Osteogenic Transcription Factors Osterix Research Articles

Vascular smooth muscle cell-derived exosomes promote osteoblast-to-osteocyte transition via β-catenin signaling

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via β-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/β-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.

Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors

BackgroundEndogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs.MethodsMSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLCResultsThe extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect.ConclusionsData suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

Inflammatory osteoclasts‐derived exosomes promote bone formation by selectively transferring lncRNA LIOCE into osteoblasts to interact with and stabilize Osterix

Bone loss is a hallmark of inflammatory bone diseases caused by aberrantly activated osteoclasts (OCLs). Studies have shown that OCLs exhibit various phenotypes and functions due to variations in the source(s) of precursor cells, cytokine expressions, and microenvironment-dependent factors. During these conditions, inflammatory osteoclasts (iOCLs) lose their immune-suppressive effect relative to OCLs under physiological conditions. This induces TNF α-producing CD4+ Tcells in an antigen-dependent manner and finally leads to cascade amplification of iOCLs. OCL-derived exosomes have been reported to regulate OCL formation and inhibit the osteoblast activity. However, the specific function and mechanism of iOCL-derived exosomes on osteoblast have not been studied yet. In the present study, we compare the osteoblast promoting activities of iOCL-derived exosomes and OCL-derived exosomes. We found that iOCLs exosomes specifically target osteoblasts through ephrinA2/EphA2. Mechanistically, the lncRNA LIOCE is enriched in iOCL exosomes and promotes the osteoblast activity after being incorporated into osteoblasts. Furthermore, our results revealed that exosomal lncRNA LIOCE stabilizes osteogenic transcription factor Osterix by interacting and reducing the ubiquitination level of Osterix. This study demonstrated that the bone loss is alleviated in the inflammatory osteolysis mice model after injection of iOCL exosomes encapsulating lncRNA LIOCE. The role of exosomes encapsulating lncRNA LIOCE in promoting bone formation was well established in the rat bone repair model. Our results indicate that iOCL-derived exosomal lncRNA LIOCE promotes bone formation by upregulating Osx expression, and thus, the exosomes encapsulating lncRNA LIOCE may be an effective strategy to increase bone formation in osteoporosis and other bone metabolic disorders.

Adiponectin inhibits vascular smooth muscle cell calcification induced by beta-glycerophosphate through JAK2/STAT3 signaling pathway

Vascular calcification is a common problem in the elderly with diabetes, heart failure and end-stage renal disease. The differentiation of vascular smooth muscle cells (VSMCs) into osteoblasts is the main feature, but the exact mechanism remains unclear. It is not clear whether adiponectin (APN) affects osteogenic differentiation of VSMCs. This study aims to explore the effect of APN on vascular calcification by using a cell model induced by beta-glycerophosphate (beta-GP). VSMCs were isolated and treated with beta-GP and APN in this study. The alkaline phosphatase (ALP) activity and expression levels of Runx2, BMP-2, collagen type I and osteocalcin were determined. The expression levels of STAT3 and p-STAT3 in nucleus and cytoplasm of VSMCs were analyzed. The results showed that APN significantly inhibited the expression of ALP, Runx2, BMP-2, collagen I, osteocalcin and the formation of the mineralized matrix in VSMCs induced by beta-GP. APN reduces the osteogenic differentiation of VSMCs induced by beta-GP and down-regulates the expression of the osteogenic transcription factor osterix by inhibiting STATS3 phosphorylation and nuclear transport. APN may be one of the potential candidates for clinical treatment of vascular calcification.

The long noncoding RNA lnc-ob1 facilitates bone formation by upregulating Osterix in osteoblasts.

Long noncoding RNAs (lncRNAs) have emerged as integral regulators of physiology and disease, but specific roles of lncRNAs in bone disease remain largely unknown. Here, we show that lnc-ob1 regulates osteoblast activity and bone formation in mice by upregulating the osteogenic transcription factor Osterix. Expression of lnc-ob1 is enriched in osteoblasts and upregulated during osteoblastogenesis. We demonstrate that osteoblast-specific knock-in of lnc-ob1 enhances bone formation and increases bone mass. Pharmacological overexpression of lnc-ob1 specifically in osteoblasts confers resistance to ovariectomy-induced osteoporosis in mice. In humans, expression of the homologue, lnc-OB1, decreases with age in osteoblasts of patients with osteoporosis. Mechanistically, lnc-ob1 upregulates the expression of Osterix in mouse and human osteoblasts, probably via inhibition of H3K27me3 methylation. Our data indicate that lnc-OB1 regulates bone formation and might be a drug target for the treatment of osteoporosis.

Characterization of the Hematopoietic Supporting Activity of Osteoblasts Derived from Bone Marrow Mesenchymal Stromal Cells

Osteoblasts (OST) found within the endosteal niche are important regulators of Hematopoietic Stem and Progenitor Cells (HSPC) under steady state and during hematopoietic reconstitution. OST are derived from mesenchymal stromal cells (MSC) following osteogenic differentiation. MSC and OST secrete a wide array of soluble factors that sustain hematopoiesis. Recently, we showed that media conditioned with OST derived from MSC (referred as M-OST) after 6 days of osteogenic differentiation were superior to MSC conditioned media (CM) for the expansion of cord blood (CB) progenitors, and CB cells expanded with M-OST CM supported a more robust engraftment of platelets in NSG mice after transplantation. These findings raised the possibility that M-OST could be superior to MSC for the ex vivoexpansion HSPC.In this study, we set out to test the hypothesis that the growth modulatory activity of M-OST would vary as a function of their maturation status. The objectives were to first monitor the impact of M-OST differentiation and maturation status on the expression of soluble factors that promote HSPC expansion and in second, to investigate the capacity of M-OST CMs prepared from M-OST at distinct stages of differentiation to support the expansion and differentiation of HSPCs in culture. M-OST at distinct stages of differentiation were derived by culturing bone marrow MSC in osteogenic medium for various length of time (3 to 21 days). All CB CD34+ enriched (92±7% purity) cell cultures were done with serum free media conditioned or not with MSC or M-OST and supplemented with cytokines SCF, TPO and FL.We first confirmed the progressive differentiation and maturation of M-OST as a function of osteogenic culture length, which was evident by the induction of the osteogenic transcription factors Osterix, Msx2 and Runx2 mRNAs, the gradual increase in osteopontin and alkaline phosphatase positive cells and quantitative increases in calcium deposit. Next, we investigated the expression in MSC and M-OSTs of genes known to collaborate for the expansion of HSPCs by Q-PCR. Transcript copy numbers for IGFBP-2 increased swiftly during osteogenic differentiation, peaking at day-3 (˃100-fold vs MSC, n=2) and returning below MSC level by day-21. In contrast, ANGPTL members (ANGPTL-1, -2, -3 and -5) remained superior in M-OSTs throughout osteogenic differentiation with expression levels peaking around day 6 (n=2).Next, we tested the capacity of media conditioned with primitive (day-3, -6), semi-mature (day-10, -14) and mature M-OST (day-21) to support the growth of CB cells. All M-OST CMs increased (p˂0.03) the growth of total nucleated cells (TNC) after 6 days of culture compared to non-conditioned medium used as control (mean 2.0-fold, n=4). Moreover, there was a positive correlation between cell growth and M-OST maturation status though differences between the different M-OST CMs tested were not significant. The capacity of M-OST CMs to increase (mean 2-fold, n=4) the expansion of CD34+ cells was also shared by all M-OST CMs (p˂0.05), as supported by significant increases with immature day-3 (mean ± SD of 18 ± 6, p˂0.02) and mature day-21 M-OST CMs (14 ± 5, p˂0.05) vs. control (8 ± 3, n=4). Conversely, expansions of TNC and CD34+ cells in MSC CM cultures were in-between that of control and M-OST CMs cultures. Interestingly, M-OST CMs also modulated the expansion of the HSPC compartment. Indeed, while the expansion of multipotent progenitors defined as CD34+CD45RA+ was promoted in control culture (ratio of 4.5 for CD34+CD45RA+/CD34+CD45RA- cells), M-OST CMs supported greater expansion of the more primitive CD34+CD45RA- HSPC subpopulation reducing the ratio to 3.3±0.4 for M-OST cultures (cumulative mean of 10 cultures, n=2). Moreover, the expansions of CD34+CD38- cells and of the long term HSC-enriched subpopulation (CD34+CD38-CD45RA-Thy1+) in M-OST CM cultures were respectively 2.7- and 2.8-fold greater than those measured in control cultures (n=2-4). Finally, the impact of M-OST CMs on the expansion of myeloid progenitors was investigated using a colony forming assay; expansion of myeloid progenitors were superior in all M-OST CM cultures (1.6±0.2 fold, n=2).In conclusion, our results demonstrate that M-OST rapidly acquire the expression of growth factors known to promote HSPC expansion. Moreover, the capacity of M-OST CMs to support the expansion of HSPCs appears to be a property shared by M-OST at various stages of maturation. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of protein kinase-D promotes cartilage repair at injured growth plate in rats

IntroductionInjured growth plate cartilage is often repaired by bony tissue, causing bone growth defects in children. Currently, mechanisms for the undesirable repair remain unclear and there are no biological treatments available to prevent the associated bone growth defects. Osterix is known as a vital transcription factor for osteoblast differentiation which is critical for normal bone formation and bone repair, and osterix is known to be regulated by protein kinase-D; however it is unknown whether protein kinase-D–osterix signalling plays any roles in the bony repair of injured growth plate. MethodsUsing a rat model, this study investigated potential roles of protein kinase-D (PKD) in regulating expression of osteogenic transcription factor osterix and the growth plate bony repair. 4 days post injury at the proximal tibial growth plate, rats received four once-daily injections of vehicle or 2.35mg/kg gö6976 (a PKD inhibitor), and growth plate tissues collected at day 10 were examined histologically and molecularly. In addition, effects of PKD inhibition on osteogenic and chondrogenic differentiation were examined in vitro using rat bone marrow mesenchymal stromal cells. ResultsCompared to vehicle control, PKD inhibition caused a decrease in bone volume (p<0.05), an increase in % of mesenchymal tissue (p<0.01), and an increase in cartilaginous tissue within the injury site. Consistently, gö6976 treatment tended to decrease expression of bone-related genes (osterix, osteocalcin) and increase levels of cartilage-related genes (Sox9, collagen-2a, collagen-10a1). In support, in vitro experiments showed that gö6976 presence in the primary rat marrow stromal cell culture resulted in a decrease of alkaline phosphatase+ CFU-f colonies formed (p<0.05) and an increase in collagen-2a expression in chondrogenic pellet culture (p<0.05). ConclusionThese studies suggest that PKD is important for growth plate bony repair and its inhibition after growth plate injury may result in less bone formation and potentially more cartilage repair.

Influence of select extracellular matrix proteins on mesenchymal stem cell osteogenic commitment in three-dimensional contexts

Growth factors have been shown to be powerful mediators of mesenchymal stem cell (MSC) osteogenic differentiation. However, their use in tissue engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically promote MSC osteogenic differentiation in the absence of exogenous growth factors via the manipulation of scaffold material properties would be beneficial. The current work examines the influence of select extracellular matrix (ECM) proteins on MSC osteogenesis toward the goal of developing scaffolds with intrinsically osteoinductive properties. Fibrinogen (FG), fibronectin (FN) and laminin-1 (LN) were chosen for evaluation due to their known roles in bone morphogenesis or bone fracture healing. These proteins were conjugated into poly(ethylene glycol) diacrylate (PEGDA) hydrogels and their effects on encapsulated 10T½ MSCs were evaluated. Specifically, following 1week of culture, mid-term markers of various MSC lineages were examined in order to assess the strength and specificity of the observed osteogenic responses. PEG–LN gels demonstrated increased levels of the osteogenic transcription factor osterix relative to day 0 levels. In addition, PEG–FG and PEG–LN gels were associated with increased deposition of bone ECM protein osteocalcin relative to PEG–FN gels and day 0. Importantly, the osteogenic response associated with FG and LN appeared to be specific in that markers for chondrocytic, smooth muscle cell and adipocytic lineages were not similarly elevated relative to day 0 in these gels. To gain insight into the integrin dynamics underlying the observed differentiation results, initial integrin adhesion and temporal alterations in cell integrin profiles were evaluated. The associated results suggest that α2, αv and α6 integrin subunits may play key roles in integrin-mediated osteogenesis.

Murine calvaria-derived progenitor cells express high levels of osterix and lose their adipogenic capacity

Though the mouse is the most widely used biomedical animal model, it is difficult to isolate murine mesenchymal stem cells (MSCs) from the bone marrow because of contamination by hematopoietic cells. The murine compact bone tissue of long bones is considered a novel and reliable source of MSCs with low hematopoietic cell contamination. We investigated whether the murine compact bone of the calvaria would be a promising source of MSCs due to its low bone marrow content. We isolated cells from both long bones and the calvaria using the same method. Although they shared morphological features and surface antigens similar to those of long bone-derived MSCs, the calvaria-derived cells highly expressed the osteogenic transcription factor osterix, lost their adipogenic capacity and gained a higher osteogenic capacity. These findings suggest that the cells that migrated from the calvaria were progenitor cells rather than MSCs and that the differentiation fate of mesenchymal stem/progenitor cells existing in different murine compact bone deposits is already committed.

A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine whose levels in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. Here, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function and therefore contributes to OB/OC uncoupling in MM.

1,25-Dihydroxyvitamin D3-induced aortic calcifications in experimental uremia: up-regulation of osteoblast markers, calcium-transporting proteins and osterix

Whether treatment with vitamin D receptor activators contributes to cardiovascular disease in patients with chronic kidney disease is a matter of debate. We studied mechanisms involved in vitamin D-related vascular calcifications in vivo and in vitro. Aortic calcifications were induced in subtotally nephrectomized (SNX) rats by treatment with a high dose (0.25 μg/kg per day) of 1,25-dihydroxyvitamin D3 (calcitriol) given for 6 weeks. Likewise, primary rat vascular smooth muscle cells (VSMCs) were incubated with calcitriol at concentrations ranging from 10 to 10 mol/l. Immunohistochemistry revealed that the aortic expression of osteopontin, osteocalcin and bone sialoprotein was significantly increased in calcitriol-treated SNX rats compared to untreated SNX controls. In addition, aortic expression of the transient receptor potential vanilloid calcium channel 6 (TRPV6) and calbindin D9k was significantly up-regulated by treatment with calcitriol. Furthermore, calcitriol significantly increased expression of the osteogenic transcription factor osterix. In-vitro studies showed similar results, confirming that these effects could be attributed to treatment with calcitriol. High-dose calcitriol treatment induces an osteoblastic phenotype in VSMC both in SNX rats and in vitro, associated with up-regulation of proteins regulating mineralization and calcium transport, and of the osteogenic transcription factor osterix.

The Orphan Receptor Tyrosine Kinase Ror2 Promotes Osteoblast Differentiation and Enhances ex Vivo Bone Formation

Ror2 is a receptor tyrosine kinase, the expression of which increases during differentiation of pluripotent stem cells to osteoblasts and then declines as cells progress to osteocytes. To test whether Ror2 plays a role in osteoblastogenesis, we investigated the effects of Ror2 overexpression and down-regulation on osteoblastic lineage commitment and differentiation. Expression of Ror2 in pluripotent human mesenchymal stem cells (hMSCs) by adenoviral infection caused formation of mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Concomitantly, Ror2 over-expression inhibited adipogenic differentiation of hMSCs as monitored by lipid formation. Ror2 shifted hMSC fate toward osteoblastogenesis by inducing osteogenic transcription factor osterix and suppressing adipogenic transcription factors CCAAT/enhancer-binding protein alpha and peroxisome proliferator activated receptor gamma. Infection with Ror2 virus also strongly promoted matrix mineralization in committed osteoblast-like MC3T3-E1 cells. Expression of Ror2 in a human preosteocytic cell line by stable transfection also promoted further differentiation, as judged by inhibited alkaline phosphatase activity, potentiated osteocalcin secretion, and increased cellular apoptosis. In contrast, down-regulation of Ror2 expression by short hairpin RNA essentially abrogated dexamethasone-induced mineralization of hMSCs. Furthermore, down-regulation of Ror2 expression in fully differentiated SaOS-2 osteosarcoma cells inhibited alkaline phosphatase activity. We conclude that Ror2 initiates commitment of MSCs to osteoblastic lineage and promotes differentiation at early and late stages of osteoblastogenesis. Finally, using a mouse calvariae ex vivo organ culture model, we demonstrate that these effects of Ror2 result in increased bone formation, suggesting that it may also activate mature osteoblasts.