RESUMEN
OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage. Some features of OA, including chondrocyte hypertrophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factor ß (TGFß) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFß-related genes are regulated during murine OA and endochondral ossification. METHODS: A list of 580 TGFß-related genes, including TGFß signaling pathway components and TGFß-target genes, was generated. Regulation of these TGFß-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6â¯weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development). RESULTS: A total of 106 TGFß-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplementary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients. CONCLUSION: By focusing on TGFß-related genes during OA and chondrocyte hypertrophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients.
Asunto(s)
Condrocitos/metabolismo , Condrocitos/patología , Bases de Datos Genéticas , Regulación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoartritis/genética , Osteoartritis/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Hipertrofia , Articulaciones/metabolismo , Masculino , Ratones Endogámicos C57BL , Reproducibilidad de los ResultadosRESUMEN
Microfracture surgery may be improved by the implantation of unidirectional collagen scaffolds that provide a template for mesenchymal stem cells to regenerate cartilage. Incorporation of growth factors in unidirectional scaffolds may further enhance cartilage regeneration. In scaffolds, immobilization of growth factors is required to prolong in vivo activity, to limit diffusion and to reduce the amount of growth factor needed for safe clinical application. We investigated the immobilization of bone morphogenetic protein 2 (BMP2) to unidirectional collagen scaffolds and the effect on in vitro chondrogenesis. C3H10T1/2 cells were seeded on unidirectional collagen scaffolds with and without covalently attached heparin, and with and without incubation with BMP2 (1 and 10 µg), or with BMP2 present in the culture medium (10-200 ng ml-1). Culturing was for 2 weeks and readout parameters included histology, immunohistochemistry, biochemical analysis and molecular biological analysis. The unidirectional pores facilitated the distribution of C3H10T1/2 cells and matrix formation throughout scaffolds. The effective dose of medium supplementation with BMP2 was 100 ng ml-1 (total exposure 1 µg BMP2), and similar production of cartilage-specific molecules chondroitin sulfate (CS) and type II collagen was found for scaffolds pre-incubated with 10 µg BMP2. Pre-incubation with 1 µg BMP2 resulted in less cartilage matrix formation. The conjugation of heparin to the scaffolds resulted in more CS and less type II collagen deposition compared to scaffolds without heparin. In conclusion, unidirectional collagen scaffolds pre-incubated with 10 µg BMP2 supported chondrogenesis in vitro and may be suitable for prolonged cartilage matrix synthesis in vivo.
Asunto(s)
Proteína Morfogenética Ósea 2/química , Condrocitos/citología , Colágeno/química , Ingeniería de Tejidos/métodos , Animales , Cartílago/química , Diferenciación Celular , Condrogénesis/efectos de los fármacos , Sulfatos de Condroitina/química , Medios de Cultivo/química , Heparina/química , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C3H , Microscopía Electrónica de Rastreo , Reacción en Cadena de la Polimerasa , Polímeros/química , Regeneración , Andamios del Tejido/química , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (N = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (N = 3 donors) and isolated adult human BMSCs (N = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (N = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture.
Asunto(s)
Diferenciación Celular , Condrocitos/metabolismo , Condrogénesis , Células Madre Mesenquimatosas/metabolismo , Proteínas Nucleares/genética , Proteína 1 Relacionada con Twist/genética , Anciano , Anciano de 80 o más Años , Animales , Células Cultivadas , Condrocitos/citología , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Proteínas Nucleares/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Proteína 1 Relacionada con Twist/metabolismoRESUMEN
To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-ß (TGFß) receptor ALK5 is crucial for chondrogenesis induced by TGFß. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFß-induced chondrogenesis. Activation of TGFß signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFß signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFß-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFß-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFß-induced chondrogenesis of human BMSCs.
Asunto(s)
Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas/fisiología , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Células Cultivadas , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor ß (TGFß) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFß receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. MATERIALS & METHODS: ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFß1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. RESULTS: ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFß receptors did not clearly associate with chondrogenic induction. TGFß increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFß. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFß-induced chondrogenesis. CONCLUSION: ALK5 as well as ALK1 are required for TGFß-induced chondrogenic differentiation of BMSCs, and TGFß not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.