Involvement of JAGGED/ NOTCH pathway in human chondrocytes exposed to LPS: effect of high molecular weight hyaluronan treatment

Purpose: Since previous studies revealed the activation of JAGGED/NOTCH signaling in inflammatory processes, including cell treatment with lipopolysaccharides (LPS), we aimed to investigate the involvement of JAGGED/NOTCH pathway and the effects of high molecular weight hyaluronan (HMW-HA) treatment in human chondrocytes stimulated by (LPS). Methods: Chondrocytes were stimulated with LPS and then the mRNA expression of TLR-4, NOTCH1/2, JAGGED1, MAPK, RBP-J, IL-6, IL-1β, and MMP-13 was evaluated. A further set of plates were treated with NOTCH1/2 siRNAs before LPS treatment. NF-kB (p65) activation was also determined by a commercial assay. The same parameters were assayed after HMW-HA treatment. Results: LPS induced high expression of TLR-4, MAPK, JAGGED1, NOTCH1/2, RBP-J, IL-6, IL-1β and MMP-13, as well as NF-kB activation. The treatment of chondrocytes with HMWHA, previously stimulated with LPS, reduces significantly the inflammatory response. Discussion: Stimulation of TLR-4 triggers activation of downstream signaling molecules such as MAPK and finally NF-kB. Furthermore, this receptor may prime NOTCH pathway through JAGGED1 that in turn activated pro-inflammatory cytokine transcription andconsequently a strong inflammatory response. The treatment with HMW-HA reduced the expression of pro-inflammatory mediators, as well as NF-kB activation. In addition, HMW-HA was able to reduce MAPK, JAGGED1, NOTCH1/2, and RBP-J activity. As HMW-HA acts by blocking TLR4 activation, specific NOTCH1/2 siRNAs were used to verify if the inflammatory response could be further influenced. Results proved that IL-6, IL-1β and MMP-13 were further reduced, those suggesting that NOTCH-1/2 signaling can be also activate independently by TLR-4 activation Conclusion: These results suggest that HMW-HA exert a protective effects on human primary chondrocytes by indirectly reducing JAGGED/NOTCH pathway trough TLR-4 modulation.