【作者】 白晓卉；

【导师】 于修平； 刘传聚；

【作者基本信息】 山东大学 ， 病原生物学， 2009， 博士

【摘要】 金属蛋白酶ADAMTS-7和ADAMTS-12同属于ADAMTS家族(含TSP结构的去整合素金属蛋白酶,a disintegrin and metalloprotease with thromospondinmotifs,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thrombospondin motif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-16和ADAMTS-18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-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直接结合并降解软骨寡聚基质蛋白(cartilage oligomeric matrix protein,COMP),一种软骨最主要的非胶原组成物。另外,我们研究发现α-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。本文的研究发现,在软骨分化过程中ADAMTS-7和ADAMTS-12的表达量都显著增高,意味着在骨骼发育中ADAMTS-7和ADAMTS-12的表达量在时间、空间上都有变化。我们的研究重点关注ADAMTS-7和ADAMTS-12在软骨分化中的作用,以及涉及到的分子机制。我们研究发现ADAMTS-7和ADAMTS-12蛋白在软骨分化中被诱导表达。实时荧光定量PCR结果揭示在体外软骨分化的第5天,这两种蛋白的表达量还较低,但从第7天开始,他们的量成三倍增长,并在分化后期一直维持此高表达状态。本文中,我们首次发现ADAMTS-7和ADAMTS-12都能够抑制软骨细胞的分化及软骨成骨。我们研究发现ADAMTS-7对软骨细胞的分化及软骨成骨的抑制功能是依赖于ADAMTS-7的蛋白酶活性的。ADAMTS-7的cysteine-rich结构域对于ADAMTS-7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-末端的4个thromospondin motif结构域对于ADAMTS-7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-7对于软骨细胞分化和软骨成骨,是一个潜在的负调控因子,这种抑制功能严格地依赖于ADAMTS-7的酶切活性。而当ADAMTS-7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-7由多个功能性亚基构成,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cysteine-rich结构域(CRD)、1个spacer-1结构域、3个thrombospondin motifs、1个spacer-2结构域和C-末端4个thrombospondin motifs。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-7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-7的C-末端缺失突变体,发现ADAMTS-7的cysteine-rich结构域对于ADAMTS-7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。然而有趣的是,ADAMTS-7及一系列C-末端缺失突变体蛋白在Cos-7细胞中的检测结果显示,cysteine-rich结构域对ADAMTS-7蛋白定位的影响,是有细胞类型特异性的。另外,ADAMTS-7的酶切活性同样也受到非催化亚基的调控,尤其是C-末端的4个thrombospondinmotifs和抑制性spacer-2结构域。ADAMTS-7和ADAMTS-12是PTHrP重要的靶分子,原因如下:(1)PTHrP诱导ADAMTS-7和ADAMTS-12的表达,(2)在PTHrP基因敲除小鼠的软骨生长板上几乎检测不到ADAMTS-7和ADAMTS-12的表达,(3)沉默ADAMTS-7/ADAMTS-12或用特异性抗体封闭ADAMTS-7/ADAMTS-12的活性,则几乎阻断了PTHrP介导的抑制软骨细胞肥大化和软骨成骨的生长。研究发现ADAMTS-7和一个新的软骨生长因子GEP(Granulin-epithelin precursor)相互作用。骨骺生长板内的软骨内骨化过程,涉及多个信号传导途径。我们的研究证明:1)过表达ADAMTS-7和ADAMTS-12诱导PTHrP,抑制IHH;2)在体外软骨分化过程中,PTHrP促进ADAMTS-7和ADAMTS-12的表达:3)在小鼠生长板软骨细胞中,ADAMTS-7和ADAMTS-12的表达依赖于PTHrP信号途径。这些研究结果揭示在软骨分化过程中,ADAMTS-7、ADAMTS-12和PTHrP信号途径之间存在一个正反馈通路。另外,ADAMTS-7、ADAMTS-12是PTHrP的下游靶分子,基于以下原因:1)PTHrP抑制软骨分化过程中Col X的表达,然而当通过特异性干扰RNA抑制ADAMTS-7/ADAMTS-12或用抗ADAMTS-7/ADAMTS-12的抗体封闭后,PTHrP的抑制作用则消失了;再次表达ADAMTS-7/ADAMTS-12,则PTHrP的抑制作用又重新回复:2)ADAMTS-7/ADAMTS-12抗体的封闭,则完全中和了PTHrP所抑制的软骨细胞肥大化、矿化和骨生长。另外,研究发现PTHrP促进软骨细胞的增殖,ADAMTS-7发现也具有促进软骨细胞增殖的功能,而且此促细胞增殖作用主要依赖于C-末端的4个thrombospondin motifs结构域。另外ADAMTS-7可以作为GEP-转化酶,抵消GEP刺激诱导的软骨分化及软骨内成骨。总之,我们的试验结果证明,ADAMTS-7是PTHrP信号途径下游的非常重要的靶分子,负调控软骨内成骨过程;直接结合、转化GEP,从而失活软骨分化生长因子GEP的诱导分化作用。GEP是一种生长因子,参与组织重生、肿瘤形成和炎症过程。研究发现GEP与弹性蛋白酶结合,被弹性蛋白酶降解。弹性蛋白酶消化GEP的颗粒内连接子,产生微粒多肽;然而分泌性白细胞蛋白酶抑制因子(secretory leukocyte protease inhibitor,SLPI)直接结合到弹性蛋白酶或抑制颗粒多肽与蛋白酶的结合,继而封闭弹性蛋白酶的活性。最近研究揭示蛋白酶3也结合并降解GEP;蛋白酶3和弹性蛋白酶都通过降解抗炎症作用的GEP,从而达到促进嗜中性粒细胞依赖性的致炎症功能。本文中,我们提供了足够的试验证据表明GEP和金属蛋白酶ADAMTS-7在软骨细胞内相互作用,ADAMTS-7可以将GEP转化为小分子片段;另外,GEP能具有促进软骨细胞分化和软骨成骨,而ADAMTS-7很可能是通过失活GEP的活性而抑制软骨分化及软骨成骨的。我们以往研究发现:1)ADAMTS-7结合并降解COMP,2)COMP和GEP相互作用,促进GEP刺激的软骨分化功能。结合现在我们的结果ADAMTS-7和GEP相互作用,我们发现ADAMTS-7、GEP和COMP形成一个调节软骨细胞功能的蛋白质-蛋白质相互作用网。然而ADAMTS-7、生长因子GEP和细胞外基质COMP之间是如何相互作用,如何调控软骨细胞分化和软骨成骨的。本文研究发现ADAMTS-7和ADAMTS-12是PTHrP调控通路中的新调控因子,同时也可调控软骨细胞分化和软骨内成骨。本文对ADAMTS-7、ADAMTS-12负调节软骨分化的机制进行了初步探讨,例如金属蛋白酶ADAMTS-7和促软骨分化作用的生长因子GEP相结合,通过转化GEP成片段从而抑制GEP刺激的软骨分化作用。另外,本文也为软骨关节炎疾病提供了潜在的靶分子。更多还原

【Abstract】 ADAMTS-7 and ADAMTS-12,metalloproteinases that belongs to ADAMTS family, are important for the degradation of cartilage extracellular matrix proteins and their levels are significantly levated in arthritis.The ADAMTS(a disintegrin and metalloproteinase with thrombospondin type 1 motifs) family consists of secreted zinc metalloproteinases with a precisely ordered modular organization that includes at least one thrombospondin typeⅠrepeat.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-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.In addition,Alpha-2-Macroglobulin inhibits their degradation of COMP. Recent report revealed that ADAMTS-12 also degraded aggrecan.In this study we report that ADAMTS-7 and ADAMTS-12 are strongly upregulated during chondrogenesis and demonstrates the temporal and spatial expression pattern during skeletal development.The current study focused on the roles of ADAMTS-7 and ADAMTS-12 in chondrogenesis as well as the molecular mechanism involved. ADAMTS-7 and ADAMTS-12 proteins were highly induced in the course of chondrogenesis.Real-time PCR for measurements of ADAMTS-7 and ADAMTS-12 showed that the level of ADAMTS-7 and ADAMTS-12 mRNAs were relatively low until day 5,and at day 7 it tripled and thereafter remained at high levels during the late differential stage.In this study we found ADAMTS-7 and ADAMTS-12 inhibited chondrocyte differentiation and endochondral bone formation.ADAMTS-7 potently inhibits chondrocyte differentiation and endochondral bone formation,and this inhibition depends on the proteolytic activity of ADAMTS-7.The cysteine-rich domain of ADAMTS-7 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-7 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-7 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-7 remains unknown.To address this issue,we generated series of domain deletion mutants of ADAMTS-7 and found that The CRD is required for ADAMTS-7 binding to the cell surface and ECM in chondrocytes.Interestingly,this CRD-dependent localization appears to be cell type-specific,since ADAMTS-7 and its deletion mutants are predominately localized in the cytoplasm of Cos-7 cells.In addition,the enzymatic activities of ADAMTS-7 are also precisely regulated by its non-catalytic domains, specially its substrate-capturing C-terminal four thrombospondin motifs and an inhibitory spacer-2 domain.ADAMTS-7 and ADAMTS-12 are important targets of PTHrP signaling,since(1) PTHrP induces ADAMTS-7 and ADAMTS-12,(2) ADAMTS-7 and ADAMTS-12 are hardly detectable in PTHrP-/- growth plate chondrocytes,and(3) knocking down ADAMTS-7/ADAMTS-12 expression or blocking ADAMTS-7/ADAMTS-12 activities almost abolishes PTHrP-mediated inhibition of chondrocyte hypertrophy and endochondral bone growth.ADAMTS-7 associates with Granulin-epithelin precursor(GEP),a novel chondrogenic growth factor.Multiple signaling pathways are involved in endochondral ossification in epiphyseal growth plate.Our studies demonstrating that 1) overexpressing ADAMTS-7 and ADAMTS-12 enhanced the expression of PTHrP whereas inhibited IHH,2) PTHrP induced ADAMTS-7 and ADAMTS-12 expression in the course of chondrogenesis in vitro,and 3) ADAMTS-7 and ADAMTS-12 expression strictly depend on PTHrP in the growth plate chondrocytes in mice.These findings suggest that there exists a positive feedback loop between ADAMTS-7,ADAMTS-12 and PTHrP signaling in the course of chondrogenesis.In addition,ADAMTS-7 and ADAMTS-12 appear to be a crucial downstream molecule of PTHrP based on the facts that 1) repression of ADAMTS-7/ADAMTS-12 via siRNA approach or blocking ADAMTS-7/ ADAMTS-12 activities using its blocking antibodies almost abolished PTHrP-mediated inhibition of Col X expression,but reexpression of ADAMTS-7/ ADAMTS-12 restored PTHrP action;and 2) ADAMTS-7/ADAMTS-12 blocking antibody totally neutralized the inhibition of chondrocyte hypertrophy,mineralization, and bone growth by PTHrP.Furthermore,similar to PTHrP that is known to stimulate chondrocyte proliferation,ADAMTS-7 was also found to increase chondrocyte proliferation and its stimulation on cell proliferation largely depends on its substrate-binding C-terminal four thrombospondin motifs.In addition,ADAMTS-7 acts as a new GEP-convertase and neutralizes GEP-stimulated endochondral bone formation.Collectively,these findings demonstrate that ADAMTS-7,an important downstream molecule of PTHrP signaling, negatively regulates endochondral bone formation via associating with and inactivating GEP chondrogenic growth factor.GEP is a growth factor implicated in tissue regeneration,tumorigenesis,and inflammation.GEP was previously shown to associate with and be processed by elastase.Elastase digests GEP exclusively in the intergranulin linkers,resulting in the generation of granulin peptides;whereas the secretory leukocyte protease inhibitor(SLPI) blocks this proteolysis either by directly binding to elastase or by sequestering granulin peptides from the enzyme.Recent report reveled that proteinase 3 associated with and processed GEP,and proteinase 3 and elastase enhance neutrophil-dependent inflammation by eliminating the anti-inflammatory activity of GEP.Here we present evidences showing that GEP associates with ADAMTS-7 metalloproteinase in chondrocytes,and ADAMTS-7 is able to convert GEP into its processed fragments;in addition,ADAMTS-7-inhibits chondrocyte differentiation and endochondral bone formation probably via inactivating chondrogenic activity of GEP.Our previous report showing that 1) ADAMTS-7 binds to and degrades COMP,and 2) COMP interacts with GEP and potentiates GEP-stimulated chondrocyte functions,together with present study revealing the interaction between ADAMTS-7 and GEP,indicate that ADAMTS-7, GEP and COMP form a protein-protein interaction network in regulating chondrocyte functions.It remains to determine how the interaction network among ADAMTS-7 metallproteinase,GEP growth factor and COMP extracellular matrix molecule acts in concert in regulating chondrocyte differentiation and endochondral ossification.The study in this paper provides novel insights into the role of ADAMTS-7 and ADAMTS-12,novel mediators in PTHrP pathway,in regulating chondrocyte differentiation and endochondral bone formation,and sheds light on the molecular mechanism by which ADAMTS-7 adversely regulates chondrogenesis,i.e. ADAMTS-7 metalloproteinase associates with GEP chondrogenic grow factor,and eliminates GEP-stimulated chondrogenesis via conversion of GEP into its processed fragments.In addition,this study also provides us with potential molecule targets for treatment of cartilage disorders and arthritic conditions.更多还原