NOTCH SIGNALING ACTIVATION IS PART OF THE INFLAMMATORY RESPONSE INDUCED BY LPS AND SMALL HYALURONAN OLIGOSACCHARIDES IN HUMAN CHONDROCYTES

NOTCH signalling regulates numerous cellular processes including inflammatory response. TLRs might prime NOTCH activation indirectly through inducing the expression of NOTCH receptors and ligands. Stimulation of TLR4 leads to NF-kB and p38MAPK activation that increases JAGGED1 expression, responsible in turn of NOTCH receptors activation which through mediation of NICD, together with NF-kB, leads to the transcription of the pro-inflammatory cytokines and other detrimental mediators1. Previous investigations reported the enhancement of NOTCH pathway in patients with rheumatoid arthritis and other pathologies involving cartilage injury 2. The present study aimed to investigate the involvement of NOTCH signalling in an experimental model in which inflammation was induced by the treatment of LPS and hyaluronan oligosaccharides (6-mer HA) in human chondrocytes. Changes in TLR-4, p38MAPK, JAGGED1, NOTCH1, NOTCH2, NOTCH3, RBP-J, IRF8, HES1, IL-1beta and MMP-13 mRNA expression of LPS and 6-mer HA-stimulated chondrocytes were examined by PCR RealTime. Protein levels of TLR-4, p38MAPK, JAGGED1, NOTCH1, NOTCH2, NOTCH3, HES1 were evaluated by western blot, while IL-1beta, MMP-13, and p-NF-kB p65 subunit levels were assayed by ELISA kits. Specific NF-kB inhibitor and NOTCH1 siRNA were also used to clarify the inflammatory pathway. Treatment of chondrocytes with LPS produced NF-kB activation, as well as a significant increment in all the parameters reported above. The expression of NOTCH receptors was also increased with more significance for NOCH1. Stimulation of chondrocytes with 6-mer HA produced the same effect but with less intensity. The exposition of cells to both LPS and 6-mer HA resulted in an additive effect on the considered parameters. Specific siRNAs blocking NOTCH1 revealed the involvement of this pathway. The use of a selective NF-kB inhibitor clarify that NF-kB pathway was more effective than NOTCH pathway in the transcription of inflammatory mediators. In conclusion, the activation of TLR4 induced by both LPS and HA oligosaccharides leads to the acute inflammatory response mediated both through the direct NF-kB/MAPK pathway and indirectly by the JAGGED1/NOTCH1 pathway.