RESUMO
We aimed to determine the longitudinal effects of low-energy (generally considered non-injurious) impact loading on (1) chondrocyte proliferation, (2) chondroprogenitor cell activity, and (3) EGFR signaling. In an in vitro study, we assessed 127 full-thickness, cylindrical osteochondral plugs of bovine cartilage undergoing either single, uniaxial unconfined impact loads with energy densities in the range of 1.5-3.2mJ/mm3 or no impact (controls). We quantified cell responses at two, 24, 48, and 72 h via immunohistochemical labeling of Ki67, Sox9, and pEGFR antibodies. We compared strain, stress, and impact energy density as predictors for mechanotransductive responses from cells, and fit significant correlations using linear regressions. Our study demonstrates that low-energy mechanical impacts (1.5-3.2mJ/mm3) generally stimulate time-dependent anabolic responses in the superficial zone of articular cartilage and catabolic responses in the middle and deep zones. We also found that impact energy density is the most consistent predictor of cell responses to low-energy impact loading. These spatial and temporal changes in chondrocyte behavior result directly from low-energy mechanical impacts, revealing a new level of mechanotransductive sensitivity in chondrocytes not previously appreciated.
Assuntos
Cartilagem Articular , Condrócitos , Animais , Bovinos , Transdução de Sinais , Estresse MecânicoRESUMO
Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP production in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. The dephosphorylation requires a PPP-family phosphatase. Thus FGF signaling lowers cyclic GMP production in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth.
Assuntos
Desenvolvimento Ósseo , Fatores de Crescimento de Fibroblastos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores do Fator Natriurético Atrial/metabolismo , Animais , GMP Cíclico/metabolismo , Camundongos , Fosforilação , Transdução de SinaisRESUMO
Bone morphogenetic proteins (BMPs) are involved in multiple aspects of limb development including regulation of cartilage differentiation. Several BMPs bind strongly to heparin, and heparan sulfate proteoglycans (HSPGs) at the cell surface or in the extracellular matrix have recently been implicated as modulators of BMP signaling in some developing systems. Here we have explored the role of HSPGs in regulating BMP activity during limb chondrogenesis by evaluating the effects of exogenous heparan sulfate (HS), heparitinase treatment, and overexpression of the HSPG syndecan-3 on the ability of BMP2 to modulate the chondrogenic differentiation of limb mesenchymal cells in micromass culture. Exogenous HS dramatically enhances the ability of BMP2 to stimulate chondrogenesis and cartilage specific gene expression, and reduces the concentration of BMP2 needed to stimulate chondrogenesis. Furthermore, HS stimulates BMP2-mediated phosphorylation of Smad1, Smad5, and Smad8, transcriptional mediators of BMP2 signaling, indicating that HS enhances the interaction of BMP2 with its receptors. Pretreatment of micromass cultures with heparitinase to degrade endogenous HSPGs also enhances the chondrogenic activity of BMP2, and reduces the concentration of BMP2 needed to promote chondrogenesis. Taken together these results indicate that exogenous HS or heparitinase enhance the chondrogenic activity of BMP2 by interfering with its interaction with endogenous HSPGs that would normally restrict its interaction with its receptors. Consistent with the possibility that HSPGs are negative modulators of BMP signaling during chondrogenesis, we have found that overexpression of syndecan-3, which is one of the major HSPGs normally expressed during chondrogenesis, greatly impairs the ability of BMP2 to promote cartilage differentiation. Furthermore, retroviral overexpression of syndecan-3 inhibits BMP2-mediated Smad phosphorylation in the regions of the cultures in which chondrogenesis is inhibited and in which ectopic syndecan-3 protein is highly expressed. These results indicate that syndecan-3 interferes with the interaction of BMP2 with its receptors, and that this interference results in an inhibition of chondrogenesis. Taken together these results indicate that HSPGs including syndecan-3 normally modulate the strength of BMP signaling during limb cartilage differentiation by limiting the effective concentration of BMP available for signaling.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/fisiologia , Diferenciação Celular/fisiologia , Condrogênese/fisiologia , Extremidades/crescimento & desenvolvimento , Proteoglicanas de Heparan Sulfato/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteína Morfogenética Óssea 2 , Células Cultivadas , Embrião de Galinha , Condrócitos/citologia , Condrócitos/fisiologia , Extremidades/anatomia & histologia , Heparitina Sulfato/metabolismo , Mesoderma/citologia , Mesoderma/fisiologia , Proteínas Smad/metabolismo , Sindecana-3RESUMO
Humans with X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of the disease, develop severe osteoarthropathy and the precise factors that contribute to this joint degeneration remain largely unknown. Fibroblast growth factor 2 (FGF2) is a key regulatory growth factor in osteoarthritis. Although there are multiple FGF2 isoforms the potential involvement of specific FGF2 isoforms in joint degradation has not been investigated. Mice that overexpress the high molecular weight FGF2 isoforms in bone (HMWTg mice) phenocopy Hyp mice and XLH subjects and Hyp mice overexpress the HMWFGF2 isoforms in osteoblasts and osteocytes. Given that Hyp mice and XLH subjects develop osteoarthropathies we examined whether HMWTg mice also develop knee joint degeneration at 2, 8, and 18 mo compared with VectorTg (control) mice. HMWTg mice developed spontaneous osteoarthropathy as early as age 2 mo with thinning of subchondral bone, osteophyte formation, decreased articular cartilage thickness, abnormal mineralization within the joint, increased cartilage degradative enzymes, hypertrophic markers, and angiogenesis. FGF receptors 1 and 3 and fibroblast growth factor 23 were significantly altered compared with VectorTg mice. In addition, gene expression of growth factors and cytokines including bone morphogenetic proteins, Insulin like growth factor 1, Interleukin 1 beta, as well as transcription factors Sex determining region Y box 9, hypoxia inducible factor 1, and nuclear factor kappa B subunit 1 were differentially modulated in HMWTg compared with VectorTg. This study demonstrates that overexpression of the HMW isoforms of FGF2 in bone results in catabolic activity in joint cartilage and bone that leads to osteoarthropathy.
Assuntos
Cartilagem Articular/metabolismo , Raquitismo Hipofosfatêmico Familiar/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Densidade Óssea/fisiologia , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Raquitismo Hipofosfatêmico Familiar/diagnóstico por imagem , Raquitismo Hipofosfatêmico Familiar/genética , Raquitismo Hipofosfatêmico Familiar/patologia , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Isoformas de Proteínas/genética , Microtomografia por Raio-XRESUMO
The IGF axis is important for long bone development, homeostasis and disease. The activities of IGF-I and IGF-II are regulated by IGF binding proteins (IGFBPs). IGF-I and IGFBP2 are co-expressed in dynamic fashions in the developing long bones of the chick wing, and we have found that IGF-II is present in the cartilage model and surrounding perichondrium, proliferative and hypertrophic chondrocytes and developing periosteum. To gain insight into endogenous roles of IGF-I, IGF-II and IGFBP2 in long bone development, we have overexpressed IGFBP2 in the developing skeletal elements of the embryonic chick wing in vivo, using an RCAS retroviral vector. IGFBP2 overexpression led to an obvious shortening of the long bones of the wing. We have investigated, at the cellular and molecular levels, the mechanism of action whereby IGFBP2 overexpression impairs long bone development in vivo. At an early stage, IGFBP2 excess dramatically inhibits proliferation by the chondrocytes of the cartilage models that prefigure the developing long bones. Later, IGFBP2 excess also reduces proliferation of the maturing chondrocytes and attenuates proliferation by the perichondrium/developing periosteum. IGFBP2 excess does not affect morphological or molecular indicators of chondrocyte maturation, osteoblast differentiation or cell/matrix turnover, such as expression of Ihh, PTHrP, type X collagen and osteopontin, or distribution and relative abundance of putative clast cells. We also have found that IGFBP2 blocks the ability of IGF-I and IGF-II to promote proliferation and matrix synthesis by wing chondrocytes in vitro. Together, our results suggest that the mechanism of action whereby IGFBP2 excess impairs long bone development is to inhibit IGF-mediated proliferation and matrix synthesis by the cartilage model; reduce the proliferation and progression to hypertrophy by the maturing chondrocytes; and attenuate proliferation and formation of the periosteal bony collar. These actions retard the growth and longitudinal expansion of the developing long bones, resulting in shortened wing skeletal elements. Our results emphasize the importance of a balance of IGF/IGFBP2 action at several stages during normal long bone development.
Assuntos
Desenvolvimento Ósseo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Fator de Crescimento Insulin-Like II/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Animais , Animais Geneticamente Modificados , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Cartilagem/química , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Proliferação de Células , Galinhas , Lâmina de Crescimento/química , Lâmina de Crescimento/citologia , Lâmina de Crescimento/efeitos dos fármacos , Humanos , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/análise , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/análise , Fator de Crescimento Insulin-Like II/metabolismo , Fator de Crescimento Insulin-Like II/farmacologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
A role for choline during early stages of mammalian embryogenesis has not been established, although recent studies show that inhibitors of choline uptake and metabolism, 2-dimethylaminoethanol (DMAE), and 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), produce neural tube defects in mouse embryos grown in vitro. To determine potential mechanisms responsible for these abnormalities, choline metabolism in the presence or absence of these inhibitors was evaluated in cultured, neurulating mouse embryos by using chromatographic techniques. Results showed that 90%-95% of 14C-choline was incorporated into phosphocholine and phosphatidylcholine (PtdCho), which was metabolized to sphingomyelin. Choline was oxidized to betaine, and betaine homocysteine methyltransferase was expressed. Acetylcholine was synthesized in yolk sacs, but 70 kDa choline acetyltransferase was undetectable by immunoblot. DMAE reduced embryonic choline uptake and inhibited phosphocholine, PtdCho, phosphatidylethanolamine (PtdEtn), and sphingomyelin synthesis. ET-18-OCH3 also inhibited PtdCho synthesis. In embryos and yolk sacs incubated with 3H-ethanolamine, 95% of recovered label was PtdEtn, but PtdEtn was not converted to PtdCho, which suggested that phosphatidylethanolamine methyltransferase (PeMT) activity was absent. In ET-18-OCH3 treated yolk sacs, PtdEtn was increased, but PtdCho was still not generated through PeMT. Results suggest that endogenous PtdCho synthesis is important during neurulation and that perturbed choline metabolism contributes to neural tube defects produced by DMAE and ET-18-OCH3.
Assuntos
Sistema Nervoso Central/embriologia , Colina/metabolismo , Acetilcolina/biossíntese , Animais , Betaína/metabolismo , Células Cultivadas , Sistema Nervoso Central/metabolismo , Ceramidas/metabolismo , Deanol/farmacologia , Diglicerídeos/metabolismo , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal , Gástrula/metabolismo , Camundongos , Modelos Neurológicos , Fosfatidilcolinas/biossíntese , Éteres Fosfolipídicos/farmacologia , Fosforilcolina/metabolismo , Esfingomielinas/biossínteseRESUMO
During endochondral ossification, the skeletal elements of vertebrate limbs form and elongate via coordinated control of chondrocyte and osteoblast differentiation and proliferation. The role of signaling by the ErbB family of receptor tyrosine kinases, which consists of ErbB1 (epidermal growth factor receptor or EGFR), ErbB2, ErbB3 and ErbB4, has been little studied during cartilage and bone development. Signaling by the ErbB network generates a diverse array of cellular responses via formation of ErbB dimers activated by distinct ligands that produce distinct signal outputs. Herstatin is a soluble ErbB2 receptor that acts in a dominant negative fashion to inhibit ErbB signaling by binding to endogenous ErbB receptors, preventing functional dimer formation. Here, we examine the effects of Herstatin on limb skeletal element development in transgenic mice, achieved via Prx1 promoter-driven expression in limb cartilage and bone. The limb skeletal elements of Prx1-Herstatin embryos are shortened, and chondrocyte maturation and osteoblast differentiation are delayed. In addition, proliferation by chondrocytes and periosteal cells of Prx1-Herstatin limb skeletal elements is markedly reduced. Our study identifies requirements for ErbB signaling in the maintenance of chondrocyte and osteoblast proliferation involved in the timely progression of chondrocyte maturation and periosteal osteoblast differentiation.
Assuntos
Osso e Ossos/embriologia , Cartilagem/embriologia , Receptores ErbB/fisiologia , Receptor ErbB-2/fisiologia , Transdução de Sinais/fisiologia , Animais , Extremidades/embriologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Transgênicos , Receptor ErbB-2/deficiência , Receptor ErbB-2/genética , Transdução de Sinais/genéticaRESUMO
The homeodomain transcription factor Dlx5 has been implicated in the regulation of chondrocyte and osteoblast differentiation during endochondral ossification in the developing limb. In a gain-of-function approach to directly investigate the role of Dlx5 in chondrocyte maturation, we have used cartilage-specific Col2a1-Dlx5 promoter/enhancer constructs to target overexpression of Dlx5 to the differentiating cartilage models of the limbs of transgenic mice. Targeted overexpression of Dlx5 in cartilage rudiments results in the formation of shortened skeletal elements containing excessive numbers of hypertrophic chondrocytes and expanded domains of expression of Ihh and type X collagen, molecular markers of hypertrophic maturation. This suggests that hypertrophic differentiation is enhanced in response to Dlx5 misexpression. Skeletal elements overexpressing Dlx5 also exhibit a marked reduction in the zone of proliferation, indicating that overexpression of Dlx5 reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together these results indicate that Dlx5 is a positive regulator of chondrocyte maturation during endochondral ossification, and suggest that it regulates the process at least in part by promoting the conversion of immature proliferating chondrocytes into hypertrophying chondrocytes; a critical step in the maturation process.