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Progesterone suppresses an oxytocin-stimulated signal pathway in COS-7 cells transfected with the oxytocin receptor.

ABSTRACT: The present study was conducted to determine if progesterone (P4) would inhibit oxytocin-stimulated phosphoinositide hydrolysis in COS-7 cells expressing transfected ovine oxytocin receptor (OTR) with little or no nuclear P4 receptor (nPR) protein present. The relative absence of nPR in these cells was confirmed by immunocytochemistry and RT-PCR. To investigate the effects of P4 on oxytocin (OT) signaling, cells were transiently transfected with the ovine OTR. Radioreceptor assay for [(3)H]-OT binding confirmed the presence of a high affinity binding site for OT in transfected cells, while treatment with P4 and GTPgammaS (which uncouples the OTR from the heterotrimeric G-protein) increased the K(d) for OT binding slightly. Cells were then assayed for inositol phosphate hydrolysis 48 h post-transfection. Pre-treatment of cells with P4 for 10 min significantly interfered with rapid (20 min) OT-stimulated inositol trisphosphate (IP(3)) production. This inhibition was specific to P4, because pre-treatment of cells with promegestone (R5020), testosterone, mifepristone (RU 486), or cortisol did not decrease OT-stimulated IP(3) levels. By radioreceptor assay for PR, no measurable specific binding of R5020 was observed for either transfected or non-transfected cells. We conclude that P4 can inhibit OTR-mediated phosphoinositide hydrolysis in COS-7 cells that express little or no nPR protein. These data support a role for a non-genomic action for P4 in OTR signaling via some mechanism other than by binding to a membrane progestin receptor in an immortalized, transfected cell.

SUBMITTER: Bishop CV

PROVIDER: S-EPMC2630247 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Progesterone suppresses an oxytocin-stimulated signal pathway in COS-7 cells transfected with the oxytocin receptor.

Bishop Cecily V CV Filtz Theresa T Zhang Yong Y Slayden Ov O Stormshak Fredrick F

Steroids 20080716 14

The present study was conducted to determine if progesterone (P4) would inhibit oxytocin-stimulated phosphoinositide hydrolysis in COS-7 cells expressing transfected ovine oxytocin receptor (OTR) with little or no nuclear P4 receptor (nPR) protein present. The relative absence of nPR in these cells was confirmed by immunocytochemistry and RT-PCR. To investigate the effects of P4 on oxytocin (OT) signaling, cells were transiently transfected with the ovine OTR. Radioreceptor assay for [(3)H]-OT b ...[more]

PMID: 18674552

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Project description:IntroductionBasal phenotype breast cancers (BPBC) are often associated with apparent epithelial to mesenchymal transition (EMT). The role of progesterone (P4) in regulating EMT of BPBC has not been reported.MethodsThe EMT relevant biology was investigated in vitro using human BPBC cell models (MDA-MB468 and MDA-MB231) with P4, PR agonist (RU486), and PR antagonist (R5020) treatments. The essential role of membrane progesterone receptor alpha (mPRalpha) in the P4-regulated EMT was demonstrated by knocking down the endogenous gene and/or stably transfecting exogenous mPRalpha gene in the BPBC cell models.ResultsThe expression of snail and down-stream EMT proteins such as occludin, fibronectin, and E-cadherin was significantly regulated by P4 incubation, which was accompanied by cell morphological reversion from mesenchymal to epithelial phenotypes. In searching for the cell mediator of P4' action in the MDA-MB468 (MB468) cells, it was found that mPRalpha but not the nuclear PR has an essential role in the P4 mediated EMT inhibition. Knocking down the expression of mPRalpha with specific siRNA blocked the P4's effects on expression of the EMT proteins. In another BPBC cell line--MDA-MB231 (MB231), which is mPRalpha negative by Western blotting--P4 treatment did not alter cell proliferation and EMT protein expressions. Introduction of the exogenous mPRalpha cDNA into these cells caused cell proliferation, but not EMT, to become responsive to P4 treatment. In further studies, it was found that activation of the PI3K/Akt pathway is necessary for the P4-induced EMT reversion. To define the potential inter-mediate steps between mPRalpha and PI3K, we demonstrated that mPRalpha, caveolin-1 (Cav-1), and epidermal growth factor receptor (EGFR) are colocalized in the membrane of caveolar vesicle and the P4-repressed EMT in MB468 cells can be blocked by EGFR inhibitor (AG1478) and PI3K inhibitor (wortmannin).ConclusionsOur data suggest that the signaling cascade of P4 induced mesenchymal repression is mediated through mPRalpha and other caveolae bound signaling molecules namely Cav-1, EGFR, and PI3K. This novel finding may have great impact on fully understanding the pathogenesis of BPBC and provide an essential clue for developing a targeted therapeutic strategy for treatment of BPBC.

Dataset Information

Progesterone suppresses an oxytocin-stimulated signal pathway in COS-7 cells transfected with the oxytocin receptor.

Publications

Progesterone suppresses an oxytocin-stimulated signal pathway in COS-7 cells transfected with the oxytocin receptor.

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