The mitogenic potential of heparin-binding epidermal growth factor in the human endometrium is mediated by the epidermal growth factor receptor and is modulated by tumor necrosis factor-alpha

Abstract

Heparin-binding epidermal growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is implicated in a variety of biological processes, including reproduction. Previous studies describe increased levels of HB-EGF in the human endometrium during the midsecretory stage of the menstrual cycle, suggesting a function for HB-EGF in implantation of the human blastocyst. Here we have investigated the expression and function of the soluble and transmembrane forms of HB-EGF in the human endometrium. We show that the expression of the transmembrane form of HB-EGF in the human endometrium is modulated according to the stage of the menstrual cycle. We present data demonstrating that both the soluble and transmembrane forms of HB-EGF induce DNA synthesis in human endometrial stromal cells. Furthermore, TNFalpha has a cooperative effect on HB-EGF, EGF, TGFalpha, and betacellulin-induced DNA synthesis in stromal cells, suggesting roles for the EGF family and TNFalpha in regeneration and maturation of human endometrium. Induction of DNA synthesis by HB-EGF and its modulation by TNFalpha in endometrial stromal cells are mediated by the EGF receptor and not the HB-EGF receptor ErbB4. Our data suggest key functions for HB-EGF, TNFalpha, and the EGF receptor in endometrial maturation, via autocrine/paracrine and juxtacrine pathways, in preparation for embryo implantation.

Fig. 1

Expression of tm-HB-EGF in the human endometrium. Tissue sections derived from proliferative (A and C) and secretory (B) endometrium were stained with anti tm-HB-EGF antibodies. Control staining was performed with antibodies preincubated with the appropriate control peptide (D). The region adjacent to the lumenal surface is marked by an arrow. Scale bars, 50 μm (in A–C) and 125 μm (D).

Fig. 2

Cultured endometrial stromal cells express both HB-EGF receptors, EGFR and ErbB4, and tm-HB-EGF. Stromal cells were grown on coverslips and stained for tm-HB-EGF (A), EGFR (C), and ErbB4 (D). Control staining was performed with either goat or mouse IgG (B and E, respectively). Scale bars, 10 μm.

Fig. 3

Sol- and HB-EGF-precursor stimulate DNA synthesis in human endometrial stromal cells. Stromal cell lines (n = 15), each derived from a different human endometrium, were cultured with or without 10 ng/ml sol-HB-EGF in serum-free medium, and DNA synthesis assays were performed at passages 1–6 (A). Stromal cells were cultured with or without 10 ng/ml sol-HB-EGF in serum-free medium or in medium supplemented with 5% FCS (n = 7; B). Crude membrane preparations from CHO/vector and CHO/HB-EGF were analyzed by Western blot with antibodies to HB-EGF precursor (C). Stromal cells cultures overlaid with medium, fixed CHO/HB-EGF, or fixed CHO/vector and cultured in serum-free medium (n = 6; D). DNA synthesis was assessed by measurement of the incorporation of [³H]thymidine into the cells. Incorporation of [³H]thymidine into cells in the absence of HB-EGF was taken as 100% (control). Each bar represents the mean ± sem of at least four independent experiments with each individual cell line. *, Significant difference (P < 0.05) compared with the control.

Fig. 4

HB-EGF induces phosphorylation of EGFR, but not ErbB4. Serum-starved endometrial stromal cells were stimulated with either sol-HB-EGF (A) or HB-EGF-precursor (B). Control cells were treated with medium alone. Whole cell lysates were prepared and immunoprecipitated (IP) with either anti-EGFR or anti-ErbB4. Samples were separated by 7.5% SDS-PAGE and subjected to Western blotting (WB) with antiphosphotyrosine antibodies (A and B), anti-EGFR, or anti-ErbB4 antibodies (C). Molecular weight markers are indicated.

Fig. 5

Induction of DNA synthesis in endometrial stromal cells by HB-EGF is mediated by EGFR. DNA synthesis in endometrial stromal cells induced by sol-HB-EGF (10 ng/ml) is suppressed by PD153035 (2 μm; HB-EGF/PD). Each bar represents the mean ± sem of four replicates in each of three independent experiments.

Fig. 6

Cooperativity of TNFα and HB-EGF and EGFR ligands, EGF, TGFα, and BTC, in the induction of DNA synthesis in endometrial stromal cells. Stromal cells were stimulated with 10 ng/ml HB-EGF (HB), EGF, TGFα, and BTC alone (□) or in the presence of 10 ng/ml TNFα (▪). Control cells (ctrl) were cultured with (▪) or without (□) 10 ng/ml TNFα. Incorporation of [³H]thymidine in cells in the absence of the exogenous factors was taken as 100% (ctrl; □). Each bar represents the mean ± sem of four replicates in each of four independent experiments with different cell lines.

Fig. 7

TNFα increases levels of EGFR in endometrial stromal cells. Flow cytometry analysis of stromal cells incubated in the absence (A) or presence (B) of 10 ng/ml TNFα, labeled with mouse anti-EGFR antibodies (▪) or mouse IgG (□). The graphs are representative of data obtained from 11 independent stromal cell lines.