Impact Of PR-M On Mitochondrial Membrane Potential Of Uterine Smooth Muscle Cells

(1) The first affiliated hospital zhengzhou university, Obstetrics and Gynecology, Henan Zhengzhou 450052(2) Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Duke University Medical Center, Durham, N.C., USAProgesterone has a broad range of functions in reproductive biology, including follicular growth, induction of oocyte maturation, ovulation, implantation, and maintenance of pregnancy. In addition, progesterone is a major regulatory factor in the growth of the normal breast and breast cancer. Typically, steroids act via a nuclear receptor to regulate gene transcription. However, progesterone action may result in rapid non-genomic actions in the plasma membrane or cytosol. Many rapid non genomic actions of progesterone/progestin have been identified, including induction of oocyte maturation, modulation of reproductive signaling in the brain, rapid activation of breast cancer cell signaling and induction of the acrosomal reaction. The physiological action of progesterone is mediated via two specific nuclear receptors named PR-B and PR-A. Other putative progesterone receptors have been identified including PR-C, PR-M, membrane PR (mPR) and progesterone receptor membrane component 1(PMRC 1). Transcripts for two other potential receptors, PR-S, PR-T, have been identified but not a specific protein. The function of these non-nuclear receptors continues to be investigated. PR-A and PR-B are traditional nuclear receptors. The structure is characterized by an amino domain with the main transcription activator region (AF-1, AF-3), a ligand-binding domain (LBD), a hinge region(H) and a DNA binding domain (DBD). Typically, PR-A and PR-B are bound to a heat shock protein (HSP) in the cytoplasm. Ligand binding leads to dissociation of the HSP, translocation to the nucleus, receptor dimerization and binding to specific DNA recognition sequences. Subsequent binding of co-activators and co-repressors leads to transcription regulation.Yet, studies also suggest that the nuclear PR (nPR) may function in non-genomic regulation of cell function. As an example, a polyproline SH3 region in amino acid position 421-428, interacts with cytoplasmic tyrosine kinase Src receptors and activates Src by an SH3-domain displacement mechanism. Other PRs mainly localize to the plasma membrane and affect cellular processes via the control of ion fluxes such as calcium or activation of other second messenger systems. There are three receptor candidates for mediating rapid progestin actions:(1) membrane progestin receptors (mPRs) mediate signaling via G-protein coupled pathways (2) progestin receptor membrane components (PGRMCs) have a single transmembrane domain with a putative Src homology domain for potential activation of second messengers (3) nuclear progesterone receptors (nPRs) interacting with cytpoplasmic signaling peptide Src via an N-terminal proline-rich domain to activate Src/Ras/MAPK, PI3 kinase/Akt,JAK2/Stat3 and other signaling pathways.The laboratory of Professor Price has previously cloned a novel PR, termed PR-M, from human adipose and aortic cDNA libraries. The cDNA is approximately 2230 bp. PR-M is a truncated version of the nuclear PRs. Sequence analysis of PR-M suggests a protein of 314 amino acids. Initiation of transcription of PR-M occurs in intron 3, with the initial exon consisting of 16 novel amino terminal amino acids derived from sequence at the end of the 3rd intron. This is followed by sequence identical to exons 4-8 of the nuclear PRs., The unique 16 novel amino-terminus amino acids are consistent with a mitochondrial localization sequence (MLS). Thus, the PR-M isoform lacks both a DNA binding domain and a nuclear localization signal (NLS). Expression of PR-M in Sf9 insect cells shows a 38 kDa protein by western blot analysis. The transcript and protein for PR-M has been identified in multiple cells including human aortic endothelial cells, MCF-10A breast epithelial cells, T47D breast cancer cells and human sperm. Immunofluorescent confocal microscopy of green fluorescent protein (GFP) tagged PR-M expressed in Hela cells localizes PR-M to the mitochondrion.Mitochondria are vital intracellular organelles involved in cellular energy production and programmed cell death. Apoptosis is mediated by two distinct pathways:an extrinsic pathway and an intrinsic pathway. The extrinsic pathway is initiated by the binding of the death receptors (Fas, TNF and TRAIL) to their ligands in the plasma membrane with subsequent activation of the death-inducing signaling complex (DISC) signaling pathways or activation of caspase 8. This results in a loss of mitochondrial transmembrane potential (MTP) and release of cytochrome c into the cytoplasm with activation of the caspase cascade.The key event in regulation of intrinsic apoptosis or the mitochondrial pathway is mitochondrial permeability which is determined by the ratio of pro-apoptotic factors and antiapoptotic members of the Bcl-2 family. The death signal (such as DNA damage) induces a conformational change of the pro-apoptotic BH3 domain proteins which triggers their translocation into the outer mitochondrial membrane (OMM) forming the mitochondrial permeability transition pore (PTP) or voltage-dependent anion channels (VDAC). This results in the rapid loss of MTP increasing mitochondrial membrane permeability, leading to matrix swelling, rupture of the OMM with release of pro-apoptotic substances into the cytosol, principally cytochrome c, smac/DIABLO. Cytochrome c and the apoptotic protease activating factor-1(Apaf-1) ultimately activate the effector caspases (caspase 3 and 7) leading to cell disintegration. Smac/DIABLO neutralize the caspase-inhibitory properties of the inhibitor of apoptosis proteins (IAPs). Both apoptotic pathways lead to definitive cell destruction through affecting the mitochondrial membrane potential.PR-M may play a unique role in the modulation of mitochondrial membrane potential. Protection from apoptosis by PR-M was previously demonstrated by the Price TM group in the benign breast epithelial cell line MCF 10A, known to lack expression of nuclear PR 5, but to express PR-M. Treatment with progesterone or the specific PR agonist, R5020, resulted in a dose-dependent increase in MTP with concordant increase in cellular ATP. The reaction was inhibited by the concomitant addition of the PR antagonist, RTI-6413-049b. Pre-treatment with the translation inhibitor cycloheximide had no effect on the reaction, showing that the progestin-induced increase in MTP is not dependent upon protein synthesis. Treatment with the glucocorticoid Dexamethasone resulted in no increase in MTP, showing that the progestin-induced increase in MTP is not due to cross reactivity with the glucocorticoid receptor.Further experiments demonstrated that the R5020-induced increase in MTP resulted in a decrease in FasL mediated apoptosis with a decrease in Caspase-3 and caspase-7 activity. Additional evidence that PR-M mediates MTP has been obtained with the use of RNA interference (RNAi) studies. PR-M expression was diminished in T47D breast cancer cells by transfection with small interfering RNA (siRNA) duplexes. Progestin-induced increase in MTP was inhibited after siRNA silencing of PR-M expression. Silencing of nuclear PR-B and A expression had no effect on progestin-induced MTP.The above data support a progesterone/progestin mediated increase in mitochondrial membrane potential via PR-M. This increase in MTP corresponds to an increase in cellular ATP consistent with an increase in cellular respiration. Enhanced cellular respiration may improve cell survival under conditions predisposing to apoptosis. The molecular mechanisms of PR-M action remain to be determinedTissue distribution studies show that the abundance of PR-Mcorresponds with the quantity of mitochondrial in a tissue. Thus, high levels of PR-M are found in the heart, liver and myometrium. This study focuses on the function of PR-M in the myometrium. Specifically, the study had three specific aims:1) To compare the expression of PR-M in leiomyomata and adjacent normal myometrium.2) To determine the modulation of mitochondrial membrane potential by progesterone/progestin in isolated human uterine smooth muscle cells and in an immortalized myometrial-cell line (hTert-HM).3) To study the mechanism of mitochondrial membrane hyperpolarization of.hTert-HM cells induced by Progesterone. Aim 1:To compare the expression of PR-M and porin in leiomyomata and adjacent normal myometrium.Methods: 1. Protein isolation and western blot analysis.Human tissue procurement was approved by the Institutional Review Board of Duke University. Tissue from 5 patients undergoing hysterectomy was obtained. Specifically, tissue was obtained from the edge of a leiomyomata (FE) and from adjacent myometrium (MA) with no evidence of abnormality. Western blot analysis was used to investigate the expression of PR-M, PR-A, PR-B and porin protein in leiomyoma and myometrium. Samples were stored at-80℃in OCT prior to processing. Total protein was isolated by homogenization and sonication of tissue in RIPA buffer with protease inhibitors. Total protein concentration was determined by a Bradford reagent using known concentrations of BSA for a standard curve. For western blot analysis,30-70μg protein was separated on a 10% polyacrylamide gel in Tris/Glycine/SDS buffer. Protein was transferred to PDVF membrane in Tris/Glycine buffer. The membrane was blocked with 5% milk for 1 hr at RT, incubated overnight at 4C with either a rabbit polyclonal anti-PR antibody (C19, Santa Cruz Biotechn ology) or a mouse monoclonal anti-PR antibody (MAB 462, Chemicon International). Membranes were stripped and subsequently hybridized with a monoclonal antibody to voltage-dependent anion channel 3 (VDAC-3 or porin) and a monoclonal antibody to GAPDH. After the primary antibody, membranes were hybridized with the appropriate anti-rabbit or anti-mouse peroxidase conjugated secondary antibnody for 1 hr at RT. The membrane was developed with ECL Reagent (Amersham, Buckinghamshire, UK) per manufacture's recommendation and exposed to film. Western blot analysis of protein from the 5 subjects was performed in quadruplicate.2. Semi-quantitative analysis of protein expressionWestern blots were analyzed semi-quantitatively by densitometry performed on a'AlphaImager 2200 scanner (Alpha Innotech). Images were scanned to obtain Integrated Density Value (IDV). The IDV value of PR-M, PR-A, PR-B, porin'was corrected by the IDV value of GAPDH. Results were expressed as a ratio of FE/MA densitometry values. Statistical analysis was performed with SPSS software.Results:1. The expression of PR-M,PR-A and PR-B protein levels in leiomyomas and myometrium.As a group, PR-M protein levels were greater in FE compared to MA (P=0.007). Inspection of the results of each subject showed PR-M protein to be greater in FE compared to MA in 5 subjects. PR-A and PR-B levels tended to be greater in FE but did not reach statistical significance (P=0.153, P=0.052).2. The expression of porin protein in leiomyomas and myometrium.Porin protein levels in FE were higher than in MA (P=0.003). This pattern was seen in all 5 subjects.3. Correlation Analysis for PR-M, PR-A,PR-B and porinSpearman's correlation analysis showed that there was correlation between PR-M and porin (t=0.359,.P=0.023), but there was no correlation between PR-M, PR-A, PR-B and porin (rs=0.068,-0.009, P=0.777,0.970)Aim 2:To determine the modulation of mitochondrial membrane potential by progesterone/progestin in isolated human uterine smooth muscle cells and in an immortalized myometrial cell line (hTert-HM).Methods:1. Western blot was used to investigate the expression of PR-M and porin protein in primary cultured uterine smooth muscle cells and immortalized uterine smooth muscle cells (hTert-HM).Uterine smooth muscle cells for primary culture were obtained from patients undergoing hysterectomy due to leiomyomas at Duke University Medical Center. Myometrium tissue was incubated in hepes balanced salt solution (HBSS, no Mg or Ca) and digested with 0.1% type I collagenase in serum-free culture medium. The cells were cultured in DMEM/F12 medium (10% fetal bovine serum+penicillin+ streptomycin+0.5%Fungizone). Proteins were extracted respectively from cells which were immediately digested after hysterectomy, cells grown for 5 days, cells before passage and after the third passage of cells. Wester blot analysis was performed as described above.Immortalized uterine smooth muscle cells (hTert-HM) were obtained from the lab of Reproductive Endocrinology and Fertility of Duke University. Western blotting was used to investigate the expression of PR-M and porin in hTert-HM cells.2. RT-PCR was used to investigate the expression of PR-M at mRNA levels in hTert-HM cells.Total RNA was isolated from cells using TRIzol reagent. cDNA was generated' by treatment with reverse Transcriptase (RT). PCR was performed.The product of the PCR was analyzed after SDS-PAGE electrophoresis. T47D breast cancer cells were used as a positive control. A negative control was performed in which the cDNA was replaced with water.3. Using JC-1 to determine mitochondrial membrane potential (MMP) of primary cultured uterine smooth muscle cells pretreated with R5020 and P4.Normal uterine smooth muscle cells were cultured in DMEM/F12 medium (10% fetal bovine serum+penicillin+streptomycin+Fungizone). Primary cultured uterine smooth muscle cells (120k/well) were grown for 4 days in 48 well plates in media. Cells at 80% confluence were placed in Krebs-Ringer-HEPES buffer (KRH) containing 25 mM Na-HEPES,115 mM NaCl,5 mM KC1,1 mM KH2PO4,1.2 mM MgSO4,0.5 mM CaCl2 and 5 mM glucose at pH 7.4 at 37℃for 2 hrs and then treated for 60 mins with P4 (10-6,10-8 and 10-10 M). The passage 2 cells (40k/well) from 7 days of primary cultured cells were grown for 3 days in 48 well plates in media, then incubated in KRH for 2 hrs, then treated for 60 mins with R5020 (10-6 and 10-7 M). JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazole-carbocyanide iodine) was used to determine Mitochondrial membrane potential (MMP) after treatment with progesterone/progestin.4. Using JC-1 to determine mitochondrial membrane potential(MMP)after hTert-HM cells were pretreated with R5020,MPA and P4.Cells were cultured in DMEM/F12 medium (10% fetal bovine serum+penicillin +streptomycin). Cells (50K /well) were grown for 24 hours in 48 well plates in media. Cells at 80% confluence were placed in buffer for 2 hrs and then treated with R5020 (10-6,10-8 and 10-10 M), MPA (10-8M) and P4 (10-8M) for 60 mins. JC-1 was used to determine Mitochondrial membrane potential(MMP) after treatment.Results:1. The expression of PR-M, porin andα—SMA protein in primary cultured uterine smooth muscle cellsWestern blot analysis showed that PR-M and porin protein were expressed in each group of cells. The molecular weight of PR-M was 38 kDa while the molecular weight of porin was 31 kDa.2.The expression of PR-M at mRNAin hTert-HM cellsA specific transcript for PR-M was identified by RT-PCR in hTert-HM cells.3. Mitochondrial membrane hyperpolarization after primary cultured uterine smooth muscle cells pretreated with P4 and R5020.There was a dose dependent increase in Mitochondrial membrane hyperpolarization with P4 at concentrations of 10-6,10-8 and 10-10 M in primary cultured uterine smooth muscle cells (rs=-0.781, P＜0.0001), with significant differences compared to control seen at the 10-6 and 10-8 M concentrations(Z=3.838, P=0.000; Z=2.914,P=0.004).Treatment with R5020 (10-6M,10-7M) also induced Mitochondrial membrane hyperpolarization of the cells, but with no significant differences compared to control group (F=1.511,P=0.253)4. Mitochondrial membrane hyperpolarization after hTert-HM cells pretreated with R5020,MPA and P4.Treatment with R5020, MPA and P4 at concentrations of 10-8 induced Mitochondrial membrane hyperpolarization of the cells(P=0.031,0.001,0.203). There was a significant increae in Mitochondrial membrane hyperpolarization with synthetic progesterone R5020 and MPA.Aim 3:To study the mechanism of mitochondrial membrane hyperpolarization of hTert-HM cells induced by Progesterone Methods:1. Using JC-1 to determine mitochondrial membrane potential(MMP) in hTert-HM cells pretreated with the PR antagonist (RTI)hTert-HM Cells were obtained from the lab of Reproductive Endocrinology and Fertility of Duke University. Cells were cultured in DMEM/F12 medium(10% fetal bovine serum+penicillin+streptomycin). Cells (50K/well) were grown 24 hours in 48 well plates in media. Cells at 80% confluence were placed in KRH buffer for 2 hrs and then treated with R5020 (10-8M) with or without the PR antagonist, RTI-6413-049b (10-6 M) for 1 hr. JC-1 was used to determine Mitochondrial membrane potential(MMP) as above.2. Using JC-1 to determine mitochondrial membrane potential(MMP) in hTert-HM cells pretreated with DexamethasoneCells (50K/well) were grown for 24 hours in 48 well plates in media. Cells at 80%confluence were placed in buffer for 2 hrs, then treated for 60min with Dexamethasone (10-8,10-9,10-10 M). JC-1 was used to determine Mitochondrial membrane potential (MMP) as above.3. Using JC-1 to determine mitochondrial membrane potential(MMP) in hTert-HM cells pretreated with Cycloheximide (CHX)Cells (50K/well) were grown for 24 hours in 48 well plates in media. Cells at 80% confluence were placed in KRH buffer with or without CHX (5μg/ml)for 2 hrs, then treated for 60 mins with 10-6 M R5020 with or without CHX (5μg/ml). JC-1 was used to determine Mitochondrial membrane potential (MMP) as above. 4. Inhibition of translation by CHX was shown by western blot analysis of the induction of Id-1 protein in hTert-HM cells by treatment with TgF-β1hTert-HM cells (800K/well) were incubated in KRH buffer with or without CHX (5μg/ml)for 2 hrs, then treated for 60 mins with 5ng/mL TgF-β1. Western blot was employed to investigate the expression of Id-1.Results:1. The change of mitochondrial membrane potential (MMP) after hTert-HM cells were pretreated with the PR antagonist (RTI)MMP was compared in treatment groups of R5020 (10-8 M), R5020+ RTI(10-6 M), RTI (10-6 M) and EtOH control. MMP was highest in the R5020 treatment (P=0.033,0.017,0.005)with no significant difference in group R5020+RTI(10-6 M), RTI (10-6 M) compared to control (P=0.394,0.549). These results demonstrate inhibition of the R5020-induced mitochondrial membrane hyperpolarization by a specific PR antagonist.2. The change of mitochondrial membrane potential (MMP) after hTert-HM cells were pretreated with DexamethasoneThere was no significant difference:in MMP between the treatment groups of Dexamethasone 10-8,10-9,and 10-10 M and EtOH control (F=0.722,P=0.541) Dexamethasone can not induce the hyperpolarization of mitochondrial membrane potential(MMP) of hTert-HM cells. This suggests that progesterone/progestin induction of MMP is not mediated by Glucocorticoid receptors(GRs).3. The change of mitochondrial membrane potential(MMP)after hTert-HM cells were pretreated with CHXTreatment groups included R5020 (10-6 M), R5020 (10-6 M)+CHX (5μg/ml), CHX (5μg/ml) and EtOH control. The results showed that there was a significant difference in the R5020 groups with and without CHX (5μg/ml) when compared to control group (P=0.026,0.006) There was no significant difference in the CHX (5μg/ml) group when compared to the control group (P=0.246). There was no significant difference between group CHX(5μg/ml) and group R5020(10-6M) combination of CHX(5μg/ml) (P=0.600). CHX is an inhibitor of translation. These results suggest that the R5020-induced increase in MMP is not dependent upon protein synthesis.4. The expression of Id-1 after hTert-HM cells were pretreated with CHX and TgF-β1To prove that the concentration of CHX was adequate to block translation, we investigated the TgF-β1 induction of Id-1 expression. hTERT-HM cells were incubated with CHX (5μg/mL) for 2 hrs and then treated with TgFβ1(5ng/mL) for 60 mins. Western blot analysis showed a significant inhibition of Id-1 expression with CHX pretreatment.Statistical analysisStatistical analysis was performed with SPSS 16.0 software. Nomal distribution of data are expressed as错误!不能通过编辑域代码创建对象。±s. Unormal distribution of data are expressed as M±Q. T-test was used to evaluate differences between two matched groups. All data were tested with Test of Normality and Homogeneity of Variance Test. Least significant difference (LSD) method was used to compare two groups and Kruskal-Wallis Rank sum test was used to compare multiple groups. Spearman was used to evaluate correlation between treatment groups. Statistical significance was considered a P≤0.05.Conclusions:1. The mitochondrial progesterone receptor, PR-M, is highly expressed in human myometrial tissue. Expression in leiomyomata is greater than in adjacent myometrium.2. Using primary culture and immortalized uterine smooth muscle cells as a model, progestin induces a significant dose-dependent increase in mitochondrial membrane potential. This reaction is blocked by a PR antagonist and is not seen with a glucocorticoid and cycloheximide, obviating the possible involvement of the glucocorticoid receptor and inhibition of protein biosynthesis.3. With these observations, we propose that progesterone/progestin action via non genomic action of PR-M enhances cellular energy production. This increase in cellular energy supports abnormal cell replication in leiomyomata.