Murine Female Reproductive Tract Research Articles

Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) elicits multiple cytokines from human cells and has a minor effect on bacterial persistence in the murine female reproductive tract

ABSTRACT Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analyzed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1β, IL-6, macrophage inflammatory protein (MIP)-1, tumor necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10–28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract. One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and affects bacterial persistence in the female reproductive tract.

IL-36γ induces a transient HSV-2 resistant environment that protects against genital disease and pathogenesis

Herpes simplex virus 2 (HSV-2) causes a persistent, lifelong infection that increases risk for sexually transmitted infection acquisition. Both the lack of a vaccine and the need for chronic suppressive therapies to control infection presents the need to further understand immune mechanisms in response to acute HSV-2 infection. The IL-36 cytokines are recently identified members of the IL-1 family and function as inflammatory mediators at epithelial sites. Here, we first used a well-characterized three-dimensional (3-D) human vaginal epithelial cell (VEC) model to understand the role of IL-36γ in the context of HSV-2 infection. In 3-D VEC, IL-36γ is induced by HSV-2 infection, and pretreatment with exogenous IL-36γ significantly reduced HSV-2 replication. To assess the impact of IL-36γ treatment on HSV-2 disease pathogenesis, we employed a lethal genital infection model. We showed that IL-36γ treatment in mice prior to lethal intravaginal challenge significantly limited vaginal viral replication, delayed disease onset, decreased disease severity, and significantly increased survival. We demonstrated that IL-36γ treatment transiently induced pro-inflammatory cytokines, chemokines, and antimicrobial peptides in murine lower female reproductive tract (FRT) tissue and vaginal lavages. Induction of the chemokines CCL20 and KC in IL-36γ treated mice also corresponded with increased polymorphonuclear (PMN) leukocyte infiltration observed in vaginal smears. Altogether, these studies demonstrate that IL-36γ drives the transient production of immune mediators and promotes PMN recruitment in the vaginal microenvironment that increases resistance to HSV-2 infection and disease. Our data indicate that IL-36γ may participate as a key player in host defense mechanisms against invading pathogens in the FRT.

Circulating memory is sufficient for protection against Chlamydia muridarum in the murine female reproductive tract

Abstract Chlamydia trachomatis is the most prevalent bacterial STI worldwide and yet no vaccine currently exists to protect the public from this reproductive tract pathogen. Recent studies show that Tissue Resident Memory T cells (Trm) are essential for vaccine-induced protection against Chlamydia trachomatis in mice. Moreover, Trm have been implicated in protective immunity to other reproductive tract pathogens including Herpes Simplex Virus. Here, we examine the requirement of Trm T cells in protection against the murine pathogen Chlamydia muridarum (Cm), commonly used to model human Chlamydia infection. Using parabiosis to test whether circulating memory from mice immune to Cm infection can protect naïve mice from reproductive tract challenge with Cm, we found that naïve mice receiving circulating immunity were fully protected from Cm challenge. Ongoing studies with MHC class-II tetramers will examine the phenotype of protective circulating Chlamydia-specific CD4 memory T cells and whether migration to the reproductive tract is a requirement for protection against secondary challenge.

ZIKA virus exhibits tropism to the ovary and increases follicular apoptosis in a mouse model

An adapted strain of Zika virus (ZIKV) has been shown to negatively impact male reproductive tissues in a mouse model, specifically the epididymis and testes.1 We were interested in potential effects of ZIKV in the murine female reproductive tract, specifically the ovary, which is resistant to damage by most viral pathogens.

Estrogen Receptor α Antagonists Mediate Changes in CCL20 and CXCL1 Secretions in the Murine Female Reproductive Tract

Estradiol regulates chemokine secretion from uterine epithelial cells, but little is known about estradiol regulation in vivo or the role of estrogen receptors (ERs). CCL20 and CXCL1 present in reproductive washes following treatment with selective estrogen receptor modulators (SERMs) were compared with that during estrous and following estradiol-treated ovariectomized BALB/c mice. Cellular regulation was determined using isolated vaginal and uterine epithelial/stromal cells in vitro. Uterine and vaginal chemokine secretion is cyclically regulated with CCL20 at low levels but CXCL1 at high levels during high estradiol, generally mimicking estradiol effect in vivo. ERα but not ERβ regulated CCL20/CXCL1 secretion by uterine epithelial cells in vitro and vaginal CCL20 in vivo. Estradiol/SERMs failed to alter uterine CCL20 secretion in ovariectomized mice. Diminished uterine epithelial ERα staining following ovariectomy corresponded with estradiol unresponsiveness of uterine tissue. Estrogen receptors α regulates CCL20/CXCL1 secretion in the female reproductive tract, and ERα antagonists directly oppose the regulation by estradiol. Understanding ER-mediated antimicrobial chemokine expression is important to elucidate cyclic susceptibility to sexually transmitted pathogens.

Human seminal plasma fosters CD4(+) regulatory T-cell phenotype and transforming growth factor-β1 expression.

Semen mediates expansion of CD4(+) regulatory T cells (Treg) in the murine female reproductive tract. The impact of semen on Treg in humans, however, remains unclear. Using seminal plasma (SP) from 20 healthy donors, we investigated the impact of human semen on CD4(+) T-cell expression of CD127 and CD49d as well as CD4(+) CD127(low) CD49d(low) Treg proliferation, apoptosis, and intracellular expression of FoxP3, TGF-β1, and IL-10. SP reduced CD4(+) T-cell expression of CD127 and CD49d and increased the proportion of CD127(low) CD49d(low) Treg. This increase was non-proliferative, mediated in part via the conversion of CD4(+) helper T cells into FoxP3(-) but not FoxP3(+) Treg. Additionally, SP induced an increase in intracellular expression of the immunosuppressive cytokine TGF-β1 by the FoxP3(-) but not FoxP3(+) Treg. SP had no impact on Treg intracellular expression of IL-10. Human seminal plasma fosters the non-proliferative increase in the proportion and immunoregulatory activity of FoxP3(-) Treg.

Estradiol and selective estrogen receptor modulators (SERMs) regulate CCL20 and CXCL1 secretion in the murine female reproductive tract

Estradiol and selective estrogen receptor modulators (SERMs) regulate CCL20 and CXCL1 secretion in the murine female reproductive tract

Changes in innate immune parameters in the murine female reproductive tract throughout the estrous cycle

Changes in innate immune parameters in the murine female reproductive tract throughout the estrous cycle

Estradiol and selective estrogen receptor modulators (SERMs) alter antimicrobial production in the murine female reproductive tract in vivo

Temporal dynamics of the gut microbiota potentially limit the identification of microbial features associated with health status. Here, we used whole-genome metagenomic and 16S rRNA gene sequencing to characterize the intra- and inter-individual variations of gut microbiota composition and functional potential of a disease-free Swedish population (n = 75) over one year. We found that 23% of the total compositional variance was explained by intra-individual variation. The degree of intra-individual compositional variability was negatively associated with the abundance of Faecalibacterium prausnitzii (a butyrate producer) and two Bifidobacterium species. By contrast, the abundance of facultative anaerobes and aerotolerant bacteria such as Escherichia coli and Lactobacillus acidophilus varied extensively, independent of compositional stability. The contribution of intra-individual variance to the total variance was greater for functional pathways than for microbial species. Thus, reliable quantification of microbial features requires repeated samples to address the issue of intra-individual variations of the gut microbiota.

Prenatal exposure to bisphenol a at environmentally relevant doses adversely affects the murine female reproductive tract later in life.

BackgroundExposure to endocrine-disrupting chemicals during critical developmental periods causes adverse consequences later in life; an example is prenatal exposure to the pharmaceutical diethylstilbestrol (DES). Bisphenol A (BPA), an environmental estrogen used in the synthesis of plastics, is of concern because its chemical structure resembles that of DES, and it is a “high-volume production” chemical with widespread human exposure.ObjectivesIn this study we investigated whether prenatal BPA causes long-term adverse effects in female reproductive tissues in an experimental animal model previously shown useful in studying effects of prenatal DES.MethodsTimed pregnant CD-1 mice were treated on days 9–16 of gestation with BPA (0.1, 1, 10, 100, or 1,000 μg/kg/day). After delivery, pups were held for 18 months; reproductive tissues were then evaluated.ResultsOvarian cysts were significantly increased in the 1-μg/kg BPA group; ovarian cyst-adenomas were seen in the other three BPA-treated groups but not in corn-oil controls. We observed increased progressive proliferative lesions of the oviduct after BPA treatment, similar to those described in response to DES. Further, although not statistically different from the controls, prominent mesonephric (Wolffian) remnants and squamous metaplasia of the uterus, as well as vaginal adenosis, were present in BPA-treated mice, similar to lesions reported following DES treatment. More severe pathologies observed in some BPA-treated animals included atypical hyperplasia and stromal polyps of the uterus; sarcoma of the uterine cervix; and mammary adenocarcinoma. We did not observe these lesions in controls.ConclusionsThese data suggest that BPA causes long-term adverse reproductive and carcinogenic effects if exposure occurs during critical periods of differentiation.

Regulation of surfactant protein D in the mouse female reproductive tract in vivo

Surfactant protein D (SP-D) plays a role in innate immunity in the lung and is expressed at many other mucosal surfaces throughout the human body. In this study, we show that SP-D mRNA and protein are present in the murine female reproductive tract; i.e. in the vagina, cervix, uterus and oviduct. SP-D protein is primarily localized to epithelial cells lining the genital tract and is also present in secretory material within the lumen of the uterus and cervix. The levels of SP-D mRNA in the uterus vary by a factor of 10 during the estrous cycle with peak levels present at estrus and the lowest levels at diestrus. In contrast, SP-D mRNA levels in the lung do not change during the estrous cycle. Since SP-D is an innate host defense protein present in the mouse reproductive tract, we studied the influence of infection on SP-D levels in vivo. We found that Chlamydia muridarum infection caused an increase in the SP-D protein content of reproductive tract epithelial cells. These data are suggestive that SP-D may play a role in innate immunity in the female reproductive tract in vivo.

Prostaglandin E2 modulates dendritic cell function during chlamydial genital infection

Inflammatory responses mediated by antigen-presenting dendritic cells (DCs), can be modulated by the presence of prostaglandins (PG), including prostaglandin E2 (PGE2). PGE2 modifies the production of an immune response by altering DC function through PGE2 receptors. PGE2 is produced by epithelial cells lining the murine female reproductive tract during Chlamydia muridarum infection and likely manipulates the antichlamydial immune response during antigen uptake in the genital mucosa. Our data demonstrate that the PGE2 present locally in the genital tract upon chlamydial genital infection enhanced the recruitment of CD11b+ conventional DCs, but not CD45R+ plasmacytoid DCs, to infected genital tract tissue and draining lymph nodes in vivo. Furthermore, exposure to PGE2 in vitro during infection of murine bone-marrow-derived conventional DCs (cDCs) boosted interleukin-10 mRNA and protein while not influencing interleukin-12p40 production. Infection of cDCs markedly increased mRNA production of the costimulatory molecules CD86, CD40 and a member of the C-type lectin family, DEC-205, but addition of PGE2 increased other costimulatory molecules and C-type lectins. Also, exposure of PGE2 to infected cDCs increased FcgammaRIII and FcgammaRIIb, suggesting that PGE2 enhances the uptake and presentation of C. muridarum and augments production of the antichlamydial adaptive immune response. Taken together, the data suggest that exposure of infected cDCs to PGE2 drives production of a diverse adaptive immune response with implications for regulating tissue inflammation.

Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract

The developing fetus is uniquely sensitive to perturbation by chemicals with hormone-like activity. The adverse effects of prenatal diethylstilbestrol (DES) exposure are a classic example. Since concern has been mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical with estrogenic activity used in the synthesis of plastics, we investigated its long-term effects in an experimental animal model that was previously shown useful in studying the adverse effects of developmental exposure to DES. Outbred female CD-1 mice were treated on days 1–5 with subcutaneous injections of BPA (10, 100 or 1000 μg/kg/day) dissolved in corn oil or corn oil alone (Control). At 18 months, ovaries and reproductive tract tissues were examined. There was a statistically significant increase in cystic ovaries and cystic endometrial hyperplasia (CEH) in the BPA-100 group as compared to Controls. Progressive proliferative lesion (PPL) of the oviduct and cystic mesonephric (Wolffian) duct remnants were also seen in all of the BPA groups. More severe pathologies of the uterus following neonatal BPA treatment included adenomyosis, leiomyomas, atypical hyperplasia, and stromal polyps. These data suggest that BPA causes long-term adverse effects if exposure occurs during critical periods of differentiation.

Estrogen receptor expression and neuronal nitric oxide synthase in the clitoris and prepucial gland structures of mice

Purpose ERα is the predominant form of estrogen receptor expressed in uterine and vaginal tissue of the murine female reproductive tract. Expression of both isoforms has been detected in vaginal tissue and in the ovary. Estrogen receptors were seen in basal and suprabasal cells of vaginal epithelium and epidermis of labia minora Understanding the locations of the different isoforms of the ER within genital structures may elucidate mechanisms of genital growth and development. It is well known that the sensory condition of the vulva epithelium is highly influenced by estrogen status with enlargement of the genitosensory field in response to this hormone. Estrogens may also prove crucial to the maintenance of urogential sensitivity. Their ability to promote the growth of neurons has been established. Abundant literature has reported on nitric oxide in the context of the internal female reproductive tract and penis, few studies have investigated the female external genital structure or the clitoris. The aim of this work is to study the presence of estrogen receptors (ER), as well as n-NOS in the mouse clitoris. Materials and methods Immunocytochemical studies specific for Estrogen Receptor-α, Estrogen Receptor-β, and nNOS were performed on a series of sections of the pelvic area including the prepucial glands and clitoris of ten mice. Selected sections were also stained with Masson's trichrome. Results ERα was expressed predominantly by the glandular epithelium as well as in the glandular tissue, preputial and apocrine glands. ERα was observed in the nuclei of stromal cells around the cavernous tissue and near the epithelium of the clitoris. ERβ immunostaining was only detected in few cells located in the vascular lumen of the cavernous tissue of the clitoris. nNOS immunostaining was detected in the clitoris, the preputial glans and the connective tissue. Conclusions ERα, ERβ isoforms and neuronal NOS are present in the clitoris and prepucial glands of female mice in different cellular localizations and with differing levels of receptivity. Functional studies would further elucidate the role of receptor functions and their relationship to neuronal expression of nitric oxide.

Wnt7a is a suppressor of cell death in the female reproductive tract and is required for postnatal and estrogen-mediated growth.

The murine female reproductive tract is undifferentiated at birth and undergoes pronounced growth and cytodifferentiation during postnatal life. Postnatal reproductive tract development proceeds in the absence of high levels of circulating estrogens and is disrupted by precocious exposure to estrogens. The WNT gene family is critical in guiding the epithelial-mesenchymal interactions that direct postnatal uterine development. We have previously described a role for Wnt7a in controlling morphogenesis in the uterus. In addition to patterning defects, Wnt7a mutant uteri are atrophic in adults and do not show robust postnatal growth. In the present study, we examine immature female Wnt7a mutant and wild-type uteri to assess the cellular processes that underlie this failure in postnatal uterine growth. Levels of proliferation are higher in wild-type versus Wnt7a mutant uteri. Exposure to the potent estrogen-agonist diethylstilbestrol (DES) leads to an increase in cell proliferation in the uterus in wild-type as well as in mutant uteri, indicating that Wnt7a is not required in mediating cell proliferation. In contrast, we observe that Wnt7a mutant uteri display high levels of cell death in response to DES, whereas wild-type uteri display almost no cell death, revealing that Wnt7a plays a key role as a cell death suppressor. The expression pattern of other key regulatory genes that guide uterine development, including estrogen receptor (alpha), Hox, and other WNT genes, reveals either abnormal spatial distribution of transcripts or abnormal regulation in response to DES exposure. Taken together, the results of the present study demonstrate that Wnt7a coordinates a variety of cell and developmental pathways that guide postnatal uterine growth and hormonal responses and that disruption of these pathways leads to aberrant cell death.

Distribution and regulation of ENaC subunit and CFTR mRNA expression in murine female reproductive tract.

The present study investigated the regional distribution and cyclic changes in the mRNA expression of epithelial Na+ channel (ENaC) subunit and cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in adult female mouse reproductive tract. In situ hybridization revealed that in contrast to the abundant expression of CFTR, ENaC (alpha, beta, gamma) mRNA signal was not detected throughout the estrus cycle in the ovary and oviduct. Messenger RNA for all ENaC subunits was abundantly detected in the cervical and vaginal epithelia throughout the estrus cycle but for CFTR, mRNA was found only at proestrus. In the uterine epithelium, alphaENaC mRNA was detected at diestrus but not found at any other stage, while CFTR mRNA was only detected at early estrus but not other stages. Semi-quantitative RT-PCR detected mRNA for all ENaC subunits in the uterus throughout the cycle with maximal expression at diestrus and CFTR mRNA was only found in the early stages of the cycle. The involvement of ENaC and CFTR in Na+ absorption and Cl- secretion was demonstrated in cultured endometrial epithelia using the short-circuit current technique and found to be influenced by ovarian hormones. Taken together, these data indicate a main secretory role of the ovary and oviduct and a predominantly absorptive role of the cervix and vagina. The present results also suggest an ability of the uterus to secrete and absorb at different stages of the estrus cycle. Variations in the fluid profiles may be dictated by the regional and cyclic variations in expression of ENaC and CFTR and are likely to contribute to various reproductive events in different regions of the female reproductive tract.

Murine female reproductive tract intraepithelial lymphocytes display selection characteristics distinct from both peripheral and other mucosal T cells

Despite immense effort, the development of vaccines effective at mucosal sites has proceeded at a faltering pace. Efforts concentrating on humoral immunity but neglecting cellular immunity may be misdirected by ignoring many viral mucosal pathogens. Improved understanding of the development and maintenance of lymphocytes populating the reproductive tract (rtIELs) may inform advances in vaccination strategies for sexually transmitted diseases. Recent studies highlight tissue-specific differences in the development of mucosal immunity and suggest that the local milieu may play a role in selection, maintenance and function of resident lymphocytes. Here, we describe MHC class I and thymus dependence of subpopulations of rtIELs. TCRαβ+ CD8αβ+ T cells in the periphery, intestine, and female reproductive tract are all developmentally dependent on classical class I MHC and the thymus. TCRαβ+ CD8αα+ are absent from the periphery and the rtIELs, but are present and classical MHC class I-independent, in the intestine. In contrast to intestinal TCRγδ+ cells, TCRγδ+ rtIELs are CD8 negative and thymus dependent. In contrast to peripheral TCRγδ+ cells, murine TCRγδ+ rtIELs express not a diverse array of Vδ genes, but rather, a canonical Vδ1. In summary, lymphocytes isolated from the murine female reproductive tract have characteristics distinct from both peripheral T cells and those found at other mucosal sites. Therefore, for the purpose of vaccination strategies, the female reproductive tract should be regarded neither as peripheral nor mucosal, but rather as a tissue with distinctive immunological characteristics.

Wnt-7a maintains appropriate uterine patterning during the development of the mouse female reproductive tract

The murine female reproductive tract differentiates along the anteroposterior axis during postnatal development. This process is marked by the emergence of distinct cell types in the oviduct, uterus, cervix and vagina and is dependent upon specific mesenchymal-epithelial interactions as demonstrated by earlier heterografting experiments. Members of the Wnt family of signaling molecules have been recently identified in this system and an early functional role in reproductive tract development has been demonstrated. Mice were generated using ES-mediated homologous recombination for the Wnt-7a gene (Parr, B. A. and McMahon, A. P. (1995) Nature 374, 350-353). Since Wnt-7a is expressed in the female reproductive tract, we examined the developmental consequences of lack of Wnt-7a in the female reproductive tract. We observe that the oviduct lacks a clear demarcation from the anterior uterus, and acquires several cellular and molecular characteristics of the uterine horn. The uterus acquires cellular and molecular characteristics that represent an intermediate state between normal uterus and vagina. Normal vaginas have stratified epithelium and normal uteri have simple columnar epithelium, however, mutant uteri have stratified epithelium. Additionally, Wnt-7a mutant uteri do not form glands. The changes observed in the oviduct and uterus are accompanied by a postnatal loss of hoxa-10 and hoxa-11 expression, revealing that Wnt-7a is not required for early hoxa gene expression, but is required for maintenance of expression. These clustered hox genes have been shown to play a role in anteroposterior patterning in the female reproductive tract. In addition to this global posterior shift in the female reproductive tract, we note that the uterine smooth muscle is disorganized, indicating development along the radial axis is affected. Changes in the boundaries and levels of other Wnt genes are detectable at birth, prior to changes in morphologies. These results suggest that a mechanism whereby Wnt-7a signaling from the epithelium maintains the molecular and morphological boundaries of distinct cellular populations along the anteroposterior and radial axes of the female reproductive tract.

Differential expression patterns of Wnt genes in the murine female reproductive tract during development and the estrous cycle

The murine female reproductive tract differentiates during postnatal development. This process of cytodifferentiation and morphogenesis is dependent upon specific mesenchymal–epithelial interactions as well as circulating steroid hormones (Cunha, G.R., 1976. Int. Rev. Cytol. 47, 137–194; Pavlova, A. et al., 1994. Development 120, 335–346). Members of the Wnt family of signaling molecules have been recently identified in this system (Pavlova, A. et al., 1994. Development 120, 335–346; Bui, T.D. et al., 1997. Br. J. Cancer 75, 1131–1136; Miller, C., Sassoon, D.A., 1998. Development 125, 3201–3211). We describe the expression patterns of Wnt genes in the developing and adult female reproductive tract. Additionally, we note that changes in the levels of expression occur during the estrous cycle. Wnt gene expression patterns are regulated by the presence of epithelium in tissue graft experiments, suggesting that Wnt genes may indeed play roles in the mesenchymal–epithelial interactions critical for female reproductive tract development and function.

Cross talk between peptide growth factor and estrogen receptor signaling systems.

Epidermal growth factor reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estrogen-induced growth and differentiation. The mechanism by which the actions of estrogens and epidermal growth factor (EGF) converge is unknown. The studies described herein were performed to investigate the possibility that some of the actions of EGF may be mediated through the estrogen receptor. A specific estrogen receptor (ER) antagonist inhibited estrogenlike effects of EGF in the mouse uterus, specifically induction of DNA synthesis and phosphatidylinositol turnover. In addition, EGF elicited enhanced nuclear localization of uterine ER and formation of a unique nuclear form of ER that is present after estrogen treatment. These in vivo observations indicated that EGF may elicit some of its actions by activation of nuclear ER. Thus, the effect of peptide growth factors on activation of a consensus estrogen response element was assessed in Ishikawa human endometrial adenocarcinoma cells, which contain negligible ER levels, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ER. EGF and TGF alpha induced transcriptional activation of a consensus estrogen response element (ERE) in an ER-dependent manner in both cell types. In addition, insulinlike growth factor I (IGF-I) was as potent as 17 beta-estradiol in BG-1 cells. Synergism between growth factors and estrogen was observed in both cell types, although synergism was not observed between the different classes of growth factors [i.e., transforming growth factor alpha (TGF alpha) and IGF-I] in BG-1 cells. The most potent activator of ERE-dependent transcription was a protein kinase C activator (TPA), which acted synergistically with 17 beta-estradiol. A protein kinase C inhibitor abolished the effect of TPA but not that of 17 beta-estradiol, IGF-I, or TGF alpha. A protein kinase A activator elicited ER-dependent activation of transcription and did not synergize with estrogen or growth factors. In conclusion, some physiologic actions of peptide growth factors are dependent on ER. Indeed, growth factors are capable of eliciting ER-dependent activation of an ERE. Both the protein kinase A and protein kinase C pathways can elicit ER-dependent transcriptional activation; however, it is unlikely that these pathways mediate the effects of peptide growth factors on the ER in BG-1 cells.