Abstract
BACKGROUND: Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFbeta) signaling. TGFbeta binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 beta (IL1beta) on SMAD2/3 signaling dynamics and post-translational modifications. RESULTS: Co-stimulation of MSC with TGFbeta and IL1beta did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFbeta and IL1beta compared to TGFbeta alone. Furthermore, IL1beta stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFbeta signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFbeta, IL1beta or TGFbeta and IL1beta stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1beta-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFbeta. Co-stimulation with both factors resulted in a similar kinetic profile as TGFbeta alone. Nevertheless, IL1beta did subtly alter TGFbeta-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFbeta target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1beta on TGFbeta signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1beta-induced SMAD2 linker modifications and CAGA12-luciferase activity. CONCLUSIONS: TGFbeta and IL1beta signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFbeta target genes were repressed by IL1beta independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1beta-induced TGFbeta signal modulation.
