Takashi Nishida

Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture.

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs...

Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro

Connective tissue growth factor/hypertrophic chondrocyte‐specific gene product Hcs24 (CTGF/Hcs24) promotes the proliferation and differentiation of chondrocytes and endothelial cells which are involved in endochondral ossification (Shimo et al., 1998, J Biochem 124:130–140; Shimo et al., 1999, J Biochem 126:137–145; Nakanishi et al., 2000, Endocrinology 141:264–273). To further clarify the role of CTGF/Hcs24 in endochondral ossification, here we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of osteoblastic cell lines in vitro. A binding study using 125I‐labeled recombinant CTGF/Hcs24 (rCTGF/Hcs24) disclosed two classes of specific binding sites on a human osteosarcoma cell line, Saos‐2. The apparent dissociation constant (Kd) value of each binding site was 17.2 and 391 nM, respectively. A cross‐linking study revealed the formation of 125I‐rCTGF/Hcs24‐receptor complex with an apparent molecular weight of 280 kDa. The intensity of 125I‐rCTGF/Hcs24‐receptor complex decreased on the addition of increasing concentrations of unlabeled rCTGF/Hcs24, but not platelet‐derived growth factor‐BB homodimer or basic fibroblast growth factor. These findings suggest that osteoblastic cells have specific receptor molecules for CTGF/Hcs24. rCTGF/Hcs24 promoted the proliferation of Saos‐2 cells and a mouse osteoblast cell line MC3T3‐E1 in a dose‐ and time‐dependent manner. rCTGF/Hcs24 also increased mRNA expression of type I collagen, alkaline phosphatase, osteopontin, and osteocalcin in both Saos‐2 cells and MC3T3‐E1 cells. Moreover, rCTGF/Hcs24 increased alkaline phosphatase activity in both cells. It also stimulated collagen synthesis in MC3T3‐E1 cells. Furthermore, rCTGF/Hcs24 stimulated the matrix mineralization on MC3T3‐E1 cells and its stimulatory effect was comparable to that of bone morphogenetic protein‐2. These findings indicate that CTGF/Hcs24 is a novel, potent stimulator for the proliferation and differentiation of osteoblasts in addition to chondrocytes and endothelial cells. Because of these functions, we are re‐defining CTGF/Hcs24 as a major factor to promote endochondral ossification to be called “ecogenin: endochondral ossification genetic factor.” J. Cell. Physiol. 184:197–206, 2000.

CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, stimulates proliferation and differentiation, but not hypertrophy of cultured articular chondrocytes

We previously reported that connective tissue growth factor/hypertrophic chondrocyte‐specific gene product 24 (CTGF/Hcs24) stimulated the proliferation and differentiation of rabbit growth cartilage (RGC) cells in vitro. In this study, we investigated the effects of CTGF/Hcs24 on the proliferation and differentiation of rabbit articular cartilage (RAC) cells in vitro. RAC cells transduced by recombinant adenoviruses generating mRNA for CTGF/Hcs24 synthesized more proteoglycan than the control cells. Also, treatment of RAC cells with recombinant CTGF/Hcs24 (rCTGF/Hcs24) increased DNA and proteoglycan syntheses in a dose‐dependent manner. Northern blot analysis revealed that the rCTGF/Hcs24 stimulated the gene expression of type II collagen and aggrecan core protein, which are markers of chondrocyte maturation, in both RGC and RAC cells. However, the gene expression of type X collagen, a marker of hypertrophic chondrocytes, was stimulated by rCTGF/Hcs24 only in RGC cells, but not in RAC cells. Oppositely, gene expression of tenascin‐C, a marker of articular chondrocytes, was stimulated by rCTGF/Hcs24 in RAC cells, but not in RGC cells. Moreover, rCTGF/Hcs24 effectively increased both alkaline phosphatase (ALPase) activity and matrix calcification of RGC cells, but not of RAC cells. These results indicate that CTGF/Hcs24 promotes the proliferation and differentiation of articular chondrocytes, but does not promote their hypertrophy or calcification. Taken together, the data show that CTGF/Hcs24 is a direct growth and differentiation factor for articular cartilage, and suggest that it may be useful for the repair of articular cartilage.

Involvement of CTGF, a Hypertrophic Chondrocyte-Specific Gene Product, in Tumor Angiogenesis

Connective tissue growth factor (CTGF) is a potent secreted signaling factor which functions in multiple stages of angiogenesis. In the present study, we examined the role of CTGF in tumor angiogenesis and made the following observations: (1) Histological analysis of human breast cancer (MDA231) cell and human fibrosarcoma (HT1080) cell xenografts in BALB/c nude mice showed a high level of neovascularization. Human squamous cell carcinoma (A431) xenografts induced only a low level of neovascularization. (2) CTGF mRNA was strongly expressed in MDA231 and in HT1080 cells in vivo and in vitro, but not in A431 cells. (3) CTGF protein was markedly produced in MDA231 cells and HT1080 cells and secreted into culture medium, and its production was greater during phases of growth rather than confluency. (4) Production of CTGF in bovine aorta endothelial cells was induced by CTGF, VEGF, bFGF and TGF-β. (5) Neovascularization induced by HT1080 cells or MDA231 cells on chicken chorioallantoic membrane was suppressed in the presence of neutralizing CTGF-specific polyclonal antibody. These results suggest that CTGF regulates progression in tumor angiogenesis and the release or secretion of CTGF from tumor cells is essential for the angiogenesis.

Characterization of binding properties of urinary trypsin inhibitor to cell-associated binding sites on human chondrosarcoma cell line HCS-2/8.

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP40), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP45). Here we characterize binding properties of UTI-BPs·UTI complexes in the cells. In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP40 and UTI-BP45 bind125I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP40. Additional experiments, using various reagents to block binding of 125I-UTI and NG-UTI to the UTI-BP40 and UTI-BP45 confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP45. Analysis of binding of125I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP40 (which can bind NG-UTI), and the high affinity sites are the UTI-BP45. In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.