RESUMO
SOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.
Assuntos
Medula Óssea/embriologia , Calcificação Fisiológica/genética , Cartilagem/irrigação sanguínea , Neovascularização Fisiológica/genética , Fatores de Transcrição SOX9/fisiologia , Animais , Animais Recém-Nascidos , Medula Óssea/metabolismo , Cartilagem/metabolismo , Cartilagem Articular/embriologia , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/patologia , Cartilagem Articular/fisiologia , Diferenciação Celular/genética , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/fisiologia , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Lâmina de Crescimento/embriologia , Lâmina de Crescimento/crescimento & desenvolvimento , Lâmina de Crescimento/metabolismo , Hipertrofia/genética , Camundongos , Camundongos Transgênicos , Organogênese/genética , Osteogênese/genética , Osteogênese/fisiologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
Wnt factors are involved in the regulation of all steps of cartilage development. The activity of Wnt factors is generally regulated at the extracellular level by factors like the Dkk family, sFRPs, Cerberus and Wnt inhibitory factor 1 (Wif-1). Here we report that Wif-1 is highly expressed at cartilage-mesenchyme interfaces of the early developing skeleton. In fetal and postnatal skeletal development, Wif-1 is expressed in a sharply restricted zone in the upper hyaline layer of epiphyseal and articular cartilage and in trabecular bone. Coimmunoprecipitation and pull-down assays using recombinant Wif-1 and Wnt factors show specific binding of Wif-1 to Wnt3a, Wnt4, Wnt5a, Wnt7a, Wnt9a and Wnt11. Moreover, Wif-1 was able to block Wnt3a-mediated activation of the canonical Wnt signalling pathway. Consequently, Wif-1 impaired growth of mesenchymal precursor cells and neutralised Wnt3a-mediated inhibition of chondrogenesis in micromass cultures of embryonic chick limb-bud cells. These results identify Wif-1 as a novel extracellular Wnt modulator in cartilage biology.
Assuntos
Cartilagem/embriologia , Cartilagem/metabolismo , Condrogênese , Proteínas da Matriz Extracelular/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Proliferação de Células , Embrião de Galinha , Condrogênese/genética , Desenvolvimento Embrionário , Epífises/embriologia , Epífises/metabolismo , Proteínas da Matriz Extracelular/genética , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Botões de Extremidades/citologia , Botões de Extremidades/embriologia , Botões de Extremidades/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mesoderma/citologia , Camundongos , Ligação Proteica , Transdução de Sinais , Técnicas de Cultura de Tecidos , Proteína Wnt3 , Proteína Wnt3ARESUMO
The purpose of the current study was to investigate the abnormal expression of Col X, PTHrP, TGF-beta, bFGF, and VEGF in cartilage from patients with Kashin-Beck disease (KBD) to understand the pathogenesis of chondronecrosis in KBD. Articular cartilage and growth plate cartilage collected were divided into four groups: control children (8 samples, 5 cases), KBD children (19 samples, 9 cases), control adults (8 samples, 6 cases), and KBD adults (16 samples, 15 cases). The presence of PTHrP, TGF-beta1, bFGF, VEGF, and collagen X in articular cartilage and in growth plate cartilage was analyzed by immunohistochemistry. Articular cartilage and growth plate were each divided in three zones, and the rate of positive cells was counted by light microscope for cytoplasmic and pericellular staining. Results showed that (1) in KBD children, Col X expression was lower in the deep zone of growth plate cartilage than in normal children; in articular cartilage of KBD adults, however, collagen X expression was higher in the middle zone compared to the controls; (2) staining for bFGF, PTHrP, TGF-beta1, and VEGF in KBD adult patients was prominent in the chondrocyte clusters and the eroded surface of articular cartilage, and the percentage of chondrocyte staining was significantly higher than in control samples (t = 3.64-10.34, df = 12 for children and 19 for adults, P = 0.002-0.0001); and (3) the enhanced PTHrP, TGF-beta1, and VEGF staining in the deep and middle zone of KBD articular cartilage correlated with the high incidence of chondronecrosis in the middle zone (48.5% +/- 10.2%) and deep zone (70.6% +/- 27.0%) of adult KBD cartilage. In conclusion, Col X expression was reduced in areas of chondrocyte necrosis in the deep zone of KBD articular cartilage, indicating changes in terminal chondrocyte differentiation. PTHrP, TGF-beta1, and VEGF expression was significantly altered and indicated degenerative changes in KBD cartilage, which initially resemble those occurring in osteoarthritis, but lead eventually to chondronecrosis, an event not observed in osteoarthritis.
Assuntos
Cartilagem/metabolismo , Cartilagem/patologia , Colágeno/genética , Fatores de Crescimento de Fibroblastos/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Fator de Crescimento Transformador beta/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Criança , Condrócitos/patologia , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Osteoartrite/patologiaRESUMO
We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.
Assuntos
Adesão Celular/fisiologia , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Mioblastos/metabolismo , Proteínas ADAM , Proteína ADAM12 , Processamento Alternativo/genética , Animais , Sítios de Ligação/genética , Adesão Celular/genética , Linhagem Celular , Cisteína/metabolismo , Desintegrinas/metabolismo , Células Epiteliais/ultraestrutura , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Integrinas/genética , Laminina/metabolismo , Ligantes , Manganês/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Mioblastos/ultraestrutura , Fosforilação , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Tirosina Quinases/metabolismo , Ratos , Transfecção , Tirosina/metabolismoRESUMO
Adenovirus vectors have been targeted to different cell types by genetic modification of the capsid or by using recombinant or chemically engineered adaptor molecules. However, both genetic capsid modifications and bridging adaptors have to be specifically tailored for each particular targeting situation. Here, we present an efficient and versatile strategy allowing the direct use of monoclonal antibodies against cell surface antigens for targeting of adenovirus vectors. A synthetic 33-amino-acid immunoglobulin G (IgG)-binding domain (Z33) derived from staphylococcal protein A was inserted into the adenovirus fiber protein. The fiber retained the ability to assemble into trimers, bound IgG with high affinity (Kd = 2.4 nM), and was incorporated into vector particles. The transduction efficiency of the Z33-modified adenovirus vector in epidermal growth factor receptor (EGFR)-expressing cells was strongly and dose-dependently enhanced by combination with an EGFR-specific monoclonal antibody. The antibody-mediated increase in cellular transduction was abolished in the presence of competing protein A. In targeting experiments with differentiated primary human muscle cells, up to a 77-fold increase in reporter gene transfer was achieved by preincubation of the vector with monoclonal antibodies directed against neuronal cell adhesion molecule or integrin alpha(7), respectively. The IgG-binding adenovirus vector holds promise for directed gene transfer to a wide variety of cell types by simply changing the target-specific antibody.
Assuntos
Adenoviridae/genética , Anticorpos Monoclonais/metabolismo , Capsídeo/metabolismo , Técnicas de Transferência de Genes , Imunoglobulina G/metabolismo , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Sítios de Ligação , Linhagem Celular , Receptores ErbB/análise , Receptores ErbB/imunologia , Terapia Genética , Vetores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/metabolismoRESUMO
Mucosal mast cells (MMC) or their precursors migrate through the intestinal lamina propria to reside intraepithelially, where expression of mouse mast cell protease-1 indicates the mature phenotype. Alterations in expression of integrins that govern cell adhesion to the extracellular matrix may regulate this process. As the key cytokine mediating differentiation of mouse mast cell protease-1-expressing MMC homologues in vitro, TGF-beta1 was considered a likely candidate for regulation of the integrins that facilitate intraepithelial migration of MMC. Therefore, we examined adhesion of bone marrow-derived mast cells cultured with and without TGF-beta1 to laminin-1, fibronectin, and vitronectin along with expression of integrins likely to regulate this adhesion. Adhesion of PMA-stimulated cultured mast cells to laminin-1 increased from 5.3 +/- 3.6% (mean +/- SEM) in the absence of TGF-beta1 to 58.7 +/- 4.0% (p < 0.05) when cultured mast cells had differentiated into MMC homologues in the presence of TGF-beta1. Increased adhesion of MMC homologues to laminin-1 was also stimulated by FcepsilonRI cross-linking and the calcium ionophore A23187. Expression of the laminin-binding integrin alpha(7) by MMC homologues grown in the presence of TGF-beta1 was demonstrated by RT-PCR and flow cytometry, and preincubation of MMC homologues with the alpha(7)-neutralizing Ab 6A11 inhibited adhesion to laminin-1 by 98% (p < 0.05), demonstrating a novel role for this molecule in adhesion of a hemopoietic cell to laminin-1.
Assuntos
Antígenos CD/biossíntese , Cadeias alfa de Integrinas/biossíntese , Mucosa Intestinal/citologia , Mucosa Intestinal/fisiologia , Laminina/metabolismo , Mastócitos/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Anticorpos Bloqueadores/farmacologia , Anticorpos Monoclonais/farmacologia , Antígenos/farmacologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Calcimicina/farmacologia , Bovinos , Adesão Celular/imunologia , Adesão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Meios de Cultivo Condicionados/metabolismo , Fibronectinas/metabolismo , Imunoglobulina E/farmacologia , Cadeias alfa de Integrinas/antagonistas & inibidores , Cadeias alfa de Integrinas/imunologia , Cadeias alfa de Integrinas/fisiologia , Integrinas/biossíntese , Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica/fisiologia , RNA Mensageiro/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Regulação para Cima/efeitos dos fármacos , Vitronectina/metabolismoRESUMO
The integrin alpha(7)beta(1) occurs in several cytoplasmic (alpha(7A), alpha(7B)) and extracellular splice variants (alpha(7X1), alpha(7X2)), which are differentially expressed during development of skeletal and heart muscle. The extracellular variants result from the alternative splicing of exons X1 and X2, corresponding to a segment within the putative ligand binding domain. To study the specificity and affinity of the X1/X2 variants to different laminin isoforms, soluble alpha(7)beta(1) complexes were prepared by recombinant coexpression of the extracellular domains of the alpha- and beta-subunits. The binding of these complexes to purified ligands was measured by solid phase binding assays. Surprisingly, the alternative splice variants revealed different and specific affinities to different laminin isoforms. While the alpha(7X2) variant bound much more strongly to laminin-1 than the alpha(7X1) variant, the latter showed a high affinity binding to laminins-8 and -10/11. Laminin-2, the major laminin isoform in skeletal muscle, was recognized by both variants, whereas none of the two variants were able to interact with laminin-5. A specific blocking antibody inhibited the binding of both variants to all laminins tested, indicating the involvement of common epitopes in alpha(7X1)beta(1) and alpha(7X2)beta(1). Because laminin-8 and -10/11 as well as alpha(7X1) are expressed in developing skeletal and cardiac muscle, these findings suggest that alpha(7X1)beta(1) may represent a physiological receptor with novel specificities for laminin-8 and -10.