Progesterone receptor membrane component 1 as the mediator of the inhibitory effect of progestins on cytokine-induced matrix metalloproteinase 9 activity in vitro

Abstract

Progesterone (P4) and the progestin, 17α-hydroxyprogesterone caproate, are clinically used to prevent preterm births (PTBs); however, their mechanism of action remains unclear. Cytokine-induced matrix metalloproteinase 9 (MMP-9) activity plays a key role in preterm premature rupture of the membranes and PTB. We demonstrated that the primary chorion cells and the HTR8/SVneo cells (cytotrophoblast cell line) do not express the classical progesterone receptor (PGR) but instead a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), whose role remains unclear. Using HTR8/SVneo cells in culture, we further demonstrated that 6 hours pretreatment with medroxyprogesterone acetate (MPA) and dexamethasone (Dex) but not P4 or 17α-hydroxyprogesterone hexanoate significantly attenuated tumor necrosis factor α-induced MMP-9 activity after a 24-hour incubation period. The inhibitory effect of MPA, but not Dex, was attenuated when PGRMC1 expression was successfully reduced by PGRMC1 small interfering RNA. Our findings highlight a possible novel role of PGRMC1 in mediating the effects of MPA and in modulating cytokine-induced MMP-9 activity in cytotrophoblast cells in vitro.

Keywords: inflammation; preterm delivery; progesterone; progesterone receptor.

Figure 1.

A, Western blot demonstrating that PGRMC1 but not PGR (specifically the PR-A and PR-B subtypes) is expressed in primary chorion cells (CHs) and HTR8/Svneo cells (HTR8). Both receptors are expressed in the T47D breast cancer cell line (positive control). B, Immunohistochemistry stain (×10) demonstrating that PGRMC1 is highly expressed in the chorion layer of fetal membranes. C, Negative control slide (×10). Slides were counterstained with hematoxylin and eosin staining (A, amnion; C, chorion; and D, decidua). Experiments were replicated on tissue samples from 3 patients. PGR indicates nuclear progesterone receptor; PGRMC1, progesterone receptor membrane component 1.

Figure 2.

A, A representative zymogram (top) showing the increase in MMP-9 activity following TNF-α stimulation in an unstimulated (vehicle) control in HTR8/SVneo cells. TNF-α increased relative MMP-9 activity when compared with unstimulated controls by 42% (^^P < .001 vs control). The effect of progesterone pretreatment on MMP-9 activity in HTR8/SVneo cells. B, A representative zymogram demonstrating the effects of progesterone and progestin pretreatment on TNF-α induced MMP-9 activity. In addition to the unstimulated (vehicle) control, each progesterone and progestin-treatment group is represented with both the unstimulated and the TNF-α stimulated MMP-9 activity. The relative MMP-9 activity represents activity in excess of baseline for each experimental group. The unstimulated control only is represented graphically. C, MPA pretreatment significantly reduced relative MMP-9 activity, while P4 and 17P were ineffective (*P < .05 vs TNF-α). Experiments were replicated on 5 separate occasions. Data are mean ± standard error of the mean. MMP-9 indicates matrix metalloproteinase 9; MPA, medroxyprogesterone acetate; TNF-α, tumor necrosis factor α.

Figure 3.

A, A representative zymogram showing the effect of dexamethasone pretreatment on TNF-α-induced MMP-9 activity. B, Dexamethasone pretreatment significantly reduced relative MMP-9 activity when compared with the TNF-α-stimulated group (*P < .05 vs TNF-α). The unstimulated control only is represented graphically. Experiments were replicated on 3 separate occasions. Data are mean ± standard error of the mean. MMP-9 indicates matrix metalloproteinase 9; TNF-α, tumor necrosis factor α.

Figure 4.

A, A Western blot demonstrating significant reduction in PGRMC1 expression in HTR8/SVneo cells with PGRMC1-specific siRNA treatment. B, A representative zymogram demonstrating that knocking down PGRMC1 expression reduces the efficacy of MPA pretreatment to inhibit TNF-α-induced MMP-9 activity. Both the unstimulated (vehicle) control and the MPA groups are shown in the zymogram (C). MPA pretreatment significantly reduced TNF-α-induced relative MMP-9 activity in the scramble siRNA group by 45% (P < .008 vs TNF-α) but not in the PGRMC1 siRNA groups. The relative MMP-9 activity represents activity in excess of baseline for each experimental group. The unstimulated (vehicle) control only is represented graphically for both siRNA treatments. Experiments were replicated on 5 separate occasions. Data are mean ± standard error of the mean. MMP-9 indicates matrix metalloproteinase 9; MPA, medroxyprogesterone acetate; PGRMC1, progesterone receptor membrane component 1; siRNA, small interfering RNA; TNF α, tumor necrosis factor α.

Figure 5.

A, A representative zymogram demonstrating that knocking down PGRMC1 expression in the HTR8/SVneo cells did not significantly reduce the efficacy of dexamethasone pretreatment to reduce TNF-α-induced MMP-9 activity. B, Dexamethasone pretreatment significantly reduced TNF-α-induced relative MMP-9 activity in both the scramble (P = .003 vs TNF-α) and the PGRMC1 siRNA groups (P = .005 vs TNF-α). The relative MMP-9 activity represents activity in excess of baseline for each experimental group. Experiments were replicated on 3 separate occasions. Data are mean ± standard error of the mean. MMP-9 indicates matrix metalloproteinase 9; PGRMC1, progesterone receptor membrane component 1; siRNA, small interfering RNA; TNF α, tumor necrosis factor α.