Connective tissue growth factor/CCN family 2 is a pericellular protein whose cellular response is dependent upon the TGF β type I receptor

Connective tissue growth factor (CTGF), a member of the CCN family of proteins, is expressed in skeletal cells, and the ctgf null mutation leads to neonatal lethality due to defects in skeletal development. To define the function of CTGF in the postnatal skeleton, we created transgenic mice overexpressing CTGF under the control of the human osteocalcin promoter. CTGF transgenic female and male mice exhibited a significant decrease in bone mineral density, compared with wild-type littermate controls. Bone histomorphometry revealed that CTGF overexpression caused decreased trabecular bone volume due to impaired osteoblastic activity because mineral apposition and bone formation rates were decreased. Osteoblast and osteoclast number and bone resorption were not altered. Calvarial osteoblasts and stromal cells from CTGF transgenics displayed decreased alkaline phosphatase and osteocalcin mRNA levels and reduced bone morphogenetic protein (BMP) signaling mothers against decapentaplegic, Wnt/β-catenin, and IGF-I/Akt signaling. In conclusion, CTGF overexpression in vivo causes osteopenia, secondary to decreased bone formation, possibly by antagonizing BMP, Wnt, and IGF-I signaling and activity.

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N-terminal domains of CCN family 2/connective tissue growth factor bind to aggrecan

Biochemical Journal ◽

10.1042/bj20081991 ◽

2009 ◽

Vol 420 (3) ◽

pp. 413-420 ◽

Cited By ~ 43

Author(s):

Eriko Aoyama ◽

Takako Hattori ◽

Mitsuhiro Hoshijima ◽

Daisuke Araki ◽

Takashi Nishida ◽

...

Keyword(s):

Extracellular Matrix ◽

Growth Factor ◽

Connective Tissue ◽

Connective Tissue Growth Factor ◽

Binding Protein ◽

Direct Interaction ◽

Tissue Growth ◽

Yeast Two Hybrid ◽

Ccn Family ◽

Two Hybrid

CCN2/CTGF (CCN family 2/connective tissue growth factor) is a multi-cellular protein with a broad range of activities. It modulates many cellular functions, including proliferation, migration, adhesion and extracellular matrix production, and it is thus involved in many biological and pathological processes. In particular, CCN2/CTGF is essential for normal skeletal development. To identify CCN2/CTGF-interactive proteins capable of modulating its action in cartilage, we carried out a yeast two-hybrid screening using CCN2/CTGF peptide as a bait and a cDNA library from a chondrocytic cell line, HCS-2/8. In the present paper, we report the identification of aggrecan, which is a major proteoglycan of the extracellular matrix in cartilage, as a CCN2/CTGF-binding protein. Among the four domains of CCN2/CTGF, the IGFBP [IGF (insulin-like growth factor)-binding protein-like] and/or VWC (von Willebrand factor type C) domains had a direct interaction with aggrecan in a yeast two-hybrid assay. The results of a solid-phase-binding assay using aggrecan-coated plates also showed binding to recombinant CCN2/CTGF in a dose-dependent manner. rIGFBP (recombinant IGFBP) and rVWC (recombinant VWC) module peptides had stronger binding to aggrecan compared with rTSP1 (recombinant thrombospondin type 1 repeat) and rCT (recombinant C-terminal cystine knot) module peptides. SPR (surface plasmon resonance) analysis showed the direct interaction between the CCN2/CTGF and aggrecan, and ectopically overexpressed CCN2/CTGF and AgG3 (G3 domain of aggrecan) confirmed their binding In vivo. Indirect immunofluorescence analysis indicated that CCN2/CTGF was extracellularly co-localized with aggrecan on HCS-2/8 cells. The rIGFBP–rVWC peptide effectively enhanced the production and release of aggrecan compared with the rTSP–rCT peptide in chondrocytes. These results indicate that CCN2/CTGF binds to aggrecan through its N-terminal IGFBP and VWC modules, and this binding may be related to the CCN2/CTGF-enhanced production and secretion of aggrecan by chondrocytes.

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Role of Connective Tissue Growth Factor (CTGF) in Survival and Chemosensitivity of Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.2593.2593 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2593-2593

Author(s):

Hongbo Lu ◽

Venkata Lokesh Battula ◽

Yuexi Shi ◽

Richard B Lock ◽

Suzanne Spong ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Growth Factor ◽

Connective Tissue ◽

Signaling Pathways ◽

Connective Tissue Growth Factor ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Tissue Growth ◽

Ccn Family

Abstract Abstract 2593 Connective tissue growth factor (CTGF/CCN2) is a member of the CCN family of proteins involved in extracellular matrix production, tumor cell proliferation, adhesion, migration, and metastasis. Recent studies have shown that CTGF expression is elevated in 75% of acute lymphoblastic leukemia (Br J Haematol, 2007; 138(6):740–8), and that increased expression of CTGF is associated with inferior outcome in B-ALL (Blood, 2007; 109(7):3080–3). In this study, we characterized the functional role and downstream signaling pathways of CTGF in ALL cells. First, we utilized lentiviral shRNA to knock-down CTGF in RS4;11 and REH ALL cells expressing high levels of CTGF mRNA (479.3±37.2 and 57.3±5.9 copies per 100 copies of ABL1, respectively). Silencing of CTGF (CTGF-knockdown, CTGF-kd) resulted in significant suppression of leukemia cell growth (57% in RS4;11 and by 70% in REH) compared to control vector. CTGF knockdown moderately reduced adhesion of RS4;11 to fibronectin (27%±0.1%). In the in vitro culture system, CTGF knockdown significantly enhanced growth inhibition and apoptosis induction after 48 hour exposure to chemotherapy agents (annexinV(+): Vincristine 25.8±3.5%, Vincristine/CTGF-kd 42.6±2.8%; Dexamethasone 66.3±1.8%, Dexamethasone/CTGF-kd 99.3±0.6%; Methotrexate, 17.4±0.6%, Methotrexate/CTGF-kd 39.5±3.9). Analysis of signaling pathways showed that CTGF down-regulation inhibits Src phosphorylation at Tyr416. Remarkably, phosphorylation of AKT at Ser473, and of mTOR downstream targets S6 Ribosomal Protein and 4E-BP1 were significantly inhibited in CTGF-knockdown RS4;11 cells, concomitantly with upregulation of expression of AKT targets Bim and p27. No changes in the levels of apoptotic regulators cIAP1 and Bcl-xL were found. This data suggest that CTGF regulates growth and chemosensitivity of ALL cells through Src and AKT/mTOR signaling. We previously reported that an anti-CTGF monoclonal antibody significantly extended median survival of mice implanted with xenografts derived from a primary CTGF expressing ALL sample in NOD/SCID mice. We are now investigating the effects of combining anti-CTGF treatment with cytotoxic chemotherapy in this model. Blocking CTGF signaling may represent a useful adjunct to cytotoxic therapies in acute lymphoblastic leukemia. Disclosures: Spong: Fibrogen: Employment, Equity Ownership.

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