Nuclear Factor-κB (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

Abstract

Growth hormone (GH) stimulates growth plate chondrogenesis and longitudinal bone growth with its stimulatory effects 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 GH promoting effect on IGF-1 expression and on chondrogenesis, yet it is not known whether other signaling molecules are activated by GH in growth plate chondrocytes. We have previously demonstrated that nuclear factor-B 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 inhibitor-B␣ (IB; component of the nuclear factor-B (NF-B) 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-B 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 NF-B-DNA binding and chondrocyte proliferation and differentiation and prevented chondrocyte apoptosis. The inhibition of NF-B p65 expression and activity (by NF-B 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-B-DNA binding, whereas the inhibition of NF-B p65 expression or activity prevented the GH-dependent activation of IGF-1 and bone morphogenetic protein-2 expression.

Intermittent injections of osteocalcin reverse autophagic dysfunction and endoplasmic reticulum stress resulting from diet-induced obesity in the vascular tissue via the NFκB-p65-dependent mechanism

“…The activation of NFκB followed by Akt phosphorylation plays an important role in the development of insulin resistance. 33 To confirm that NFκB pathway was implicated in the regulation of osteocalcin on autophagy and ER stress, we blocked NFκB pathway via pyrrolidinedithiocarbamate (PDTC), a known NFκB inhibitor and NFκB p65 siRNA, respectively, in mouse VECs and VSMCs in the presence of tunicamycin and osteocalcin. NFκB p65 siRNA-transfected cells exhibited reduced p65 protein expression compared with control siRNA-transfected cells (Fig.…”

Transcriptional control of chondrocyte specification and differentiation

“…This results in chondrocyte catabolic actions and Sox9 downregulation [57]. In contrast, RELA/p65 , which is expressed in healthy SSCs and GPCs, was shown in vitro to promote cell survival and proliferation in response to growth hormone [58, 59]. This finding will merit in vivo validation in future studies.…”

Minireview: Mechanisms of Growth Hormone-Mediated Gene Regulation

“…More recently, Stat5 has been shown to be acutely phosphorylated in response to GH in growthplate chondrocytes (154), consistent with the presumption that Stat5 also participates in Igf1 transcription at the growth plate based upon the similar growth phenotypes in individuals with mutations of the GH receptor and Stat5b genes (127). Moreover, a role for nuclear factor (NF)-B p65 has been also revealed in a series of experiments by Wu et al (155). Their group demonstrated that both inhibition of nuclear factor-B (NF-B) activity by the inhibitor pyrrolidine dithiocarbamate and depletion of NF-B by targeted small interfering RNA eliminated the effects of GH on Igf1 expression and chondrocyte proliferation and differentiation.…”