Shufang Wu

Role of Fibroblast Growth Factor 21 (FGF21) in Undernutrition-Related Attenuation of Growth in Mice

Reduced caloric intake in mammals causes reduced skeletal growth and GH insensitivity. However, the underlying molecular mechanisms are not fully elucidated. The aim of this study was to determine whether the increased activity of fibroblast growth factor 21 (FGF21) during chronic undernutrition in mice causes GH insensitivity and growth failure. After 4 wk of food restriction, fgf21 knockout (KO) mice exhibited greater body and tibial growth than their wild-type (WT) littermates. Daily injections of recombinant human FGF21 in a subgroup of food-restricted fgf21 KO mice prevented these differences in body and tibial growth. At the end of the 4-wk food restriction, GH binding and GH receptor expression were reduced in the liver and in the growth plate of food-restricted WT mice (compared to WT mice fed ad libitum), whereas they were similar between food-restricted and ad libitum KO mice. In addition, a single injection of GH induced greater liver signal transducer and activator of transcription 5 phosphorylation and IGF-I mRNA in food-restricted KO mice than in WT mice. Lastly, in the tibial growth plate of food-restricted WT mice, FGF21 mRNA and protein expression was greater than that of WT mice fed ad libitum. In contrast, the IGF-I mRNA and protein expression was smaller. Our findings support a causative role for FGF21 in the inhibition of skeletal growth during prolonged undernutrition. Such role may be mediated by the antagonistic effect of FGF21 on GH action in the liver and, possibly, in the growth plate.

Stanniocalcin 1 acts as a paracrine regulator of growth plate chondrogenesis.

During embryogenesis, the expression of mammalian stanniocalcin (STC1) in the appendicular skeleton suggests its involvement in the regulation of longitudinal bone growth. Such a role is further supported by the presence of dwarfism in mice overexpressing STC1. Yet, the STC 1 inhibitory effect on growth may be related to both postnatal metabolic abnormalities and prenatal defective bone formation. In our study, we used an organ culture system to evaluate the effects of STC on growth plate chondrogenesis, which is the primary determinant of longitudinal bone growth. Fetal rat metatarsal bones were cultured in the presence of recombinant human STC (rhSTC). After 3 days, rhSTC suppressed metatarsal growth, growth plate chondrocyte proliferation and hypertrophy/differentiation, and extracellular matrix synthesis. In addition, rhSTC increased the number of apoptotic chondrocytes in the growth plate. In cultured chondrocytes, rhSTC increased phosphate uptake, reduced chondrocyte proliferation and matrix synthesis, and induced apoptosis. All these effects were reversed by culturing chondrocytes with rhSTC and phosphonoformic acid, an inhibitor of phosphate transport. The rhSTC-mediated inhibition of metatarsal growth and growth plate chondrocyte proliferation and hypertrophy/differentiation was abolished by culturing metatarsals with rhSTC and phosphonoformic acid. Taken together, our findings indicate that STC1 inhibits longitudinal bone growth directly at the growth plate. Such growth inhibition, likely mediated by an increased chondrocyte phosphate uptake, results from suppressed chondrocyte proliferation, hypertrophy/differentiation, and matrix synthesis and by increased apoptosis. Last, the expression of both STC1 and its binding site in the growth plate would support an autocrine/paracrine role for this growth factor in the regulation of growth plate chondrogenesis.

Effects of Ca2+ sensing receptor activation in the growth plate

The Ca2+‐sensing receptor (CaR) is a G protein‐coupled receptor expressed in many mammalian tissues, including the long bones growth plate. CaR knockout mice exhibit growth retardation, suggesting that CaR may promote skeletal growth. However, the complex phenotype of these knockout mice, which includes hyperparathyroidism, hypercalcemia, and hypophosphatemia, may confound the effects of CaR activation. To determine whether CaR regulates growth plate chondrogenesis and longitudinal bone growth, we chose an organ culture model. Fetal rat metatarsal bones (dpc 20) were cultured in serum‐free medium for 7 days in the presence or absence of NPS‐R‐568, a CaR agonist. The addition of 10 nM NPS‐R‐568 increased the cumulative longitudinal growth of the metatarsal explants. To explore the underlying mechanisms, we then assessed the effects of NPS‐R‐568 on growth plate chondrocyte hypertrophy/differentiation and chondrocyte proliferation. After 7 days in culture, NPS‐R‐568 increased the height of the growth plate hypertrophic zone and the expression of collagen X, a marker of growth plate chondrocyte differentiation (assessed by immunohistochemistry). NPS‐R‐568 also induced a significant increase of the height of the growth plate proliferative zone and of the total thymidine incorporation in the metatarsal bone. In conclusion, our findings suggest that the activation of CaR in the growth plate accelerates longitudinal bone growth by stimulating growth plate chondrogenesis.

Nuclear Factor-κB p65 Facilitates Longitudinal Bone Growth by Inducing Growth Plate Chondrocyte Proliferation and Differentiation and by Preventing Apoptosis

NF-κB is a group of transcription factors involved in cell proliferation, differentiation, and apoptosis. Mice deficient in the NF-κB subunits p50 and p52 have retarded growth, suggesting that NF-κB is involved in bone growth. Yet, it is not clear whether the reduced bone growth of these mice depends on the lack of NF-κB activity in growth plate chondrocytes. Using cultured rat metatarsal bones and isolated growth plate chondrocytes, we studied the effects of two NF-κB 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-κB 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-κB 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-κB p65 expressed in growth plate chondrocytes facilitates growth plate chondrogenesis and longitudinal bone growth by inducing BMP-2 expression and activity.

Increased expression of Fibroblast Growth Factor 21 (FGF21) during chronic undernutrition causes Growth Hormone insensitivity in chondrocytes by inducing leptin receptor overlapping transcript (LEPROT) and leptin receptor overlapping transcript-like 1 (LEPROTL1) expression

Background: FGF21 causes GH insensitivity. Results: Increased FGF21 expression induces LEPROT and LEPROTL1 expression. Inhibition of LEPROT or LEPROTL1 in growth plate chondrocytes prevents the FGF21-mediated inhibition of the GH stimulatory effects on chondrocyte function and IGF-1 expression. Conclusion: FGF21 prevents the GH effects on chondrocytes by activating LEPROT and LEPROTL1. Significance: LEPROT and LEPROTL1 mediate the FGF21 inhibition of GH action. During calorie restriction in mice, increased expression of FGF21 causes growth attenuation and growth hormone (GH) insensitivity. Previous evidence also indicates that fasting-associated increased expression of leptin receptor overlapping transcript (LEPROT) and LEPROT-like 1 (LEPROTL1) (two proteins that regulate intracellular protein trafficking) reduces GH receptor cell-surface expression in the liver. Thus, we hypothesized that FGF21 causes GH insensitivity through regulation of LEPROT and/or LEPROTL1 expression. After 4 weeks of food restriction, LEPROT and LEPROTL1 mRNA expression in the liver and in the tibial growth plate of wild-type (WT) mice was increased compared with WT mice fed ad libitum. In Fgf21 knock-out (KO) mice, LEPROT and LEPROTL1 mRNA expression in food-restricted and fed ad libitum was similar, with the exception of a subgroup of food-restricted Fgf21 KO mice treated with recombinant human (rh) FGF21 that experienced increased LEPROT and LEPROTL1 mRNA expression compared with untreated food-restricted Fgf21 KO mice. In cultured growth plate chondrocytes, FGF21 stimulated LEPROT and LEPROTL1 mRNA expression, with such effect being prevented in chondrocytes transfected with FGFR1 siRNA or ERK1 siRNA. In cells transfected with control siRNA, GH increased [3H]thymidine incorporation, collagen X, and IGF-1 mRNA expression, with all effects being prevented by rhFGF21. In addition, rhFGF21 decreased 125I-GH binding. In LEPROT siRNA- and/or LEPROTL1 siRNA-transfected cells, rhFGF21 did not prevent the GH stimulatory effects on thymidine incorporation, collagen X, and IGF-1 expression; furthermore, rhFGF21 did not prevent 125I-GH binding. Consistent with the effects of rhFGF21, LEPROT overexpression in chondrocytes resulted in the inhibition of GH action. Our findings indicate that the increased expression of FGF21 during chronic undernutrition inhibits GH action on chondrocytes by activating LEPROT and LEPROTL1.

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

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-κB-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-κB 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-κB-p65 nuclear translocation. The inhibition of NF-κB-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-κB activation. In conclusion, our findings indicate that IGF-I stimulates growth plate chondrogenesis by activating NF-κB-p65 in chondrocytes.

Association between Oxidative Stress and Masked Hypertension in a Multi-Ethnic Population of Obese Children and Adolescents

OBJECTIVE To evaluate whether oxidative stress is correlated with adiposity, obesity-related metabolic abnormalities, and ambulatory blood pressure (ABP) in a multi-ethnic pediatric population. STUDY DESIGN We conducted a prospective study enrolling 42 obese children (age, 12.8 ± 2.4 years) and 34 non-obese children (age, 11.8 ± 3.4 years). We measured urine 8-isoprostane and hydrogen peroxide (markers of oxidative stress) in both obese and non-obese groups. In the obese group, we measured the 24-hour ABP and obtained an oral glucose tolerance test, lipid panel, interleukin-6, and tumor necrosis factor-α. RESULTS 8-isoprostane and hydrogen peroxide were correlated with body mass index standard deviation score and waist circumference. The mean 8-isoprostane and hydrogen peroxide levels of the obese group were higher than those of the non-obese group. In the subset of obese subjects who underwent ABP monitoring, 8-isoprostane was correlated with mean 24-hour systolic blood pressure: within the obese group, 8-isoprostane was higher in obese children with elevated mean 24-hour systolic blood pressure. CONCLUSIONS Our findings provide evidence of a significant correlation between oxidative stress, adiposity, and blood pressure in children. Longitudinal studies in a larger population sample are needed to validate the association between elevated urine 8-isoprostane level and cardiovascular risk factors in an obese pediatric population.

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

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-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 inhibitor-κBα (IκB; component of the nuclear factor-κB (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 GH-mediated 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-κB-DNA binding and chondrocyte proliferation and differentiation and prevented chondrocyte apoptosis. The inhibition of NF-κB p65 expression and activity (by NF-κB 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, whereas the inhibition of NF-κB p65 expression or activity prevented the GH-dependent activation of IGF-1 and bone morphogenetic protein-2 expression.

Insulin-Like Growth Factor-Independent Effects of Growth Hormone on Growth Plate Chondrogenesis and Longitudinal Bone Growth

GH stimulates growth plate chondrogenesis and longitudinal bone growth directly at the growth plate. However, it is not clear yet whether these effects are entirely mediated by the local expression and action of IGF-1 and IGF-2. To determine whether GH has any IGF-independent growth-promoting effects, we generated (TamCart)Igf1r(flox/flox) mice. The systemic injection of tamoxifen in these mice postnatally resulted in the excision of the IGF-1 receptor (Igf1r) gene exclusively in the growth plate. (TamCart)Igf1r(flox/flox) tamoxifen-treated mice [knockout (KO) mice] and their Igf1r(flox/flox) control littermates (C mice) were injected for 4 weeks with GH. At the end of the 4-week period, the tibial growth and growth plate height of GH-treated KO mice were greater than those of untreated C or untreated KO mice. The systemic injection of GH increased the phosphorylation of Janus kinase 2 and signal transducer and activator of transcription 5B in the tibial growth plate of the C and KO mice. In addition, GH increased the mRNA expression of bone morphogenetic protein-2 and the mRNA expression and protein phosphorylation of nuclear factor-κB p65 in both C and KO mice. In cultured chondrocytes transfected with Igf1r small interfering RNA, the addition of GH in the culture medium significantly induced thymidine incorporation and collagen X mRNA expression. In conclusion, our findings demonstrate that GH can promote growth plate chondrogenesis and longitudinal bone growth directly at the growth plate, even when the local effects of IGF-1 and IGF-2 are prevented. Further studies are warranted to elucidate the intracellular molecular mechanisms mediating the IGF-independent, growth-promoting GH effects.

Effects of valproic acid on longitudinal bone growth

Valproic acid is widely used in the treatment of children with epilepsy. Evidence indicates that valproic acid has teratogenic effects on the skeletal system. In addition, the use of valproic acid in children has been associated with short stature. Thus, we hypothesized that valproic acid could also affect bone growth after skeletal morphogenesis is completed. To test this hypothesis, we cultured fetal rat metatarsal bones in the presence of valproic acid. Valproic acid markedly suppressed metatarsal longitudinal growth. To determine the underlying mechanisms, we studied cell proliferation and hypertrophy in the growth plate, the site where bone growth takes place. At the end of the culture period, valproic acid—treated bones exhibited narrow growth plate proliferative and hypertrophic zones and an expanded ossification center. In addition, valproic acid suppressed chondrocyte proliferation in the metatarsal rudiments. Our results suggest that valproic acid suppresses longitudinal bone growth by inhibiting cartilage formation and accelerating ossification of the growth plate. These findings warrant a prospective study on the effects of valproic acid on growth in children treated with valproic acid. (J Child Neurol 2004;19:26—30).