Downregulation of the RNA-binding protein PUM2 facilitates MSC-driven bone regeneration and prevents OVX-induced bone loss.

BACKGROUND: Although mRNA dysregulation can induce changes in mesenchymal stem cell (MSC) homeostasis, the mechanisms by which post-transcriptional regulation influences MSC differentiation potential remain understudied. PUMILIO2 (PUM2) represses translation by binding target mRNAs in a sequence-specific manner. METHODS: In vitro osteogenic differentiation assays were conducted using human bone marrow-derived MSCs. Alkaline phosphatase and alizarin red S staining were used to evaluate the osteogenic potential of MSCs. A rat xenograft model featuring a calvarial defect to examine effects of MSC-driven bone regeneration. RNA-immunoprecipitation (RNA-IP) assay was used to determine the interaction between PUM2 protein and Distal-Less Homeobox 5 (DLX5) mRNA. Ovariectomized (OVX) mice were employed to evaluate the effect of gene therapy for postmenopausal osteoporosis. RESULTS: Here, we elucidated the molecular mechanism of PUM2 in MSC osteogenesis and evaluated the applicability of PUM2 knockdown (KD) as a potential cell-based or gene therapy. PUM2 level was downregulated during MSC osteogenic differentiation, and PUM2 KD enhanced MSC osteogenic potential. Following PUM2 KD, MSCs were transplanted onto calvarial defects in 12-week-old rats; after 8 weeks, transplanted MSCs promoted bone regeneration. PUM2 KD upregulated the expression of DLX5 mRNA and protein and the reporter activity of its 3'-untranslated region. RNA-IP revealed direct binding of PUM2 to DLX5 mRNA. We then evaluated the potential of adeno-associated virus serotype 9 (AAV9)-siPum2 as a gene therapy for osteoporosis in OVX mice. CONCLUSION: Our findings suggest a novel role for PUM2 in MSC osteogenesis and highlight the potential of PUM2 KD-MSCs in bone regeneration. Additionally, we showed that AAV9-siPum2 is a potential gene therapy for osteoporosis.

RUNX2 stabilization by long non-coding RNAs contributes to hypertrophic changes in human chondrocytes.

Background: Chondrocyte hypertrophy has been implicated in endochondral ossification and osteoarthritis (OA). In OA, hypertrophic chondrocytes contribute to the destruction and focal calcification of the joint cartilage. Although studies in this field have remarkably developed the modulation of joint inflammation using gene therapy and regeneration of damaged articular cartilage using cell therapy, studies that can modulate or prevent hypertrophic changes in articular chondrocytes are still lacking. Methods: In vitro hypertrophic differentiation and inflammation assays were conducted using human normal chondrocyte cell lines, TC28a2 cells. Human cartilage tissues and primary articular chondrocytes were obtained from OA patients undergoing total knee arthroplasty. Long non-coding RNAs (lncRNAs), LINC02035 and LOC100130207, were selected through RNA-sequencing analysis using RNAs extracted from TC28a2 cells cultured in hypertrophic medium. The regulatory mechanism was evaluated using western blotting, real-time quantitative polymerase chain reaction, osteocalcin reporter assay, RNA-immunoprecipitation (RNA-IP), RNA-in situ hybridization, and IP. Results: LncRNAs are crucial regulators of various biological processes. In this study, we identified two important lncRNAs, LINC02035 and LOC100130207, which play important roles in hypertrophic changes in normal chondrocytes, through RNA sequencing. Interestingly, the expression level of RUNX2, a master regulator of chondrocyte hypertrophy, was regulated at the post-translational level during hypertrophic differentiation of the normal human chondrocyte cell line, TC28a2. RNA-immunoprecipitation proved the potential interaction between RUNX2 protein and both lncRNAs. Knockdown (KD) of LINC02035 or LOC100130207 promoted ubiquitin-mediated proteasomal degradation of RUNX2 and prevented hypertrophic differentiation of normal chondrocyte cell lines, whereas overexpression of both lncRNAs stabilized RUNX2 protein and generated hypertrophic changes. Furthermore, the KD of the two lncRNAs mitigated the destruction of important cartilage matrix proteins, COL2A1 and ACAN, by hypertrophic differentiation or inflammatory conditions. We also confirmed that the phenotypic changes raised by the two lncRNAs could be rescued by modulating RUNX2 expression. In addition, the KD of these two lncRNAs suppressed hypertrophic changes during chondrogenic differentiation of mesenchymal stem cells. Conclusion: Therefore, this study suggests that LINC02035 and LOC100130207 contribute to hypertrophic changes in normal chondrocytes by regulating RUNX2, suggesting that these two novel lncRNAs could be potential therapeutic targets for delaying or preventing OA development, especially for preventing chondrocyte hypertrophy.

Chemically-induced osteogenic cells for bone tissue engineering and disease modeling.

Cell reprogramming can satisfy the demands of obtaining specific cell types for applications such as tissue regeneration and disease modeling. Here we report the reprogramming of human fibroblasts to produce chemically-induced osteogenic cells (ciOG), and explore the potential uses of ciOG in bone repair and disease treatment. A chemical cocktail of RepSox, forskolin, and phenamil was used for osteogenic induction of fibroblasts by activation of RUNX2 expression. Following a maturation, the cells differentiated toward an osteoblast phenotype that produced mineralized nodules. Bulk and single-cell RNA sequencing identified a distinct ciOG population. ciOG formed mineralized tissue in an ectopic site of immunodeficiency mice, unlike the original fibroblasts. Osteogenic reprogramming was modulated under engineered culture substrates. When generated on a nanofiber substrate ciOG accelerated bone matrix formation in a calvarial defect, indicating that the engineered biomaterial promotes the osteogenic capacity of ciOG in vivo. Furthermore, the ciOG platform recapitulated the genetic bone diseases Proteus syndrome and osteogenesis imperfecta, allowing candidate drug testing. The reprogramming of human fibroblasts into osteogenic cells with a chemical cocktail thus provides a source of specialized cells for use in bone tissue engineering and disease modeling.

Diabetic bone regeneration with nanoceria-tailored scaffolds by recapitulating cellular microenvironment: Activating integrin/TGF-ß co-signaling of MSCs while relieving oxidative stress.

Regenerating defective bone in patients with diabetes mellitus remains a significant challenge due to high blood glucose level and oxidative stress. Here we aim to tackle this issue by means of a drug- and cell-free scaffolding approach. We found the nanoceria decorated on various types of scaffolds (fibrous or 3D-printed one; named nCe-scaffold) could render a therapeutic surface that can recapitulate the microenvironment: modulating oxidative stress while offering a nanotopological cue to regenerating cells. Mesenchymal stem cells (MSCs) recognized the nanoscale (tens of nm) topology of nCe-scaffolds, presenting highly upregulated curvature-sensing membrane protein, integrin set, and adhesion-related molecules. Osteogenic differentiation and mineralization were further significantly enhanced by the nCe-scaffolds. Of note, the stimulated osteogenic potential was identified to be through integrin-mediated TGF-ß co-signaling activation. Such MSC-regulatory effects were proven in vivo by the accelerated bone formation in rat calvarium defect model. The nCe-scaffolds further exhibited profound enzymatic and catalytic potential, leading to effectively scavenging reactive oxygen species in vivo. When implanted in diabetic calvarium defect, nCe-scaffolds significantly enhanced early bone regeneration. We consider the currently-exploited nCe-scaffolds can be a promising drug- and cell-free therapeutic means to treat defective tissues like bone in diabetic conditions.

Cellular and Tissue Selectivity of AAV Serotypes for Gene Delivery to Chondrocytes and Cartilage.

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients. Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted. Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models. Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector.

Enhancement of Mesenchymal Stem Cell-Driven Bone Regeneration by Resveratrol-Mediated SOX2 Regulation.

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine. However, MSCs age rapidly during long-term ex vivo culture and lose their therapeutic potential before they reach effective cell doses (ECD) for cell therapy. Thus, a prerequisite for effective MSC therapy is the development of cell culture methods to preserve the therapeutic potential during long-term ex vivo cultivation. Resveratrol (RSV) has been highlighted as a therapeutic candidate for bone disease. Although RSV treatment has beneficial effects on bone-forming cells, in vivo studies are lacking. The current study showed that long-term (6 weeks from primary culture date)-cultured MSCs with RSV induction retained their proliferative and differentiation potential despite reaching ECD. The mechanism of RSV action depends entirely on the SIRT1-SOX2 axis in MSC culture. In a rat calvarial defect model, RSV induction significantly improved bone regeneration after MSC transplantation. This study demonstrated an example of efficient MSC therapy for treating bone defects by providing a new strategy using the plant polyphenol RSV.

Inhibition of STAT5A promotes osteogenesis by DLX5 regulation.

The regulation of osteogenesis is important for bone formation and fracture healing. Despite advances in understanding the molecular mechanisms of osteogenesis, crucial modulators in this process are not well-characterized. Here we demonstrate that suppression of signal transducer and activator of transcription 5A (STAT5A) activates distal-less homeobox 5 (DLX5) in human bone marrow-derived stromal cells (hBMSCs) and enhances osteogenesis in vitro and in vivo. We show that STAT5A negatively regulates expression of Dlx5 in vitro and that STAT5A deletion results in increased trabecular and cortical bone mass and bone mineral density in mice. Additionally, STAT5A deletion prevents age-related bone loss. In a murine fracture model, STAT5A deletion was found to significantly enhance bone remodeling by stimulating the formation of a fracture callus. Our findings indicate that STAT5A inhibition enhances bone formation by promoting osteogenesis of BMSCs.

Zinc Promotes Osteoblast Differentiation in Human Mesenchymal Stem Cells Via Activation of the cAMP-PKA-CREB Signaling Pathway.

The crucial trace element zinc stimulates osteogenesis in vitro and in vivo. However, the pathways mediating these effects remain poorly understood. This study aimed to investigate the effects of zinc on osteoblast differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) and to identify the molecular mechanisms of these effects. In hBMSCs, zinc exposure resulted in a dose-dependent increase in osteogenesis and increased mRNA and protein levels of the master transcriptional factor RUNX2. Analyzing the upstream signaling pathways of RUNX2, we found that protein kinase A (PKA) signaling inhibition blocked zinc-induced osteogenic effects. Zinc exposure increased transcriptional activity and protein levels of phospho-CREB and enhanced translocation of phospho-CREB into the nucleus. These effects were reversed by H-89, a potent inhibitor of PKA. Moreover, zinc exposure led to dose-dependent increases in levels of intracellular cyclic adenosine monophosphate (cAMP). These findings indicate that zinc activates the PKA signaling pathway by triggering an increase in intracellular cAMP, leading to enhanced osteogenic differentiation in hBMSCs. Our results suggest that zinc exerts osteogenic effects in hBMSCs by activation of RUNX2 via the cAMP-PKA-CREB signaling pathway. Zinc supplementation may offer a promise as a potential pharmaceutical therapy for osteoporosis and other bone loss conditions.

Measurement of Intracellular ROS in Caenorhabditis elegans Using 2',7'-Dichlorodihydrofluorescein Diacetate.

Reactive oxygen species (ROS) are generated during normal metabolic processes under aerobic conditions. Since ROS production initiates harmful radical chain reactions on cellular macromolecules, including lipid peroxidation, DNA mutation, and protein denaturation, it has been implicated in a wide spectrum of diseases such as cancer, cardiovascular disease, ischemia-reperfusion and aging. Over the past several decades, antioxidants have received explosive attention regarding their protective potential against these deleterious reactions. Accordingly, many analytical methodologies have been developed for the evaluation of the antioxidant capacity of compounds or complex biological samples. Herein, we introduce a simple and convenient method to detect in vivo intracellular ROS levels photometrically in Caenorhabditis elegans using 2',7'-dichlorofluorescein diacetate (H2DCFDA), a cell permeant tracer.

Subunits of the DNA polymerase alpha-primase complex promote Notch-mediated proliferation with discrete and shared functions in C. elegans germline.

Notch receptor signaling is a highly conserved cell communication system in most multicellular organisms and plays a critical role at several junctures in animal development. In Caenorhabditis elegans,GLP-1/Notch signaling is essential for both germline stem cell maintenance and germ cell proliferation during gonad development. Here, we show that subunits (POLA-1, DIV-1, PRI-1, and PRI-2) of the DNA polymerase alpha-primase complex are required for germ cell proliferation in response to GLP-1/Notch signaling in different tissues at different developmental stages. Specifically, genetic and functional analyses demonstrated that (a) maternally contributed DIV-1 (regulatory subunit) is indispensable non-cell autonomously for GLP-1/Notch-mediated germ cell proliferation during early larval development, whereas POLA-1 (catalytic subunit) and two primase subunits, PRI-1 and PRI-2, do not appear to be essential; (b) germline POLA-1, PRI-1, and PRI-2 play a crucial role in GLP-1/Notch-mediated maintenance of proliferative cell fate during adulthood, while DIV-1 is dispensable; and (c) germline POLA-1, DIV-1, PRI-1, and PRI-2 function in tandem with PUF (Pumilio/FBF) RNA-binding proteins to maintain germline stem cells in the adult gonad. These findings suggest that the subunits of the DNA polymerase alpha-primase complex exhibit both discrete and shared functions in GLP-1/Notch or PUF-mediated germ cell dynamics in C. elegans. These findings link the biological functions of DNA replication machineries to signals that maintain a stem cell population, and may have further implications for Notch-dependent tumors.

Triclosan Disrupts SKN-1/Nrf2-Mediated Oxidative Stress Response in C. elegans and Human Mesenchymal Stem Cells.

Triclosan (TCS), an antimicrobial chemical with potential endocrine-disrupting properties, may pose a risk to early embryonic development and cellular homeostasis during adulthood. Here, we show that TCS induces toxicity in both the nematode C. elegans and human mesenchymal stem cells (hMSCs) by disrupting the SKN-1/Nrf2-mediated oxidative stress response. Specifically, TCS exposure affected C. elegans survival and hMSC proliferation in a dose-dependent manner. Cellular analysis showed that TCS inhibited the nuclear localization of SKN-1/Nrf2 and the expression of its target genes, which were associated with oxidative stress response. Notably, TCS-induced toxicity was significantly reduced by either antioxidant treatment or constitutive SKN-1/Nrf2 activation. As Nrf2 is strongly associated with aging and chemoresistance, these findings will provide a novel approach to the identification of therapeutic targets and disease treatment.

A Phenotype-Based RNAi Screening for Ras-ERK/MAPK Signaling-Associated Stem Cell Regulators in C. elegans.

Stem cells have the ability to self-renew and to generate differentiated cell types. A regulatory network that controls this balance is critical for stem cell homeostasis and normal animal development. Particularly, Ras-ERK/MAPK signaling pathway is critical for stem cell self-renewal and differentiation in mammals, including humans. Aberrant regulation of Ras-ERK/MAPK signaling pathway results in either stem cell or overproliferation. Therefore, the identification of Ras-ERK/MAPK signaling pathway-associated regulators is critical to understand the mechanism of stem cell (possibly cancer stem cell) control. In this report, using the nematode C. elegans mutants, we developed a methodology for a phenotype-based RNAi screening that identifies stem cell regulator genes associated with Ras-ERK/MAPK signaling within the context of a whole organism. Importantly, this phenotype-based RNAi screening can be applied for other stem cell-associated signaling pathways such as Wnt/ß-catenin and Notch using the C. elegans.

Synergistic Action of IL-8 and Bone Marrow Concentrate on Cartilage Regeneration Through Upregulation of Chondrogenic Transcription Factors.

The objective of this study was to determine whether a biphasic scaffold loaded with a combination of a chemokine and bone marrow concentrate (BMC) could improve tissue regeneration in knee articular cartilage of beagles with cylindrical osteochondral defects. For this investigation, an osteochondral defect (6 mm in diameter and 8 mm deep) was created in the weight-bearing articular surface of the femoral medial condyle in beagles. Bone marrow was aspirated from the posterior iliac crests of beagles to obtain mesenchymal stem cells (MSCs) for in vitro assay. Hematoxylin and eosin (HE), Masson's trichrome (MT), safranin O/fast green staining, and immunohistochemistry were performed for histological analysis. Quantitative real-time polymerase chain reaction was performed to understand the roles of BMC in chondrogenic differentiation of MSCs. At 12 weeks after transplantation of biphasic scaffolds, we observed that interleukin-8 (IL-8) or the combination of IL-8 and BMC induced massive bone regeneration compared to saline, BMC only, and MSCs. In gross appearance, the osteochondral defect site was nearly completely filled with repair tissue in the group that received the combination of IL-8 and BMC but not in the other groups. Moreover, histological analysis showed obvious differences in cartilage regeneration among groups. HE and MT staining showed that the cartilage defect sites of the group receiving the combination of IL-8 and BMC were regenerated with cartilage-like tissues showing chondrocyte morphology. Safranin O staining showed hyaline cartilage regeneration in the group receiving IL-8 and BMC, whereas fibrous-like tissues were formed in the other groups. Furthermore, immunostaining revealed the presence of type II collagen and aggrecan in regenerated cartilage tissue of the group receiving IL-8 and BMC, whereas regenerated cartilage tissues of the other groups weakly expressed type II collagen and aggrecan. These results indicate that the combination of a chemokine IL-8 and BMC has significant positive effects on osteochondral regeneration in a beagle model through enhancing expression of the chondrogenic transcription factors and markers such as Sox9 and type II collagen.

Different effects of resveratrol on early and late passage mesenchymal stem cells through ß-catenin regulation.

Resveratrol is a sirtuin 1 (SIRT1) activator and can function as an anti-inflammatory and antioxidant factor. In mesenchymal stem cells (MSCs), resveratrol enhances the proliferation and differentiation potential and has an anti-aging effect. However, contradictory effects of resveratrol on MSC cultures have been reported. In this study, we found that resveratrol had different effects on MSC cultures according to their cell passage and SIRT1 expression. Resveratrol enhanced the self-renewal potential and multipotency of early passage MSCs, but accelerated cellular senescence of late passage MSCs. In early passage MSCs expressing SIRT1, resveratrol decreased ERK and GSK-3ß phosphorylation, suppressing ß-catenin activity. In contrast, in late passage MSCs, which did not express SIRT1, resveratrol increased ERK and GSK-3ß phosphorylation, activating ß-catenin. We confirmed that SIRT1-deficient early passage MSCs treated with resveratrol lost their self-renewal potential and multipotency, and became senescent due to increased ß-catenin activity. Sustained treatment with resveratrol at early passages maintained the self-renewal potential and multipotency of MSCs up to passage 10. Our findings suggest that resveratrol can be effectively applied to early passage MSC cultures, whereas parameters such as cell passage and SIRT1 expression must be taken into consideration before applying resveratrol to late passage MSCs.

Characterization of adipose tissue-derived stromal vascular fraction for clinical application to cartilage regeneration.

Bone marrow concentration (BMC) is the most recognized procedure to prepare mesenchymal stem cells for cartilage regeneration. However, bone marrow aspiration is highly invasive and results in low stem cell numbers. Recently, adipose tissue-derived stromal vascular fraction (AT-SVF) was studied as an alternate source of stem cells for cartilage regeneration. However, AT-SVF is not fully characterized in terms of functional equivalence to BMC. Therefore, in this study, we characterized AT-SVF and assessed its suitability as a one-step surgical procedure for cartilage regeneration, as an alternative to BMC. AT-SVF contained approximately sixfold less nucleated cells than BMC. However, adherent cells in AT-SVF were fourfold greater than BMC. Additionally, the colony-forming unit frequency of AT-SVF was higher than that of BMC, at 0.5 and 0.01%, respectively. The mesenchymal stem cell (MSC) population (CD45-CD31-CD90+CD105+) was 4.28% in AT-SVF and 0.42% in BMC, and the adipose-derived stromal cell (ASC) population (CD34+CD31-CD146-) was 32% in AT-SVF and 0.16% in BMC. In vitro chondrogenesis demonstrated that micromass was not formed in BMC, whereas it was clearly formed in AT-SVF. Taken together, uncultured AT-SVF could be used in one-step surgery for cartilage regeneration as a substitute for BMC.

In Situ Recruitment of Human Bone Marrow-Derived Mesenchymal Stem Cells Using Chemokines for Articular Cartilage Regeneration.

Bone marrow-derived mesenchymal stem cells (BMSCs) are a good cell source for regeneration of cartilage as they can migrate directly to the site of cartilage injury and differentiate into articular chondrocytes. Articular cartilage defects do not heal completely due to the lack of chondrocytes or BMSCs at the site of injury. In this study, the chemotaxis of BMSCs toward chemokines, which may give rise to a complete regeneration of the articular cartilage, was investigated. CCR2, CCR4, CCR6, CXCR1, and CXCR2 were expressed in normal BMSCs and were increased significantly upon treatment with proinflammatory cytokines. BMSC migration was increased by MIP-3α and IL-8 more than by MCP-1 or SDF-1α. IL-8 and MIP-3α significantly enhanced the chemotaxis of BMSCs compared with MCP-1, SDF-1α, or PBS. Human BMSC recruitment to transplanted scaffolds containing either IL-8 or MIP-3α significantly increased in vivo compared to scaffolds containing PBS. Furthermore, IL-8- and MIP-3α-containing scaffolds enhanced tissue regeneration of an osteochondral defect site in beagle knee articular cartilage. Therefore, this study suggests that IL-8 and MIP-3α are the candidates that induce the regeneration of damaged articular cartilage.

SIRT1 directly regulates SOX2 to maintain self-renewal and multipotency in bone marrow-derived mesenchymal stem cells.

SOX2 is crucial for the maintenance of the self-renewal capacity and multipotency of mesenchymal stem cells (MSCs); however, the mechanism by which SOX2 is regulated remains unclear. Here, we report that RNA interference of sirtuin 1 (SIRT1) in human bone marrow (BM)-derived MSCs leads to a decrease of SOX2 protein, resulting in the deterioration of the self-renewal and differentiation capacities of BM-MSCs. Using immunoprecipitation, we demonstrated direct binding between SIRT1 and SOX2 in HeLa cells overexpressing SOX2. We further discovered that the RNA interference of SIRT1 induces the acetylation, nuclear export, and ubiquitination of SOX2, leading to proteasomal degradation in BM-MSCs. SOX2 suppression by trichostatin A (TSA), a known histone deacetylase inhibitor, was reverted by treatment with resveratrol (0.1 and 1 µM), a known activator of SIRT1 in BM-MSCs. Furthermore, 0.1 and 1 µM resveratrol reduced TSA-mediated acetylation and ubiquitination of SOX2 in BM-MSCs. SIRT1 activation by resveratrol enhanced the colony-forming ability and differentiation potential to osteogenic and adipogenic lineages in a dose-dependent manner. However, the enhancement of self-renewal and multipotency by resveratrol was significantly decreased to basal levels by RNA interference of SOX2. These results strongly suggest that the SIRT1-SOX2 axis plays an important role in maintaining the self-renewal capability and multipotency of BM-MSCs. In conclusion, our findings provide evidence for positive SOX2 regulation by post-translational modification in BM-MSCs through the inhibition of nuclear export and subsequent ubiquitination, and demonstrate that SIRT1-mediated deacetylation contributes to maintaining SOX2 protein in the nucleus.

Interleukin-6 induces the lineage commitment of bone marrow-derived mesenchymal multipotent cells through down-regulation of Sox2 by osteogenic transcription factors.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a heterogeneous population of cells that differ in size and morphology. BM-MSCs become committed to the osteogenic lineage as senescence approaches and lose multipotency. Nevertheless, little is known about the effects of cell-cell interaction between different populations on stemness loss and lineage commitment. The current study aimed to identify mechanisms by which cell-cell interactions between heterogeneous BM-MSCs affect stemness and lineage commitment of multipotent subpopulation. The lineage commitment of primitive multipotent cells was strongly induced in the presence of cytokines secreted by senescent-like cells in a cell culture insert system. Senescent-like cells secreted higher levels of interleukin-6 (IL-6) than primitive multipotent cells in a human cytokine array. IL-6 induced the lineage commitment and stemness loss in multipotent cells by decreasing Sox2 expression. Furthermore, we confirmed that IL-6 decreased the transcriptional activity of Sox2 through up-regulation of Runx2 and Dlx5. We suggest a mechanism by which IL-6 modulates the expression of Sox2, resulting in decreased multipotency and causing primitive multipotent cells to undergo osteogenic lineage commitment. This is the first study to identify mechanisms in which the cell-cell interactions between the different populations play important roles in the stemness loss and lineage commitment of multipotent populations.-Yoon, D. S., Kim, Y. H., Lee, S., Lee, K.-M., Park, K. H., Jang, Y., Lee, J. W. Interleukin-6 induces the lineage commitment of bone marrow-derived mesenchymal multipotent cells through down-regulation of Sox2 by osteogenic transcription factors.

microRNA-495 inhibits chondrogenic differentiation in human mesenchymal stem cells by targeting Sox9.

The chondrogenic differentiation process of human mesenchymal stem cells (hMSCs) passes through multiple stages, which are carried out by various factors and their interactions. Recently, microRNAs that regulate chondrogenic differentiation have been reported. However, microRNA that regulates SRY-related high mobility group-box gene 9 (Sox9), a chondrogenic key factor, has not been identified in hMSC. In this study, we identified that microRNA-495 (miR-495) is an important regulator of hMSC chondrogenic differentiation. In our microarray, miR-495 was downregulated during transforming growth factor (TGF)-ß3-induced chondrogenic differentiation of hMSCs in vitro. We found that there is an miR-495 binding site in the 3' untranslated region (3'UTR) of Sox9. We confirmed opposite expression between miR-495 and Sox9 by using real-time polymerase chain reaction. Further, overexpression of miR-495 inhibited Sox9 expression, and repression of miR-495 increased expression of Sox9 in SW1353 cells and hMSCs. Additionally, luciferase analysis revealed that miR-495 directly binds to the Sox9 3'UTR, and we confirmed a seed sequence of miR-495 on the Sox9 3'UTR. Subsequently, overexpression of miR-495 repressed the expression of the extracellular matrix (ECM) protein, such as type II collagen (Col2A1), aggrecan, and proteoglycan products, whereas inhibition of miR-495 increased their expression. Collectively, this study indicates that miR-495 directly targets Sox9, ultimately leading to the regulation of chondrogenic differentiation in hMSCs.

Zinc inhibits osteoclast differentiation by suppression of Ca2+-Calcineurin-NFATc1 signaling pathway.

BACKGROUND: Zinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells. RESULTS: In BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca2+ oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca2+ oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc. CONCLUSIONS: Taken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca2+-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.