Molecular targeting of hepatocyte growth factor by an antagonist, NK4, in the treatment of rheumatoid arthritis

INTRODUCTION: Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice. METHODS: Arthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4(+ )T cells stimulated with allogeneic spleen cells. RESULTS: The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4(+ )T cells stimulated with allogeneic spleen cells. CONCLUSIONS: These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4(+ )T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA

Blocking c‐Met signaling enhances bone morphogenetic protein‐2‐induced osteoblast differentiation

We previously demonstrated that blocking hepatocyte growth factor (HGF) receptor/c‐Met signaling inhibited arthritis and articular bone destruction in mouse models of rheumatoid arthritis (RA). In the present study, we investigated the role of c‐Met signaling in osteoblast differentiation using the C2C12 myoblast cell line derived from murine satellite cells and the MC3T3‐E1 murine pre‐osteoblast cell line. Osteoblast differentiation was induced by treatment with bone morphogenetic protein (BMP)‐2 or osteoblast‐inducer reagent in the presence or absence of either HGF antagonist (NK4) or c‐Met inhibitor (SU11274). Osteoblast differentiation was confirmed by Runx2 expression, and alkaline phosphatase (ALP) and osteocalcin production by the cells. Production of ALP, osteocalcin and HGF was verified by enzyme‐linked immunosorbent assay. Runx2 expression was confirmed by reverse transcription‐PCR analysis. The phosphorylation status of ERK1/2, AKT, and Smads was determined by Western blot analysis. Both NK4 and SU11274 enhanced Runx2 expression, and ALP and osteocalcin production but suppressed HGF production in BMP‐2‐stimulated C2C12 cells. SU11274 also enhanced ALP and osteocalcin production in osteoblast‐inducer reagent‐stimulated MC3T3‐E1 cells. SU11274 inhibited ERK1/2 and AKT phosphorylation in HGF‐stimulated C2C12 cells. This result suggested that ERK and AKT were functional downstream of the c‐Met signaling pathway. However, both mitogen‐activated protein kinase/ERK kinase (MEK) and phosphatidylinositol 3‐kinase (PI3K) inhibitor suppressed osteocalcin and HGF production in BMP‐2‐stimulated C2C12 cells. Furthermore, SU11274, MEK, and PI3K inhibitor suppressed Smad phosphorylation in BMP‐2‐stimulated C2C12 cells. These results indicate that although the c‐Met‐MEK‐ERK‐Smad and c‐Met‐PI3K‐AKT‐Smad signaling pathways positively regulate osteoblast differentiation, c‐Met signaling negatively regulates osteoblast differentiation, independent of the MEK‐ERK‐Smad and PI3K‐AKT‐Smad pathways. Therefore, blocking c‐Met signaling might serve as a therapeutic strategy for the repair of destructed bone in patients with RA