Stimulatory Effects of Insulin-like Growth Factor-I on Growth Plate Chondrogenesis Are Mediated by Nuclear Factor-κB p65

Abstract

Insulin-like growth factor-I (IGF-I) is an important regulator of endochondral ossification. However, little is known about the signaling pathways activated by IGF-I in growth plate chondrocytes. We have previously shown that NF-B-p65 facilitates growth plate chondrogenesis. In this study, we first cultured rat metatarsal bones with IGF-I and/or pyrrolidine dithiocarbamate (PDTC), a known NF-B inhibitor. The IGF-I-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by PDTC. In rat growth plate chondrocytes, IGF-I induced NF-B-p65 nuclear translocation. The inhibition of NF-B-p65 expression and activity (by p65 short interfering RNA and PDTC, respectively) in chondrocytes reversed the IGF-I-mediated induction of cell proliferation and differentiation and the IGF-I-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of IGF-I on NF-B activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-B-p65 in chondrocytes. IGF-I2 is a key regulator of longitudinal bone growth, with such role being exerted both during intrauterine and extrauterine life. Knock-out mice for IGF-I exhibit intrauterine growth retardation and experience a subnormal postnatal growth rate (1). A similar growth pattern has been described in a child born with a homozygous IGF-I deletion (2).The fact that IGF-I null mice have a reduced growth plate height clearly suggests a facilitatory role for IGF-I on growth plate chondrogenesis and, in turn, on longitudinal bone growth. The presence of the IGF-I receptor in growth plate chondrocytes also suggests that IGF-I facilitates longitudinal bone growth directly at the growth plate (3, 4). However, little is known about the specific molecular mechanisms responsible for the IGF-I-mediated induction of growth plate chondrogenesis.Mammalian NF-B is a group of transcription factors, including seven members, p65 (RelA), c-Rel, RelB, p50/p105 (NF-B1), and p52/p100 (NF-B2) (5). Upon activation by a wide variety of stimuli (proinflammatory cytokines, growth factors, and viral proteins), NF-B translocates to the nucleus, where it modulates the expression of target genes involved in cell growth, survival, adhesion, and death (6, 7). These target genes include anti-apoptotic (8) as well as pro-apoptotic ones (9), suggesting that the effects of NF-B on cell growth and survival may depend on the cell type and on the nature of the extracellular stimuli.Previous evidence indicates that NF-B exerts a regulatory role in bone growth and development. Mice deficient in both the NF-B subunits p50 and p52 have retarded growth and shortened long bones (10), suggesting that NF-B may be involved in bone formation and growth. In addition, we have recently shown that the NF-B subunit p65 has a facilitatory role on growth plate chondrogenesis (11). Because experimental evidence in a number of cell types suggests a functional interaction between IGF-I and NF-B (12-...

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

“…The targeted silencing of NF-B p65 expression or activity prevented the GH stimulation of IGF-1 mRNA and protein expression, suggesting that both Stat5b and NF-B p65 are necessary to mediate the GH stimulatory effects on IGF-1 in chondrocytes. Interestingly, we have previously shown that NF-B p65 mediates the stimulatory effects of IGF-1 on chondrocyte proliferation and differentia- tion and demonstrated its preventive effects on chondrocyte apoptosis (8). Thus, our study supports a reciprocal functional interaction between NF-B p65 and IGF-1 in which the transcription factor modulates both IGF-1 synthesis and action in growth plate chondrocytes.…”

“…Whole Metatarsal Culture-The second, third, and fourth metatarsal bone rudiments were isolated from Sprague-Dawley rat embryos (20 days postcoitum) and cultured individually in 24-well plates (8). Each well contained 0.5 ml of minimum essential medium (Invitrogen) supplemented with 0.05 mg/ml ascorbic acid (Invitrogen), 1 mM sodium glycerophosphate (Sigma-Aldrich), 0.2% bovine serum albumin (Sigma), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen) without or with 10 ng/ml recombinant mouse GH and/or 1 M PDTC.…”

“…In addition, we have also shown that NF-B p65 mediates the growth-promoting effects of insulin-like growth factor-1 (IGF-1) on chondrogenesis and longitudinal bone growth (8). Along with IGF-1, growth hormone (GH) is another critical regulator of longitudinal bone growth and growth plate chondrogenesis.…”

Role of NF-κB in the skeleton

“…However, BMP2 also drives terminal differentiation, supporting the expression of collagen X. NF-κB mediates the regulation of growth plate chondrogenesis by IGF-1, which stimulates longitudinal bone growth by inducing chondrocyte proliferation and maturation and inhibiting apoptosis. The IGF-1 receptor is a receptor tyrosine kinase that activates an array of intracellular signaling pathways, and recent studies have shown that the major effects of IGF-1 on the growth plate appear to be mediated by p65 [54,55]. Chemical inhibition of NF-κB blocks the stimulatory effects of IGF-1 on chondrocyte proliferation and hypertrophy in rat metatarsal explant cultures and its inhibitory effect on apoptosis in cultured chondrocytes.…”

WISP3 - IGF1 interaction regulates chondrocyte hypertrophy

“…Other studies have reported that WISP3 interacts with BMP and components of the Wnt1 signaling pathway including LRP6 and Fz8 (Nakamura et al, 2007). In light of the fact that WISP3 harbors a potential IGF1 binding motif and IGF1 is required for cartilage/bone growth (McQuillan et al, 1986;Ohlsson et al, 1992;Wang et al, 1999;Wu et al, 2008), it is important to decipher if WISP3 also physically interacts with IGF1 and controls its mode of actions. Although, WISP3 has been demonstrated to suppress IGF1 signaling in breast cancer as well as chondrocyte cell lines (Kleer et al, 2004;Cui et al, 2007;Lorenzatti et al, 2011), WISP3-IGF1 interaction and its potential influence on chondrocyte hypertrophy has not been documented.…”