1. GEP Acts A Novel Mediator In Controlling Skeletal Muscle Differentiation 2. ADAMTS-12: A Novel Mediator Of Chondrogenesis

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.