【作者】 杨璞；

【导师】 屈伸；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 极低密度脂蛋白受体(very low density lipoprotein receptor, VLDLR)属于低密度脂蛋白受体(low density lipoprotein receptor, LDLR)超家族。VLDLR由于第16外显子的选择性剪接从而产生胞外段O-linked糖链结合域缺失的亚型,根据有无此结构域可将VLDLR分为Ⅰ型和Ⅱ型两种亚型。早期研究认为,该受体主要结合富含apoE(载脂蛋白E)的脂蛋白而参与甘油三酯的代谢;与泡沫细胞的形成及动脉粥样硬化的发生发展相关。但由于该基因敲除的小鼠仅表现体脂下降,发育迟缓,因此对该受体研究一直未引起足够重视。近年来的研究发现,因此VLDLR和LDLR家族的其他成员一样被认为是一种“瑞士军刀”样多功能受体。但是对两型受体的功能差异,特别是Ⅱ型受体的独特功能及其生物学意义尚未阐明。我们的前期工作和已有研究资料表明:⑴、两型受体的分布具有明显的组织细胞特异性:Ⅰ型受体主要分布于脂代谢旺盛的组织,而Ⅱ型受体则主要分布于肾脏、脾脏、肾上腺、睾丸、子宫、卵巢等非肌组织。⑵、在分化程度不同的细胞、组织中两型受体的表达不同:在高分化的胃腺癌细胞系中,Ⅱ型受体低表达或不表达,而在低分化的胃腺癌细胞系中Ⅱ型受体高表达;同样,在多种胃腺癌组织细胞中观察到Ⅱ型受体的表达增多;宫颈癌病变组织中也检测到VLDLRⅡ型的高表达;另外在鸡和小鼠胚胎发育过程中VLDLR两种亚型的表达可发生变化:在胚胎发育早期以Ⅱ型VLDLR的表达为主,而在发育成熟的体细胞中则以Ⅰ型VLDLR表达为主;在胚胎脑中以Ⅱ型VLDLR表达为主,而在成熟分化的脑组织中则以Ⅰ型VLDLR表达为主。⑶、在退行性病变的纤维化脾组织中,Ⅱ型VLDLR则几乎消失;而在阿尔茨海默病病人的老年斑斑块中以Ⅰ型VLDLR表达为主;⑷、最新研究报道认为VLDLR,尤其是Ⅰ型VLDLR,表达减少或不表达会导致肿瘤的形成;且Ⅰ型VLDLR在抑制肿瘤细胞生长方面作用更重要。这些现象强烈提示:VLDLR亚型与细胞的增殖、分化、迁移等细胞活动存在密切关系。但这种关系的生物学意义目前尚不清楚。uPA-PAI-1复合物和TFPI是VLDLR已知的两种配体, VLDLR与uPA-PAI-1复合物的结合可促进细胞增殖、迁移;而与TFPI结合可抑制细胞的增殖;这些表明VLDLR与不同配体结合可影响截然相反的细胞功能,但这些配体是否通过VLDLR亚型的变化来影响细胞生物学行为尚不清楚。本研究首先针对VLDLR亚型变化与细胞增殖、迁移之间的关系进行深入研究,探讨VLDLR亚型的变化与细胞生物学行为的关系和意义。为探讨胃腺癌细胞SGC7901在向不同方向诱导分化过程中VLDLR亚型的表达变化与细胞生物学行为之间的关系,本实验在体外,利用全反式维甲酸(ATRA)持续诱导SGC7901细胞建立向高分化诱导变化的模型,利用佛波酯(PMA)持续诱导SGC7901细胞建立向低分化诱导变化的模型;检测端粒酶催化亚单位(TERT) mRNA的表达量判断细胞分化程度。结果显示:SGC7901细胞在ATRA的持续作用下,细胞向高分化转化,Ⅱ型VLDLR明显减少,同时细胞增殖活性和迁移能力逐渐减弱; SGC7901细胞在PMA的持续作用下,细胞向低分化转化,Ⅱ型VLDLR明显升高,同时细胞增殖活性和迁移能力增强。上述结果表明Ⅱ型VLDLR的表达变化与肿瘤细胞分化之间存在相关性。为了深入探讨VLDLR亚型与细胞分化、增殖、迁移等的关系,本实验利用能影响细胞增殖迁移的VLDLR的配体(uPA-PAI-1、TFPI)与细胞温育,观察VLDLR亚型的表达变化与细胞生物学行为之间的关系。实验结果发现:TFPI抑制细胞增殖、迁移的同时可明显减少Ⅱ型VLDLR,对Ⅰ型VLDLR无影响,并可使Ⅱ型VLDLR与Ⅰ型VLDLR的比值逐渐降低;uPA-PAI-1复合物促进细胞增殖、迁移的同时可减少Ⅰ型VLDLR,增加Ⅱ型VLDLR,并使Ⅱ型VLDLR与Ⅰ型VLDLR的比值逐渐升高。综上所述,我们的结果证实:无论是诱导分化,还是影响细胞增殖相关配体的作用,肿瘤细胞中VLDLR亚型的变化具有如下规律:在低分化、增殖、迁移能力强的细胞中同时伴有Ⅱ型VLDLR增加,在高分化、增殖、迁移能力弱的细胞中同时伴有Ⅱ型VLDLR减少。这一变化规律说明Ⅱ型VLDLR在促进细胞增殖、迁移,抑制细胞分化中扮演着重要角色。已有的研究表明, VLDLR在神经组织发育过程中,介导Reelin-Dab1信号途径,通过SFK/PI3K-Gsk3β调节细胞骨架蛋白Tau而影响细胞骨架的重构和细胞迁移;在内皮细胞增殖和迁移相关的wnt信号途径中VLDLR发挥重要的负调控作用,表明VLDLR在细胞增殖分化的wnt信号途径中发挥重要调节作用。另外,VLDLR与uPA-PAI-1复合物的结合可维持胞内ERK的磷酸化,从而促进细胞增殖、迁移;而与TFPI结合可通过通过p16ink4a和p38/JNK信号途径抑制细胞的增殖;这些表明VLDLR与不同配体的结合,调节与细胞增殖分化相关的MAPK信号体系中不同的通路,影响截然相反的细胞功能。另有研究表明,ERK的活化可使GSK-3β磷酸化失活,导致β-catenin在胞内聚集,而β-catenin可促进下游包括MMPs在内的特异基因的转录,影响细胞增殖、迁移。这些表明VLDLR与不同配体结合影响细胞功能的信号调节作用可能与MAPK、wnt途径有关。但VLDLR亚型的变化影响细胞生物学行为的机制目前尚不清楚。根据已有研究我们推测Ⅱ型VLDLR影响细胞增殖、迁移与MAPK、wnt信号通路有关,因此本实验在诱导分化过程中和细胞增殖迁移调控配体的温育下,观察VLDLR可能涉及的信号途径的关键分子的活性及表达变化,初步探讨Ⅱ型VLDLR影响细胞生物学行为的可能机制。实验结果发现:在细胞向高分化诱导过程中和TFPI温育后Ⅱ型VLDLR减少,β-catenin的磷酸化明显增强促进其降解从而使其在胞内表达降低,同时其下游靶基因MMP-2和MMP-9的表达下调,抑制细胞增殖、迁移;而在细胞向低分化诱导过程中和uPA-PAI-1温育后Ⅱ型VLDLR增加,β-catenin的磷酸化受到抑制增加其稳定性从而促进胞内表达上调,同时其下游靶基因MMP-2和MMP-9的表达上调,促进细胞增殖、迁移。因此Ⅱ型VLDLR影响细胞生物学行为变化可能与β-catenin在胞内的表达上调,从而促进特异靶基因的转录有关。已有研究表明,VLDLR与uPA-PAI-1的结合可维持乳腺癌胞内ERK的磷酸化。我们的实验发现,uPA-PAI-1与细胞温育5 min即可明显增强ERK的磷酸化,这种作用可一直持续到30 min,对ERK1的磷酸化作用在温育60 min后仍较明显。这与前述研究一致。VLDLR与TFPI结合可通过活化p38/JNK信号途径抑制细胞增殖。我们的实验发现,TFPI可抑制ERK的活化。对LDLR表达调控的研究发现,胞外分子可通过激活p38信号途径抑制ERK的活性从而下调LDLR的表达。因此我们推测,TFPI可能通过活化p38从而抑制ERK的活化。上述结果提示,uPA-PAI-1复合物可能通过Ⅱ型VLDLR活化胞内ERK,从而促进细胞增殖、迁移;而TFPI可能通过Ⅰ型VLDLR活化p38从而抑制ERK的活化,从而抑制细胞增殖、迁移。上述结果提示,Ⅱ型VLDLR影响细胞生物学行为的变化可能与ERK的活化抑制β-catenin的磷酸化降解使其在胞内表达上调,调节特异靶基因的转录有关。综上所述,Ⅱ型VLDLR可能通过与特定配体的结合、摄取,影响细胞内增殖、分化相关的信号途径,导致相应细胞生物学行为改变。本研究的创新之处在于初步揭示了Ⅱ型VLDLR的表达变化与细胞增殖、分化、迁移之间的关系及其可能涉及的信号转导途径,扩展了VLDLR功能的多样性,并对脂蛋白受体家族成员作为“瑞士军刀”样多功能受体提供了新的认识。更多还原

【Abstract】 Very low density lipoprotein receptors (VLDLR), a member of low density lipoprotein receptor (LDLR) superfamily, consist of two subtypes, type I VLDLR and type II VLDLR. It is generally accepted that VLDLR plays a major role in the metabolism of triglyceride through binding lipoproteins enriched in apoE and has an intimate relation with atherosclerosis. There have been many attempts to study the biological phenotype in homozygous VLDLR knockout mice. However, the physiological and pathological importance of these receptors has not been clearly identified. Recently, VLDLR and many other members of the low density lipoprotein receptor family are found to bind different ligands besides lipoproteins, causing endocytosis and affecting many cellular functions. For example, VLDLR impacts the immigration and location of nerve cells during the early stages of embryonic development by binding the signaling molecule Reln. It can also inhibit cell proliferation by interacting with tissue factor pathway inhibitor (TFPI). In addition, VLDLR plays a certain role in the invasion and metaptosis of tumor cells. And now, VLDLR and the other members of LDLR are known as the multifunctional receptor like“Swiss army knife”. Nevertheless, the functional differences between the two VLDLR subtypes need to be further clarified, especially the distinctive biological function of typeⅡVLDLR lacking the o-linked sugar domain has not been illuminated.Studies about the distribution of these two VLDLR subtypes suggested that their distribution presents obvious tissue- and cell-specificity. Type I VLDLR is most highly expressed in heart, skeletal muscle and adipose tissue with active fatty acid metabolism, while type II VLDLR is predominant in non-muscle tissue, including kidney, spleen, adrenal gland, et al. Recent studies have shown that the two VLDLR subtypes are associated with the differentiation and development of tissues and cells. The expression pattern of the two VLDLR subtypes can be changed during embryonic development of chicken and human brain. The type II VLDLR has been found to be the major receptor expressed in early phase of embryonic or fetal brain development, whereas type I VLDLR is mainly present in adult tissues. Other reports indicate that some tumor tissues and cells also express two VLDLR subtypes with inhomogeneity. The expression of the type II VLDLR increases obviously in poorly differentiated adenocarcinomas. Our previous study has also found that the type II VLDLR is mainly expressed in poorly- or moderately- differentiated human gastric adenocarcinoma cell lines, but its expression is relatively low or even can not be detected in well differentiated human gastric adenocarcinoma cell lines. Our recent studies also found that the expression of type II VLDLR is higher in uterine cervix cancer tissues than that in adjacent tissues. Type I VLDLR is the major receptor in senile plaques of Alzheimer diseased brain and type II VLDLR in congestive fibrotic spleen disappeared from the patients with liver cirrhosis. Genomic loss and epigenetic silencing of very-low-density lipoprotein receptor involved in gastric carcinogenesis and the O-linked sugar domain of VLDLR was demonstrated to relate with cell growth inhibition. These studies suggest that the type II VLDLR activities may be related to certain cellular functions other than its involvement in lipoprotein metabolism.Tissue factor pathway inhibitor (TFPI) and urokinase-type plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex are the ligands of VLDLR, which affect different cell function through VLDLR. But whether VLDLR affect cellular function via the expression variability of two VLDLR subtypes is still unknown.Thus, we explored the expression and function of type II VLDLR during the induction of human gastric adenocarcinoma cell line SGC7901 and in cells treated with two ligands of VLDLR to explore the relationship of type II VLDLR with cell function. To investigate the relationship between the expression variability of two VLDLR subtypes and cellular functions during the induction of SGC7901 cells, we use all-trans retinoic acid (ATRA) to induce SGC7901 differentiation and phorbol-12-myristate-13-acetate (PMA) to induce a change of differentiation to relatively lower. The mRNA expression of Telomerase reverse transcriptase (TERT) acts as a marker to identify the extent of cellular differentiation. The expression of two subtypes of VLDLR after treatment was detected by western blotting. The cells became well differentiated when induced by ATRA, accompanied by decrease in expression of type II VLDLR and gradually attenuated cell proliferation and migration. However, the cells became poorly differentiated when induced by PMA. These cells had increased receptor expression, and enhanced cell proliferation and migration. Our data indicate that the increased expression or the activity of type II VLDLR may be associated with the poor differentiation, and the enhanced proliferation and migration of the cells. Then, in order to further understand the expression of two VLDLR subtypes and the cellular function, we use two VLDLR ligands, uPA-PAI-1 complex and TFPI, which can affect cell proliferation and migration, to incubate with SGC7901 cells. In this study, we showed that cell proliferation and migration were inhibited by TFPI, but promoted by uPA-PAI-1 complex. In addition, we also demonstrated that TFPI treatment caused a decrease, but uPA-PAI-1 complex caused an increase, in the expression of type II VLDLR, suggesting that increasing type II VLDLR activity might be associated with augmenting cell proliferation and migration. In conclusion, the expression of type II VLDLR had a general phenomenon during the differentiation of cancer cells: the expression of type II VLDLR increased in lowly-differentiated cells with high proliferation and migration, but it decreased in highly-differentiated cells with low proliferation and migration. These indicate that the increased expression or the activity of type II VLDLR may be associated with the poor differentiation, and the enhanced proliferation and migration of the cells.It was reported that during the development of nervous tissue, VLDLR mediates Reelin-Dab1 signal pathway, modulates tau phosphorylation through glycogen synthase kinase-3beta cascade and affects tissue remodeling and cell migration. Recent study indicates that VLDLR is a negative regulator of the wnt signaling pathway. These studies suggest that VLDLR may play an important role in wnt signal pathway. In addition VLDLR can bind with different ligands relative with proliferation, regulate different signal pathway, and affect cell function. The binding of VLDLR and urokinase-type plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex can sustain the phosphorylation of extracellular signal-regulates kinase (ERK) to promote cell proliferation and migration. But VLDLR binding with TFPI can inhibit cell proliferation through activating p38 signal pathway. The two ligands appear to have quite different effects on cell function through VLDLR. It was reported that the activation of ERK can induce the expression ofβ-catenin, which promote the transcription of certain target genes inclding matrix metalloproteinase (MMPs). These studies suggest that the effect of VLDLR on cell function may be related with mitogen-activated protein kinases (MAPK) signal pathway and wnt signal. But it was unclear whether the two VLDLR subtypes were regulated differently and affected cell function through dinstinct signal pathway.It was speculated that the role of type II VLDLR may be related with MAPK and wnt signal pathway. So we observe the possible signal pathway involved in the role of type II VLDLR. Our results indicated that the well differentiated cells induced by ATRA and the cells treated with TFPI with a significant decrease in type II VLDLR expression accompanied by a gradually attenuatedβ-catenin and MMP-2 and MMP-9 expression, but in the poorly differentiated cells induced by PMA and in the cells treated with uPA-PAI-1 complex with an increase in type II VLDLR expression showed an increase inβ-catenin and MMP-2 and MMP-9 expression. These suggested that the role of type II VLDLR could be related with the aggregation of intracellularβ-catenin, which promotes some specific target genes transcription.In our study, uPA-PAI-1 complex can rapidly activate the ERK phosphorylation of SGC7901 cells after 5 min incubation and it can sustain for at least 30 min. The phosphorylation of ERK1 can last for 1 h obviously. Thise was agreed with previous study. But TFPI can inhibit the phosphorylation of ERK. Studies about LDLR found that stress-activated p38 MAPK regulates LDL receptor expression via negatively modulation of p42/44 MAPK cascade. So it was speculated that TFPI inhibited ERK through activating p38 MAPK. These results suggest that the ligand promoting cell proliferation and migration can activate ERK through type II VLDLR; but the ligand inhibiting cell proliferation and migration can reduce the phosphorylation of ERK through type I VLDLR. Our studies suggested that the role of type II VLDLR on cell function may be related with the activation of ERK, then induce the aggregation ofβ-catenin, promoting the transcription of some specific down-stream genes to affect cell function. These suggested that type II VLDLR may play an important role in promoting cell proliferation and migration, and inhibiting cell differentiation.In conclusion, type II VLDLR may bind and internalize specific ligand, mediate the relative signal pathway, and affect cell function. Our study revealed the relationship between type II VLDLR and cellular function and the possible related signal pathway, which provided profound views of VLDLR function and new cognition of lipoprotein receptor as a cellular Swiss army knife.更多还原