RESUMO
Bone morphogenetic protein 7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues. Signal transduction occurs through the activation of intracellular Smad proteins. In this paper, using a yeast two-hybrid screen, Smad1 was found to interact with the cytoplasmic domain of CD44, a receptor for the extracellular matrix macromolecule hyaluronan. Coimmunoprecipitation experiments confirmed the interaction of Smad1 with full-length CD44-interactions that did not occur when CD44 receptors truncated within the cytoplasmic domain were tested. Chondrocytes overexpressing a truncated CD44 on a background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation. Further, pretreatment of chondrocytes with Streptomyces hyaluronidase to disrupt extracellular hyaluronan-cell interactions inhibited BMP-7-mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4-luciferase reporter activation. These results support a functional link between the BMP signaling cascade and CD44. Thus, changes in hyaluronan-cell interactions may serve as a means to modulate cellular responsiveness to BMP.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/metabolismo , Condrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/genética , Células COS , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Bovinos , Condrócitos/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Genes Reporter/genética , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/farmacologia , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad , Proteína Smad1 , Proteína Smad4 , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.
Assuntos
Botões de Extremidades/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Células Cultivadas , Embrião de Galinha , Condrócitos/efeitos dos fármacos , Condrócitos/fisiologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Botões de Extremidades/efeitos dos fármacos , Botões de Extremidades/embriologia , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/efeitos dos fármacos , Homologia de Sequência , Fatores de Tempo , Tretinoína/farmacologiaRESUMO
Articular cartilage, the load-bearing tissue of the joint, has limited repair and regeneration potential. The scarcity of treatment modalities for large chondral defects has motivated attempts to engineer cartilage tissue constructs that can meet the functional demands of this tissue in vivo. Cartilage tissue engineering requires three components: cells, scaffold, and environment. Adult stem cells, specifically multipotent mesenchymal stem cells, are considered the cell type of choice for tissue engineering, because of the ease with which they can be isolated and expanded and their multilineage differentiation capabilities. Successful outcome of cell-based cartilage tissue engineering ultimately depends on the proper differentiation of stem cells into chondrocytes and the assembly of the appropriate cartilaginous matrix to achieve the load-bearing capabilities of the natural articular cartilage. Multiple requirements, including growth factors, signaling molecules, and physical influences, need to be met. Adult mesenchymal stem-cell-based tissue engineering is a promising technology for the development of a transplantable cartilage replacement to improve joint function.