Relaxin-Induced Proliferation of Epithelial and Stromal Cells in the Mouse Cervix Requires Estrogen Receptor-Alpha Within Stromal Cells Binding to Estrogen Response Elements.

Abstract

Relaxin is secreted by the corpora lutea during the second half of pregnancy in mice, rats and pigs. Relaxin stimulates marked growth and extensibility of the lower reproductive tract and thereby facilitates birth in these species. Relaxin-dependent growth of the cervix and vagina is accompanied by increases in both stromal and epithelial cells. Recently, we reported that relaxin receptor-expressing cells in the stroma are necessary and sufficient for relaxin to promote proliferation and inhibit apoptosis in both stromal and epithelial cells of mouse cervix and vagina. Estrogen is secreted in increasing amounts by developing follicles as pregnancy progresses in rats and mice. An understanding of relaxin's growth-promoting actions on the lower reproductive tract in rodents requires knowledge of the cellular and molecular actions of estrogen, because relaxin-induced growth of the cervix and vagina is obligatorily dependent upon estrogen signaling through estrogen receptor alpha (ERα). There were two objectives in this study. The objective of experiment 1 was to determine whether stromal and/or epithelial ERα is required for relaxin to promote proliferation of stromal and epithelial cells in the mouse cervix. Tissue recombinants were prepared with stroma (St) and epithelium (Ep) from wild type (wt) and ERα knockout (ko) mice: wt-St+wt-Ep, wt-St+ko-Ep, ko-St+wt-Ep and ko-St+ko-Ep. Tissue recombinants were grafted under the renal capsule of intact syngeneic female mice. Following 3 weeks of transplant growth, hosts were ovariectomized and fitted with silicon implants containing estradiol-17β (designated day 1 of treatment). Animals were injected sc with relaxin or relaxin vehicle PBS at 6-h intervals from 0600 h on day 8 through 0600 h on day 10 of treatment. To enable morphometric immunohistochemical analysis of cell proliferation, 5-bromo-2'-deoxyuridine (BrdU) was injected sc 10 h before cervices were collected at 1000 h on day 10. Relaxin markedly increased proliferation of epithelial and stromal cells in tissue recombinants containing wt stroma (P< 0.001), but not in tissue recombinants prepared with ko stroma, regardless of whether epithelium was derived from wt or ko mice. The objective of experiment 2 was to determine if ERα enables relaxin's proliferative effects on the cervix by acting through the classical signal transduction pathway in which the estrogen-ERα complex binds to specific estrogen response elements (ERE) in regulatory regions of target genes or through a non-classical pathway in which ERα binding to an ERE is not required. Wild type mice (ERERα+/+), mutant ERα DNA binding domain knock-in (NERKI) mice (ERα+/AA & ERα-/AA) and ERα null mice (ERα-/-) were ovariectomized at 4 weeks of age and treated with estrogen or estrogen plus relaxin as described for experiment 1. Following the administration of relaxin, the rate of cell proliferation increased dramatically in the two groups that contained wt ERα (ERα+/+ and ERα+/AA) but not in groups ERα-/AA and ER-/-. that lack a classical ER signaling pathway. In conclusion, this study demonstrates that estradiol-17β acting through ERα-expressing cells in the stroma, using a classical signaling pathway, is necessary for relaxin to promote marked proliferation in both stromal and epithelial cells of the mouse cervix. (platform)