Activation of NF-κB/p65 Facilitates Early Chondrogenic Differentiation during Endochondral Ossification

Abstract

BackgroundNF-κB/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-κB/p65 signaling.Methodology/Principal FindingsThe role of NF-κB/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-κB/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-κB/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65. NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways.Conclusions/SignificanceThe presented data demonstrate that NF-κB/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.

The Novel Small Leucine-rich Protein Chondroadherin-like (CHADL) Is Expressed in Cartilage and Modulates Chondrocyte Differentiation

“…RelA (p65) was predicted to be activated (activation z score of 2.125) and is known to up-regulate Sox9 (25), the master regulator of chondrogenesis. In addition, other differentiation regulators were predicted to be activated: NF-B, STAT1, STAT3, and p38 MAPK (acti- 4.…”

Impairment of the chondrogenic phase of endochondral ossification in vivo by inhibition of cyclooxygenase-2

“…A challenge is to define at which stage of EO COX-2 is involved and to clarify why EO seemed to be delayed in fracture healing and growth plate development, while being inhibited in the PEO model. EO can be divided into 4 stages: 1) initiation of chondrogenic differentiation of chondroprogenitor cells, 2) chondrocyte proliferation, 3) hypertrophic differentiation, 4) vascularisation and apoptosis (Caron et al, 2012b;Caron et al, 2014). Previous work from our group shows a bi-phasic COX-2 expression pattern during chondrogenic differentiation in vitro (Caron et al, 2016;Welting et al, 2011).…”

“…EO can be divided into 4 stages: 1) initiation of chondrogenic differentiation of chondroprogenitor cells, 2) chondrocyte proliferation, 3) hypertrophic differentiation, 4) vascularisation and apoptosis (Caron et al, 2012b;Caron et al, 2014). Previous work from our group shows a bi-phasic COX-2 expression pattern during chondrogenic differentiation in vitro (Caron et al, 2016;Welting et al, 2011). The two stages, corresponding to COX-2 expression peaks, resemble early differentiation and chondrocyte hypertrophy.…”

“…We found that no distinctive ectopic cartilage tissue was formed in the celecoxib-treated group. Ectopic periosteal cartilage formation and the formation of cartilaginous fracture callus are de novo initiated cellular processes, requiring an initial inflammatory environment (Caron et al, 2012b;Gerstenfeld et al, 2003a;Gerstenfeld et al, 2003b). With the systemic celecoxib-dependent reduction of COX-2 activity, an essential inflammatory reaction may be dampened, resulting in an impaired initiation of chondrogenic differentiation or inflammationdriven osteogenic remodelling of fracture callus.…”

“…During the final phase, the newly-formed bone is remodelled to its original shape (Hadjiargyrou and O'Keefe, 2014;Kronenberg, 2003). In addition, cartilaginous tissue, ectopically generated for the purpose of cartilage repair, forms from progenitor cells by an EO process (Caldwell and Wang, 2014;Caron et al, 2014). One of the main problems in many cartilage repair strategies is the premature hypertrophic differentiation of the generated cartilage (Dickhut et al, 2009;van Osch et al, 2009).…”