RESUMO
The overall mechanisms governing the role of laminins during osteogenic differentiation of human mesenchymal stem cells (hMSC) are poorly understood. We previously reported that laminin-332 induces an osteogenic phenotype in hMSC and does so through a focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK) dependent pathway. We hypothesized that this is a result of integrin-ECM binding, and that it occurs via the known alpha3 LG3 integrin binding domain of laminin-332. To test this hypothesis we cultured hMSC on several different globular domains of laminin-332. hMSC adhered best to the LG3 domain, and this adhesion maximally activated FAK and ERK within 120 min. Prolonged culturing (8 or 16 days) of hMSC on LG3 led to activation of the osteogenic transcription factor Runx2 and expression of key osteogenic markers (osterix, bone sialoprotein 2, osteocalcin, alkaline phosphatase, extracellular calcium) in hMSC. LG3 domain binding did not increase matrix mineralization, demonstrating that the LG3 domain alone is not sufficient to induce complete osteogenic differentiation in vitro. We conclude that the LG3 domain mediates attachment of hMSC to laminin-332 and that this adhesion recapitulates most, but not all, of the osteogenic differentiation associated with laminin-5 binding to hMSC.
Assuntos
Moléculas de Adesão Celular/química , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/citologia , Calcificação Fisiológica/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrina alfa3beta1/metabolismo , Osteoblastos/metabolismo , Peptídeos/farmacologia , Estrutura Terciária de Proteína , Fator de Transcrição Sp7 , Fatores de Transcrição/metabolismo , CalininaRESUMO
The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/enzimologia , Osteogênese/fisiologia , Sequência de Bases , Adesão Celular , Diferenciação Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Primers do DNA/genética , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Humanos , Sistema de Sinalização das MAP Quinases , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Vitronectina/metabolismoRESUMO
Focal adhesion kinase (FAK) is a key integrator of integrin-mediated signals from the extracellular matrix to the cytoskeleton and downstream signaling molecules. FAK is activated by phosphorylation at specific tyrosine residues, which then stimulate downstream signaling including the ERK1/2 pathway, leading to a variety of cellular responses. In this study, we examined the effects of FAK point mutations at tyrosine residues (Y397, Y925, Y861, and Y576/7) on osteogenic differentiation of human mesenchymal stem cells exposed to collagen I and cyclic tensile strain. Our results demonstrate that FAK signaling emanating from Y397, Y925, and to a lesser extent Y576/7, but not from Y861, controls osteogenic differentiation through an ERK1/2 pathway, as measured by expression levels of key osteogenesis marker genes and subsequent matrix mineralization. These data indicate that FAK is a critical decision maker in extracellular matrix/strain-enhanced osteogenic differentiation.
Assuntos
Diferenciação Celular , Colágeno Tipo I/metabolismo , Quinase 2 de Adesão Focal/genética , Quinase 2 de Adesão Focal/metabolismo , Mecanotransdução Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Calcificação Fisiológica , Epitopos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sialoproteína de Ligação à Integrina , Células-Tronco Mesenquimais/metabolismo , Osteocalcina/genética , Fosforilação , Mutação Puntual , Proteínas Proto-Oncogênicas c-myc/genética , Retroviridae/genética , Sialoglicoproteínas/genética , Fator de Transcrição Sp7 , Resistência à Tração , Fatores de Transcrição/genéticaRESUMO
The laminin family of proteins is critical for managing a variety of cellular activities including migration, adhesion, and differentiation. In bone, the roles of laminins in controlling osteogenic differentiation of human mesenchymal stem cells (hMSC) are unknown. We report here that laminin-5 is found in bone and expressed by hMSC. hMSC isolated from bone synthesize laminin-5 and adhere to exogenous laminin-5 through alpha3beta1 integrin. Adhesion to laminin-5 activates extracellular signal-related kinase (ERK) within 30 min and leads to phosphorylation of the osteogenic transcription factor Runx2/CBFA-1 within 8 d. Cells plated on laminin-5 for 16 d express increased levels of osteogenic marker genes, and those plated for 21 d deposit a mineralized matrix, indicative of osteogenic differentiation. Addition of the ERK inhibitor PD98059 mitigates these effects. We conclude that contact with laminin-5 is sufficient to activate ERK and to stimulate osteogenic differentiation in hMSC.
Assuntos
Moléculas de Adesão Celular/metabolismo , Expressão Gênica , Células-Tronco Mesenquimais/citologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteogênese , Biomarcadores , Western Blotting , Cálcio/análise , Cálcio/metabolismo , Adesão Celular , Moléculas de Adesão Celular/genética , Diferenciação Celular , Movimento Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Imuno-Histoquímica , Integrina alfa3beta1/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Fosforilação , Testes de Precipitina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Tempo , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismo , CalininaRESUMO
The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin > collagen I >/= collagen IV >/= vitronectin > laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells.
