Estrogen mediated epithelial proliferation in the uterus is directed by stromal Fgf10 and Bmp8a.

To define endometrial stromal-derived paracrine mediators that participate in estradiol-17ß (E2)-induced epithelial proliferation, microarray analysis of gene expression was carried out in mouse uterine epithelial-stromal co-culture systems under the condition of E2 or vehicle (control). Our results demonstrated gene alteration by E2: in epithelial cells, we found up-regulation of 119 genes and down-regulation of 28 genes, while in stroma cells we found up-regulation of 144 genes and down-regulation of 184 genes. A functional enrichment analysis of the upregulated epithelial genes implicated them for proliferation, while upregulated stromal genes were associated with extracellular functions. Quantitative RT-PCR and in situ hybridization results confirmed differential gene expression in both cell cultures and ovariectomized uteri after the above treatments. Based on our identification of stromal secretory factors, we found evidence that suppression by siRNA specifically for Bmp8a and/or Fgf10 in the stromal layer caused significant inhibition of proliferation by E2 in the co-culture system, suggesting Bmp8a and Fgf10 act as paracrine mediators during E2-dependent control of uterine proliferation. The localization of receptors and receptor activation signaling in epithelial cells in both the co-culture system and uteri was consistent with their involvement in ligand-receptor signaling. Interestingly, loss of Bmp8a or Fgf10 also caused abrogation of E2-regulated epithelial receptor signaling in co-culture systems, suggesting that stroma-derived Fgf10 and Bmp8a are responsible for epithelial communication. Overall, stromal Fgf10 and Bmp8a serve as potential paracrine factors for E2-dependent regulation of epithelial proliferation in the uterus.

Epigenetic changes through DNA methylation contribute to uterine stromal cell decidualization.

Embryo-uterine interaction during early pregnancy critically depends on the coordinated expression of numerous genes at the site of implantation. The epigenetic mechanism through DNA methylation (DNM) plays a major role in the control of gene expression, although this regulatory event remains unknown in uterine implantation sites. Our analysis revealed the presence of DNA methyltransferase 1 (Dnmt1) in mouse endometrial cells on the receptive d 4 of pregnancy and early postattachment (d 5) phase, whereas Dnmt3a had lower abundant expression. Both Dnmt1 and Dnmt3a were coordinately expressed in decidual cells on d 6-8. 5-Methycytosine showed a similar expression pattern to that of Dnmt1. The preimplantation inhibition of DNM by 5-aza-2'-deoxycytodine was not antagonistic for embryonic attachment, although endometrial stromal cell proliferation at the site of implantation was down-regulated, indicating a disturbance with the postattachment decidualization event. Indeed, the peri- or postimplantation inhibition of DNM caused significant abrogation of decidualization, with concomitant loss of embryos. We next identified decidual genes undergoing alteration of DNM using methylation-sensitive restriction fingerprinting. One such gene, Chromobox homolog 4, an epigenetic regulator in the polycomb group protein family, exhibited hypomethylation in promoter DNA and increased expression with the onset of decidualization. Furthermore, inhibition of DNM resulted in enhanced expression of hypermethylated genes (Bcl3 and Slc16a3) in the decidual bed as compared with control, indicating aberration of gene expression may be associated with DNM-inhibition-induced decidual perturbation. Overall, these results suggest that uterine DNM plays a major role for successful decidualization and embryo development during early pregnancy.

Nucleolar Sik-similar protein (Sik-SP) is required for the maintenance of uterine estrogen signaling mechanism via ERα.

Sik-similar protein (Sik-SP), a small nucleolar ribonucleoprotein, has been shown to be primarily involved in ribosome biogenesis. However, its role in the hormone-directed nuclear receptor signaling is largely unknown. Here, we provide novel evidence that Sik-SP is required for appropriate regulation of estrogen receptor (ER)α-mediated estradiol-17ß (E2)-dependent uterine physiologic responses in mice. Studies by Western blotting using the newly developed antibodies for Sik-SP showed that this protein is up-regulated in both the ovariectomized wild-type and ERα null uteri by E2. Immunohistochemical analyses in uterine sections showed that this protein is induced in the epithelial and stromal cells. Coimmunoprecipitation studies revealed that E2 directs molecular interaction between Sik-SP and ERα. Furthermore, gel-mobility shift and chromatin immunoprecipitation analyses provided evidence that Sik-SP is recruited with ERα to estrogen-responsive uterine gene promoters. Overexpression of Sik-SP in vitro demonstrated a role for Sik-SP in cellular growth and viability. In a primary uterine epithelial-stromal coculture system, E2 exhibited early induction of Sik-SP in both the epithelial and stromal cells. Interestingly, suppression of Sik-SP in this coculture model, for the stromal but not epithelial cells, caused perturbation of E2-dependent proliferation in the epithelial cell layer. Similarly, in vivo uterine suppression of Sik-SP also caused inhibition of epithelial cell proliferation and aberrant prolongation of water imbibition in the late phase by E2. Finally, studies showed that Sik-SP is physiologically important during the onset of implantation by E2. In conclusion, Sik-SP, an early E2-responsive nucleolar protein, is necessary to induce E2-dependent ERα-mediated appropriate physiologic responses in the uterus.

Mouse primary uterine cell coculture system revisited: ovarian hormones mimic the aspects of in vivo uterine cell proliferation.

Previously, the uterine epithelial-stromal coculture system had limited success mimicking in vivo ovarian hormone-dependent cell-specific proliferation. Here, we established a mouse primary uterine coculture system, in which cells collected in pseudopregnancy specifically on d 4 are conducive to supporting hormone-induced cell-specific proliferation. When two cell types are placed in coculture without direct contact via cell culture inserts (nonadjacent), as opposed to with contact (adjacent), epithelial cells exhibit significant proliferation by estradiol-17ß (E2), whereas progesterone in combination with E2 caused inhibition of epithelial cell proliferation and a major shift in proliferation from epithelial to stromal cells. Epithelial cell integrity, with respect to E-cadherin expression, persisted in nonadjacent, but not adjacent, conditions. In subsequent studies of nonadjacent cocultures, localization of estrogen receptor (ER)α and progesterone receptor (PR), but not ERß, appeared to be abundant, presumably indicating that specific ER or PR coregulator expression might be responsible for this difference. Consistently, an agonist of ERα, but not ERß, was supportive of proliferation, and antagonists of ER or PR totally eliminated cell-specific proliferation by hormones. RT-PCR analyses also revealed that hormone-responsive genes primarily exhibit appropriate regulation. Finally, suppression of immunoglobulin heavy chain binding protein, a critical regulator of ERα signaling, in epithelial and/or stromal cells caused dramatic inhibition of E2-dependent epithelial cell proliferation, suggesting that a molecular perturbation approach is applicable to mimic in vivo uterine control. In conclusion, our established coculture system may serve as a useful alternative model to explore in vivo aspects of cell proliferation via communication between the epithelial and stromal compartments under the direction of ovarian hormones.

TRPC1, STIM1, and ORAI influence signal-regulated intracellular and endoplasmic reticulum calcium dynamics in human myometrial cells.

To explore the relationship between signal-stimulated increases in intracellular calcium ([Ca(2+)](i)) and depletion and refilling of the endoplasmic reticulum (ER) Ca(2+) stores ([Ca(2+)](L)) in human myometrial cells, we measured simultaneous changes in [Ca(2+)](i) and [Ca(2+)](L) using Fura-2 and Mag-fluo-4, respectively, in PHM1-41 immortalized and primary cells derived from pregnant myometrium and in primary cells derived from nonpregnant tissue. Signal- and extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and ER refilling stimulated by oxytocin and cyclopiazonic acid were not inhibited by voltage-operated channel blocker nifedipine or mibefradil, inhibition of Na(+)/Ca(2+) exchange with KB-R7943, or zero extracellular Na(+) in PHM1-41 cells. Gadolinium-inhibited oxytocin- and cyclopiazonic acid-induced SRCE and slowed ER store refilling. TRPC1 mRNA knockdown specifically inhibited oxytocin-stimulated SRCE but had no statistically significant effect on ER store refilling and no effect on either parameter following cyclopiazonic acid treatment. Dominant negative STIMΔERM expression attenuated oxytocin- and thapsigargin-stimulated SRCE. Both STIM1 and ORAI1-ORAI3 mRNA knockdowns significantly attenuated oxytocin- and cyclopiazonic acid-stimulated SRCE. The data also suggest that reduction in STIM1 or ORAI1-ORAI3 mRNA can impede the rate of ER store refilling following removal of SERCA inhibition. These data provide evidence for both distinct and overlapping influences of TRPC1, STIM1, and ORAI1-ORAI3 on SRCE and ER store refilling in human myometrial cells that may contribute to the regulation of myometrial Ca(2+) dynamics. These findings have important implications for understanding the control of myometrial Ca(2+) dynamics in relation to myometrial contractile function.

Attenuation of canonical transient receptor potential-like channel 6 expression specifically reduces the diacylglycerol-mediated increase in intracellular calcium in human myometrial cells.

An increase in intracellular Ca(2+) ([Ca(2+)](i)) as a result of release of Ca(2+) from intracellular stores or influx of extracellular Ca(2+) contributes to the regulation of smooth muscle contractile activity. Human uterine smooth muscle cells exhibit receptor-, store-, and diacylglycerol (OAG)-mediated extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and express canonical transient receptor potential-like channels (TRPC) mRNAs (predominantly TRPC1, -4, and -6) that have been implicated in SRCE. To determine the role of TRPC6 in human myometrial SRCE, short hairpin RNA constructs were designed that effectively targeted a TRPC6 mRNA reporter for degradation. One sequence was used to produce an adenovirus construct (TC6sh1). TC6sh1 reduced TRPC6 mRNA but not TRPC1, -3, -4, -5, or -7 mRNAs in PHM1-41 myometrial cells. Compared with uninfected cells or cells infected with empty vector, the increase in [Ca(2+)](i) in response to OAG was specifically inhibited by TC6sh1, whereas SRCE responses elicited by either oxytocin or thapsigargin were not changed. Similar findings were observed in primary pregnant human myometrial cells. When PHM1-41 cells were activated by OAG in the absence of extracellular Na(+), the increase in [Ca(2+)](i) was partially reduced. Furthermore, pretreatment with nifedipine, an L-type calcium channel blocker, also partially reduced the OAG-induced [Ca(2+)](i) increase. Similar effects were observed in primary human myometrial cells. These findings suggest that OAG activates channels containing TRPC6 in myometrial cells and that these channels act via both enhanced Na(+) entry coupled to activation of voltage-dependent Ca(2+) entry channels and a nifedipine-independent Ca(2+) entry mechanism to promote elevation of intracellular Ca(2+).

Potential role for oxidative stress in 2,2'-dichlorobiphenyl-induced inhibition of uterine contractions but not myometrial gap junctions.

Previously, we reported that 2,2'-dichlorobiphenyl (2,2'-DCB)-induced decreases of amplitude and synchronization of uterine contractions are dependent on MAPK-induced phosphorylation of Connexin43 (Cx43) and inhibition of myometrial gap junctions. Recent studies show that oxidative stress inhibits uterine contractions and myometrial gap junctions also. The present study examines the hypothesis that 2,2'-DCB-induced modification of uterine contraction is dependent on oxidative stress-mediated inhibition of myometrial gap junctions via activation of mitogen-activated protein kinase (MAPK) and phosphorylation of Cx43. In uterine strips treated with alpha-tocopherol (100 microM), deferoxamine mesylate (Def, 50 microM), or superoxide dismutase (SOD, 1000 U) after a 1-h exposure to 100 microM 2,2'-DCB, modification of uterine contractions reversed 1 h after initiating antioxidant treatment. Treatment of uterine strips with 100 microM 2,2'-DCB for 1 h lowered total SOD activity and also induced a surge of superoxide generation after 5 min of exposure. However, myometrial cells exposed in culture to 100 microM 2,2'-DCB did not produce reactive oxygen species as determined by the lack of superoxide anion generation measured by the cytochrome c reduction assay, reactive species by the formazan assay, hydrogen peroxide by the 2',7'-dichlorofluorescein assay, and lipid peroxidation by the thiobarbituric acid-reactive substance assay. Furthermore, cotreatment with SOD or Def was unable to prevent 2,2'-DCB-induced phosphorylation of Cx43, activation of MAPK, and inhibition of myometrial gap junctions. Although antioxidants reversed 2,2'-DCB-induced inhibition of uterine contraction force and synchronization, the myometrial cell culture experiments failed to support oxidative stress as a mechanistic link between 2,2'-DCB-induced inhibition of myometrial gap junctions and modification of uterine contraction.

2,2'-Dichlorobiphenyl decreases amplitude and synchronization of uterine contractions through MAPK1-mediated phosphorylation of GJA1 (connexin43) and inhibition of myometrial gap junctions.

The present study examined the hypothesis that inhibition of myometrial gap junctions through MAPK1-induced phosphorylation of GJA1 (connexin43) leads to inhibition of spontaneous phasic uterine contractions by 2,2'-dichlorobiphenyl (2,2'-DCB). Uterine strips from Gestation Day 10-pregnant rats exposed in muscle baths to 2,2'-DCB exhibited increased oscillatory frequency and decreased amplitude and synchronization of contractions. To assess effects on gap junctions, Lucifer yellow was injected into myometrial cells and transfer to adjacent cells was scored. After a 1-h treatment, 100 microM 2,2'-DCB decreased Lucifer yellow intercellular transfer in a concentration-dependent manner. The MAP2K1 inhibitor PD98059 increased percentage of dye transfer to adjacent myometrial cells from 18% in cultures exposed for 1 h to 100 microM 2,2'-DCB alone to 48% in cultures cotreated with 50 microM PD98059 and 100 microM 2,2'-DCB. In contrast, the conventional PRKC inhibitor Gö6976 (10 microM) had no significant effect on 2,2'-DCB-induced inhibition of dye transfer. Western blotting showed about a 4.5-fold increase in phosphorylation of GJA1 at S255, a MAPK1 site, after exposure to 100 microM 2,2'-DCB compared to untreated and solvent controls. However, there was no difference in phosphorylation of GJA1 at S368, a PRKC site. Cells treated with 2,2'-DCB increased phosphorylated MAPK1, implicating the increase of activation of MAPK1. Cotreatment with 100 microM 2,2'-DCB and 5 microM PD98059 reversed 2,2'-DCB-induced modification of uterine contractions and increase of pGJA1(S255) in uterine strips. Therefore, this study suggests that 2,2'-DCB decreases amplitude and synchronization of uterine contractions mediated through MAPK1-mediated phosphorylation of GJA1 and subsequent inhibition of myometrial gap junctions.