Activation of NF-κB/p65 facilitates early chondrogenic differentiation during endochondral ossification

Background: NF-kappa B/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-kappa B/p65 signaling. Methodology/Principal Findings: The role of NF-kappa B/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of 6 weeks old mice. NF-kappa B/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents. Gene expression and protein expression analysis, and (immuno) histochemical stainings were employed to determine the role of NF-kappa B/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-kappa B/p65. NF-kappa B/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways. Conclusions/Significance: The presented data demonstrate that NF-kappa B/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification. Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques.