L-Sox5 and Sox6 proteins enhance chondrogenic miR-140 microRNA expression by strengthening dimeric Sox9 activity

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.     

Transient down-regulation of cbfa1/Runx2 by RNA interference in murine C3H10T1/2 mesenchymal stromal cells delays in vitro and in vivo osteogenesis, but does not overtly affect chondrogenesis

In order to ensure that MSCs designed for in vivo cartilage repair do not untowardly differentiate into osteoblasts and mineralize in situ, we tested whether siRNA-induced suppression of cbfa1/Runx2 affected the osteogenic and chondrogenic differentiation potential of the murine cell line C3H10T1/2. Anti-cbfa1/Runx2 siRNA decreased the levels of cbfa1/Runx2 mRNA and protein by 65-80%, and also markedly reduced the expression of osteoblast-related genes such as Dlx5, osterix, collagen type I, alkaline phosphatase (AP), osteocalcin, SPARC/osteonectin and osteopontin, leading to a temporal expression of AP enzyme activity and mineralization potential delayed by at least some 7-9 days. Furthermore, siRNA-transfected cells, grown under chondrogenic conditions did not display biologically significant changes in the expression of aggrecan, collagen type II or type X, or histology when grown in micropellets or monolayer cultures. Finally, when cells were propagated in osteogenic medium and injected into the tibial muscles of SCID mice, no overtly mineralized bone tissue emerged. These experiments indicate that a major transient reduction of cbfa1/Runx2 expression in MSCs is sufficient to delay osteoblastic differentiation, both in vitro and in vivo, while chondrogenesis seemed to be sustained.     

Noggin is novel inducer of mesenchymal stem cell adipogenesis: implications for bone health and obesity

Noggin is a glycosylated-secreted protein known so far for its inhibitory effects on bone morphogenetic protein (BMP) signaling by sequestering the BMP ligand. We report here for the first time a novel mechanism by which noggin directly induces adipogenesis of mesenchymal stem cells independently of major human adipogenic signals through C/EBPδ, C/EBPα and peroxisome proliferator-activated receptor-γ. Evaluation of a possible mechanism for noggin-induced adipogenesis of mesenchymal stem cells identified the role of Pax-1 in mediating such differentiation. The relevance of elevated noggin levels in obesity was confirmed in a preclinical, immunocompetent mouse model of spontaneous obesity and in human patients with higher body mass index. These data clearly provide a novel role for noggin in inducing adipogenesis and possibly obesity and further indicates the potential of noggin as a therapeutic target to control obesity.     

MicroRNA-140 Promotes Adipocyte Lineage Commitment of C3H10T1/2 Pluripotent Stem Cells via Targeting Osteopetrosis-associated Transmembrane Protein 1

BMP4 has been shown to induce C3H10T1/2 pluripotent stem cells to commit to adipocyte lineage. In addition to several proteins identified, microRNAs also play a critical role in the process. In this study, we identified microRNA-140 (miR-140) as a direct downstream component of the BMP4 signaling pathway during the commitment of C3H10T1/2 cells to adipocyte lineage. Overexpression of miR-140 in C3H10T1/2 cells promoted commitment, whereas knockdown of its expression led to impairment. Additional studies indicated that Ostm1 is a bona fide target of miR-140, which is significantly decreased during commitment, and Ostm1 was also demonstrated to function as an anti-adipogenic factor.     

Expression patterns and function of chromatin protein HMGB2 during mesenchymal stem cell differentiation

The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2(-/-) mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2(-/-) MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2(-/-) MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/β-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations.     

Delta-like 1 serves as a new target and contributor to liver fibrosis down-regulated by mesenchymal stem cell transplantation

Chronic liver injury always progresses to fibrosis and eventually to cirrhosis, a massive health care burden worldwide. Delta-like 1 (Dlk1) is well known as an inhibitor of adipocyte differentiation. However, whether it is involved in liver fibrosis remains unclear. Here, we provide the first evidence that Dlk1 is a critical contributor to liver fibrosis through promoting activation of hepatic stellate cells (HSCs) during chronic liver injury. We found that upon liver injury, Dlk1 was dramatically induced and initially expressed in hepatocytes and then into the HSCs by a paracrine manner. It leads to the activation of HSCs, which is considered to be a pivotal event in liver fibrogenesis. Two forms (～50 and ～25 kDa) of the Dlk1 protein were detected by Western blot analysis. In vitro administration of Dlk1 significantly promoted HSC activation, whereas in vivo knockdown of Dlk1 dramatically inhibited HSC activation and the subsequent fibrosis. The large soluble form (～50 kDa) of Dlk1 was shown to contribute to HSC activation. We were encouraged to find the Dlk1-promoted HSC activation and liver fibrosis can be depressed by transplantation of bone marrow-mesenchymal stem cells (BM-MSCs). Furthermore, we demonstrated that FGF2 was up-regulated in BM-MSCs under injury stimulation, and it probably participated in the inhibition of Dlk1 by BM-MSCs. Our findings provide a novel role of Dlk1 in liver fibrosis leading to a better understanding of the molecular basis in fibrosis and cirrhosis and also give insights into the cellular and molecular mechanisms of MSC biology in liver repair.     

Conditioned medium of IGF1-induced synovial membrane mesenchymal stem cells increases chondrogenic and chondroprotective markers in chondrocyte inflammation

Recently, mesenchymal stem cells (MSCs) have been the most explored cells for cell therapy for osteoarthritis (OA) that can be obtained from various sources. Synovial membrane MSCs (SMMSCs) provide best potential for OA therapy, however they are not widely explored. Conditioned medium of SMMSCs (SMMSCs-CM) rich in growth factors and cytokines can inhibit apoptosis and increase chondrocytes cell proliferation. The aim of the present study was to determine growth factors content in SMMSCs-CM as well as the chondrogenic and chondroprotective markers expression in OA model after insulin-like growth factor (IGF)1-induced and non-induced SMMSCs-CM treatments. Chondrocyte cell line (CHON002) was induced by IL1β as OA model (CHON002 with IL1β (IL1β-CHON002)) and treated with SMMSCs-CM with or without IGF1 induction to determine its effectiveness in repairing OA cells model. ELISA was used to assay BMP2, fibroblast growth factor 18 (FGF18) and transforming growth factor (TGF) β1 (TGFβ1) levels in SMMSCs-CM, matrix metalloproteinase (MMP) 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS4) levels in OA cells model treated with SMMSCs-CM. RT-qPCR analyses were used to investigate the gene expression of SOX9, COL2, and COL10. CM from SMMSCs cultured and induced by IGF1 150 ng/mL was the most effective concentration for increasing the content of growth factor markers of SMMSCs-CM, which had successfully increased negative cartilage hypertrophy markers (SOX9 and COL2) and reduced hypertrophy markers (COL10, MMP13, and ADAMTS4). Preconditioning with IGF1 has better and very significant results in lowering MMP13 and ADAMTS4 levels. The present study supports IGF1 pre-conditioned SMMSCs-CM to develop a new therapeutic approach in OA improvement through its chondrogenic and chondroprotective roles.     

Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3

RUNX1 regulates formation of the definitive hematopoietic stem cell and its subsequent lineage maturation, and mutations of RUNX1 contribute to leukemic transformation. Phosphorylation of Ser-48, Ser-303, and Ser-424 by cyclin-dependent kinases (cdks) increases RUNX1 trans-activation activity without perturbing p300 interaction. We now find that endogenous RUNX1 interacts with endogenous HDAC1 or HDAC3. Mutation of the three RUNX1 serines to aspartic acid reduces co-immunoprecipitation with HDAC1 or HDAC3 when expressed in 293T cells; mutation of these three serines to alanine increases HDAC interaction, and mutation of each serine individually to aspartic acid also reduces these interactions. GST-RUNX1 isolated from bacterial extracts bound in vitro translated HDAC1 or HDAC3, and these interactions were weakened by mutation of Ser-48, Ser-303, and Ser-424 to aspartic acid. The ability of RUNX1 phosphorylation and not only serine to aspartic acid conversion to reduce HDAC1 binding was demonstrated using wild-type GST-RUNX1 phosphorylated in vitro using cdk1/cyclinB and by exposure of 293T cells transduced with RUNX1 and HDAC1 to roscovitine, a cdk inhibitor. Finally, RUNX1 or RUNX1(tripleD), in which Ser-48, Ser-303, and Ser-424 are mutated to aspartic acid, stimulated proliferation of transduced, lineage-negative murine marrow progenitors more potently than did RUNX1(tripleA), in which these serines are mutated to alanine, suggesting that stimulation of RUNX1 trans-activation by cdk-mediated reduction in HDAC interaction increases marrow progenitor cell proliferation.