Design of Group IIA Secreted/Synovial Phospholipase A2 Inhibitors: An Oxadiazolone Derivative Suppresses Chondrocyte Prostaglandin E2 Secretion

Group IIA secreted/synovial phospholipase A2 (GIIAPLA2) is an enzyme involved in the synthesis of eicosanoids such as prostaglandin E2 (PGE2), the main eicosanoid contributing to pain and inflammation in rheumatic diseases. We designed, by molecular modeling, 7 novel analogs of 3-{4-[5(indol-1-yl)pentoxy]benzyl}-4H-1,2,4-oxadiazol-5-one, denoted C1, an inhibitor of the GIIAPLA2 enzyme. We report the results of molecular dynamics studies of the complexes between these derivatives and GIIAPLA2, along with their chemical synthesis and results from PLA2 inhibition tests. Modeling predicted some derivatives to display greater GIIAPLA2 affinities than did C1, and such predictions were confirmed by in vitro PLA2 enzymatic tests. Compound C8, endowed with the most favorable energy balance, was shown experimentally to be the strongest GIIAPLA2 inhibitor. Moreover, it displayed an anti-inflammatory activity on rabbit articular chondrocytes, as shown by its capacity to inhibit IL-1β-stimulated PGE2 secretion in these cells. Interestingly, it did not modify the COX-1 to COX-2 ratio. C8 is therefore a potential candidate for anti-inflammatory therapy in joints

Peroxisome Proliferator-activated Receptor-{gamma} Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

Interleukin-1{beta} (IL-1{beta}) induces degradation via hyperexpression of an array of genes, including metalloproteinases (MMP), in cartilage cells during articular degenerative diseases. In contrast, natural ligands for peroxisome proliferator-activated receptors (PPARs) display protective anti-cytokine effects in these cells. We used the PPAR agonist rosiglitazone (Rtz) to investigate PPAR-{gamma} isotype on IL-1{beta}-target genes. Immunocytochemistry, electrophoretic mobility shift, and transient transfection assays revealed a functional PPAR-{gamma} in chondrocytes in vitro. Rtz displayed significant inhibition of IL-1{beta} effects in chondrocytes. Low Rtz concentrations (close to Kd values for PPAR-{gamma}, 0.1 to 1 µM) inhibited the effects of IL-1{beta} on 35S-sulfated proteoglycan production and gelatinolytic activities and downregulated MMP1 expression at mRNA and protein levels. We have investigated the mechanism of action of Rtz against IL-1{beta}-mediated MMP1 gene hyperexpression. Rtz effect occurs at the transcriptional level of the MMP1 promoter, as observed in transiently transfected cells with pMMP1-luciferase vector. Transient expression of wild type PPAR-{gamma} enhanced Rtz inhibitory effect in chondrocytes, whereas a mutated dominant negative PPAR-{gamma} abolished it, supporting the role of PPAR-{gamma} in this effect. MMP1 gene promoter analysis revealed the involvement of a cis-acting element located at -83 to -77, shown to be a composite PPRE/AP1 site. Gel mobility and supershift assays demonstrated that PPAR-{gamma} and c-Fos/c-Jun proteins bind this cis-acting element in a mutually exclusive way. Our data highlight a new PPAR-{gamma}-dependent inhibitory mechanism on IL-1{beta}-mediated cartilage degradation occurring through DNA binding competition on the composite PPRE/AP1 site in the MMP1 promoter

Modulation of proteoglycan production by cyclic tensile stretch in intervertebral disc cells through a post-translational mechanism

Proteoglycan production is one of the major extracellular matrix components implicated in the dynamic process of intervertebral disc degeneration. Mechanical stress is an important modulator of the degeneration, but the underlying molecular mechanism at the proteoglycan level remains unclear. The aim of this work was to study the regulation of proteoglycan production by cyclic tensile stretch applied to intervertebral disc annulus fibrosus cells. Matrix metalloproteinases do not seem to be implicated in the regulation of proteoglycan production. By contrast, nitrite oxide production is induced by cyclic tensile stretch, in a time, intensity, and frequency dependant manner. Using a non-specific nitric oxide synthases inhibitor [NG-methyl-L-arginine (L-NMA)], we suppress totally the inhibition of proteoglycan production induced by cyclic tensile stretch suggesting the implication of nitric oxide synthases in the observed phenomenon. Introducing the transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole or a more specific inhibitor of nitric oxide synthases II [N-iminoethyl-L-lysine (L-NIL)] did not affect the decreased proteoglycan production, which suggests a post-translational regulation. In contrast, N-omega nitro-L-arginine (L-NNA) a more specific inhibitor of NOS I and III abrogated the cyclic tensile stretch-dependant inhibition of proteoglycan production. These results suggest that cyclic tensile stretch regulates proteoglycan production through a post-translational mechanism involving nitrite oxide. This result could be of interest in the development of local therapeutic strategies aimed at controlling intervertebral disc degeneration