{"paper_id":"da4c1e2d-0b58-41d6-8505-9b8201bde839","body_text":"Endometriosis is a hormonal and inflammatory disease primarily affecting the pelvis ( 1 ),\nfeaturing estrogendependent cell growth and increased local estrogen production. This state\nis due to aberrations in steroidogenesis enzymes ( 2 ) and increased pro-inflammatory\ncytokines and chemokines production ( 1 ). Clinical observations show that the biosynthesis of\nestrogen is essential for the development of endometriosis in endometriotic tissues ( 3 ,  4 ).\nExpression of steroidogenic enzymes capacitates endometriotic tissue to synthesize estradiol\n(E2), The biologically active estrogen, de novo from cholesterol ( 5 ). Through the\nbiosynthesis of estrogen, the rate-limiting enzyme is cytochrome P450 aromatase (P450arom\nencoded by  CYP19A1 ), which converts the androstenedione to estrone ( 6 ).\nKitawaki et al. ( 7 ) showed that the expression of the aromatase gene is significantly higher\nin endometriosis implants compared to eutopic endometrium in endometriosis women. In\ncontrast, aromatase gene expression was undetectable in the normal endometrium of women\nwithout endometriosis.\nAromatase gene expression is tissue-specific and under\nthe control of several alternative promoters ( 8 ). In endometriosis tissues, aromatase is expressed in the stromal\ncells, and its expression is regulated primarily by promoter II ( 9 ). Promoter II of aromatase is largely regulated\nvia cyclic adenosine 3′, 5′-monophosphate (cAMP) dependent mechanisms by cAMP response element (CRE)\nbinding protein (CREB) family transcription factors ( 10 ).\nIt has been reported that CREB family members binds\nto two distinct CRE-like sequences, namely CRE1 and\nCRE2 (approximately 80 bp. upstream of CRE1), within\nthe promoter II region of the  CYP19A1  gene and regulates\nthis promoter ( 10 ).\nCREB family is a member of a large family of transcription factors containing basic leucine\nzipper (bZIP), comprising three members of CREB, cAMP response element modulator (CREM)\n( 11 ), and Activating transcription factors (ATF), which stimulate target gene expression at\npromoters that contain CRE ( 12 ). CRE is known as an 8-base-pair Palindrome, TGACGTCA ( 13 ),\nand also as a half-site motif (TGACG or CGTCA), which is less active than the full CRE\npalindrome for cAMP binding and responsiveness ( 14 ). There is a high degree of similarity\nbetween CREB and CREM, especially in the bZIP domain, which is consistent with the finding\nthat these agents can form homo and heterodimers and can bind to the same cis-regulatory\nelement ( 15 ). CRE-mediated gene transcription depends on the competitive binding of several\ndimerized transcription factors, including activators and repressors of gene transcription.\n CREM  gene contains several exons and encodes both transcriptional\nactivator and repressor proteins generated by alternative splicing ( 16 ), thus,\nTranscriptional activation by CREB/CREM is greatly influenced by the expression of CREM\nisoforms which include DNA-binding and dimerization domains but lacks transactivation\ndomains ( 17 ). Inducible cAMP early repressor (ICER) encoded from an alternative intronic\npromoter in the  CREM  gene. ICER is a potent endogenous repressor of\nCRE-mediated gene transcription and is highly inducible by a variety of stimuli. ICER\nrepresses transcription through its binding to the CRE sites of target gene promoters or by\nthe formation of inactive heterodimers with CREB or other associated transactivators\n( 17 ).\nThe transactivation potential of CREB is largely mediated by phosphorylation upon stimulation with cAMP in\nthe kinase inducible domain, at Ser133, by protein kinase\nA (PKA) ( 18 ), but that is not sufficient to stimulate the activation of all CREB target genes ( 14 ). A family of CREB\nco-activators, termed CREB-regulated transcription co-activators (CRTCs) has been identified ( 19 ,  20 ). The highly\nconserved N-terminal CREB binding domain of the CRTCs\nis known to interact with the bZIP domain of CREB ( 19 ).\nFurthermore, CRTCs are known to act as co-activators of\nCREB independent of their phosphorylation status at Ser133 ( 19 ). Beyond their role in transcription, CRTCs appear\nto modulate the alternative splicing of certain CREB target\ngenes through a conserved Pro-rich domain ( 21 ,  22 ).\nIn 2019 a group of researchers designed research to investigate the effect of Farnesoid X on disruption of the\nCREB-CRTC2 complex in endometriosis. A receptor\ncalled Farnesoid X can disrupt the CREB-CRTC2 complex and inhibit the transcriptional activity of CREB ( 23 ).\nThey revealed that activation of the Farnesoid X receptor\n(have a noticeable expression in endometriotic tissue) decreases aromatase expression ( 24 ).\nDue to the role of  CREB, CREM,  and  CRTC2  genes in\nestrogen biosynthesis and the role of estrogen in pathogenesis of endometriosis, in this\nstudy, we decided to evaluate the gene expression levels of  CREB, CREM , and\n CRTC2  and also the binding of ICER to the promoter II of aromatase gene\nin eutopic and ectopic tissues of women with endometriosis compared to the control\nendometrial tissues.\n\nIn this case-control study, 24 women with and without endometriosis were enrolled (12 women in each group). This\nstudy was approved by the Institutional Ethics Committee\nof Royan Institute (IR.ACECR.ROYAN.REC.1398.006).\nAll women signed the informed consent form.\nThe inclusion criteria were age between 20-45 years,\nnot receiving any hormonal treatments during the last\nthree months, regular menstrual cycle, and not having any\nother endometrial disease. Control endometrial samples\ncollected from women who had no evidence of endometriosis during diagnostic laparoscopy surgery. Ectopic\nsamples were obtained during laparoscopy surgery from\nendometriosis women while eutopic endometrial samples\nof these women were obtained by pipelle.\nTo study the gene expression, 12 eutopic, 12 ectopic,\nand 12 control endometrial samples were examined. All\nendometriosis women were in stage III and IV of the disease, as classified by the American Society for Reproductive Medicine (ASRM, 1997).\nAfter endometrial tissue collection, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific,\nUSA) according to the manufacturer’s instructions. The\npurity 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\nwas reverse transcribed to complementary DNA (cDNA)\naccording to the protocol of the kit (TaKara Bio, Japan).\nReal-time quantitative polymerase chain reaction (RT-qPCR) was performed to study the\nrelative mRNA expression of the  CREB, CREM  and  CRTC2 \ngenes using Step One Plus™ Real-time PCR System (Applied Biosystems International, Inc.,\nSwitzerland). Glyceraldehyde-3-phosphate dehydrogenase gene ( GAPDH ) was\nused as an endogenous control. Relative quantification was analyzed using the comparative\nthreshold cycle (Ct) method. All samples were normalized to  GADPH  mRNA\nlevels. The fold change in gene expression was calculated using the 2 -ΔΔCt \nmethod. The primer sequences and the size of amplicons are shown in Table 1. The expected\nRT-PCR product size was confirmed by ethidium bromide stained 2% agarose gel\nelectrophoresis.\nChromatin immunoprecipitation (ChIP) assay was used\nfor assessing the amount of ICER transcription factor\nbinding to the CRE regulatory region in the promoter II\nof the  CYP19A1  gene. ChIP experiment was performed\nusing anti-ICER antibody (Abcam, Massachusetts), following the manufacturer’s instructions as previously described ( 25 ). The relative level of ICER binding to the\npromoter II of the  CYP19A1  gene was analyzed by realtime PCR (Applied Biosystems International, Inc., Switzerland) on DNA recovered from the ChIP and the total\nchromatin input with specific primer set listed in Table 1.\nThe IP/INPUT ratio of the target sequence was calculated\nusing the following formula: (% IP/ INPUT = 2[(Ct (x %\ninput) − log (x %) /log 2) − Ct (IP)] × 100).\nThe IBM SPSS Statistics (Version 27, IBM, America)\nsoftware was used for data entry and statistical analysis.\nDifferences 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\nstatistically significant.\n\nIn this study, 12 women were examined in each group: control and endometriosis group. The\ndemographic and clinical characteristics of the studied women, including age, body mass\nindex (BMI), and menstrual phase distribution are summarized in Table 2. There were no\nstatistical differences between two groups in regard to age and BMI. The expression of the\ngenes CREB,  CREM, CRTC2 , and  GAPDH  was analyzed using\nrealtime PCR.\nPrimer sequence and length of PCR product\nPCR; Polymerase chain reaction.\nCharacteristics of both studied groups\nData are presented as mean ± SD. BMI; Body mass index.\nCREB  gene expression was significantly increased in ectopic tissues of\nwomen with endometriosis compared to both the control group (P=0.006) and the eutopic\ntissues of women with endometriosis (P=0.008). In contrast, although there was a slight\nincrease in  CREB  expression in eutopic tissues of women with\nendometriosis compared to the control group, this difference was not statistically\nsignificant (P=0.992,  Fig .1A ).\nThe expression level of the  CREM  gene was significantly higher in\nectopic tissues of women with endometriosis compared to both the control group (P=0.001)\nand eutopic tissues from the same patients (P=0.007). On the other hand, the comparison of\n CREM  expression between eutopic tissues and the control group showed\nonly a slight increase. However, this increase was not statistically significant (P=0.664,\n Fig .1B ).\nThe results also showed an increase in  CRTC2  gene expression in both\nectopic and eutopic tissues of women with endometriosis compared to the control group.\nThere was a significant increase in  CRTC2  gene expression in ectopic\ntissues compared to the control group (P=0.005), but this increase was not significant in\nthe eutopic tissues compared to controls (P=0.211). Additionally, while  CRTC2\n expression was higher in ectopic tissues compared to eutopic tissues, this\ndifference was not statistically significant (P=0.243,  Fig .1C ).\nExpression profile of genes in eutopic and ectopic endometrial tis- sues of women with\nendometriosis compared to control endometrial tissues. Real-time polymerase chain\nreaction (PCR) data of  A.  CREB ,  B.\n CREM , and  C.  CRTC2 . Different\nletters indicate a significant difference.\nThe alterations in ICER binding to promoter II of the\n CYP19A1  gene were assessed using a ChIP assay. Results\nshowed that ICER incorporation was significantly decreased in both ectopic and eutopic tissues of women with\nendometriosis compared to the control group (P=0.001 and\nP<0.001, respectively). Although ICER incorporation was\nlower in eutopic tissues than in ectopic tissues, this difference was not statistically significant (P=0.96,  Fig .2 ).\nIncorporation of ICER into PII promoter of CYP19A1 in eutopic and ectopic tissues vs. normal\nendometrium using Chromatin immunoprecipi- tation real-time polymerase chain reaction\n(ChIP-qPCR).\n\nEndometriosis is an estrogen-dependent disease that is\ndefined by the presence of endometrial glands and stroma\noutside the uterine cavity, mainly in the pelvic cavity ( 26 ).\nIn this case-control study, we evaluated the gene expression of  CREB,\nCREM , and  CRTC2  in endometriosis and control endometrial tissues\nas well as the binding of ICER to the regulatory region of the  CYP19A1 \ngene. According to the results,  CREB, CREM , and  CRTC2  have\nalmost the same gene expression profile and they increased in the endometriosis tissues\ncompared to non-endometriosis samples. In addition, incorporation of ICER into the PII\npromoter of  CYP19A1  in eutopic and ectopic tissues of women with\nendometriosis was decreased compared to the controls.\nSince endometriosis is an estrogen-dependent disease,\naromatase, 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.\nPromoter II aromatase has two CRE-like sequences,\nnamely CRE1 and CRE2 for CRE binding proteins and\nare largely regulated via cAMP-dependent mechanisms\n( 10 ). Bulun et al. ( 2 ) proposed that pro-inflammatory cytokines through activation of cyclooxygenase-2 (COX-2)\nincrease prostaglandin E2 (PGE2) production which is a\npotent 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\ninduction of COX-2. PGE2 by elevating intracellular levels of cAMP activate PKA signaling pathway ( 9 ), which\nregulates the steroidogenic enzyme expression through\nphosphorylation induction of the CREB family transcription factors ( 27 ) and nuclear localization of CRTC2 ( 28 ).\nThese processes lead to increased inflammation due to\nelevated PGE2 levels and also increased lesion growth\nbecause of the potent mitogenic effect of E2.\nCREB family members, most notably CREM, have an\nimportant 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\nproteins to induce steroid hormone biosynthesis in response\nto cAMP ( 29 ). Also, Morales et al. ( 30 ) have reported that\nICER can repress rat ovarian  CYP19A1  promoter.\ncAMP through another pathway regulate aromatase gene\nexpression. In the basal state, CRTCs are sequestered in\nthe cytoplasm through phosphorylation-dependent interactions. Exposure of the cell to cAMP causes CRTCs to be\ndephosphorylated and transported to the nucleus, which\nthen binds to CREB over relevant promoters. The binding\nof CRTCs to the bZIP domain of CREB leads to increased\nCREB occupancy over cognate binding sites ( 31 ).\nAccording to the results of this study, increased gene expression of  CREB \nand  CREM  in ectopic tissues of women with endometriosis through their\nbindings to the CRE elements and thus expression activation of aromatase could be a reason\nfor the presence of significant expression levels of aromatase. Also, decreased ICER\nincorporation to the promoter region of aromatase, as an inhibitor of the CREB family, in\nectopic and eutopic endometriosis tissue may be a cause for increased aromatase expression\nin endometriosis tissue.\nEven though endometriosis is a benign disease, many studies have suggested that\nendometriosis represents the early stages of neoplastic processes. Endometriosis probably\nrepresents a form of transition from benign disease to cancer. Common features of\nendometriosis and cancer include the ability to escape apoptosis, angiogenesis, growth,\nadult stem cell-like dysregulation, and implantation in distant locations and to create an\nenvironment that functions independently of the immune system. Some studies have suggested\nthat alterations in the eutopic endometrial characteristics of women with endometriosis (for\nexample unusual secretion of different cytokines, growth factors, or angiogenesis factors)\nmay contribute to the development and maintenance of the disease. Another possibility may be\na change in the expression of specific cancer-related genes ( 32 ). Overexpression of\n CREB  has been reported in many types of solid tumors such as breast\ncancer, and estrogen dependent cancers, compared with adjacent normal tissues ( 33 ,  34 ) as\nwell as in hematopoietic malignancies ( 35 ). In addition, its overexpression is associated\nwith clinicopathologic parameters including grade, tumor stage, increased recurrence,\nmetastasis, worse prognosis, and decreased survival of tumor patients ( 34 ,  36 ,  37 ). There is\nevidence of a causal relationship between CREB activation, tumor initiation and progression\n( 38 ). This is due to the involvement of CREB in the upregulation of target gene expression\nwith CRE sequences ( 38 ).\nAlso, many studies have reported that CRTCs in signaling networks regulate cell growth and differentiation,\nproliferation, survival, DNA damage repair, and apoptosis. Therefore, several links between aberrant activation\nof CRTC and carcinogenesis have been identified with\na growing list of different cancers. Direct links between\nactivated CRTCs and the biological capabilities of malignant cells have been identified ( 39 ). Therefore, increased\nexpression of CRTC2, both as CREB co-activator and as\nits carcinogenesis role, could be important in pathogenesis of endometriosis.\n\nAccording to these results, the increased expression of CREB, CREM and\n CRTC2  transcription factor genes in ectopic and eutopic tissues of women\nwith endometriosis may indicate their role in the pathogenesis of endometriosis through\ntheir regulatory effect on the expression of downstream genes such as enzymes involved in\nestrogen biosynthesis (aromatase), and cancer-related genes. On the other hand, decreased\nbinding of ICER to the promoter II region of aromatase in endometriosis tissues could be a\nreason for increased expression of aromatase in this disease.","source_license":"CC0","license_restricted":false}