NF-κB p65 interacts with Stat5b in growth plate chondrocytes and mediates the effects of Growth Hormone on chondrogenesis and on the expression of Insulin-like Growth Factor-1 and Bone Morphogenetic Protein-2

Growth Hormone (GH) stimulates growth plate chondrogenesis and longitudinal bone growth, with its stimulatory effects being primarily mediated by Insulin-like Growth Factor-1 (IGF-1), both systemically and locally in the growth plate. It has been shown that the transcription factor Stat5b mediates the GHpromoting effect on IGF-1 expression and on chondrogenesis: yet, it is not known whether other signalling molecules are activated by GH in growth plate chondrocytes. We have previously demonstrated that Nuclear Factor-κB p65 is a transcription factor expressed in growth plate chondrocytes, where it facilitates chondrogenesis. We have also shown that fibroblasts isolated from a patient with growth failure and a heterozygous mutation of IκBα (a component of the NF-κB signaling pathway) exhibit GH insensitivity. In this study, we cultured rat metatarsal bones in the presence of GH and/or pyrrolidine dithiocarbamate (PDTC), a known NF-κB inhibitor. The GHmediated stimulation of metatarsal longitudinal growth and growth plate chondrogenesis was neutralized by PDTC. In cultured chondrocytes isolated from rat metatarsal growth plates, GH induced NFκB-DNA binding, chondrocyte proliferation and differentiation, and prevented chondrocyte apoptosis. The inhibition of NF-κB p65 expression and activity (by NFκB p65 siRNA and PDTC, respectively) in chondrocytes reversed the GH-mediated effects on chondrocyte proliferation, differentiation, and apoptosis. Lastly, the inhibition of Stat5b expression in chondrocytes prevented the GH-promoting effects on NF-κB-DNA binding, while the inhibition of NF-κB p65 expression or activity prevented the GH-dependent activation of IGF-1 and BMP-2 expression.