NF-kappaB is a group of transcription factors involved in cell proliferation, differentiation, and apoptosis. Mice deficient in the NF-kappaB subunits p50 and p52 have retarded growth, suggesting that NF-kappaB is involved in bone growth. Yet, it is not clear whether the reduced bone growth of these mice depends on the lack of NF-kappaB activity in growth plate chondrocytes. Using cultured rat metatarsal bones and isolated growth plate chondrocytes, we studied the effects of two NF-kappaB inhibitors (pyrrolidine dithiocarbamate (PDTC) or BAY11-7082 (BAY)), p65 short interference RNA (siRNA), and of the overexpression of p65 on chondrocyte proliferation, differentiation, and apoptosis. To further define the underlying mechanisms, we studied the functional interaction between NF-kappaB p65 and BMP-2 in chondrocytes. PDTC and BAY suppressed metatarsal linear growth. Such growth inhibition resulted from decreased chondrocyte proliferation and differentiation and from increased chondrocyte apoptosis. In cultured chondrocytes, the inhibition of NF-kappaB p65 activation (by PDTC and BAY) and expression (by p65 siRNA) led to the same findings observed in cultured metatarsal bones. In contrast, overexpression of p65 in cultured chondrocytes induced chondrocyte proliferation and differentiation and prevented apoptosis. Although PDTC, BAY, and p65 siRNA reduced the expression of BMP-2 in cultured growth plate chondrocytes, the overexpression of p65 increased it. The addition of Noggin, a BMP-2 antagonist, neutralized the stimulatory effects of p65 on chondrocyte proliferation and differentiation, as well as its anti-apoptotic effect. In conclusion, our findings indicate that NF-kappaB p65 expressed in growth plate chondrocytes facilitates growth plate chondrogenesis and longitudinal bone growth by inducing BMP-2 expression and activity.

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-...

In many normal tissues, proliferation rates decline postnatally causing somatic growth to slow. Previous evidence suggests that this decline is due in part to declining expression of growth-promoting imprinted genes including Mest, Plagl1, Peg3, Dlk1, and Igf2. Embryonal cancers are composed of cells that maintain embryonic characteristics and proliferate rapidly in childhood. We hypothesized that the abnormal persistent rapid proliferation in embryonal cancers occurs in part because of abnormal persistent high expression of growth-promoting imprinted genes. Analysis of microarray data showed elevated expression of MEST, PLAGL1, PEG3, DLK1, and IGF2 in various embryonal cancers, especially rhabdomyosarcoma, compared to non-embryonal cancers and normal tissues. Similarly, mRNA expression, assessed by real-time PCR, of MEST, PEG3, and IGF2 in rhabdomyosarcoma cell lines was increased compared to non-embryonal cancer cell lines. Furthermore, siRNA-mediated knockdown of MEST, PLAGL1, PEG3, and IGF2 expression inhibited proliferation in Rh30 rhabdomyosarcoma cells. These finding suggest that the normal postnatal downregulation of growth-promoting imprinted genes fails to occur in some embryonal cancers, particularly rhabdomyosarcoma, and contributes to the persistent rapid proliferation of rhabdomyosarcoma cells, and, more generally, that failure of the mechanisms responsible for normal somatic growth deceleration can promote tumorigenesis.