摘要
孕激素和多种生殖过程的调节有关,如：卵泡的生长、卵母细胞成熟、排卵、种植及妊娠的维持等,也是正常乳腺和乳腺癌的重要调节因子。传统的观念认为甾体激素通常通过其核受体发挥其基因转录的功能,然而,孕激素也可以在细胞核外的浆膜和胞浆发挥其快速的非基因组作用。已经发现孕激素有多种快速的非基因组调节功能,涉及到卵母细胞成熟的诱导、大脑生殖信号的调节、乳腺癌细胞信号的快速激活、顶体反应的诱导等。孕激素通过与孕激素受体(progesterone receptor, PR)结合发挥其生理作用。分布在胞核的PR主要有两种：PR-A和PR-B。近些年来发现了一些新的PR受体类型,包括PR-C、PR-M、膜PR (membrane progestin receptor, mPR)和孕激素受体膜元件1(progesteronereceptor membrane component 1, PGRMC1)。还有另外两个转录本PR-S、PR-T。这些非核受体的功能正处于研究之中。传统的核受体PR-A和PR-B的结构中含有与核受体转录功能相关的重要部分,包括配体结合结构域(Ligand-binding domain, LBD)、铰链区(hinge region,H、DNA结合结构域(DNA binding domain,DBD)和转录激活功能(activation function, AF)区域AF-1、AF-3。PR-A和PR-B在胞浆与热休克蛋白结合,和配体结合后与热休克蛋白分离,形成二聚体进入胞核与核内特定DNA上的类固醇激素反应元件结合,启动并调控相关基因的转录。
研究发现核孕激素受体(nuclear progestin receptorsn, nPRs)也可以发挥非基因组调节的功能。例如其N末端可变区的421—428个氨基酸含有一个多聚脯氨酸SH3结构域的反应基序,该基序可以以SH3结构域替代机制直接调节nPR与非受体酪氨酸激酶Src的SH3结构域的结合,激活Src信号途径。其它的PR主要分布在细胞核外的浆膜,通过调节离子如钙的流动或激活第二信使影响细胞内的过程。发挥孕激素快速作用的受体主要有三种：(1)mPRs;通过G蛋白耦联受体信号途径发挥作用。(2)PGRMCs;通过其跨膜结构中的Src同源结构域激活第二信使(cAMP)。(3)nPRs;通过PR-B氨基末端富含脯氨酸的结构域与Src的SH3结合激活Src/Ras/MAPK、PI3 kinase/Akt和JAK2/Stat3等信号途径。
Thomas M. Price实验室从人主动脉和脂肪组织的cDNA文库中克隆到一个新的孕激素受体,命名为PR-M。cDNA长约2230 bp。PR-M是一个截短的PR,含有314个氨基酸,转录起始于npr的外显子4起始部的内含子3,其蛋白质结构的氨基末端包含有一个16个aa的独特的线粒体定位序列(mitochondrial localization sequence，MLS)。经测序鉴定这16个aa与nPR的外显子4～8相连,但是这个PR的亚型缺乏DNA结合结构域和核定位序列(nuclear location signal,NLS)。Westernblot分析PR-M在Sf9昆虫细胞中显示表达为一个38kDa的蛋白。在人主动脉上皮细胞、MCF-10A乳腺上皮细胞、T47D乳腺癌细胞和人的精子等多种细胞中均发现有PR-M的转录本和蛋白。采用激光共聚焦显微镜发现标记PR-M的绿色荧光蛋白(green fluorescen protein,GFP)存在于Hela细胞的线粒体。
线粒体是细胞内必不可少的细胞器,与细胞内能量的产生和细胞的程序性死亡(PCD, programmed cell death) (即凋亡)有关。诱导细胞凋亡的途径主要有两种：外在途径和内在途径。死亡受体(Fas, TNF and TRAIL)和浆膜上的配体结合后形成死亡诱导信号复合物(death-inducing signalling complex, DISC)或激活caspase8启动外在途径,使线粒体膜势能(mitochondrial transmembrane potential, MTP)减弱,释放细胞色素C进入胞浆,激活caspase级联反应。
内在途径即线粒体凋亡途径,线粒体的渗透性为内在性凋亡调节的重要环节,它取决于Bc1-2家族促凋亡和抗凋亡因子之间的比率。死亡信号(如DNA损伤)诱导促凋亡蛋白BH3结构域发生结构上的改变,然后转移到线粒体外膜(outermitochondrial membrane, OMM),形成线粒体通透性转换孔(permeability transition pore, PTP)或电压依赖性阴离子通道(voltage-dependent anion channels,VDAC),导致MTP的快速丢失,膜渗透性增加,基质水肿,OMM破裂并释放细胞色素C、Smac/DIABLO等促凋亡物质释放入胞浆,通过激活Apaf-1,最终激活caspase-9、caspase-3效应因子,导致细胞的碎裂；同时解除了IAPs对凋亡的抑制。两种途径均通过影响线粒体膜电位(mitochondrial membrane potential, MMP)而发挥作用。
PR-M可能在调节线粒体膜电势方面发挥其独特的作用,这种作用已经在(?)Price TM小组对良性乳腺癌细胞系MCF10A一系列的实验中显示出来。MCF10A是一种缺乏孕激素核受体表达但却有PR-M表达的乳腺非肿瘤上皮细胞。用孕激素(P4)或特异的孕激素受体激动剂R5020处理MCF10A细胞后,细胞MMP呈计量依赖性增加,与细胞内ATP的增加一致,应用孕激素受体拮抗剂RTI-6413-049b可以抑制孕激素诱导的线粒体超极化反应。实验显示孕激素诱导的MMP超极化与地塞米松和放线菌酮Cycloheximide (CHX)的作用无关,表明孕激素诱导的线粒体膜电位的增加并非糖皮质激素受体(Glucocorticoid receptors, GRs)的交叉反应,而且不依赖于蛋白质的合成。
进一步实验证明R5020可以抑制FasL诱导的MCF10A细胞的凋亡,同时伴随有caspase-3、caspase-7活性的下调。另外,采用RNAi技术对PR-M的调节作用进行研究发现,敲除T47D乳腺癌细胞中的PR-M后,PR-M的表达减弱,孕激素诱导线粒体膜电位增加的作用减弱；但敲除PR-A和PR-B后却没有这种变化。
上述结果表明,孕激素可能通过PR-M增加了细胞线粒体的膜电位及细胞内ATP,从而增加了细胞内呼吸。孕激素可能通过增加细胞呼吸促进了即将凋亡细胞的生长,抑制了线粒体调节的凋亡。但孕激素通过何种机制影响了线粒体的膜电位,以及其具体的抑制凋亡的途径尚不清楚。
研究表明PR-M的富集与线粒体的分布有关,PR-M在心脏、肝脏和子宫平滑肌中有高表达。本研究着重于PR-M对子宫平滑肌的作用,其目的为：(1)比较PR-M在子宫肌瘤及其邻近正常子宫平滑肌组织中的表达；(2)了解孕激素对原代培养的子宫平滑肌细胞以及人永生化子宫平滑肌(hTert-HM)细胞线粒体膜电位的调节作用；(3)研究孕激素诱导的hTert-HM细胞线粒体膜电位超极化反应的机制。本研究分为以下三部分：
第一部分孕激素受体亚型PR-M和线粒体porin蛋白在子宫肌瘤及其邻近正常子宫平滑肌组织中的表达
方法
1.蛋白提取和Western blotting分析
选取美国杜克大学医学院妇产科因子宫肌瘤行手术治疗的5例患者的组织标本(组织获取经杜克大学伦理委员会审核批准),术中分别取子宫肌瘤组织近边缘部分(fibroid edge,FE) (肌瘤组)及其邻近子宫肌瘤的正常平滑肌组织(myometrium adjacent,MA) (正常组)各5例,共10例。采用Western blotting检测孕激素受体PR-M、PR-A和PR-B和线粒体porin蛋白在子宫肌瘤和正常子宫平滑肌组织中的表达。标本采集后—80℃保存于OCT冻存液中待用。将组织置于含蛋白酶抑制剂的RIPA缓冲液中进行高速组织粉碎仪搅拌和超声裂解,用Bradford微孔板检测试剂盒和读板器检测总蛋白浓度(以已知的BSA浓度为标准曲线)。Western blotting分析采用10%的聚丙烯酰胺于Tris/Glycine/SDS缓冲液中电泳分离蛋白,组织上样量为每孔30～70μg,Tris/Glycine缓冲液中转膜,5%牛奶常温封闭1h,按照一定浓度加入一抗4℃孵育过夜。PR-M用MAB462鼠单克隆抗体检测,PR-A和PR-B用C-19兔多克隆抗体检测。Porin采用鼠单克隆porin或VDAC-3抗体检测。以GAPDH做为系统对照。一抗反应后TBST洗膜,分别加入5%牛奶稀释的一定浓度的抗兔或抗鼠二抗,常温下摇床孵育1h。将膜用ECL增强化学发光剂快速摇洗1min后显影。
每次检测后用蛋白印迹膜再生液洗膜。重复取样,PR-M蛋白检测实验重复4次,共行20例次；PR-A和PR-B重复2次,共行10例次。以乳腺癌T47D细胞做为阳性对照。
2.蛋白表达的半定量分析
采用多图像光柜过滤系统(MultilmageTM Light Cabinet Filter Position) Alpha lamagerTM 2200行半定量分析。图片扫描获得IDV (Integrated Density Value)值,分别将PR-M. PR-A, PR-B、porin的IDV值与GAPDH的IDV值相比。将两组PR-M、PR-A、PR-B、porin的IDV与GAPDH的IDV比值均值进行统计分析。
结果1. PR-M、PR-A和PR-B在子宫肌瘤及其邻近正常子宫平滑肌组织中的表达
子宫肌瘤组PR-M/GAPDH的IDV比值高于正常组,差异有统计学意义(P=0.007)。分别将PR-A、PR、PR-M的IDV值与GAPDH的IDV值相比,并设定正常组的IDV的比值为1,5例患者中4例PR-M/GAPDH的IDV比值明显大于1。子宫肌瘤组PR-A/GAPDH和PR-B/GAPDH的IDV比值也高于正常组,但差异无统计学意义(P=0.153,P=0.052)。
2. porin在子宫肌瘤及其邻近正常子宫平滑肌组织中的表达
子宫肌瘤组porin/GAPDH的IDV比值高于正常组,差异有统计学意义(P=0.003)。分别将porin的IDV值与GAPDH的IDV值相比,并设定正常组的IDV的比值为1。5例porin/GAPDH的IDV比值均大于1。
3. PR-M, PR-A和PR-B和porin的相关性
Spearman's相关性分析时,PR-M/GAPDH与porin/GAPDH之间呈正相关(rs=0.359,P=0.023)。但PR-A/GAPDH、PR-B/GAPDH与porin/GAPDH之间无相关性(rs=0.068、-0.009,P=0.777、0.970)
第二部分孕激素对原代子宫平滑肌细胞和永生化子宫平滑肌细胞(hTert-HM)线粒体膜电位的调节
方法
1. Western blotting检测PR-M和porin蛋白在原代培养子宫平滑肌细胞和hTert-HM细胞中的表达
选取美国杜克大学医学中心因子宫肌瘤行手术治疗患者的正常子宫组织行子宫平滑肌原代细胞培养。手术后将标本立即置入无钙无镁加双抗的HBSS液体中,用加双抗的无血清DMEM/F12、0.1% I A型胶原蛋白酶Ⅰ消化细胞,用适量DMEM/F12培养基(+10%FBS+1%双抗和0.5%Fungizone)进行细胞培养。分别对手术后立即消化的原代子宫平滑肌细胞、生长了5天的原代子宫平滑肌细胞、传代前的子宫平滑肌细胞和第三代培养子宫平滑肌细胞进行Western blotting检测(方法同上述)。PR-M用MAB462、C262抗体检测。
hTert-HM永生化子宫正常平滑肌细胞株为美国杜克大学生殖内分泌实验室保存提供。Westem blotting检测PR-M和porin蛋白在hTert-HM细胞中的表达。2.RT-PCR检测PR.M mRNA在hTert-HM细胞中的表达
用TRIzol试剂对永生化子宫平滑肌细胞进行总RNA的提取,提取物进行逆转录为cDNA,行PCR扩增反应。PCR产物行聚丙烯酰胺凝胶电泳,显色分析。T47D乳腺癌细胞为阳性对照。阴性对照用水替代cDNA。3.JC-1实验检测P4和R5020对原代培养子宫平滑肌细胞线粒体膜电位的作用
用适量DMEM/F12培养基(+10%FBS+1%双抗和0.5%Fungizone)对切除的正常子宫平滑肌组织进行原代细胞培养,细胞按120k/孔铺于48孔板,生长4天,至80%密度时放入pH 7.4的KRH缓冲液(含25 mM Na-HEPES,115 mM NaCl, 5 mM KCl,1 mM KH2P04,1.2 mM MgSO4,0.5 mM CaCl2 and 5 mM glucose)在无C02的37℃孵育箱中孵育2h后,分别用10-6M、10-8M和10-10M的P4诱导细胞60min。第一代细胞生长7天后传代,第二代细胞按40000/每孔铺于48孔板,3天后分别应用10-6M和10-7M的R5020诱导细胞60min。采用Jc-1(5,5',6,6'-tetrachloro- 1,1',3,3'-tetraethyl-benzimidazole- carbocyanide iodine,5,5',6,6'-四氯-1,1',3,3'-四乙基-苯并咪唑-碳氰碘化物)实验检测孕激素作用后细胞MMP的变化。
4.JC-1实验检测R5020、MPA和P4对hTert-HM细胞线粒体膜电位的作用
用适量DMEM/F12培养基(加10%胎牛血清和青霉素、链霉素双抗)对hTert-HM细胞进行细胞培养,细胞按50K/每孔的密度铺于48孔板,生长24h至80%密度时行JC-1实验。分别应用10-8M的R5020、MPA和P4,以及10-6M、10-8M和10-10M的R5020诱导细胞60min后采用JC-1检测细胞MMP的变化。
结果
1.PR-M、porin和α—SMA蛋白在原代培养子宫平滑肌细胞中的表达
Westem blotting结果显示手术后立即消化的原代子宫平滑肌细胞、生长了5天的原代子宫平滑肌细胞和传代前的子宫平滑肌细胞中均有PR-M蛋白和Porin蛋白的表达,PR-M为一个38kDa的条带,ponn为一个31kDa的条带。
2. PR-M mRNA在hTert-HM细胞的表达
结果显示hTert永生化子宫平滑肌细胞株中有PR-M mRNA的表达。
3.P4和R5020对原代培养子宫平滑肌细胞线粒体膜电位的超极化作用
结果显示10-6M,10-8M和10-10M的P4均引起原代培养子宫平滑肌细胞MMP的超极化反应,但10-6M,10-8M与对照组比较差异有统计学意义(Z=3.838,p=0.000; Z=2.914, P=0.004)。Progesterone以浓度依赖的形式引起MMP的增加,浓度越高,其引起的MMP超极化作用越强(rs=-0.781,P<0.0001)。10-6M和10-7M的R5020引起第二代原代培养子宫平滑肌细胞MMP的超极化反应,但与对照组比较差异无统计学意义(F=1.511，p=0.253)
4.R5020、MPA和P4对hTert-HM细胞线粒体膜电位的超极化作用
R5020、MPA和P4三种孕激素在10-8M的浓度时均引起hTert-HM细胞MMP的超极化改变(p=0.031,0.001,0.203)。人工合成的孕激素R5020和MPA可以明显引起hTert细胞MMP的超极化反应。
第三部分孕激素诱导的hTert-HM细胞线粒体膜电位超极化反应的机制
方法
1.JC-1实验检测孕激素受体拮抗剂RTI-6413-049b作用后的hTert-HM细胞线粒体膜电位
hTert-HM永生化子宫正常平滑肌细胞株为杜克大学生殖内分泌实验室提供。用适量DMEM/F12培养基(加10%胎牛血清和青霉素、链霉素双抗)对hTert-HM细胞进行细胞培养。细胞按50K/每孔的密度铺于48孔板,生长24h至80%,然后放入KRH缓冲液2h,分别应用加有和不加10-6M的孕激素受体拮抗剂(RTI-6413-049b),以及10-6M的RTI诱导hTert-HM细胞60min后采用JC-1检测细胞MMP的变化。
2.JC-1实验检测地塞米松(Dexamethasone)作用后的hTert-HM细胞线粒体膜电位
细胞按50K/每孔的密度铺于48孔板,生长24h至80%,放入缓冲液2h后,分别应用10-8M,10-9M,10-10M的Dexamethasone诱导hTert-HM细胞60min后采用JC-1检测细胞MMP的变化。
3.JC-1实验检测放线菌酮Cycloheximide(CHX)作用后的hTert-HM细胞线粒体膜电位
hTert-HM细胞按50K/每孔的密度铺于48孔板,生长24h至80%,分别放入含有5μg/mL CHX缓冲液和不含CHX缓冲液中2h后,分别用加有和不加CHX(5μg/ml)的10-6M的R5020、CHX(5μg/ml)诱导细胞60min,应用JC-1检测MMP的变化。
4. Western blotting检测TGF-β1作用于hTert-HM细胞后CHX对Id-1蛋白表达的抑制作用
细胞按800k/每孔铺于6孔板上,分别加含有5μg/mL的CHX的缓冲液和不含CHX的缓冲液中孵育2h后,加入5ng/mL TgF-β1处理60min后收集细胞,应用western blotting检测Id-1蛋白的表达。
结果
1.RTI作用后hTert-HM细胞线粒体膜电位的改变
10-8M R5020引起的超极化反应与联合应用10-8M R5020和10-6M RTI组、单用10-6MRTI组、对照组之间相比差异均有统计学意义(P=0.033,0.017,0.005)。联合应用10-8M R5020和10-6M RTI组,以及单用10-6MRTI组,与对照组比较时差异无统计学意义(P=0.394,0.549)。表明,R5020引起的细胞MMP的超极化反应被RTI所抑制。
2. Dexamethasone作用后hTert-HM细胞线粒体膜电位的改变
10-8，10-9，10-10浓度的地塞米松与对照组各组间比较均无统计学意义(F=0.722，p=0.541)。表明,地塞米松对hTert细胞的MMP没有作用。提示,孕激素引起的hTert-HM细胞线粒体膜电位的超极化可能与糖皮质激素受体的作用无关。
3.CHX作用后hTert-HM细胞线粒体膜电位的改变
10-6M R5020组、以及联合使用10-6M R5020和CHX(5μg/ml)组,与对照组比较差异均有统计学意义(P=0.026,0.006)；单用CHX组与对照组之间比较差异无统计学意义(p=0.246)；10-6M R5020组与联合使用10-6M R5020和CHX组之间比较差异也无统计学意义(p=0.600)。表明,转录抑制剂CHX对hTert细胞MMP没有影响,应用CHX后不影响10-6M R5020引起的hTert细胞MMP的超极化作用,R5020的作用与细胞蛋白合成无关。
4.CHX及TGF-β1作用后hTert-HM细胞中Ib-1蛋白的表达
TgFβ-1作用于细胞后,在一定时间内(1h左右)可以诱导细胞产生Id-1,CHX可以抑制该过程。为证明CHX对hTert细胞抑制作用的有效性,我们采用CHX (5μg/ml)孵育细胞2h后再用TgFβ-1 (5ng/ml)处理细胞60min,Westernblotting显示CHX处理后Id-1蛋白的表达减弱,表明CHX发挥了抑制作用。
统计学处理
采用SPSS 16.0对数据录入分析,正态分布数据以x±s表示,非正态分布数据以M±Q表示。配对比较采用配对t检验。数据服从正态分布且方差齐多个样本均数比较采用方差分析法,两两比较采用最小显著差异法(least significant difference,LSD),否则多组比较采用Kruskal-Wallis秩和检验。采用Spearman进行等级资料数据的相关分析。检验水准α=0.05。
结论
1.线粒体孕激素受体PR-M在人子宫平滑肌组织中呈高表达,其在子宫肌瘤组织中的表达高于临近的正常子宫平滑肌组织。
2.孕激素以剂量依赖的形式诱导了原代培养和永生化子宫平滑肌细胞线粒体膜电位的增加。这种反应可以被孕激素受体拮抗剂所抑制,但与糖皮质激素和放线菌酮的诱导无关,排除了糖皮质激素受体作用和蛋白合成抑制的可能性。
3.研究提示,孕激素可能通过PR-M的非基因组作用增加了细胞的能量代谢,细胞内能量的增加导致了子宫肌瘤的异常增殖。
(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., USA
Progesterone 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 determined
Tissue 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 expression
Western 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 porin
Spearman'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 cells
Western 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 cells
A 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 Dexamethasone
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, 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-β1
hTert-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 Dexamethasone
There 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 CHX
Treatment 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-β1
To 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 analysis
Statistical 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.
引文
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