PGRMC1 as a mediator of the protective effect of Progestins on cytokine induced MMP 9 activity.
Abstract Number: BP-2
Abstract Type: Original Research
Introduction: Elevated levels of TNFα and proteolytic enymes such as matrix metalloproteinase 9 (MMP 9) have been identified in the amniotic fluid of women who develop preterm premature rupture of membranes (PPROM). Cytokine induced MMP 9 activity in the chorion can be partially attenuated by progestin analogues such as medroxyprogesterone acetate (MPA). The mechanism of this protective effect still remains unclear. We hypothesize that this effect may be mediated through a novel membrane associated progesterone receptor: progesterone membrane receptor 1 (PGRMC1). Our objective was to determine if the protective effect of MPA was attenuated when PGRMC1 expression was depleted with PGRMC1 small interfering RNA (siRNA) in a human cytotrophoblast cell line (HTR8/SVneo) known to express the PGRMC1 receptor but not the classical nuclear progesterone receptor.
Methods: HTR8/SVneo cells were transfected with pre-validated scramble control siRNA or PGRMC1 specific siRNA (10 nM final concentration) using Lipofectamine RNAiMAX for 72 h. Cells were then pre-treated with MPA (1µM) or Ethanol (vehicle) for 6 h followed by treatments with and without TNFα 10 ng/ml for an additional 24h. Culture media were harvested for MMP 9 activity assessed using gelatin zymography. In parallel, cell lysates were harvested to confirm PGRMC1 knockdown by western blotting. Image J software was used for densitometry analysis. Experiments were replicated on 3 separate occasions. Data were pooled and expressed as mean ± standard error. The different treatment groups were compared with ANOVA with post hoc Tukey’s test for pairwise comparisons.
Results: Western blot analysis confirmed knockdown of PGRMC1 expression. MPA pretreatment reduced the relative MMP 9 activity in both the control and PGRMC1 siRNA groups in response to TNFα stimulation (figure 1). However in cells depleted of PGRMC1 there was significantly less protection (greater mean relative MMP 9 activity) from MPA pretreatment followed by TNFα compared to controls treated with MPA followed by TNFα (74% vs 40%, p<0.001) (figure 1).
Conclusion: Our results demonstrate that the reduction of cytokine induced MMP 9 activity by progestins may partially be mediated through PGRMC1 and provides some insight into possible therapeutic mechanisms for preventing PPROM.