mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation

Abstract

The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen. Estrogen acts as an activator of mTOR signaling but its role in vaginal epithelial homeostasis is unknown. We analyzed reproductive tract-specific Rptor or Rictor conditional knockout mice to reveal the role of mTOR signaling in estrogen-dependent vaginal epithelial cell proliferation and differentiation. Loss of Rptor but not Rictor in the vagina resulted in an aberrant proliferation of epithelial cells and failure of keratinized differentiation. As gene expression analysis indicated, several estrogen-mediated genes, including Pgr and Ereg (EGF-like growth factor) were not induced by estrogen in Rptor cKO mouse vagina. Moreover, supplementation of EREG could activate the proliferation and survival of vaginal epithelial cells through YAP1 in the absence of Rptor. Thus, mTORC1 signaling integrates estrogen and growth factor signaling to mediate vaginal epithelial cell proliferation and differentiation, providing new insights into vaginal atrophy treatment for post-menopausal women.

Fig. 1. Estrogen mediates activation of mTORC1 signaling in the vaginal epithelium.

A GSEA enrichment plots of mTORC1 signaling in vaginal biopsies transcriptome from 19 menopausal women with vaginal dryness pre and post 3-month E2 treatment (GSE11622). B Serum E2 level in mice during proestrus (n = 5), estrus (n = 8), metestrus (n = 15), and diestrus (n = 10). Values are expressed as the mean ± SEM. C Cytological assessment of vaginal smears using the crystal violet staining method for estrous cycle determination in 8-wk-old WT mice. n = 5 in each group. Microscopy with magnification ×20. Scale bars: 100 μm (Upper). Representative images of the immunofluorescence staining for Raptor (Middle) and Phospho-S6 (Ser235/236) (lower) in mouse vagina during proestrus, estrus, metestrus, and diestrus. n = 5 in each group. Nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. D, E Representative images of the Raptor (D) or p-S6(Ser235/236) (E) immunofluorescence staining in the vagina of OVX mice with sesame oil (n = 3) and E2 administration (n = 3). The experiments were repeated three times. Nuclei were stained with DAPI. Microscopy with magnification ×20 (Upper) and ×63 (Lower). Scale bars: 75 μm.

Fig. 2. Rptor deficiency disrupts estrous cycle homeostasis while maintaining normal ovarian function.

A Representative images for cytological assessment of vaginal smears of control and Rptor cKO mice using crystal violet staining method for estrous cycle determination. n = 5 mice in each group. Microscopy with magnification ×20. Scale bars: 100 μm. B Morphologic changes in the vaginal mucosa during the mouse estrus cycle by HE staining. n = 5 mice at each stage. Microscopy with magnification ×10. Scale bars: 200 μm. C Estrous cyclings in 8-week-old control (n = 5) and Rptor cKO (n = 7) mice were determined daily by vaginal lavage cytology for 13 days. Values are expressed as the mean ± SEM. D Representative HE-stained ovarian sections of control and Rptor cKO mice at 8~10 weeks of age. n = 5 mice in each group. Microscopy with magnification ×4. Scale bars: 1 mm. E Representative HE-stained ovarian sections of control (n = 8) and Rptor cKO (n = 6) mice at 8~10 weeks of age after superovulation. Microscopy with magnification ×4. Scale bars: 1 mm. F Oocytes were collected from the oviducts of superovulated control (n = 8) and Rptor cKO (n = 6) mice. Microscopy with magnification ×10. Scale bars: 100 μm. G Scatter plot shows the statistics of oocyte number between the two groups of mice in F. Values are expressed as the mean ± SEM. H, I Serum E2 (H) and P4 (I) levels in control and Rptor cKO mice during proestrus, estrus, metestrus, and diestrus. n = 5 (control mice at proestrus stage), n = 8 (control mice at estrus stage), n = 15 (control mice at metestrus stage), n = 10 (control mice at diestrus stage), and n = 9 (Rptor cKO mice). Values are expressed as the mean ± SEM.

Fig. 3. Rptor depletion leads to vaginal atrophy via promoting cell death and inhibiting cell proliferation.

A Representative images of the external vaginal orifice in control (n = 5) and Rptor cKO (n = 5) female mice. B 3D renderings of the female reproductive tract in control (n = 5) and Rptor cKO (n = 5) mice based on MRI T2 weighted images. Scale bars: 5 mm. C Accurate max and min diameter statistics of the vagina were measured based on B. Values are expressed as the mean ± SEM. (D) Representative HE-stained vaginal sections of control and Rptor cKO mice at 8 weeks (left) (n = 5 mice in each group) and postnatal day 28 (right) (n = 3 mice in each group). The experiments were repeated 3 times. Microscopy with magnification ×10. Scale bars: 200 μm. E, F Control and Rptor cKO mice were ovariectomized and rested for 2 weeks. E2 was administrated for 3 consecutive days. Representative images of the PAS (E) or Ki67 (F) staining of the vagina in control and Rptor cKO mice. Nuclei were stained with hematoxylin. n = 3 in each group. The experiments were repeated 3 times. Microscopy with magnification ×20. Scale bars: 50 μm. G Representative images of the TUNEL immunofluorescence staining in the vagina of OVX mice following E2 administration. Nuclei were stained with DAPI. n = 3 in each group. The experiments were repeated 3 times. Microscopy with magnification ×20. Scale bars: 75 μm.

Fig. 4. Rptor depletion impairs the expression of genes involved in the development and differentiation of vaginal epithelium.

A RNAseq analysis was performed using the vagina of OVX WT mice and Rptor cKO mice with or without 3 day-E2 administration. The number of the differentially expressed genes of the above four groups are plotted. B Heatmap of significantly changing genes in the vagina among the above four groups in A belonging to six major classes as indicated. C The GO terms of significantly changed genes in the green frame in B were enriched using the Cytoscape plug-in ClueGO. D GSEA analysis using the GO terms. E Heatmap of the expression level of keratinocyte differentiation-related genes from the significantly changing genes in the green frame in B. F Expression of Krt6a, Krt6b, Krt10, Krt13, Krt16 was quantified using qPCR in the vagina of the mice indicated in A. n = 7 (OVX control mice), n = 5 (OVX Rptor cKO mice), n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2). Values are expressed as the mean ± SEM. G GSEA enrichment analysis of the transcriptome of vaginal biopsies from post-menopausal women with vaginal dryness (n = 4), and post-menopausal controls without vaginal dryness (n = 6) (GSE26761). H GSEA enrichment analysis of the transcriptome of vaginal biopsies from post-menopausal women suffering from vaginal dryness pre and post 3-month E2 therapy (n = 19) (GSE11622).

Fig. 5. Rptor depletion leads to estrogen unresponsiveness of the vagina.

A, B Immunoblotting analysis of PR and ER protein levels in the vagina of control (n = 3) and Rptor cKO (n = 3) mice. β-Actin was used as the loading control. C Representative images of the immunofluorescence staining of PR in the vagina of OVX control and Rptor cKO mice administrated with E2 for 3 consecutive days. Nuclei were stained with DAPI. n = 3 in each group. The experiments were repeated three times. Microscopy with magnification ×20. Scale bars: 75 μm. D Heatmap of the expression level of estrogen-regulated target genes in the vagina of control and Rptor cKO mice in the presence or absence of E2 administration for 3 days. E Estrogen-regulated target genes in D were validated by qPCR. n = 7 (OVX control mice), n = 5 (OVX Rptor cKO mice), n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2). Values are expressed as the mean ± SEM.

Fig. 6. EREG acts as a potential factor for vaginal epithelial cell proliferation and differentiation.

A Heatmap of the expression levels of ErbB signaling pathway signature genes in the vagina of control and Rptor cKO mice in the presence or absence of E2 administration. B qPCR was performed to verify the genes in the frame in A. n = 7 (OVX control mice), n = 5 (OVX Rptor cKO mice), n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2). Values are expressed as the mean ± SEM. C Representative images of the immunofluorescence staining of EREG in the vagina in OVX control and Rptor cKO mice administrated with or without E2. Nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. D OVX control and Rptor cKO mice were administrated with E2 and/or EREG. The vaginas were harvested for PAS staining, Ki67 immunofluorescence staining, and TUNEL staining. In PAS staining, nuclei were stained with hematoxylin. Scale bars: 50 μm. In Ki67 immunofluorescence staining and TUNEL staining, nuclei were stained with DAPI. Microscopy with magnification ×20. Scale bars: 75 μm. E Expression levels of Krt6a, Krt6b, Krt10, Krt13, Krt16 in the vagina of the mice were quantified using qPCR. n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2), n = 4 (OVX Rptor cKO mice treated with E2 and EREG). Values are expressed as the mean ± SEM. F The vaginas as indicated in D were harvested for immunofluorescence staining of YAP1, nuclei were stained with DAPI. n = 6 (OVX control mice treated with E2), n = 5 (OVX Rptor cKO mice treated with E2), n = 4 (OVX Rptor cKO mice treated with E2 and EREG). The experiments were repeated three times. Microscopy with magnification ×20. Scale bars: 75 μm.

Fig. 7. mTORC2 signaling plays no role in the development of vaginal epithelium.

A The breeding strategy used to generate Rictor cKO female mice. B Expression of Rictor was quantified using qPCR in the vagina of control (n = 6) and Rictor cKO (n = 5) mice. C Rictor protein levels in control (n = 4) and Rictor cKO (n = 4) vagina tissues were determined by immunoblotting. β-Actin was used as the loading control. The experiments were repeated two times. D Gross morphology of external vaginal orifice and reproductive tracts of control (n = 5) and Rictor cKO (n = 5) mice. E 3D renderings of the female reproductive tract in control (n = 4) and Rictor cKO (n = 4) mice based on MRI T2 weighted images. Scale bars: 5 mm. F Accurate max and min diameters of the vagina were measured based on E. Values are expressed as the mean ± SEM. G Representative images for cytological assessment of vaginal smears of control and Rictor cKO mice using crystal violet staining method for estrous cycle determination. n = 5 mice in each group. Microscopy with magnification ×20. Scale bars: 100 μm. H Representative HE-stained vaginal sections of OVX control and Rictor cKO mice are shown. Nuclei were stained with hematoxylin. n = 3 mice in each group. The experiments were repeated three times. Microscopy with magnification ×20. Scale bars: 50 μm.

Fig. 8. A possible scheme depicting the role of mTORC1 signaling in estrogen-induced epithelial cell proliferation and differentiation in mouse vagina.

A mTORC1 signaling participates in the proliferation and differentiation of vaginal epithelium by promoting the expression level of PR and EREG-YAP1 in Rptor^fl/fl mice. B Loss of Rptor compromises the estrogen-induced proliferation and differentiation of vaginal epitheliums through down-regulating the expression of PR and EREG.