estrogen receptor action and the pregnant myometrium

Journal Pre-proof Estrogen/Estrogen Receptor action and the Pregnant Myometrium Jennifer C Condon, Chandrashekara Kyathanahalli, Prashanth Anamthathmakula, Pancharatnam Jeyasuria

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S2468-8673(19)30158-0

DOI:

https://doi.org/10.1016/j.cophys.2019.10.017

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COPHYS 241

To appear in:

Current Opinion in Physiology

Please cite this article as: Condon JC, Kyathanahalli C, Anamthathmakula P, Jeyasuria P, Estrogen/Estrogen Receptor action and the Pregnant Myometrium, Current Opinion in Physiology (2019), doi: https://doi.org/10.1016/j.cophys.2019.10.017

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Estrogen/Estrogen Receptor action and the Pregnant Myometrium. Jennifer C. Condon, Ph.D, Chandrashekara Kyathanahalli, Ph.D, Prashanth Anamthathmakula, Ph.D, Pancharatnam Jeyasuria, Ph.D Corresponding Author: Jeyasuria Pancharatnam, Ph.D Wayne State University School of Medicine Detroit, Michigan UNITED STATES

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Abstract Estrogen and estrogen action in the myometrium are necessary for a successful pregnancy. Estrogen is required for both the myometrial hyperplastic phase of gestation as well as the contractile response that leads to parturition at term. The excessive levels of circulating estrogens during pregnancy, pose a signalling conundrum. How do different tissues that have varying spatial and temporal needs for estrogen deal with this high estrogen ligand level? Insulin like growth factor (IGF1) and its receptor are important to the process of hyperplasia and are estrogen responsive. Although IGF1 is necessary for a successful pregnancy, myometrial production of IGF1 seems to be redundant for myometrial expansion. This review discusses the newly investigated functionality of estrogen receptor alpha splice isoforms in uterine myometrial contractility and quiescence whilst also covering the role of estrogen and IGF1 in uterine myometrial hyperplasia during pregnancy.

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Introduction Estrogens are steroid ligands that are the product of the enzyme aromatase that converts androgens to estrogens. Estrogens are unique steroids in that they have lost the C19 methylation of the A ring resulting in a saturated phenolic A ring. The ancestral estrogen receptor (ER) evolved in protochordates (amphioxus) 570 million years ago and most probably predates estrogen as it ligand, using other more easily synthesized androgens/steroids as ligands [1]. Genomic estrogen transcriptional action is mediated through ER(estrogen receptor  ESR1) and ER (estrogen receptor , ESR2)that bind estrogens, translocate to the nucleus and function as transcription factors by binding genomic DNA at estrogen responsive elements (ERE’s) on the promoters of estrogen responsive genes. ER’s then recruit co-activator complexes such as p160/SRC, TRAP/DRIP, CBP/p300 which in turn recruits RNA polymerase II, promoting the transcription of estrogen sensitive genes. CBP/p300 is an example of a coactivator complex that acts as histone acetyl transferases (HATs) that causes acetylation of histones that then open chromatin to transcription [2]. It is important to note that the estrogen receptors (ESR1 and ESR2) can signal through non-genomic pathways and that estrogen can also signal through membrane G-protein coupled estrogen receptor (GPER, GPR30). Through these nongenomic pathways estrogen stimulate mitogen activated protein kinases (MAPK), ERK-1 and ERK-2 [3]. In this current review, we limit our focus to genomic action of estrogen signaling in pregnant uterine myometrium.

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Estrogen plays a critical role in pregnancy starting early with changes in the endometrial lining that allow for embryo implantation and endometrial gland formation. During gestation estrogen is necessary for proliferation of the myometrium as well as for myometrial contractions associated with labor. Csapo and Schofield elucidated the important role estrogen plays in pregnancy with respect to contractility of the myometrium as early as the 1950s [4, 5]. Diethylstilbestrol (DES), an estrogen agonist that was prescribed to pregnant women as a preventative for spontaneous abortion in the late 1940s, increases the risk of clear cell carcinoma (cervix, vagina) and breast cancer [6-8]. The DES findings have inadvertently placed a constraint on furthering our knowledge of estrogen in the pregnant myometrium and as a consequence the mechanisms controlling uterotonic action of estrogen in the myometrium during pregnancy. However to overcome this limitation we have continued to study estrogen action in the myometrium using ER as a target for therapy. Estrogen and its counterpart, progesterone (P4), have long been known to play a critical and opposing roles in pregnancy especially with respect to myometrial quiescence [4, 5, 9]. The levels of estrogens rise continuously throughout pregnancy in the rat, mouse as well as in the human whereas P4 levels fall in rodents prior to labor. P4 however persists until term and during labor in the human. A functional P4 withdrawal is however considered to be the mechanism by which the myometrium is signaled to go into labor and the mechanisms for progesterone withdrawal have common aspects to both human and mouse pregnancy [10, 11]. Estrogen

action, in the pregnant uterus, drives decidualization, implantation and uterine receptivity through its receptor action in the endometrium. Estrogens have known roles in both the expansion of the myometrial segment during pregnancy as well as functionally in the contractile response prior to and at parturition. As discussed earlier in this section, estrogen action is mediated by nuclear ER and ER. We have limited our review of estrogen function in myometrium to ER as others and our laboratory have previously demonstrated that it is the dominant ER utilized by the myometrium in pregnancy [9, 12]. Furthermore, ER knockout mice demonstrate normal term parturition albeit at reduced fertility [13]. We will discuss the newly investigated functionality of alternatively spliced isoforms of the ER and their novel functions in myometrial contractility and quiescence [12].

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The main source of estrogens in the non-pregnant woman is the ovary and later by the placenta during pregnancy. The estrogen content in the human maternal serum are the products of aromatization of maternal and fetal adrenal androgens, fetal testosterone (male fetus) and androgens produced from thecal or corpus luteum of the maternal ovary. The placenta is also the main source of aromatase enzyme activity thus reducing androgen overproduction that effect parturition outcomes. It is thus conceivable that the large access of estrogens is a byproduct of the need to reduce androgen excess. The estrogens produced are estradiol (E2), estriol (E1), estrone and estetrol [14]. It is of interest that there is a shift between E2 and E1 at term with E1 becoming greater in concentration. High levels of serum estrogens throughout gestation create a conundrum. There are multiple tissues that use estrogen action for their function in pregnancy nonetheless the levels of estrogens make estrogen signaling through its ligand alone seem improbable. We will discuss this later with reference to the use of splice and promoter driven isoforms as a solution to this conundrum. The role of estrogen in the myometrium is twofold; proliferation of the myometrium prior to term and the contractile response at term. We will discuss both these roles with reference to the genomic function of estrogen and ER. We will also introduce a the novel concept of alternative isoforms of the estrogen receptor and their role in myometrial quiescence.

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Estrogen and Hyperplasia of The Pregnant Myometrium. Sex steroids in particular estrogen, regulate the proliferation and differentiation of the myometrium during pregnancy. The uterus undergoes dramatic changes throughout pregnancy, with a massive increase in both size and numbers of myometrial smooth muscle cells. The weight of the uterus increases up to 24 fold and consist mostly of myometrium by mass. Among its other functions, the myometrium is the structural organ that physically supports the fetus during gestation. In early pregnancy the myometrial mass undergoes a proliferative phase followed by an intermediate phase of hypertrophy and finally transitioning into a contractile phenotype at term. To date its established that growth factors, acting in an autocrine and or paracrine manner, exert their effects on myometrial cells through cell surface receptors and play a critical role in regulating the cellular proliferation rate. Multiple growth factors have been demonstrated in the myometrial cell, and the mitogenic action of circulating steroids on the myometrial cell is thought to be mediated by the local production of these growth factors which act as effectors of steroid hormone action locally in the myometrial compartment. In the uterus, IGFs have been demonstrated to be essential for normal uterine development and play a central role during implantation and the establishment of pregnancy in all species studied [15-18]. During human pregnancy IGF1 concentrations increased in maternal serum compared with preconception values, from 30 to 50 nmol/l whereas IGF2 (Insulin-like Growth Factor 2) concentrations remain unchanged [19]. In both the porcine and rat myometrium, Igf-1 expression is upregulated during the first half of gestation signifying an evolutionary conservation [20]. Uterine Igf1 gene expression is regulated by the steroid hormones estrogen and P4 and is thought to mediate many of the actions of these steroid hormones on uterine growth and development during pregnancy [21, 22]. IGF-1 through the insulin like growth factor 1 receptor in myometrium upregulate DNA synthesis in human and rat primary myometrial smooth muscle cells in vitro [23, 24]. There is evidence that epidermal growth factor (EGF) and IGF-1 are necessary for estrogen induced growth of uterus. In the rat model the level of uterine Igf1 mRNA was significantly reduced following ovariectomy, but it increased to levels higher than those in intact animals following administration of estrogen alone [25]. Similarly, ovariectomized mice lose IGF1 expression but the levels increase to beyond normal levels when E2 or the combination of both E2 and P4 are reintroduced [26]. The paracrine overproduction of IGF1 causes lengthening of the longitudinal layers of the uterus and intestine suggesting that smooth muscles of the uterus are not homogenous in function [27]. Elevated Igf1 expression seems necessary for the early proliferative phase of myometrial expansion and is driven in part by estrogen. ER and not ER drive the mitogenic responses through E2 and IGF1 [28]. In a recent study, deletion of an enhancer that binds ER distal to the

transcription start site of igf1 discovered through ChiP-Seq analysis resulted in complete loss of Igf1 expression particularly from the uterus without any changes in hepatic Igf1 expression. The loss of the enhancer did not affect fertility or uterine growth. The loss of Igf1 in the uterus by conditional knockout using pgr-cre;igf1fl/fl showed decreased fertility but normal estrogen induced uterine growth implicating systemic IGF1 as the driver for uterine growth and also linking other growth factors with the estrogen growth response in the uterus [29]. These experiments give us pause to rethink the role of uterine IGF1 expression in uterine growth and that the role may be redundant.

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Estrogen Action and the Contractile Myometrium The myometrium is the functionally contractile organ during parturition and its quiescence throughout pregnancy prior to labor is necessary for successful timed pregnancy. It is known that estrogen, through ERα, upregulates contractile associated proteins (CAPs). Estrogen action has been shown to upregulate cyclooxygenase2 [9] (COX2) enzyme that is required for prostaglandin production, connexin 43 [30] (CX43) a gap junction protein required to coordinate electrical impulses between smooth muscle cells of the myometrium and the oxytocin receptor (OXTR) [31], which is involved in downstream contractile protein signaling. Estrogen action also mediates an inflammatory response that is a requisite for parturition [32, 33]. MicroRNAs (miRNA) are hormonally regulated small (22 nucleotide) non-coding RNA that are recently recognized to modulate uterine contractility [34] through the transcriptional control of CAP proteins. There is evidence that estrogen through ER action suppresses zinc finger E-box binding homeobox-1 (ZEB-1) mediated expression of miRNA199a-3p/214 which permits upregulation of COX2. The decline in ZEB-1 also allows for de-repression of OXTR and CX43 that collectively contributes to the induction of uterine contractility leading to labor. Estrogen action thus directly or indirectly causes the upregulation of CAPs [35].

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We have previously defined the opposed function P4 and E2 with a functional withdrawal of progesterone resulting in an upregulation of ER and the upregulation of pro-inflammatory as well as contractile associated proteins that herald labor [9, 11]. Conversely, as explained earlier estrogen levels are high throughout pregnancy leaving us with tissue specific estrogen regulation conundrum. How do different tissues regulate estrogen-mediated transcription given a high level of the ligand? The myometrium itself requires estrogen action earlier in pregnancy for proliferative function and maintenance of the uterine mass while at the same time preventing a contractile response. We believe this is achieved through the use of alternative splicing of ESR1 producing multi-functional estrogen receptor variants.

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Pregnancy: the Role of Alternative Splicing Alternative splicing allows cell and tissue type to change phenotype by greatly increasing the biodiversity of available proteins in a relatively energy efficient manner and to quickly adapt to changing requirements. It has been proposed that alternative splicing is a crucial adaptation towards higher efficiency, as information can be stored and accessed much more economically. A single gene can encode several proteins, rather than requiring a separate gene for each protein, and thus allowing a more varied proteome from a genome of limited size [36]. Rapid changes in pregnancy require major myometrial modifications across gestation, which we believe uses alternative splicing heavily to allow increased, and rapid plasticity in responses. The myeloid cell leukemia -1 (MCL-1) gene, a member of the BCL-2 family, undergoes alternative splicing resulting in two functionally distinct proteins, MCL-1L and MCL-1S that modulate downstream cellular function. The inclusion of exon 2 of the MCL-1 gene encodes the anti-apoptotic MCL-1L protein, whereas exon 2 exclusion produces the apoptotic MCL-1S protein [37]. There are high levels of cleaved caspase 3 (CASP3) in the pregnant uterus that specifically cleave proteins involved in the contractile architecture of the myometrium working as a endogenous tocolytic [38, 39]. Anti-apoptotic MCL-1L allows these high levels of cleaved caspase 3 and uterine quiescence without the associated cell death. MCL-1L however is downregulated during the uterine involution process postpartum causing apoptosis [40]. MCL-1 is a functional example of an alternatively spliced protein that has opposing functions derived from a single gene. There is evidence for a stringent control of alternative splicing events by splice factors such as serine-arginine rich splice factors (SRSFs) and heterogenous ribonucleoproteins (hnRNPs). SRSFSs bind intron and exon splicing enhancer sites (ESEs and ISEs) and bias the inclusion of adjacent exons, whereas hnRNPs tend to exclude adjacent exons by binding exon and intron splicing inhibitor sites (ISI and ESI). With respect to MCL-1, splice factors SRSF1 and SRSF5 regulates alternative splicing event. It has been demonstrated that selective knockdown of hnRNPK and hnRNPF/H results in splicing of MCL-1 towards the apoptotic MCL-1S isoform [41,

42]. Studies by Pollard et al. demonstrated that splice factors, SRSF1 and hnRNPA1/1B are gestationally and spatially regulated in the pregnant human myometrium. The levels of SRSF1 were substantially increased in the lower uterine segment while there was a concomitant decrease in hnRNPA1/1B [43]. In a subsequent study they demonstrated that adenylyl cyclase stimulatory GTP-binding protein (Gαs) is targeted by hnRNPA1/1B and SRSF1, which alternatively splices the Gαs protein to a Gαs-long and Gαs-short form by excluding or including exon 3 [44]. The upregulation of both Gαs spliced isoforms are thought to be involved with myometrial quiescence through elevating cAMP levels [45, 46]. Other genes that are alternatively spliced and use alternate isoforms to regulate pregnancy through their contractile, transcriptional, gap junction and channel functions during pregnancy includes PR, ERα, OTR, cyclooxygenase (COX-1 and COX2), CX43, prostaglandin E2 receptors 1 and 3 (EP1 and EP3), prostaglandin F2 receptors, caldesmon and corticotrophin releasing hormone receptors [47]. These data suggest that indeed during pregnancy the uterus may rely on a robust program of alternate splicing to maintain homeostasis and an appropriate gestational length.

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Multiple alternatively spliced ER isoforms have been identified both in mouse and human tissues. The original ERα knockout (ERKONeo) where a Neo cassette was placed in exon 1 resulted in a mouse that did not produce the canonical 66 kDa protein (ER66) but retained a truncated 46 kDa ER protein (ER46) that resulted from an alternative start site (ATG) in the second exon. This truncated form of ER lacked the activation function domain in the N-terminal region of the protein but was still able to elicit a decidualization response and produce eNOS in endothelial cells. Functional analysis of ER46 by Denger et al. revealed that the human ER46 had the capacity to dimerize with the full length ER66 and could also bind DNA. Denger et al. also demonstrated that ER46 is a strong inhibitor of ER66 and when co-expressed in bone cells limits the action of the full length ER66 isoform. ERα46 is normally expressed in osteoblast [48] and endothelial cells where ER46 alone can mediate acute activation of endothelial nitric oxide synthase (eNOS) in response to estrogen stimulation [48, 49].

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We have recently published data indicating that there are three isoform variants of ER that are expressed in the pregnant myometrium. These isoforms are gestationally regulated. ER46 isoform is expressed throughout human gestation, while the ERΔ7 (missing exon 7) isoform, is highly expressed in the pregnant uterus earlier in gestation and its expression declines towards term. ERΔ7, a 51kD protein, is the product of an exon 7 skip, causing a frameshift in exon 8 with a precocious truncation resulting with in the loss of the ligand binding domain (LBD) and activation function 2 (AF2) domains. We demonstrated that the ERΔ7 isoform is functionally dominant negative, blocking estrogen mediated ERα action. ER7 when overexpressed results in the downregulation of CX43. Our data demonstrates that hnRNPG is expressed highly earlier in pregnancy and that the in vitro overexpression of hnRNPG results in higher levels ER7 while the shRNA knockdown of hnRNPG resulted in the loss of ER7. We have also determined that very high levels of estrogen that are commiserate with levels seen in the pregnant uterus at term causes the downregulation of hnRNPG which in turn results in lower levels of ER7 isoform. Gel contraction assays performed in human uterine myometrial cell line hTERTHM demonstrate that the overexpression of ER7 permit uterine myocytes to adapt a more quiescent phenotype in vitro [12]. It is of interest to note that in early gestation the P4 receptor (PR) has not gone through withdrawal and that the ER isoform, ER7, has a powerful counter effect against estrogens contractile role. Later in pregnancy when the progesterone functional withdrawal is well underway, estrogen action through CAP expression is controlled through the full length ER66. Are ERisoforms controlled by P4 action? It would be interesting to see how these alternative isoforms of estrogen receptors are associated with miRNA and IGF1 function in the myometrium. We have in this minireview explained the importance of IGF-1 in myometrial expansion that is important structurally in supporting the fetus but also necessary for parturition. The myometrium is crucial in maintaining gestational timing and it quiescence is maintained through the actions of progesterone. We have introduced an important molecular mechanism, alternative splicing that has been overlooked in pregnancy. We believe that the ER7 estrogen receptor isoforms plays a crucial role in maintaining myometrial quiescence and that changes in the splice factor milieu at different gestation times contribute not only to estrogen receptor isoforms but generate other alternatively spliced protein isoforms. The global changes in protein isoforms associated with changes in splice factors give the myometrium a mechanism by which it can produce proteins from single genes that have enhanced functions, longer half lives or alternate functions. It

would be essential to appreciate this important post-transcriptional phenomenon to develop tocolytic interventions for the prevention of preterm birth. To the editors of Current Opinions in Physiology. The authors (Drs, Jennifer Condon, Prashanth Anamthatmakula, Chandrashekara Kyanthahalli and Pancharatnam Jeyasuria) of the article “Estrogen/Estrogen Receptor action and the Pregnant Myometrium”

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have no conflicts of interest.

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