Indian hedgehog requires additional effectors besides Runx2 to induce osteoblast differentiation

AbstractIndian hedgehog (Ihh) is indispensable for osteoblast differentiation during embryonic development of the endochondral skeleton. In the absence of Ihh, cells of the osteoblast lineage fail to activate the expression of Runx2, a transcription factor integral to osteoblast differentiation. However, it is hitherto unclear whether the lack of Runx2 expression is solely responsible for the failure of osteoblast formation in Ihh-null embryos. Here, by creating a mouse allele that expresses Runx2 in a Cre-dependent manner, we show that force-expression of Runx2 in the skeletogenic cells restores bone formation in the Runx2-null, but not in the Ihh-null embryo. Thus, the mechanism through which Ihh induces osteoblast differentiation requires other effectors in addition to Runx2

Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress in vitro and in vivo in female mice

Endoplasmic reticulum (ER) stress is associated with increased reactive oxygen species (ROS), results from accumulation of misfolded/unfolded proteins, and can trigger apoptosis. ER stress is alleviated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein translation allowing the ER to recover, thus promoting cell viability. We investigated whether osteoblastic cell apoptosis induced by glucocorticoids (GCs) is due to induction of ROS/ER stress and whether inhibition of eIF2α dephosphorylation promotes survival opposing the deleterious effects of GC in vitro and in vivo. Apoptosis of osteocytic MLO-Y4 and osteoblastic OB-6 cells induced by dexamethasone was abolished by ROS inhibitors. Like GC, the ER stress inducing agents brefeldin A and tunicamycin induced osteoblastic cell apoptosis. Salubrinal or guanabenz, specific inhibitors of eIF2α dephosphorylation, blocked apoptosis induced by either GC or ER stress inducers. Moreover, GC markedly decreased mineralization in OB-6 cells or primary osteoblasts; and salubrinal or guanabenz increased mineralization and prevented the inhibitory effect of GC. Furthermore, salubrinal (1 mg/kg/day) abolished osteoblast and osteocyte apoptosis in cancellous and cortical bone and partially prevented the loss of BMD at all sites and the decreased vertebral cancellous bone formation induced by treatment with prednisolone for 28 days (1.4 mg/kg/day). We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells is mediated through ER stress, and that inhibition of eIF2α dephosphorylation protects from GC-induced apoptosis of osteoblasts and osteocytes in vitro and in vivo and from the deleterious effects of GC on the skeleton

Parathyroid hormone receptor signaling induces bone resorption in the adult skeleton by directly regulating the RANKL gene in osteocytes

PTH upregulates the expression of the receptor activator of nuclear factor κB ligand (Rankl) in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell responsible for RANKL-mediated stimulation of bone resorption remains undefined. We report that constitutive activation of PTH receptor signaling only in osteocytes in transgenic mice (DMP1-caPTHR1) was sufficient to increase Rankl expression and bone resorption. Resorption in DMP1-caPTHR1 mice crossed with mice lacking the distal control region regulated by PTH in the Rankl gene (DCR(-/-)) was similar to DMP1-caPTHR1 mice at 1 month of age, but progressively declined to reach values undistinguishable from wild-type (WT) mice at 5 months of age. Moreover, DMP1-caPTHR1 mice exhibited low tissue material density and increased serum alkaline phosphatase activity at 5 month of age, and these indices of high remodeling were partially and totally corrected in compound DMP1-caPTHR1;DCR(-/-) male mice, and less affected in female mice. Rankl expression in bones from DMP1-caPTHR1 mice was elevated at both 1 and 5 months of age, whereas it was high, similar to DMP1-caPTHR1 mice at 1 month, but low, similar to WT levels at 5 months in compound mice. Moreover, PTH increased Rankl and decreased Sost and Opg expression in ex vivo bone organ cultures established from WT mice, but only regulated Sost and Opg expression in cultures from DCR(-/-) mice. PTH also increased RANKL expression in osteocyte-containing primary cultures of calvarial cells, in isolated murine osteocytes, and in WT but not in DCR(-/-) osteocyte-enriched bones. Thus, PTH upregulates Rankl expression in osteocytes in vitro, ex vivo and in vivo, and resorption induced by PTH receptor signaling in the adult skeleton requires direct regulation of the Rankl gene in osteocytes.We thank Dr. Keith Condon and Ms. Naomie Olivos for technical assistance and Dr Munro Peacock for measurement of alkaline phosphatase. This research was supported by the National Institutes of Health (R01DK076007 and American Recovery and Reinvestment Act supplement S10-RR023710 to T.B.) and the Veterans Administration (Merit Review I01BX002104 to T.B.). Disclosure Summary: The authors declare that no conflict of interest exists

Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone

Osteocytes, >90% of the cells in bone, lie embedded within the mineralized matrix and coordinate osteoclast and osteoblast activity on bone surfaces by mechanisms still unclear. Bone anabolic stimuli activate Wnt signaling, and human mutations of components along this pathway underscore its crucial role in bone accrual and maintenance. However, the cell responsible for orchestrating Wnt anabolic actions has remained elusive. We show herein that activation of canonical Wnt signaling exclusively in osteocytes [dominant active (da)βcat(Ot) mice] induces bone anabolism and triggers Notch signaling without affecting survival. These features contrast with those of mice expressing the same daß-catenin in osteoblasts, which exhibit decreased resorption and perinatal death from leukemia. daßcat(Ot) mice exhibit increased bone mineral density in the axial and appendicular skeleton, and marked increase in bone volume in cancellous/trabecular and cortical compartments compared with littermate controls. daßcat(Ot) mice display increased resorption and formation markers, high number of osteoclasts and osteoblasts in cancellous and cortical bone, increased bone matrix production, and markedly elevated periosteal bone formation rate. Wnt and Notch signaling target genes, osteoblast and osteocyte markers, and proosteoclastogenic and antiosteoclastogenic cytokines are elevated in bones of daßcat(Ot) mice. Further, the increase in RANKL depends on Sost/sclerostin. Thus, activation of osteocytic β-catenin signaling increases both osteoclasts and osteoblasts, leading to bone gain, and is sufficient to activate the Notch pathway. These findings demonstrate disparate outcomes of β-catenin activation in osteocytes versus osteoblasts and identify osteocytes as central target cells of the anabolic actions of canonical Wnt/β-catenin signaling in bone

A Highly Selective GSK-3β Inhibitor CHIR99021 Promotes Osteogenesis by Activating Canonical and Autophagy-Mediated Wnt Signaling

The discovery and application of small molecules is one of the practical strategies of safe osteogenic drugs. The small molecule CHIR99021 (C91) is a highly specific, safe, and most effective GSK-3β Inhibitor. This study found that it efficiently activates the canonical Wnt signaling of bone marrow stromal cell ST2 and promotes osteoblast differentiation and mineralization. C91 increases the production and biochemical activity of osteoblast marker alkaline phosphatase, the expression of osteoblast marker genes Alpl, Bglap, Runx2, and Sp7, and the formation of bone nodules. Triptonide is a transcription inhibitor of Wnt target gene, which diminishes C91-induced osteoblast differentiation in a dose-dependent manner. Meanwhile, C91 also induces autophagy through autophagosome formation and conversion of autophagy biomarker LC-3I into LC-3II. Autophagy inhibitor 3MA partially reduces C91-induced osteoblast differentiation and mineralization; autophagy inducer Rapamycin increases the expression of β-catenin to promote osteogenic differentiation, but cannot alleviate the inhibition of Triptonide on C91-induced osteogenic differentiation, indicating the crosstalk of canonical Wnt signaling and autophagy regulates C91-induced osteoblast differentiation. Furthermore, in order to simulate the in vivo detection of C91 in osteogenesis process, we made a C91 slow-release hydrogel with our newly established polycaprolactone and cell-integrated 3D printing system (PCCI3D module). The sustained release C91 promotes the differentiation and mineralization of ST2 cells. C91 can also enhance the proliferative activity of ST2 cells. The release rate of C91 from hydrogel gradually decreases within 7 days. During this period, the C91 is released by 83.0% and the cell viability maintained at 96.4%. Therefore, the small molecule Wnt agonist C91 promotes osteogenesis through caonical and autophagy-mediated Wnt signaling pathway with an option for translational application

HCV core protein inhibits polarization and activity of both M1 and M2 macrophages through the TLR2 signaling pathway

Hepatitis C virus (HCV) establishes persistent infection in most infected patients, and eventually causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma in some patients. Monocytes and macrophages provide the first line of defense against pathogens, but their roles in HCV infection remains unclear. We have reported that HCV core protein (HCVc) manipulates human blood-derived dendritic cell development. In the present study, we tested whether HCVc affects human blood-derived monocyte differentiating into macrophages. Results showed that HCVc inhibits monocyte differentiation to either M1 or M2 macrophages through TLR2, associated with impaired STATs signaling pathway. Moreover, HCVc inhibits phagocytosis activity of M1 and M2 macrophages, M1 macrophage-induced autologous and allogeneic CD4+ T cell activation, but promotes M2 macrophage-induced autologous and allogeneic CD4+ T cell activation. In conclusion, HCVc inhibits monocyte-derived macrophage polarization via TLR2 signaling, leading to dysfunctions of both M1 and M2 macrophages in chronic HCV infected patients. This may contribute to the mechanism of HCV persistent infection, and suggest that blockade of HCVc might be a novel therapeutic approach to treating HCV infection

Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-κB-dependent manner

The microRNA miR-155 has been implicated in regulating inflammatory responses and tumorigenesis, but its precise role in linking inflammation and cancer has remained elusive. Here, we identify a connection between miR-155 and Notch signaling in this context. Loss of Notch signaling in the bone marrow (BM) niche alters hematopoietic homeostasis and leads to lethal myeloproliferative-like disease. Mechanistically, Notch signaling represses miR-155 expression by promoting binding of RBPJ to the miR-155 promoter. Loss of Notch/RBPJ signaling upregulates miR-155 in BM endothelial cells, leading to miR-155-mediated targeting of the nuclear factor κB (NF-κB) inhibitor κB-Ras1, NF-κB activation, and increased proinflammatory cytokine production. Deletion of miR-155 in the stroma of RBPJ(-/-) mice prevented the development of myeloproliferative-like disease and cytokine induction. Analysis of BM from patients carrying myeloproliferative neoplasia also revealed elevated expression of miR-155. Thus, the Notch/miR-155/κB-Ras1/NF-κB axis regulates the inflammatory state of the BM niche and affects the development of myeloproliferative disorders