Activin receptor-like kinase receptors ALK5 and ALK1 are both required for TGFβ-induced chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells

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 beta; (TGFbeta;) 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 TGFbeta; 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 TGFβ1. 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 TGFbeta; receptors did not clearly associate with chondrogenic induction. TGFbeta; 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 TGFbeta;. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFbeta;-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFbeta;-induced chondrogenic differentiation of BMSCs, and TGFbeta; 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

Dynamic Regulation of TWIST1 Expression during Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

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

Identification of TGFβ-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets

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 β (TGFβ) 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 TGFβ-related genes are regulated during murine OA and endochondral ossification. Methods: A list of 580 TGFβ-related genes, including TGFβ signaling pathway components and TGFβ-target genes, was generated. Regulation of these TGFβ-related genes was a

Identification of TGF beta-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets

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 beta (TGF beta) 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 TGF beta-related genes are regulated during murine OA and endochondral ossification. Methods: A list of 580 TGF beta-related genes, including TGF beta signaling pathway components and TGF beta-target genes, was generated. Regulation of these TGF beta-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 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 TGF beta-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 TGF beta-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

Low RUNX3 expression alters dendritic cell function in patients with systemic sclerosis and contributes to enhanced fibrosis

Objectives Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology.Methods In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre: Runx3(f/f) mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model.Results Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis.Conclusions We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease