Fig. 4.

Estrogen modulation of MMP-7 secretion and expression. A–C, Conditioned media were obtained from the luminal compartment of estrogen-depleted cells (SFM); estrogen-treated cells (Est); or estrogen-treated cells that were cotreated for 2 d with 10 μM ERα ASO before assays (Est+ASO/ERα). Samples were concentrated by centrifugation, and aliquots containing equal amounts of proteins were separated by gel electrophoresis and immunoblotted with MMP-7 Nab antibody (A), MMP-7 B-2 antibody (B), or MMP–7 F-12 antibody (C). Results of three to six experiments are summarized in the graphs; densitometries of MMP-7 per GAPDH were normalized to an arbitrary unit (A.U.) of 10 of the SFM category in A. a, P < 0.01, compared with SFM and Est+ASO ERα. D, Gelatin zymography of secreted proteins from cervical stromal fibroblasts and hEVECs. Stromal cells (grown in cultures plates) and hEV-ECs (grown on filters) were shifted to SFM for 3 d. Stromal cells were treated with 10 nM 17β-estradiol plus 1 μM progesterone for 2 d (E+P) or 10 nM 17β-estradiol plus 1 μM progesterone for 2 d and then with SFM for additional 24 h[E+P/withdrawal (Wdr) to stimulate secretion of MMPs]. hEVECs were treated with 10 nM 17β-estradiol for 3 d (E). Aliquots of conditioned medium (10–20 μl) containing equal amounts of proteins were assayed by gelatin zymography under nonreducing conditions. Conditioned media of the stromal cells revealed the presence of 92/85- and 65/45-kDa bands, corresponding, respectively, to secreted pro-MMP-9/activated MMP-9 and pro-MMP-2/activated MMP-2. Conditioned media of hEVECs revealed the presence of 29/21-kDa bands, corresponding, respectively, to secreted pro-MMP-7/activated MMP-7. E, Total homogenates of the hEVECs from which conditioned media of D were obtained were fractionated by gel electrophoresis and immunoblotted with the MMP-7 Nab, MMP-7 B-2 or MMP–7 F-12 antibodies. The experiments in D and E were repeated three times with similar trends.