【作者】 王大伟；

【导师】 金星； 刘传聚；

【作者基本信息】 山东大学 ， 外科学， 2009， 博士

【摘要】 GEP(granulin-epithelin precursor)蛋白是一种自分泌生长因子,也被称为progranulin,proepithelin,acrogranin或者PC细胞衍生生长因子,是一个68.5-kDa大小的分泌性生长因子。它的序列高度保守,几乎在所有真核生物中均表达。该蛋白高度糖基化,在SDS-PAGE上呈现为分子量大小约90-kDa。结构上,它属于生长因子家族。GEP可以以完整蛋白形式分泌,或者被蛋白水解,以多肽的形式分泌,即组成GEP蛋白的多肽片段一颗粒体蛋白(granulin)。GEP蛋白作为一种具有较大分子量的自分泌生长因子,参与多种生理性和病理性过程。GEP蛋白在代谢较快的上皮细胞,免疫系统的各种细胞和神经细胞中丰富表达。同时,在人类几种癌组织中也有报道GEP蛋白高度表达,有理由相信,GEP蛋白会导致乳腺癌,透明细胞肾癌,侵袭性卵巢癌,成胶质细胞瘤,脂肪畸胎瘤,多发性骨髓瘤和骨肉瘤。同时,越来越多的证据表明GEP蛋白也参与分化、发育和病理过程中的调节。本文主要研究GEP蛋白在骨骼肌分化中的作用和机制。骨骼肌分化过程是受一些转录因子所调控的(成肌因子家族),包括MyoD,Myf5,myogenin和MRF4。成肌因子在肌分化过程中起着重要的作用,如果在任何非成肌细胞(例如10T1/2成纤维细胞)中异位表达这些成肌因子,将会导致多种肌分化基因的表达,促使其向肌管分化。本研究以GEP蛋白为主要研究对象,系统的探讨了GEP蛋白在成肌细胞分化中的调节作用及机制,首次揭示了GEP蛋白在成肌细胞分化中的调节作用及其相应的分子机制:1.我们利用免疫组织化学染色的方法,显示GEP蛋白在胚胎肌肉组织中表达,应用细胞免疫荧光和实时荧光定量PCR方法,证明GEP在C2C12细胞向肌管分化过程中,GEP表达量是增高的,无论是mRNA水平还是蛋白水平。提示GEP在调节骨骼肌分化中起作用。2.应用细胞免疫荧光和实时荧光定量PCR方法,证明MyoD可以诱导内源性GEP的表达,无论在转录水平还是蛋白水平都增加。3.通过对GEP基因核酸序列的分析,发现其转录调控区有5个潜在的MyoD结合位点,我们使用了4个GEP特异性报告基因质粒,含有不同数量的MyoD结合位点,共转染上述报告基因与MyoD的真核表达质粒,通过检测荧光素酶表达活性研究MyoD对于GEP表达的影响。结果显示,MyoD可以激活GEP特异报告基因,MyoD在成肌细胞分化过程中具有调节GEP转录的功能。4.应用点突变技术,我们在报告基因271GEP-luc中GEP的转录调控区引入了MyoD结合位点的点突变,结果发现MyoD失去激活作用,同时应用凝胶电泳迁移实验及染色质免疫共沉淀,证明在成肌细胞分化过程中MyoD对GEP基因转录调控的作用通过MyoD直接结合到GEP基因转录调控区的MyoD结合位点上实现。5.我们研究了GEP过表达与低水平对成骨细胞分化的影响。结果显示,在成肌细胞分化过程中,高水平GEP能够抑制成肌细胞的分化,而在低GEP的细胞环境中,分化被促进了。6.为了揭示GEP抑制骨骼肌分化的分子机制,我们进一步研究了GEP在肌分化过程中,对成肌因子家族的作用。实验证明GEP在成肌细胞分化过程中抑制MyoD、Myf5、Myogenin、MHC基因的表达。7.JunB可以抑制骨骼肌分化。我们在肌分化的早期,应用实时荧光定量PCR,免疫印迹和细胞免疫荧光检测GEP对JunB表达的调控,发现GEP诱导JunB的表达。成功构建JunB表达质粒及JunB siRNA质粒,通过检测荧光素酶表达活性及MHC的表达证明GEP抑制成肌细胞分化的作用部分依赖于JunB。本研究针对骨骼肌发生中的分子调节,首次阐述了GEP蛋白抑制骨骼肌分化的作用和分子机制,进一步揭示骨骼肌生长与修复的机理,为提出更多的治疗肌肉疾病、肌肉创伤修复的方法奠定生物学基础。金属蛋白酶ADAMTS-12属于ADAMTS家族(含TSP结构的去整合素金属蛋白酶，a disintcgrin and metalloproteas~with thromospondin motifs，ADAMTS)，其对降解软骨细胞外基质蛋白和关节炎具有非常重要的作用。ADAMTS家族含有分泌性锌指蛋白，并且有较严谨的分子结构，至少包括一个thrombospondinmotifs重复结构。这个家族具有重要的功能，ADAMTS-1、ADAMTS-4、ADAMTS-5、ADAMTS-8、ADAMTS-9、ADAMTS-1 6和ADAMTS-1 8可以降解多聚蛋白糖，ADAMTS-5在小鼠关节炎模型中蛋白多聚糖的丢失上起着重要的作用。ADAMTS-2、ADAMTS-3和ADAMTS-14胶原前肽。ADAMTS-2突变会导致皮肤脆裂症，一种严重的皮肤遗传病。ADAMTS-13是VW因子清除蛋白酶，它的突变会导致遗传性的致命疾病，血小板减少性紫癜。ADAMTS-7和ADAMTS-12具有相同的结构域组成，构成ADAMTS家族的亚型。我们最初报道ADAMTS-7和ADAMTS-12直接结合降解软骨寡聚基质蛋白(cartilageoligomcric matrix protein，COMP))，一种软骨最主要的非胶原组成物。COMP是由五个l 10-kDa的多结构域糖蛋白亚基，通过二硫键构成一个524-kDa的复合体。COMP基因突变和常染色体显性肢断短小症…-假性软骨发育不全和多发性骨骺发育不良…-的发生有关。在膝关节损伤、创伤、原发性骨性关节炎(osteoarthritis，OA)和风湿性关节炎(rheumatoid arthritis，RA)的患者软骨、关节滑液和血清中，都可以检测到COMP的片段。我们知道只要能够抑制降解软骨的蛋白酶类就可以滞后或阻止疾病的进展，因此有人从病理生理学或治疗学的观点上提出隔离蛋白酶或使用抑制剂来抑制病情的进展。研究发现，纯化的COMP蛋白在体外可以被几种基质蛋白酶(matrix metalloproteinases，MMPs)所降解，包括间质胶原酶-1(interstitial collagenase-1，MMP-1)、胶原蛋白酶-3(collagenase-3，MMP-13)、基质溶解素-1(stromelysin-1，MMP-3)、明胶酶B(gelatinase-B，MMP-9)、MMP·19和釉质溶解素(enamelysin，MMP-20)。另外，ADAMTS家族中的ADAMTS-4被发现也具有体外酶切COMP的功能。然而我们体外酶切试验中所应用的蛋白酶和底物的浓度，都比人体生理、病理条件下的浓度要高。虽然体内COMP的量和ADAMTS的水平之间没有相关的数据支持他们的关联性，但是很有可能COMP的降解正是由这些金属蛋白酶调节的，至少部分受他们调节。另外，我们研究发现a-2-巨球蛋白可以抑制COMP蛋白的降解。最近还有研究发现ADAMTS-12也能够降解软骨聚集蛋白多糖。研究发现，在软骨分化过程中ADAMTS-12的表达量显著增高，意味着在骨骼发育中ADAMTS-12的表达量在时间、空间上都有变化。ADAMTS-12蛋白不但在体外软骨分化中被诱导表达，研究发现在体内的生长板软骨细胞中同样可以发现ADAMTS-12的表达增高。ADAMTS-12能够抑制软骨细胞的分化及软骨成骨，此抑制分化的功能是依赖于ADAMTS-12的蛋白酶活性的。ADAMTS-12的cysteine-fich结构域对于ADAMTS-12与细胞外基质相互作用和细胞膜表面定位是必不可少的，C一末端的4个TSP结构域对于ADAMTS-12的蛋白水解酶活性和抑制软骨细胞分化功能也是必需的。因此，ADAMTS-12对于软骨细胞分化和软骨成骨起负调控功能，这种抑制功能严格地依赖于ADAMTS-12的酶切活性。而当ADAMTS-12发生点突变后，则完全丧失了酶切活性-同时也丧失了对软骨细胞的抑制作用。ADAMTS-12由多个功能性亚基构成，包括：ADAMTS-12包括一个锌离子催化结构域和其他几个非催化结构域，包括：1个disintegrin结构域、1个thrombospondin结构域、1个cystdne-dch结构域(CRD)、1个spaccr-I结构域、3个thrombospondin motifs、1个space~-2结构域和C-末端4个thrombospondinmotifs。研究发现C-末端的4个thrombospondin motifs具有结合底物的功能，如结合COMP，而ADAMTS-12的其他功能结构域的生物学功能却还都不清楚。为了揭示各个结构域的功能，我们构建了一系列的ADAMTS-12的C-末端缺失突变体，发现ADAMTS-12的cysteine-rich结构域对于ADAMTS-12与软骨细胞外基质相互作用和细胞膜表面定位是必不可少的。ADAMTS-12的酶切活性受到非催化亚基的调控，尤其是C-末端的4个thrombospondin motifs和抑制性spacer-2结构域。另外，ADAMTS-12的抑制软骨分化功能同样也受到非催化亚基的调控，尤其是C-末端的4个thrombospondin motifs重复结构。本文研究发现ADAMTS-12作为软骨分化的重要负调控因子，影响软骨细胞分化和软骨内成骨。这种抑制软骨分化的功能严格地依赖于ADAMTS-12的酶切活性，而当ADAMTS-12发生点突变后，则完全丧失了酶切活性，同时也丧失了对软骨细胞的抑制作用。ADAMTS—12的酶切活性受到非催化亚基的调控，尤其是C。末端的4个thrombospondin motifs和抑制性spac~-2结构域。另外，ADAMTS-12的抑制软骨分化功能同样也受到非催化亚基的调控，尤其是C-术端的4个thrombospondin motifs重复结构。更多还原

【Abstract】 Granulin-epithelin precursor(GEP),an autocrine growth factor,also referred to as progranulin,proepithelin,PC cell derived growth factor,or acrogranin,is a 68.5-kDa secreted growth factor.It is highly conserved and ubiquitously expressed in eukaryotes.It is heavily glycosylated and appears as an～90-kDa protein on SDS-PAGE.Structurally,it belongs to one of the well established growth factor families.GEP is secreted in an intact form or undergoes proteolysis,eading to the release of its constituent peptides,the granulins.GEP itself is a secreted growth factor with high molecular weight that is involved in various biological and pathological processes.GEP is abundantly expressed in rapidly cycling epithelial cells,in cells of the immune system,and in neurons.High levels of GEP expression have been reported from several human cancers and are believed to contribute to tumorigenesisin breast cancer,clear cell renal carcinoma,invasive ovarian arcinoma, glioblastoma,adipocytic teratoma,multiple myeloma,and osteosarcoma.Increasing evidence has also implicated GEP in the regulation of differentiation,development, and pathological processes.These studies are mainly about the role of GEP in skeletal muscle differentiation and mechanism of regulating skeletal muscle differentiation.Myoblast differentiation is coordinated by a family of muscle-specific transcription factors(myogenic factors) that includes MyoD,Myf5,myogenin and MRF4.Ectopic expression of any of the myogenic factors in some nonmyogenic cell types(e.g.,10T1/2 fibroblasts) results in increases in the expression of various muscle differentiation genes and possibly in the fusion of the myoblasts to form myotubes.Our research takes GEP as the main object. We study the role of GEP during muscle differentiation and the mechanism of GEP regulating muscle differentiation.We firstly report that the effectiveness and molecular mechanism of GEP during skeletal muscle differentiation:1.To test the expression of GEP in muscle tissue,18-day embryo muscle was examined by immunohistochemistry.It shows that GEP is expressed in muscle tissue. We determined the endogenous GEP expression through real-time PCR and immunofluorescent cell staining on a time course of differentiation with C2C12 cells. During differentiation,GEP increases not only at mRNA leve but also at protein level. These data suggest that GEP expression is coordinately controlled with the process of myogenic differentiation.2.We determined the effect of MyoD on GEP expression through real-time PCR and immunofluorescent cell staining.It shows that MyoD induces endogenous GEP mRNA and protein expression.3.We analysis the transcriptional control region of GEP gene and find A 1.9-kb segment from the 5’-flanking region of the GEP gene contains at least five MyoD-specific sequences.Four reporter gene plasmids were used in which segments with MyoD-specific sequences.Different quantities of MyoD expression plasmid, GEP-specific reporter gene plasmids and PCMV-β-gal plasmid(internal control) were cotrnasfected into 10T1/2 cells.We found that luciferase gene was activated and expressed.These data shows that MyoD can drive the expression of GEP-specific reporter genes.4.GEP reporter plasmid -271GEP-luc was mutated 3 base pair.MyoD protein lost the ability of activating luciferase gene.It shows that MyoD Activates GEP-Specific Reporter Genes directly,depending on MyoD-Specific Binding Sequences in the 5’-Flanking Region of the GEP Gene Driving the Expression of GEP.We also use EMSA and ChIP to establish the sequence-specific binding of MyoD to the MyoD-specific sequences in the 5’-flanking region of GEP,MyoD activates GEP gene directly.5.We studied the Effects of Overexprssion and Knockdown of GEP on Myoblast Cells Differentiation.These data shows that GEP inhibits myotube information. SiRNA-targeting GEP stimulats myotube information.6.Differentiation of mhyoblasts is regulated by a family of muscle-specific transcription factors such as MyoD,Myf5,Myogenin and MRF4.To investigate the molecular mechanism of GEP inhibiting muscle differentiation,we used real-time PCR and western blotting to detect the expression of MyoD,Myf5,Myogenin and MHC.The data shows that GEP inhibits the expression of MyoD,Myf5,Myogenin and MHC during muscle differentiation.7.It was reported that JunB can inhibit muscle differentiation.We use real-time PCR, Western blot and cell immunofluorescent cell staining to detect the change of JunB regulated by GEP at early stage of muscle differentiaton.GEP induces the expression of JunB.We construct JunB expression plasmid and pSuser-JunB plasmid and used luciferase and real-time PCR detecting MHC mRNA expression.It was found that GEP inhibits myogenesis partly through JunB.In this study,we fist report that GEP inhibit muscle differentiation and the molecular mechanism of GEP regulating muscle differentiation.It reveals the mechanism of skeletal muscle growth and repair.It establish the biological basis of raising more methods of curing muscle disease and repairing muscle trauma. ADAMTS-12, a metalloproteinase that belongs to ADAMTS family, is important for the degradation of cartilage extracellular matrix proteins and its level is significantly levated in arthritis. The ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) family consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least one thrombospondin type I repeat. Important functions have been established for several members of the ADAMTS family. ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, ADAMTS-16 and ADAMTS-18 degrade aggrecan and ADAMTS-5 plays a primary role in aggrecan loss in murine arthritis. ADAMTS-2, ADAMTS-3, and ADAMTS-14 are procollagen N-propeptidases. ADAMTS-2 mutations cause dermatosparaxis, an inherited disorder characterized by severe skin fragility. ADAMTS-13 is a von Willebrand factor-cleaving protease, and its mutations lead to heritable life-threatening thrombocytopenic purpura. ADAMTS-7 and ADAMTS-12 share the same domain organization and form a subgroup with unique properties within ADAMTS family. Our previously reports demonstrate that ADAMTS-7 and ADAMTS-12 directly associate with and degrade cartilage oligomeric matrix protein, COMP), a prominent noncollagenous component of cartilage. COMP is a 524-kDa disulfide-bonded, multidomain glycoprotein composed of five 110-kDa subunits. Mutations in the human COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia, which are autosomal-dominant forms of short-limb dwarfism. Fragments of COMP have been detected in the diseased cartilage, synovial fluid, and serum of patients with knee iniuries. nost-traumatic. nrimarv osteoarthritis fOA^ and rheumatoid arthritis (H.AY Since the inhibition of cartilage degradative enzymes should slow or block disease progression, the isolation of these enzymes and their inhibitors is of great interest from both a pathophysiological and a therapeutic standpoint. Purified COMP has been reported to be digested by several matrix metalloproteinases (MMPs) in vitro, including interstitial collagenase-1 (MMP-1), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), gelatinase-B (MMP-9)[l], MMP-19, and enamelysin (MMP-20). In addition, a member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family ADAMTS-4, was reported to cleave purified COMP in vitro. All these assays, however, were performed using an in vitro digestion system with higher concentrations of enzymes and substrates than are found in physiological and pathological conditions. It is likely that COMP degradation in vivo is mediated, at least in part, by these metalloproteinases, although no relationship between COMP degradation and ADAMTS levels has been found to date. In addition, Alpha-2-Macroglobulin inhibits their degradation of COMP. Recent report revealed that ADAMTS-12 also degraded aggrecan.We found that ADAMTS-12 is strongly upregulated during chondrogenesis and demonstrates the temporal and spatial expression pattern during skeletal development. ADAMTS-12 protein was highly induced in the course of chondrogenesis in vitro and also demonstrated prominent expression in the growth plate chondrocytes in vivo. ADAMTS-12 potently inhibits chondrocyte differentiation and endochondral bone formation, and this inhibition depends on the proteolytic activity of ADAMTS-12. The cysteine-rich domain of ADAMTS-12 is required for its interaction with extracellular matrix and cell surface localization, and the C-terminal four thrombospondin motifs is necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. ADAMTS-12 appears to be a potent negative regulator of chondrocyte differentiation and endochondral bone growth, and its inhibitory activities strictly depend on its enzymatic activities, since its point mutant lacking enzymatic activity completely lost these inhibitions.ADAMTS-12 is composed of multiple functional domains, including a prodomain, a catalytic domain, a disintegrin domain, a thrombospondin motif, a cysteine-rich domain, a spacer-1 domain, three thrombospondin motifs, a spacer-2 domain, and a C-terminal four thrombospondin motifs. In addition to its C-terminal four thrombospondin motifs known to bind to substrates, including COMP, the role of individual domain in regulating the biochemical properties of ADAMTS-12 remains unknown. To address this issue, we generated series of domain deletion mutants of ADAMTS-12 and found that The CRD is required for ADAMTS-12 binding to the cell surface and ECM in chondrocytes. The enzymatic activities of ADAMTS-12 are precisely regulated by its non-catalytic domains, specially its substrate-capturing C-terminal four thrombospondin motifs and an inhibitory spacer-2 domain. In addition, the ADAMTS-12-mediated chondrocyte differentiation are also precisely regulated by its non-catalytic domains, specially its substrate-capturing C-terminal four thrombospondin motifs.The study in this paper provides novel insights into the role of ADAMTS-12, a novel negative mediator in chondrogenesis, in regulating chondrocyte differentiation and endochondral bone formation. Its inhibitory activities strictly depend on its enzymatic activities, since its point mutant lacking enzymatic activity completely lost these inhibitions. The enzymatic activities of ADAMTS-12 are precisely regulated by its non-catalytic domains, specially its substrate-capturing C-terminal four thrombospondin motifs and an inhibitory spacer-2 domain. In addition, the ADAMTS-12-mediated chondrocyte differentiation are also precisely regulated by its non-catalytic domains, specially its substrate-capturing C-terminal four thrombospondin motifs.更多还原