SPA0355, a thiourea analogue, inhibits inflammatory responses and joint destruction in fibroblast-like synoviocytes and mice with collagen-induced arthritis.

BACKGROUND AND PURPOSE: NF-κB has been implicated as a therapeutic target for the treatment of rheumatoid arthritis. We previously synthesized a thiourea analogue, SPA0355, which suppressed NF-κB activity. Here we have assessed the anti-inflammatory and anti-arthritic effects of SPA0355. EXPERIMENTAL APPROACH: We evaluated the effects of SPA0355 on human rheumatoid fibroblast-like synoviocytes in vitro and on collagen-induced arthritis (CIA) in mice in vivo. KEY RESULTS: In vitro experiments demonstrated that SPA0355 suppressed chemokine production, matrix metalloproteinase secretion and cell proliferation induced by TNF-α in rheumatoid fibroblast-like synoviocytes. In addition, SPA0355 inhibited osteoclast differentiation induced by macrophage colony-stimulating factor and the receptor activator of NF-κB ligand, in bone marrow macrophages. Mice with CIA that were pretreated with SPA0355 had a lower cumulative disease incidence and severity of arthritis, based on hind paw thickness, radiological and histopathological findings, and inflammatory cytokine levels, than mice treated with vehicle. Mice treated with SPA0355, after the onset of CIA, also showed significantly decreased disease incidence and joint oedema. The in vitro and in vivo protective effects of SPA0355 were mediated by inhibition of the NF-κB signalling pathway. CONCLUSION AND IMPLICATIONS: Taken together, these results suggested that using SPA0355 to block the NF-κB pathway in rheumatoid joints reduced both the inflammatory responses and tissue destruction. Therefore, SPA0355 may have therapeutic value in preventing or delaying joint destruction in patients with rheumatoid arthritis.

Participation of protein kinase C beta in osteoclast differentiation and function.

Protein kinase C (PKC) proteins have been shown to be involved in diverse cellular responses of various cell types. In experiments to identify genes regulated during osteoclast differentiation by a cDNA microarray approach, we found that the gene expression of PKC-betaII was upregulated in differentiated cells. Reverse transcription-polymerase chain reaction and Western blotting analyses also showed an increase in PKC-betaI as well as PKC-betaII during osteoclast formation in mouse bone marrow cell cultures in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB ligand (RANKL). Use of an antisense oligonucleotide to PKC-betaII resulted in a reduction in the RANKL-driven osteoclastogenesis. Pharmacological intervention with PKC-beta activity by the specific inhibitor CG53353 suppressed cellular differentiation and fusion processes during osteoclastogenesis and inhibited bone-resorbing function of mature osteoclasts. PKC-beta inhibition abolished the ERK and MEK activation by macrophage-colony stimulating factor and RANKL in osteoclast precursor cells whereas the cytokine-induced NF-kappaB activation was not hampered by the PKC-beta inhibition. Our findings indicate that PKC-beta has a role in regulation of osteoclast formation and function potentially by participating in the ERK signaling pathway of M-CSF and RANKL.

Tumor necrosis factor-alpha supports the survival of osteoclasts through the activation of Akt and ERK.

Differentiated osteoclasts have a short life span. We tested various cytokines and growth factors for the effects on the survival of purified mature osteoclasts. In the absence of any added factors, osteoclasts exhibited the survival rate of less than 25% after a 24-h incubation. Among the tested factors, tumor necrosis factor-alpha (TNF-alpha) was found to increase the survival rate to approximately 80%. The TNF-alpha-enhanced survival of osteoclasts appeared to be associated with reduction in apoptosis and suppression of caspase activation. The antiapoptotic signaling pathways involved in the TNF-alpha-induced osteoclast survival were investigated. TNF-alpha treatment increased the phosphorylation of Akt in osteoclasts, which was suppressed by a phosphatidylinositol 3-kinase inhibitor LY294002 and an Src family kinase-selective inhibitor PP1. These inhibitors also attenuated the TNF-alpha stimulation of osteoclast survival. In addition an increase in the phosphorylation of ERK was observed upon TNF-alpha stimulation. PD98059, a specific inhibitor of the ERK-activating kinase MEK-1, abolished the TNF-alpha-induced ERK phosphorylation and osteoclast survival, and in these responses the involvement of Grb2 and ceramide was observed. These results suggest that TNF-alpha promotes the survival of osteoclasts by engaging the phosphatidylinositol 3-kinase Akt and MEK/ERK signaling pathways.