EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells

Abstract

Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

Figure 1.

Secretion of intact full-length extracellular matrix metalloproteinase inducer (EMMPRIN) from human uterine epithelial (HES) cells. Both N- and C-termini of EMMPRIN are detectable in concentrated (75×) but not in unconcentrated (1×) HES conditioned medium (CM) after 24 hours of cell culture. Both HeLa and HES cell lysates (CL) were used as positive controls.

Figure 2.

Steroid hormone and cytokine stimulation of extracellular matrix metalloproteinase inducer (EMMPRIN) release by human uterine epithelial (HES) cells. A, The HES cells were treated with estradiol (0, 10, or 25 nmol/L), progesterone (25 or 100 nmol/L) or estradiol + progesterone (25/25 or 25/100 nmol/L) or B, with interleukin (IL)-1β at 0.5 or 2 ng/mL for 24 hours and then the conditioned medium was concentrated 75-fold and assayed for EMMPRIN by immunoblotting. The HES cell lysates served as the positive control (+); n = 3.

Figure 3.

The human uterine epithelial (HES) cells release extracellular matrix metalloproteinase inducer (EMMPRIN) by microvesicle shedding. The HES cells were serum starved for 24 hours, then received fresh medium which was collected at 0, 8, 12, or 24 hours of cell culture and then ultracentrifuged for microvesicle isolation. Supernatant ([S] 1×) and microvesicle pelleted ([P] 80×) fractions were analyzed by immunoblotting for EMMPRIN (top panel) and integrin β-1 (bottom panel). The EMMPRIN was detected in the microvesicle pellet fraction at 8, 12, and 24 hours but only at 24 hours in the supernatant (soluble) fraction (see circled band). The HES cell lysate was used as a positive control (+) for EMMPRIN.

Figure 4.

The phorbyl 12-myristate-13-acetate (PMA) stimulates shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) containing microvesicles. A, Treatment of human uterine epithelial (HES) cells with PMA for 2 or 4 hours in a dose-dependent manner did not alter EMMPRIN protein expression in HES cell lysates (CL) but did stimulate EMMPRIN shedding into the conditioned medium (CM). B, Treatment of HES cells with 100 ng/mL of PMA increased the shedding of EMMPRIN-containing microvesicles such that EMMPRIN was detected in the supernatant (S) and microvesicle pellet (P) fraction after 2 and 4 hours of treatment. CL+, HES cell lysates; CM+, HES conditioned medium. Both treatments served as positive controls.

Figure 5.

Immunodepletion of extracellular matrix metalloproteinase inducer (EMMPRIN) from human uterine epithelial (HES) cell conditioned medium reduces matrix metalloproteinase (MMP) stimulation in human uterine fibroblast (HUF) cells. A, Immunodepletion of EMMPRIN (75×-ID) from 75-fold concentrated HES cell conditioned medium (75× CM) was monitored by immunoblotting of fractions (1, first round of depletion; 2, second round depletion) and then used for (B) treatment of HUF cells to determine relative fold induction of MMP messenger RNAs (mRNAs). All samples were normalized to control untreated cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. Statistical significance was compared relative to controls (P < .05), n = 4. 1×, unconcentrated HES-conditioned media.

Figure 6.

Interleukin-1α/β increases matrix metalloproteinase (MMP) expression and secretion in human uterine fibroblast (HUF) cells. The HUF cells were treated with 0, 10, 25, or 100 pg/mL of either interleukin (IL)-1α or -1β for 24 hours and MMP messenger RNA (mRNA) levels and protein expression were analyzed by qualitative polymerase chain reaction (q-PCR) and immunoblotting, respectively. A, The IL-1α increased MMP-1 and -3 mRNA levels (top panel) but only MMP-3 protein secretion (bottom panel) at 25 and 100 pg/mL. B, IL-1β increased MMP-1 and -3 mRNA levels and protein secretion in a dose-dependent manner. All samples were normalized to control untreated samples. Statistical significance was compared relative to control values for each gene or protein (P < .05), n = 4. 0 hrC, 0 hour of culture; UT, untreated 24 hour of culture; BSA, vehicle treatment.