Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1alpha and its dual block by ovarian steroids

Abstract

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1alpha (IL-1alpha) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1alpha and repress MMP-1 production by IL-1alpha-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1alpha and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1alpha is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1alpha, thereby exerting an effective control via a "double-block" mechanism.

Figure 1

Collagenase production by endometrial fibroblasts depends on paracrine interactions and is repressed by ovarian steroids. (A) Endometrial epithelial cells (▵) and fibroblasts (▿) were maintained either in monocultures, in mixed cocultures (▪), or in cocultures separated by a permeable membrane. The latter were treated (○, +H) or not (•, −H) by 1 nM 17β-estradiol and 100 nM progesterone. Supernatants were collected daily and replaced by fresh medium. (B) Confluent endometrial fibroblasts were cultured either with fresh medium (lane 2); with medium conditioned by epithelial cells for 24 h (CM, lane 3); with CM that had been preincubated with either 10 μg/ml polyclonal anti-IL-1α antibodies (lane 4), or with a mixture of 10 μg/ml anti-IL-1α, 10 μg/ml anti-TNFα, and 100 μg/ml anti-EGF polyclonal antibodies (lane 5). Collagenase activity was measured in culture supernatants after 24 h. Both A and B are representative of three experiments performed in triplicate on cells obtained by endometrial biopsies from different patients. Values are means ± SEM (n = 3).

Figure 2

MMP-1 expression in endometrial fibroblasts stimulated by rhIL-1α. (A) Confluent fibroblast monolayers were cultured in the presence of 1 ng/ml rhIL-1α, and total cellular RNA was extracted after the indicated intervals. Twenty micrograms of total RNA were subjected to an RNase protection assay using riboprobes generated from MMP-1 and from 36B4 cDNAs. MMP-1 mRNA expression levels were normalized to 36B4 gene expression as loading control. (B) Cumulative collagenase activity was measured at increasing intervals of culture with the indicated effective concentrations of rhIL-1α. Values are means ± SEM (n = 3).

Figure 3

Ovarian steroids suppress MMP-1 production in rhIL-1α- stimulated fibroblasts. Confluent endometrial fibroblasts were cultured either without (lanes 1 and 2) or with (lanes 3 and 4) 1 ng/ml rhIL-1α, and in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 nM 17β-estradiol and 100 nM progesterone. Collagenase activity was measured in culture supernatants after 24 h. Values are representative of four experiments on cells from different patients and are means ± SEM (n = 3).

Figure 4

IL-1α release from endometrial explants during the menstrual cycle and its repression by ovarian steroids. (A) Biopsies were collected from 30 women at the indicated phases of the menstrual cycle, and an average of 9 (±4) groups of 6 explants from each biopsy were cultured for 24 h in the absence of ovarian steroids. Collagenase activity (○) and IL-1α concentration (•) were measured in pooled conditioned media after 24 h. (B) Explants from nonperimenstrual biopsies were cultured in parallel in the absence (−H) or presence (+H) of 1 nM 17β-estradiol and 100 nM progesterone, and IL-1α concentrations were measured in media conditioned during the second day of culture. Values are means of two measurements per condition with <12% variation.