Hes1, a new target for interleukin 1β in chondrocytes

Objectives To investigate the effects of interleukin 1 beta (IL1 beta) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. Methods Mouse articular chondrocytes in primary culture were challenged with IL1 beta, alone or combined with Notch1 and IL1 beta pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q) PCR. IL1 beta-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes. Results Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1 beta transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1 beta induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)kappa B activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the gamma-secretase inhibitor N-(N-(3,5-difluorophenacetyl)l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1 beta-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1 beta induction of matrix metalloproteinase (MMP) 13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1 beta. Conclusion Hes1 is a novel IL1 beta target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation.