USA). Total RNA was reverse transcribed to complementary DNA (cDNA) according to the protocol of the kit (TaKara Bio, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to study the relative mRNA expression of the CREB, CREM and CRTC2 genes using Step One Plus™ Real-time PCR System (Applied Biosystems International, Inc., Switzerland). Glyceraldehyde-3-phosphate dehydrogenase gene ( GAPDH ) was used as an endogenous control. Relative quantification was analyzed using the comparative threshold cycle (Ct) method. All samples were normalized to GADPH mRNA levels. The fold change in gene expression was calculated using the 2 -ΔΔCt method. The primer sequences and the size of amplicons are shown in Table 1. The expected RT-PCR product size was confirmed by ethidium bromide stained 2% agarose gel electrophoresis. Chromatin immunoprecipitation (ChIP) assay was used for assessing the amount of ICER transcription factor binding to the CRE regulatory region in the promoter II of the CYP19A1 gene. ChIP experiment was performed using anti-ICER antibody (Abcam, Massachusetts), following the manufacturer’s instructions as previously described ( 25 ). The relative level of ICER binding to the promoter II of the CYP19A1 gene was analyzed by realtime PCR (Applied Biosystems International, Inc., Switzerland) on DNA recovered from the ChIP and the total chromatin input with specific primer set listed in Table 1. The IP/INPUT ratio of the target sequence was calculated using the following formula: (% IP/ INPUT = 2[(Ct (x % input) − log (x %) /log 2) − Ct (IP)] × 100). The IBM SPSS Statistics (Version 27, IBM, America) software was used for data entry and statistical analysis. Differences between the control, eutopic, and ectopic tissue groups were evaluated using One-Way ANOVA followed by Post Hoc Tukey test. P<0.05 was considered statistically significant.