Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells.
ABSTRACT: We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs) and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5), which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine.
Project description:Previous studies indicated that let-7 enhances osteogenesis and bone formation of human adipose-derived mesenchymal stem cells (MSCs). We also have confirmed that let-7f-5p expression was upregulated during osteoblast differentiation in rat bone marrow-derived MSCs (BMSCs) and was downregulated in the vertebrae of patients with glucocorticoid (GC)-induced osteoporosis (GIOP). The study was performed to determine the role of let-7f-5p in GC-inhibited osteogenic differentiation of murine BMSCs <i>in vitro</i> and in GIOP <i>in vivo</i>. Here, we report that dexamethasone (Dex) inhibited osteogenic differentiation of BMSCs and let-7f-5p expression, while increasing the expression of transforming growth factor beta receptor 1 (TGFBR1), a direct target of let-7f-5p during osteoblast differentiation under Dex conditions. In addition, let-7f-5p promoted osteogenic differentiation of BMSCs, as indicated by the promotion of alkaline phosphatase (ALP) staining and activity, Von Kossa staining, and osteogenic marker expression (<i>Runx2</i>,<i>Osx</i>, <i>Alp</i>, and <i>Ocn</i>), but decreased TGFBR1 expression in the presence of Dex. However, overexpression of TGFBR1 reversed the upregulation of let-7f-5p during Dex-treated osteoblast differentiation. Knockdown of TGFBR1 reversed the effect of let-7f-5p downregulation during Dex-treated osteogenic differentiation of BMSCs. We also found that glucocorticoid receptor (GR) mediated transcriptional silencing of let-7f-5p and its knockdown enhanced Dex-inhibited osteogenic differentiation. Further, when injected <i>in vivo</i>, agomiR-let-7f-5p significantly reversed bone loss induced by Dex, as well as increased osteogenic marker expression (<i>Runx2</i>, <i>Osx</i>, <i>Alp</i>, and <i>Ocn</i>) and decreased TGFBR1 expression in bone extracts. These findings indicated that the regulatory axis of GR/let-7f-5p/TGFBR1 may be important for Dex-inhibited osteoblast differentiation and that let-7f-5p may be a useful therapeutic target for GIOP.
Project description:Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation, and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues. The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells. Initially, we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells. Moreover, Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line. In addition, micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo. Unexpectedly, Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2, Dlx5, Msx2, and Osterix, but led to upregulation of Alp and Osteocalcin (OCN), both of which play critical roles in promoting osteoblast maturation. Importantly, luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity. Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis, we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes. Based on these findings, we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene, suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
Project description:BACKGROUND:Osteogenic differentiation is an essential process for bone regeneration involving bone marrow mesenchymal stem cells (BMSCs). BMSC-secreted extracellular vesicles (EVs) enriched with microRNAs (miRs) have vital roles to play in mediating osteogenic differentiation. Therefore, this study aimed to explore the effect of BMSC-derived EVs loaded with miR-15b on osteogenic differentiation. METHODS:Human BMSCs (hBMSCs) were cultured and treated with plasmids overexpressing or knocking down KLF2, WWP1, and miR-15b to define the role of derived EVs in osteogenic differentiation in vitro. The expression of osteogenic differentiation-related marker was measured by Western blot analysis. The interaction among miR-15b, WWP1, and ubiquitination of KLF2 was investigated by dual-luciferase reporter, immunoprecipitation, and GST pull-down assays. Moreover, EVs from hBMSCs transfected with miR-15b inhibitor (EV-miR-15b inhibitor) were injected into ovariectomized rats to verify the effect of miR-15b on bone loss in vivo. RESULTS:WWP1 was downregulated, and KLF2 was upregulated during osteogenic differentiation. After co-culture with EVs, miR-15b expression was elevated and WWP1 expression was reduced in hBMSCs. Upregulation of miR-15b or KLF2 or downregulation of WWP1 or NF-?B increased ALP activity and cell mineralization, as well as osteogenic differentiation-related marker expression in hBMSCs. Mechanistically, miR-15b targeted and inhibited WWP1, thus attenuating KLF2 degradation and inhibiting NF-?B activity. Co-culture of EVs increased the bone volume and trabecular number, but decreased bone loss in ovariectomized rats, which could be reversed after treatment with EV-miR-15b inhibitor. CONCLUSION:Collectively, BMSC-derived EVs loaded with miR-15b promoted osteogenic differentiation by impairing WWP1-mediated KLF2 ubiquitination and inactivating the NF-?B signaling pathway.
Project description:To study the expression patterns of two Eph family molecules, the receptor EphA5, and the ligand ephrin-A5, during spinal cord development.The receptor expression was analyzed using beta-galactosidase knockin mice, and affinity ligand probe binding. The ligand expression was assessed using two different affinity probes, and knockout mouse tissues as controls.EphA5 was expressed in the ventral spinal cord, while ephrin-A5 was located in the dorsolateral regions of the spinal cord throughout development.These results show that EphA5 and ephrin-A5 are expressed over broad developmental stages and may play important roles in establishing the dorsoventral organization of the spinal cord.
Project description:Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cells but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up-regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.
Project description:Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca2+ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.
Project description:Multipotent mesenchymal stem cells (MSCs) maintain the ability to differentiate into adipogenic, chondrogenic, or osteogenic cell lineages. There is increasing concern that exposure to environmental agents such as aryl hydrocarbon receptor (AhR) ligands, may perturb the osteogenic pathways responsible for normal bone formation. The objective of the current study was to evaluate the potential of the prototypic AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to disrupt osteogenic differentiation of human bone-derived MSCs (hBMSCs) in vitro. Primary hBMSCs from three donors were exposed to 10?nM TCDD and differentiation was interrogated using select histological, biochemical, and transcriptional markers of osteogenesis. Exposure to 10?nM TCDD resulted in an overall consistent attenuation of alkaline phosphatase (ALP) activity and matrix mineralization at terminal stages of differentiation in primary hBMSCs. At the transcriptional level, the transcriptional regulator DLX5 and additional osteogenic markers (ALP, OPN, and IBSP) displayed attenuated expression; conversely, FGF9 and FGF18 were consistently upregulated in each donor. Expression of stem cell potency markers SOX2, NANOG, and SALL4 decreased in the osteogenic controls, whereas expression in TCDD-treated cells resembled that of undifferentiated cells. Coexposure with the AhR antagonist GNF351 blocked TCDD-mediated attenuation of matrix mineralization, and either fully or partially rescued expression of genes associated with osteogenic regulation, extracellular matrix, and/or maintenance of multipotency. Thus, experimental evidence from this study suggests that AhR transactivation likely attenuates osteoblast differentiation in multipotent hBMSCs. This study also underscores the use of primary human MSCs to evaluate osteoinductive or osteotoxic potential of chemical and pharmacologic agents in vitro.
Project description:Mesenchymal stem cells (MSCs), which have multipotential differentiation and self-renewal potential, are possible cells for tissue engineering. Transforming growth factor ?1 (TGF?1) can be produced by MSCs in an inactive form, and the activation of TGF?1 functions as an important regulator of osteogenic differentiation in MSCs. Recently, studies showed that Glycoprotein A repetitions predominant (GARP) participated in the activation of latent TGF?1, but the interaction between GARP and TGF?1 is still undefined. In our study, we successfully isolated the MSCs from bone marrow of rats, and showed that GARP was detected in bone mesenchymal stem cells (BMSCs). During the osteogenic differentiation of BMSCs, GARP expression was increased over time. To elucidate the interaction between GARP and TGF?1, we downregulated GARP expression in BMSCs to examine the level of active TGF?1. We then verified that the downregulation of GARP decreased the secretion of active TGF?1. Furthermore, osteogenic differentiation experiments, alkaline phosphatase (ALP) activity analyses and Alizarin Red S staining experiments were performed to evaluate the osteogenic capacity. After the downregulation of GARP, ALP activity and Alizarin Red S staining significantly declined and the osteogenic indicators, ALP, Runx2, and OPN, also decreased, both at the mRNA and protein levels. These results demonstrated that downregulated GARP expression resulted in the reduction of TGF?1 and the attenuation of osteoblast differentiation of BMSCs in vitro.
Project description:BACKGROUND:The proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMScs) are modulated by a variety of microRNAs (miRNAs). SATB homeobox 2 (SATB2) is a critical transcription factor that contributes to maintain the balance of bone metabolism. However, it remains unclear how the regulatory relationship between miR-103 and SATB2 on HBMScs proliferation and osteogenic differentiation. METHODS:HBMScs were obtained from Cyagen Biosciences and successful induced osteogenic differentiation. The proliferation abilities of HBMScs after treatment with agomiR-103 and antagomiR-103 were assessed using a cell counting Kit-8 (CCK-8) assay, and osteogenic differentiation was determined using alizarin red S staining and alkaline phosphatase (ALP) activity assay. The expression levels of miR-103, SATB2, and associated osteogenic differentiation biomarkers, including RUNX family transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), and secreted phosphoprotein 1 (SPP1), were evaluated using real-time qPCR and Western blot. The regulatory sites of miR-103 on SATB2 were predicted using bioinformatics software and validated using a dual luciferase reporter assay. The underlying mechanism of miR-103 on SATB2-medicated HBMScs proliferation and osteogenic differentiation were confirmed by co-transfection of antagomiR-103 and SATB2 siRNA. RESULTS:The expression of miR-103 in HBMScs after induction of osteogenic differentiation was reduced in a time-dependent way. Overexpression of miR-103 by transfection of agomiR-103 suppressed HBMScs proliferation and osteogenic differentiation, while silencing of miR-103 by antagomiR-103 abolished these inhibitory effects. Consistently, RUNX2, BGLAP and SPP1 mRNA and protein expression were decreased in agomiR-103 treated HBMScs compared with those in agomiR-NC group. Meanwhile, antagomiR-103 upregulated the mRNA and protein expression levels of RUNX2, BGLAP and SPP1 in HBMScs. Further studies revealed that SATB2 was a direct target gene of miR-103. BMSCs transfected with agomiR-103 exhibited significantly downregulated protein expression level of SATB2, whereas knockdown of miR-103 promoted it. Additionally, rescue assays confirmed that silencing of SATB2 partially reversed the effects of antagomiR-103 induced HBMScs proliferation and osteogenic differentiation. CONCLUSIONS:The present results suggested that miR-103 negatively regulates SATB2 to serve an inhibitory role in the proliferation and osteogenic differentiation of HBMScs, which sheds light upon a potential therapeutic target for treating bone-related diseases.
Project description:The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll ?-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J-K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs.