Transcriptional Regulation Of Corin Expression In The Pregnant Uterus

Chapter One: Identification of Key Promoter Elements that Regulate CORIN Expression in Uterine CellsObjective:Corin is a type II transmembrane serine protease discovered in the heart,where it activates atrial natriuretic peptide(ANP)to regulate blood volume and pressure.In addition to the heart,corin expression was identified in other organs,including the kidney,brain,bone and skin.Moreover,increased corin m RNA and protein levels were observed in the decidua of the pregnant uterus.The uterine corin expression is important for spiral artery remodeling and normal blood pressure in pregnancy.The increased corin m RNA levels in the pregnant uterus indicate a regulatory mechanism at the transcription level.Human CORIN gene 5'-flanking regions contain several conserved binding sites for TBX5,GATA,NKX2.5,and Krüppel-like transcription factors.In the heart,corin expression is controlled primarily by GATA-4,a cardiac transcription factor.To date,the transcriptional mechanism of corin expression in the pregnant uterus remains unknown.The goal of this study is to understand the molecular mechanism underlying CORIN expression in the pregnant uterus.Methods:Plasmids expressing firefly luciferase driven by serially truncated human corin promoter fragments from-1297 to-161(h Cp1297 Luc,h Cp405 Luc,h Cp236 Luc and h Cp161Luc)were transiently transfected into murine cardiac HL-1 cells and human endometrium adenocarcinoma AN3-CA cells,which express endogenous corin.The p RL-SV40 plasmid expressing Renilla luciferase was co-transfected to serve as a control for transfection efficiency.The transfected cells were assayed for firefly and Renilla luciferase activities.The normalized firefly luciferase activity was used as an indicator for CORIN promoter activity.Results:1.When tested in cardiac HL-1 cells,similar human CORIN promoter activities were observed when plasmids h Cp1297,405,236 and 161 were transfected.2.Mutation of the GATA sequence to CTTA in the h Cp405 fragment of human CORIN gene abolished the promoter activity in HL-1 cells,indicating the importance of the GATA element in CORIN expression in cardiomyocytes.3.In uterine endometrial AN3-CA cells transfected with plasmids h Cp1297,405 and 236,CORIN promoter activities were similar.The activity was reduced in the cells transfected with the plasmid h Cp161.There are two conserved Krüppel-like factor(KLF)-binding sites between-161 to-236 bp.Deletion of or point mutations within the core KLF-binding sites reduced CORIN promoter activity in AN3-CA cells.Conclusions:These results indicate that CORIN promoter activity in the heart is mediated primarily by the GATA element.In contrast,the CORIN promoter activity in the uterus is mediated primarily by two KLF-binding sites.These results suggest that the uterine and cardiac regulation mechanisms for CORIN expression may be different and that KLF transcription factor(s)may play a key role in regulating CORIN expression in the uterus.Chapter Two: Expression of KLF Transcription Factors in the Pregnant Uterus and Their Potential Roles in Regulating CORIN TranscriptionObjective:In Chapter One,we found that the KLF-binding sites in the CORIN promoter are critical for the promoter activity in uterine cells,suggesting that member(s)of the KLF family are involved in regulating CORIN expression in the uterus.Human KLF family has 17 members that participate in a variety of biological processes,including cell proliferation,differentiation,embryonic development,stem cell pluripotency,epithelial-mesenchymal transition,and programmed cell death.To date,no reports indicate a role of KLFs in CORIN expression.The goal of this Chapter is to identfy the KLF family member(s)that may regulate CORIN expression in the uterus.Methods:1.Mice were ovariectomized by surgery and treated with estrogen,progestogen or estrogen and progestogen.Corin and KLF m RNA levels in mouse uteruses were analyzed by q RT-PCR.2.Uterus tissues were collected from normal non-pregnant(Non-P)female mice and pregnant mice at different gestational(G)stages.Corin and KLF m RNA levels in the tissue were analyzed by q RT-PCR.3.Corin and KLF m RNA levels in human uteruses from normal Non-P and early stages of pregnancy(6-7 week,w)were analyzed by q RT-PCR.4.CORIN promoter activities were analyzed in cultured cells after overexpression of recombinant candidate KLF proteins.Results:1.Compared with the vehicle control,progestogen injection induced corin m RNA expression in the uterus of ovariectomized mice.Such an effect was not observed in the mice treated with estrogen or estrogen and progestogen.2.Increased uterine m RNA levels of KLF2,9,15 and 17,but not the other KLF members,were detected in progestogen-treated ovariectomized mice.3.In normal pregnant mice,increased levels of KLF2,9,15 and 17 m RNA expression were observed at different gestational stages.In particular,the increase of KLF2 and 17 expression occurred earlier than the increase of corin expression in the uterus.4.Compared with that in Non-P women,corin levels were higher in the early pregnant uterus,where KLF2 and 17 expression levels were also high and positively correlated with corin levels.5.In cultured uterine endometrial AN3-CA cells,overexpression of KLF2 and 17,but not KLF9 and 15,increased the CORIN promoter activity.When the KLF-binding sites in the CORIN promoter were deleted or mutated,overexpression of KLF2 and 17 did not increase the promoter activity.6.Unlike in AN3-CA cells,overexpression of KLF2 and 17 did not increase the CORIN promoter activity in cardiac HL-1 cells.Conclusions:These results show that among the KLF family members,KLF2,9,15 and 17 expression levels were up-regulated in the pregnant uterus.In particular,theup-regulation of KLF2 and 17 correlated with the increased corin expression.In transfected AN3-CA cells,overexpression of KLF2 and 17,but not KLF9 and 15,enhanced the CORIN promoter activity.These results suggest that KLF2 and/or 17 may act as key regulator(s)in CORIN transcription.Chapter Three: Role of KLF17 in CORIN Transcriptional Regulation in the Pregnant UterusObjective:In Chapter Two,we identified KLF2 and 17 as potential transcription factors in regulating CORIN expression in the pregnant uterus.The goal of this Chapter is to study the function of KLF2 and 17 in regulating CORIN expression in uterine cells and tissues to understand how CORIN transcription is controlled in the pregnant uterus.Methods:1.Human endometrial stromal cells(HESCs)were induced for decidualization in culture.Levels of prolactin,a decidual marker,and corin m RNAs were analyzed by q RT-PCR.2.Immunocytochemistry,immunofluorenscent and Western blotting were performed to examine the expression level and subcellular distribution of corin,KLF2 and KLF17 in the decidualized HESCs.3.Immunohistochemistry was used to examine the expression level and cellular distribution of corin and KLF17 in the pregnant mouse uterus of different gestational stages.4.Chromatin immunoprecipitation(Ch IP)assay was used to verify KLF-binding in the CORIN promoter in HESCs before and after the decidualization.5.Two HESC cell lines(KLF17-KO)were established,in which the KLF17 gene were disrupted by CRISPR-Cas9 techniques.The disruption of the KLF17 gene was verified by PCR-based genotyping and Western blotting.6.Parental HESC cells and the KLF17-KO cells were decidualized and corin m RNA expression was measured by q RT-PCR.7.Corin and KLF17 m RNA levels in the uterus from non-pregnant(Non-P),normal pregnant and preeclamptic(PE)women were measured by q RT-PCR.8.Gene targeting technique was used to create KLF17 gene knockout mice.Initialgenotyping and characterization were carried out.Results:1.By q RT-PCR,increased prolactin(a decidualization marker)and corin m RNA levels were detected in HESCs 12-72 h after the induction.The results indicate that corin expression was up-regulated in the decidualized HESCs.Thus,the HESCs can be used as a uterine cell model to study the regulation of CORIN expression.2.In decidualized HESCs,increased KLF17 protein levels were detected by Western blotting.Immunocytochemistry and immunofluorescent staining revealed the increased KLF17 protein levels,first in the cytoplasm and the nucleus and later mostly in the nucleus,whereas increased corin expression was mostly in the cytoplasm and the cell surface.In contract,no up-regulation of KLF2 protein expression was detected by Western blotting and immunocytochemistry under similar experimental conditions.3.In the uterus of non-pregnant mice,corin expression levels in endometrial epithelial cells were low and KLF17 expression was barely detectable.By gestational(G)day 4.5,increased corin and KLF17 expression levels were detected by immunohistochemistry in endometrial and glandular epithelial cells in pregnant mice.Such up-regulation continued throughtout the pregnancy.4.Ch IP analysis detected the specific binding of KLF17,but not KLF2 and 9,to the KLF binding sites in the CORIN promoter in decidualized,but not non-decidualized,HESCs.The results were confirmed by quantitative PCR.5.Western blotting confirmed the lack of KLF17 protein expression in the KLF17-KO HESCs,indicating that the KLF17 gene was disrupted in those cells.6.In decidualized HESCs,corin m RNA levels were significantly lower in two KLF17-KO cell lines compared with those in the parental HESCs,as measured by q RT-PCR,indicating a critical role KLF17 in corin expression in decidualized human uterine cells.7.In human uteruses,corin m RNA levels were higher in samples from normal pregnant women than those from non-pregnant women or patients with preeclampsia.Similarly reduced KLF17 expression was found in uterus samples from preeclamptic patients compared with those in normal pregnant women.8.KLF17 knockout(KO)mice were created.Preliminary genotyping and analysis confirmed the survival of homozygous KLF17 KO mice.More analysis to determine the effects of KLF17 gene disruption on embryonic development,long-term survival,uterine corin expression and pregnancy will be carried out when enoughnumbers of the KO mice are available.Conclusions:These results indicate that KLF17 was up-regulated in decidualized HESCs,which correlated with corin expression.Immunostaining of KLF17 protein in the nucleus was consistent with the finding of KLF17-binding to the CORIN promoter in Ch IP analysis.In decidualized HESCs,lack of KLF17 expression inhibited corin expression.In preeclamptic patients,both KLF17 and corin expression levels were reduced in the pregnant uterus,supporting a role of KLF17 in regulating uterine corin expression.In summary,corin expression is up-regulated in the pregnant uterus to promote uterine spiral artery remodeling,which is critical for enhancing maternal-fetal blood flow and preventing pregnancy-induced hypertension such as preeclampsia.Previously,reduced corin m RNA levels were found in preeclamptic patients,suggesting that impaired transcriptional regulation may be an underlying mechanism in preeclampsia.In this study,we seek to understand the mechanism underlying the transcriptional regulation of the CORIN gene.In biochemical and cellular experiments,mouse model studies and human uterus sample analysis,we have identified KLF17 as a key transcription factor that regulates corin expression.This conclusion is based on the following lines of experimental evidence:1)The CORIN promoter contains two conserved KLF binding sites,which were required for corin expression in uterine cells,indicating that KLF(s)are involved in CORIN gene transcription in the uterus;2)In the uterus from ovariectomized mice treated with progestogen or normal pregnant mice,KLF17 expression was up-regulated and the up-regulation proceeded the increased corin expression.Moreover,KLF17 and corin had similar expression patterns in uterine endometrial and glandular epithelial cells;3)In human endometrial AN3-CA cells,KLF17 overexpression increased the CORIN promoter activity;4)In decidualized HESCs,KLF17 expression was increased and KLF17 was shown to bind to the KLF-binding sites in the CORIN promoter;5)In decidualized HESCs,disrupting the KLF17 gene prevented the incuded corin expression;6)In normal early pregnant human uteruses,both KLF17 and corin expression were up-regulated and the expression correlated positively;7)In uterus samples from preeclamptic patients,both KLF17 and corin expression levels were reduced.Together,our results indicate that KLF17 is a key transcription factor for corin expression in the pregnant uterus.Reduced KLF17 expression or functional defects may prevent corin expression in the pregnant uterus,thereby impairing spiral artery remodeling and contributing to preeclampsia.Our findings should be of great biological and clinical significance in understanding the biology of corin and its role in hypertensive diseases.