A4.02 Linking inhibition of chondrogenesis and inflammation through the smad linker domain

Abstract

Background and objectivesCartilage repair using mesenchymal stem cells is a promising new strategy for the treatment of osteoarthritis. However, the inflammatory environment of the joint inhibits the repair process. Previously, it has been shown that signalling via transforming growth factor β family members is crucial for cartilage homeostasis. Therefore we evaluated the effect of inflammatory mediators on downstream mediators of TGFβ signalling: the R-SMAD transcription factors.Materials and methodsHuman mesenchymal stem cells were differentiated in monolayer or pellet cultures in chondrogenic differentiation medium. Differentiation was assessed at day 7 using GAG measurements. On day 3 of differentiation inflammatory stimuli were added. To determine the effect on TGFβ signalling the CAGA-luciferase reporter assay and immuno blotting for SMAD phospho-isoforms were used. A human phospho-kinase antibody array was performed to identify kinases activated by inflammation.ResultsAddition of IL1β and OAC-med to differentiating MSC at day 3, inhibited chondrogenic differentiation as measured by GAG deposition at day 7. Transcriptome analyses at 8 h post stimulation with inflammatory mediators revealed deregulation of TGFβ-related and regulated genes. TGFβ signalling was inhibited by inflammation as evidenced by inhibition of the SMAD3 dependant CAGA-luciferase reporter.SMAD2/3 activation by TGFβ is largely determined by C-terminal phosphorylation through the ALK5 receptor. However C-terminal phosphorylation was not affected after addition of inflammatory stimuli. SMAD proteins contain a highly variable linker region that can be phosphorylated by various kinases. These linker modifications are known to modulate SMAD activation and degradation. Stimulation with either IL1β or OAC-med resulted in serine phosphorylation of the SMAD2 linker region within 1 h.To determine which kinase might be responsible for SMAD linker modification a human phospho-kinase antibody array was performed. The MAP kinase pathway was strongly activated within 30 min as evidenced by a > 1.5 fold increase in pERK1/2 and pP38 compared to control pellets.ConclusionsInhibition of TGFβ signalling in differentiating MSC by IL1β and OAC-med appears to be mediated through SMAD serine linker modifications. The hot favourite kinases for phosphorylation of these serines are members of the MAP kinase family.