【作者】 王凯；

【导师】 辛毅；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 妊娠其间,胎盘新生血管的形成和血管的扩张是增加胎盘血流量的关键机制,也直接决定着胎儿的生长及存活。血管内皮生长因子（vascular endothelial growth factor, VEGF）和成纤维细胞生长因子2（fibroblast growth factor, FGF2）作为两种重要的促血管生成因子,与妊娠其间胎盘的血管形成及血流量调节密切相关。VEGF和FGF2通过与相应的受体结合并激活其胞内酪氨酸激酶,从而进一步起动细胞内的一系列信号传导通路,包括丝裂原活化蛋白激酶的激酶1/2（mitogen-activated protein kinase kinase 1/2 , MAP2K1/2）/丝裂原活化蛋白激酶3/1 （mitogen-activated protein kinase 3/1,MAPK3/1）和磷脂酰肌醇3-激酶（phosphoinositide 3-kinase, PI3K）/蛋白激酶B （ protein kinase B , PKB;又称作v-akt murine thymoma viral oncogen homolog 1, AKT1）通路。MAPK3/1和AKT1为体内重要的丝氨酸和苏氨酸蛋白激酶,通过磷酸化并进一步激活下游底物来参与介导对细胞增殖和分化等功能的调节。我们曾报道在羊胎盘动脉血管内皮细胞（ovine fetoplacental artery endothelial [OFPAE] cells）中, MAP2K1/ 2/MAPK3/1和PI3K/AKT1这两条信号通道都参与了VEGF和FGF2诱导的细胞增殖过程。相对于蛋白质的磷酸化过程,蛋白质的去磷酸化过程同样发挥着重要的作用,而这一过程主要是由蛋白磷酸酶来完成。蛋白磷酸酶3（protein phosphatase 3,PPP3;也称作蛋白磷酸酶2B [protein phosphatase2B, PP2B]或钙调神经磷酸酶[calcineurin, Cn]）是迄今发现的唯一受Ca²⁺/钙调素（calmodulin, CaM）调节的丝/苏氨酸蛋白磷酸酶,是由一个催化亚单位（α,β,γ亚基,又称作PPP3CA, PPP3CB,PPP3CC）和一个调节亚单位（B1,B2亚基,又称作PPP3R1,PPP3R2）组成的异源二聚体,由于其分布的广泛性和功能的多样性,越来越受到人们的关注。近期研究表明, PPP3介导的信号通路在心血管的形态发生中起重要作用,并参与了免疫应答,心肌肥大、血管平滑肌细胞分化等功能的调节。而且研究发现在人脐静脉血管内皮细胞和真皮微动脉血管内皮细胞中,PPP3的抑制剂环孢素A（cyclosporineA,CsA）抑制了VEGF而不是FGF2诱导的细胞增殖,这种抑制作用主要是减弱了MAPK3/1的活性所致,从而进一步表明了PPP3和MAPK3/1之间存在着某种相互的作用。同时在心肌细胞和B淋巴细胞中,也发现了CsA对MAPK3/1活性的抑制作用。但CsA对MAPK3/1的激活作用也在绒毛膜滋养层细胞和大鼠肾小管上皮细胞中有报道。相对于MAPK3/1,PPP3对蛋白激酶AKT1的作用却报道甚少,仅发现在A549细胞中,抑制PPP3的活性,并不影响AKT1的磷酸化水平。因此在本实验中,我们验证了抑制PPP3CA的表达,是否影响了VEGF和FGF2诱导的OFPAE细胞增殖,而且这一作用是否是通过作用于FGF2和VEGF激活的MAPK3/1和AKT1的磷酸化水平来实现的.本实验利用RNA干扰技术（RNA interference, RNAi）设计并化学合成了针对PPP3CA的特异性小干扰RNA（short interfering RNA, siRNA）,其对PPP3CA蛋白表达的抑制率（p < 0.05）达到97%左右,但不影响蛋白磷酸酶2的调节亚基α（protein phosphatase 3 regulatory subunitα, PPP2CA）,MAPK3/1,AKT1和3-磷酸甘油醛脱氢酶（glyceraldehyde 3-phosphate dehydrogenase, GAPDH）的蛋白水平.而且这种抑制效果至少可以持续72小时。进一步采用紫结晶法检测PPP3CA siRNA对VEGF和FGF2诱导的细胞增殖的影响,发现转染了PPP3CA和scrambled siRNA的OFPAE细胞, VEGF和FGF2都剂量依赖性（p < 0.05）地诱导了细胞的增殖。与scrambled siRNA转染组相比较,PPP3CA siRNA显著（p < 0.05）地促进了VEGF,而不是FGF2诱导的细胞增殖。为进一步验证PPP3CA siRNA对VEGF和FGF2激活的MAPK3/1和AKT1的影响,我们利用蛋白印迹分析技术检测了MAPK3/1和AKT1的磷酸化水平。结果显示相对于scrambled siRNA转染组,PPP3CA siRNA并未（p < 0.05）改变VEGF对MAPK3/1和AKT1的激活作用,但显著（p < 0.05）地抑制了FGF2激活的MAPK3/1和AKT1的磷酸化水平。因此本实验结果说明PPP3CA在VEGF,但不是FGF2诱导的OFPAE细胞增殖过程中发挥着重要的作用,同时也影响了FGF2,而不是VEGF激活的MAPK3/1和AKT1的磷酸化水平。对于PPP3参与了VEGF,而不是FGF2诱导的细胞增殖是与先前的报道相一致的。而且都是以内皮细胞作为研究对象。所不同的是其它报道认为抑制PPP3的活性减弱了VEGF诱导的细胞增殖,而我们发现抑制PPP3CA增强了VEGF诱导的细胞增殖。这很可能是由于在以前文献的报道中,一般都是利用PPP3的抑制剂CsA作为研究手段。CsA可以通过与PPP3的调节亚基和催化亚基相结合由此产生的空间位阻使得PPP3的蛋白质底物无法接近其磷酸酶活性区域,从而阻止了PPP3对底物的脱磷酸化作用。因此被广泛地用于对PPP3功能方面的研究。但近期的研究发现, PPP3并不是该抑制剂的唯一底物, CsA可以抑制线粒体内膜通透转变孔道的开放,而此抑制过程并没有PPP3的参与。因此我们认为,CsA抑制VEGF诱导的促血管生成作用可能并不仅仅通过PPP3一个底物来完成。另一方面,在本实验中,VEGF对OFPAE细胞增殖的诱导过程是发生在抑制了PPP3CA之后至少16个小时（血清饥饿时间）。而文献报道的用CsA来抑制PPP3CA的时间一般是30分钟到60分钟,因此相对于这种短期的过程,相对长期的蛋白质表达的抑制过程势必会导致一系列信号传导通路在持续时间和强度方面的改变。最后需要提出的是在本实验中我们所设计的siRNA是仅针对PPP3催化亚基中的一个亚型,它只能具有部分PPP3的生物学活性,本实验并不能排除PPP3CB也在VEGF诱导的细胞增殖中发挥着一定的作用。尽管我们前期工作发现了在OFPAE细胞中,MAP2K1/2/MAPK3/1和PI3K/AKT1两条信号通路参与了对于VEGF和FGF2诱导的细胞增殖过程的调节。但对于PPP3CA siRNA不同地影响了VEGF和FGF2诱导的细胞增殖提示在抑制PPP3CA表达之后,很可能又会激活其它信号传导途径来参与对此过程的调节。事实上,我们的前期工作也发现了在MAPK家族中,除了MAPK3/1以外, MAPK11 （也称作p38 MAPK）也参与对VEGF和FGF2诱导的OFPAE细胞增殖过程的调节。另外对于PPP3CA siRNA所引起的对VEGF和FGF2诱导的MAPK3/1和AKT1磷酸化水平的不同影响,机制仍然不清,很可能是由于抑制PPP3CA后所引起的对其它种类的蛋白磷酸化酶（如丝裂原活化蛋白激酶磷酸酶[mitogen-activated protein kinase phosphatase, MAPKP]和同源性磷酸酶-张力蛋白磷酸酶[phosphatase and tensin homolog ,PTEN]）的不同调节所导致,有关这方面的调节机理还有待于我们进一步去探讨。总而言之,本实验第一次证实了在OFPAE细胞中, PPP3CA在VEGF诱导的细胞增殖和FGF2激活的MAPK3/1和AKT1蛋白激酶中发挥着重要的作用。进一步说明了与胎盘内皮细胞功能调节相关的信号传导通路的复杂多样性,为更好地通过这些信号途径调节胎盘的血流和血供提供更多的理论依据。更多还原

【Abstract】 During pregnancy, angiogenesis and vasodilatation are two key mechanisms essential for increasing placental blood flows, which are directly correlated with fetal growth and survival as well as neonatal weights and survivability. Vascular endothelial growth factor （VEGF） and fibroblast growth factor （FGF2） are two of the most potent angiogenic factors. Expression of FGF2 and VEGF in placentas is positively associated with the placental vascular growth and blood flows during pregnancy. It is well established that the biological actions of VEGF and FGF2 are initiated by binding to their specific receptors thereby activating the cytoplasmic tyrosine kinase domains. Upon activation, these receptor-tyrosine kinases initiate multiple downstream cellular protein kinase signaling pathways, including mitogen-activated protein kinase kinase 1/2 （MAP2K1/2）/ mitogen-activated protein kinase 3/1 （MAPK3/1） and phosphoinositide 3-kinase （PI3K）/v-akt murine thymoma viral oncogen homolog 1 （AKT1） pathways. MAPK3/1 and AKT1, as major threo- nine/tyrosine protein kinases, are involved in regulating cell survival, proliferation, and migration by phosphorylation and activation their downstream substrates. Recently, we have reported that the VEGF- and FGF2-stimulated OFPAE cell proliferation is mediated at least partly via the MAP2K1/2//MAPK3/1 and PI3K/AKT1 signaling pathways.Protein dephosphorylation is the same important process with phosphorylation. This process is tightly controlled by protein phosphatases. Protein phosphatase 3 （PPP3, also term as PP2B or calcineurin） is the only Ca²⁺/calmodulin dependent member of PPP, which is composed of catalytic and regulatory subunits. Three PPP3 catalytic （α,β,γ, also named as PPP3CA, PPP3CB, PPP3CC） and two regulatory （B1 and B2, also named as PPP3R1 and PPP3R2） subunits have been identified. More and more people focused on PP2B due to its wide distribution and multiply functions. Recent studies showed PP2B plays important role in the vascular development as well as cardiac hypertrophy and vascular smooth musclar differentiation. It also indicated that suppression of PPP3 activity by its pharmacological inhibitor cyclosporine A （CsA, a widely used immunosuppressant） inhibits VEGF-, but not FGF2-induced angiogenesis in human umbilical vein endothelial and intestinal microvascular endothelial cells. This inhibition can be mediated via blocking or attenuating endothelial MAPK3/1 activation, implicating a reciprocal relationship between PPP3 and MAPK3/1 activations in endothelial cells similar to that observed in cardiomyocytes and B cells. In contrast, CsA is also capable of promoting MAPK3/1 activation in human first-trimester trophoblasts and canine kidney epithelial cells. Little is known about the role of PPP3 in the PI3K/AKT1 pathway, except that one recent report showed that PPP3 inhibition by FK506 （another selective PPP3 inhibitor） had no effect on activation of the PI3K/AKT pathway in A549 cells. In this study, we determined that if suppression of PPP3CA inhibited FGF2- and VEGF- stimulated cell proliferation and attenuated FGF2- and VEGF- induced activation of MAPK3/1 and AKT1.In this study, small interfering RNA （siRNA） specifically targeting human PPP3CA was used to suppress PPP3CA protein expression in OFPAE cells. PPP3CA siRNA decreased PPP3CA protein levels by 97%, while failing to alter protein levels of PPP2 catalytic subunitα（PPP2CA）, total MAPK3/1 and AKT1, and GAPDH （glyceraldehyde 3-phosphate dehydrogenase）, as compared with the scrambled siRNA. This inhibitory effect of PPP3CA siRNA on PPP3CA protein expression maintained at least for 3 days after the transfection. Violet crystal method was used to determined the effect of PPP3CA on VEGF- and FGF2- induced cell proliferation. Both VEGF and FGF2 dose-dependently stimulated （p < 0.05） cell proliferation. Western Blot was performed to further demonstrate effect of PPP3CA on VEGF- and FGF2- induced MAPK3/1 and AKT1 phosphorylation. Suppression of PPP3CA protein expression did not significantly affect VEGF-induced MAPK3/1 and AKT1 phosphorylation, whereas attenuating（p < 0.05） FGF2-induced MAPK3/1 and AKT1 phosphorylation. Thus, these data suggest that PPP3CA plays an important role in VEGF-, but not FGF2- stimulated OFPAE cell proliferation and in FGF2-, but not VEGF-induced MAPK3/1 and AKT1 activation.Our current observations that PPP3CA participates VEGF-, but not FGF2-stimulated endothelial proliferation are consistent with the previous reports using other endothelial cell types. Meanwhile, in contrast to these previous reports showing that inhibition of PPP3 activity attenuates VEGF-induced angiogenesis, knockdown of PPP3CA protein expression enhances OFPAE cell proliferation. It is noteworthy that in both of previous studies, inhibition of PPP3 activity is carried out by its pharmacological inhibitor CsA, which executes its action via binding to the interface of the catalytic and regulatory subunits of PPP3. Being generally considered to be a selective inhibitor of PPP3 and widely used in investigating roles of PPP3, this drug, however, can also function independent of PPP3. For example, it has been shown that CsA inhibition of the opening of mitochondrial inner membrane permeability transition pore is not mediated via PPP3. Thus, it is possible that PPP3 might not be the only major target in the CsA-inhibited VEGF-stimulated in vitro and in vivo angiogenic responses. On the other hand, OFPAE cells were exposed to VEGF at least 16 hr （serum starvation period） after knockdown of PPP3CA protein expression. This relatively chronic knockdown of PPP3CA might lead to extensive, different changes in signaling network as compared with the relatively acute （ 30 min to 1 hr） inhibition of PPP3 by CsA before VEGF stimulation. Moreover, in the current study, the siRNA designed targets only PPP3CA, which may contribute portion of total PPP3 activity in OFPAE cells. Our current study cannot exclude the possibility that the catalyticβsubunit plays a role in VEGF-stimulated endothelial proliferation.Although we have demonstrated that in OFPAE cells that activation of the MAP2K1/2/MAPK3/1 and PI3K/AKT1 pathways is critical for VEGF- and FGF2-stimulated cell proliferation. The increased VEGF-stimulated and unchanged FGF2-stimulated cell proliferation in OFPAE cells in this study suggest that after knockdown of PPP3CA signaling cascades other than the MAP2K1/2/MAPK3/1 and PI3K/AKT1 might emerge as major players in mediating the VEGF- and FGF2-stimulated cell proliferation. Indeed, we have also observed that MAPK11 （also termed as p38 MAPK） is a key signaling in mediating VEGF- and FGF2-induced OFPAE cell proliferation. Alternatively, differential regulation of other protein hosphatases （i.e. MAPK phosphatase [MAPK], and Phosphatase and TENsin Homolog [PTEN]） might contribute to differential activation of MAPK3/1 and AKT1 by VEGF and FGF2 in OFPAE cells transfected with PPP3CA siRNA.Taken together, our current data demonstrate that PPP3CA plays a critical role in VEGF-stimulated cell proliferation and FGF2-induced MAPK3/1 and AKT1 activation in fetoplacental endothelial cells. Our current findings further advance our understanding of the complex signaling mechanism controlling placental endothelial function. Future studies are needed to dissect these signaling networks, which may provide fundamental information for modulating placental vasculature and blood flows by altering activation of signaling cascades by angiogenic factors.更多还原