Osteoblasts Research Articles

Transgenerational bone toxicity in F3 medaka (Oryzias latipes) induced by ancestral benzo[a]pyrene exposure: Cellular and transcriptomic insights

Benzo[a]pyrene (BaP), a ubiquitous pollutant, raises environmental health concerns due to induction of bone toxicity in the unexposed offspring. Exposure of F0 ancestor medaka (Oryzias latipes) to 1 µg/L BaP for 21 days causes reduced vertebral bone thickness in the unexposed F3 male offspring. To reveal the inherited modifications, osteoblast (OB) abundance and molecular signaling pathways of transgenerational BaP-induced bone thinning were assessed. Histomorphometric analysis showed a reduction in OB abundance. Analyses of the miRNA and mRNA transcriptomes revealed the dysregulation of Wnt signaling (frzb/ola-miR-1–3p, sfrp5/ola-miR-96–5p/miR-455–5p) and bone morphogenetic protein (Bmp) signaling (bmp3/ola-miR-96–5p/miR-181b-5p/miR-199a-5p/miR-205–5p/miR-455–5p). Both pathways are major indicators of impaired bone formation, while the altered Rank signaling in osteoclasts (c-fos/miR-205–5p) suggests a potentially augmented bone resorption. Interestingly, a typical BaP-responsive pathway, the Nrf2-mediated oxidative stress response (gst/ola-miR-181b-5p/miR-199a-5p/miR-205), was also affected. Moreover, mRNA levels of epigenetic modification enzymes (e.g., hdac6, hdac7, kdm5b) were found dysregulated. The findings indicated that epigenetic factors (e.g., miRNAs, histone modifications) may directly regulate the expression of genes associated with transgenerational BaP bone toxicity and warrants further studies. The identified candidate genes and miRNAs may serve as potential biomarkers for BaP-induced bone disease and as indicators of historic exposures in wild fish for conservation purposes.

CD73/Adenosine Pathway Involvement in the Interaction of Non-Small Cell Lung Cancer Stem Cells and Bone Cells in the Pre-Metastatic Niche.

Adenosinergic signaling is an important regulator of tissue homeostasis and extracellular accumulation of adenosine (Ado) and is associated with different pathologies, such as cancer. In non-small-cell lung cancer (NSCLC), a subset of CD133/CXCR4+ cancer stem cell (CSCs) has been demonstrated to initiate bone metastases. Here we investigated how NSCLC CSCs interact with osteoclasts (OCs) and osteoblasts (OBs) by modulating Ado production and OC activity. We proved that CSC-spheres, generated in vitro from NSCLC cell lines, express CD38, PC-1, and CD73, enzymes of the non-canonical adenosinergic pathway, produce high level of Ado, and down-regulate A1R and A3R inhibitory receptors, while expressing A2AR and A2BR. To address the Ado role and modulation of the in-bone pre-metastatic niche, we performed co-cultures of CSC-spheres with OCs and OBs cells. Firstly, we verified that active OCs do not activate non-canonical the adenosinergic pathway, conversely to OBs. OCs co-cultured with CSC-spheres increase Ado production that is related to the OC resorption activity and contributes to T-cell suppression. Finally, we proved the efficacy of anti-CD73 agents in blocking NSCLC cell migration. Overall, we assessed the importance of adenosinergic signaling in the interaction between CSCs and OCs at the pre-metastatic niche, with therapeutic implications related to Ado production.

Vhl deficiency in Dmp1-expressing cells affects myeloid development and erythropoiesis: possible effects on myeloerythroid metabolism

Abstract The specific types of bone marrow (BM) stromal cells and local BM microenvironments that contribute to the regulation of myelopoiesis are not fully understood. The von-Hippel Lindau protein (VHL) controls responses to hypoxia and could regulate immune metabolism. We investigated this in Dmp1-Cre; Vhl conditional knockout mice (cKO), where Vhl is deleted in mesenchymal stem cells, osteoblasts (OBs) and osteocytes. Similar to previous findings when Vhl is deleted in OBs, we observed low blood glucose levels in the cKO mice, which may reflect an increase in OB cellular glycolysis and increased glucose uptake in the bone. Erythropoietin (Epo) treatment in vivo reduced blood glucose levels in rats. In preliminary studies, we found elevated Epo levels in cKO peripheral blood serum and BM fluid by 6 weeks of age, and evidence for dysregulated erythropoiesis. Moreover, the cKO displayed an increased frequency of common myeloid progenitors, CD11b+ Gr1− monocytes, and CD11b+ Gr1+ granulocytes by 10 weeks of age. We hypothesize that alterations in skeletal glucose metabolism directly affect myeloerythroid development in the BM through increased Epo-receptor (EpoR) signaling. EpoR is expressed on osteoprogenitors, hematopoietic stem cells, and B cells, but whether it is expressed on myeloid progenitors is unclear. Studies are in progress to test the hypothesis that overproduction of Epo by Vhl-deficient Dmp1+ cells results in elevated EpoR signaling in myeloid progenitors and lineages; this in turn, results in an increase in myeloid cell glycolysis and Glut1 expression and proliferation. These studies are relevant for the understanding how BM microenvironmental changes can trigger adaptations in immunometabolism of myeloid cells. Supported by grants from NIH (R15 AI154245-01, F31 AI154815), and University of California, Merced

The Role Of BMPs in the Regulation of Osteoclasts Resorption and Bone Remodeling: From Experimental Models to Clinical Applications.

In response to mechanical forces and the aging process, bone in the adult skeleton is continuously remodeled by a process in which old and damaged bone is removed by bone-resorbing osteoclasts and subsequently is replaced by new bone by bone-forming cells, osteoblasts. During this essential process of bone remodeling, osteoclastic resorption is tightly coupled to osteoblastic bone formation. Bone-resorbing cells, multinuclear giant osteoclasts, derive from the monocyte/macrophage hematopoietic lineage and their differentiation is driven by distinct signaling molecules and transcription factors. Critical factors for this process are Macrophage Colony Stimulating Factor (M-CSF) and Receptor Activator Nuclear Factor-κB Ligand (RANKL). Besides their resorption activity, osteoclasts secrete coupling factors which promote recruitment of osteoblast precursors to the bone surface, regulating thus the whole process of bone remodeling. Bone morphogenetic proteins (BMPs), a family of multi-functional growth factors involved in numerous molecular and signaling pathways, have significant role in osteoblast-osteoclast communication and significantly impact bone remodeling. It is well known that BMPs help to maintain healthy bone by stimulating osteoblast mineralization, differentiation and survival. Recently, increasing evidence indicates that BMPs not only help in the anabolic part of bone remodeling process but also significantly influence bone catabolism. The deletion of the BMP receptor type 1A (BMPRIA) in osteoclasts increased osteoblastic bone formation, suggesting that BMPR1A signaling in osteoclasts regulates coupling to osteoblasts by reducing bone-formation activity during bone remodeling. The dual effect of BMPs on bone mineralization and resorption highlights the essential role of BMP signaling in bone homeostasis and they also appear to be involved in pathological processes in inflammatory disorders affecting bones and joints. Certain BMPs (BMP2 and -7) were approved for clinical use; however, increased bone resorption rather than formation were observed in clinical applications, suggesting the role BMPs have in osteoclast activation and subsequent osteolysis. Here, we summarize the current knowledge of BMP signaling in osteoclasts, its role in osteoclast resorption, bone remodeling, and osteoblast–osteoclast coupling. Furthermore, discussion of clinical application of recombinant BMP therapy is based on recent preclinical and clinical studies.

Mesenchymal stromal cells from a progressive pseudorheumatoid dysplasia patient show altered osteogenic differentiation

BackgroundProgressive pseudorheumatoid dysplasia (PPRD) is a rare autosomal recessive non-inflammatory skeletal disease with childhood onset and is characterized by a progressive chondropathy in multiple joints, and skeletal abnormalities. To date, the etiopathological relationship between biological modification occurring in PPRD and genetic mutation remains an open issue, partially due to the limited availability of biological samples obtained from PPRD patients for experimental studies.Case presentationWe describe the clinical features of a PPRD patient and experimental results obtained from the biological characterization of PPRD mesenchymal stromal cells (MSCs) and osteoblasts (OBs) compared to normal cell populations. Phenotypic profile modifications were found in PPRD compared to normal subjects, essentially ascribed to decreased expression of CD146, osteocalcin (OC) and bone sialoprotein in PPRD MSCs and enhanced CD146, OC and collagen type I expression in PPRD OBs. Gene expression of Dickkopf-1, a master inhibitor of WNT signaling, was remarkably increased in PPRD MSCs compared to normal expression range, whereas PPRD OBs essentially exhibited higher OC gene expression levels. PPRD MSCs failed to efficiently differentiate into mature OBs, so showing a greatly impaired osteogenic potential.ConclusionsSince all regenerative processes require stem cell reservoirs, compromised functionality of MSCs may lead to an imbalance in bone homeostasis, suggesting a potential role of MSCs in the pathological mechanisms of PPRD caused by WNT1-inducible signaling pathway protein-3 (WISP3) mutations. In consideration of the lack of compounds with proven efficacy in such a rare disease, these data might contribute to better identify new specific and effective therapeutic approaches.

Network Pharmacology Deciphers the Action of Bioactive Polypeptide in Attenuating Inflammatory Osteolysis via the Suppression of Oxidative Stress and Restoration of Bone Remodeling Balance.

Oxidative stress involves enormously in the development of chronic inflammatory bone disease, wherein the overproduction of reactive oxygen species (ROS) negatively impacts the bone remodeling via promoting osteoclastogenesis and inhibiting osteogenesis. Lacking effective therapies highlights the importance of finding novel treatments. Our previous study screened a novel bioactive peptide D7 and demonstrated it could enhance the cell behaviors and protect bone marrow mesenchymal stem cells (BMSCs). Since BMSCs are progenitor cells of osteoblast (OB), we therefore ask whether D7 could also protect against the progress of inflammatory osteolysis. To validate our hypothesis and elucidate the underlying mechanisms, we first performed network pharmacology-based analysis according to the molecule structure of D7, and then followed by pharmacological evaluation on D7 by in vitro lipopolysaccharide(LPS)-induced models. The result from network pharmacology identified 20 candidate targets of D7 for inflammatory osteolysis intervention. The further analysis of Gene Ontology (GO)/KEGG pathway enrichment suggested the therapeutic effect of D7 may primarily affect osteoclast (OC) differentiation and function during the inflammatory osteolysis. Through validating the real effects of D7 on OC and OB as postulated, results demonstrated suppressive effects of D7 on LPS-stimulated OC differentiation and resorption, via the inhibition on OC marker genes. Contrarily, by improving the expression of OB marker genes, D7 displayed promotive effects on OB differentiation and alleviated LPS-induced osteogenic damage. Further mechanism study revealed that D7 could reduce LPS-induced ROS formation and strengthen antioxidants expressions in both OC and OB precursors, ameliorating LPS-triggered redox imbalance in bone remodeling. Taken together, our findings unveiled therapeutic effects of D7 against LPS-induced inflammatory osteolysis through the suppression of oxidative stress and the restoration of the bone remodeling process, providing a new therapeutic candidate for chronic inflammatory bone diseases.

Biomechanical behavior of diamond lattice scaffolds obtained by two different design approaches with similar porosity; a numerical investigation with FEM and CFD analysis.

Scaffolds provide a suitable environment for the bone tissue to maintain its self-healing ability and help new bone-cell formation by creating structures with similar mechanical properties to the surrounding tissue. In the modeling of the scaffolds, an optimum environment is tried to be provided by changing the geometrical properties of the cell architecture such as porosity, pore size, and specific surface area. For this purpose, different design approaches have been used in studies to change these properties. This study aims to determine whether scaffolds with similar porosities modeled by different design approaches exhibit distinct biomechanical behaviors or not. By using the Diamond lattice architecture, two different design approaches were constituted. The first approach has constant wall thickness and variable cell size, whereas the second approach contains variable wall thickness and constant cell size. The usage of different design approaches affected the amount of specific surface area in models with similar porosity. Mechanical compression tests were conducted via finite element analysis, while the permeability performance of configurations with similar porosities (50%, 60%, 70%, 80%, and 90%) was evaluated by using computational fluid dynamics. The mechanical results revealed that the structural strength decreased with increasing porosity. Since their higher specific surface area causes lower pressure drops, the second group exhibits better permeability. In addition, it was found that to evaluate the wall shear stresses occurring on the scaffold surfaces properly, it is essential to consider the stress distributions within the scaffold rather than the maximum values.

Hereditary retinoblastoma iPSC model reveals aberrant spliceosome function driving bone malignancies

The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSC–derived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3a–regulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.

Mycobacterium tuberculosis Induced Osteoblast Dysregulation Involved in Bone Destruction in Spinal Tuberculosis.

Disturbance of bone homeostasis caused by Mycobacterium tuberculosis (Mtb) is a key clinical manifestation in spinal tuberculosis (TB). However, the complete mechanism of this process has not been established, and an effective treatment target does not exist. Increasing evidence shows that abnormal osteoclastogenesis triggered by an imbalance of the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) axis may play a key role in the disturbance of bone homeostasis. Previous studies reported that RANKL is strongly activated in patients with spinal TB; however, the OPG levels in these patients were not investigated in previous studies. In this study, we investigated the OPG levels in patients with spinal TB and the dysregulation of osteoblasts caused by Mtb infection. Inhibition of the Mce4a gene of Mtb by an antisense locked nucleic acid (LNA) gapmer (Mce4a-ASO) was also investigated. Analysis of the serum OPG levels in clinical samples showed that the OPG levels were significantly decreased in patients with spinal TB compared to those in the group of non-TB patients. The internalization of Mtb in osteoblasts, the known major source of OPG, was investigated using the green fluorescent protein (GFP)-labeled Mycobacterium strain H37Ra (H37RaGFP). The cell-associated fluorescence measurements showed that Mtb can efficiently enter osteoblast cells. In addition, Mtb infection caused a dose-dependent increase of the CD40 mRNA expression and cytokine (interleukin 6, IL-6) secretion in osteoblast cells. Ligation of CD40 by soluble CD154 reversed the increased secretion of IL-6. This means that the induced CD40 is functional. Considering that the interaction between CD154-expressing T lymphocytes and bone-forming osteoblast cells plays a pivotal role in bone homeostasis, the CD40 molecule might be a strong candidate for mediating the target for treatment of bone destruction in spinal TB. Additionally, we also found that Mce4a-ASO could dose-dependently inhibit the Mce4a gene of Mtb and reverse the decreased secretion of IL-6 and the impaired secretion of OPG caused by Mtb infection of osteoblast cells. Taken together, the current finding provides breakthrough ideas for the development of therapeutic agents for spinal TB.

Photobiomodulation treatments drive osteogenic versus adipocytic fate of bone marrow mesenchymal stem cells reversing the effects of hyperglycemia in diabetes.

Diabetes mellitus (DM) is a chronic metabolic disease that affects bone metabolism, which can be related to a reduced osteogenic potential of bone marrow mesenchymal stem cells (BM-MSCs). MSCs from diabetic rats (dBM-MSC) have shown a tendency to differentiate towards adipocytes (AD) instead of osteoblasts (OB). Since photobiomodulation (PBM) therapy is a non-invasive treatment capable of recovering the osteogenic potential of dBM-MSCs, we aimed to evaluate whether PBM can modulate MSC's differentiation under hyperglycemic conditions. BM-MSCs of healthy and diabetic rats were isolated and differentiated into osteoblasts (OB and dOB) and adipocytes (AD and dAD). dOB and dAD were treated with PBM every 3days (660nm; 5J/cm2; 0.14J; 20 mW; 0.714W/cm2) for 17days. Cell morphology and viability were evaluated, and cell differentiation was confirmed by gene expression (RT-PCR) of bone (Runx2, Alp, and Opn) and adipocyte markers (Pparγ, C/Ebpα, and C/Ebpβ), production of extracellular mineralized matrix (Alizarin Red), and lipid accumulation (Oil Red). Despite no differences on cell morphology, the effect of DM on cells was confirmed by a decreased gene expression of bone markers and matrix production of dOB, and an increased expression of adipocyte and lipid accumulation of dAD, compared to heatlhy cells. On the other hand, PBM reversed the effects of dOB and dAD. The negative effect of DM on cells was confirmed, and PBM improved OB differentiation while decreasing AD differentiation, driving the fate of dBM-MSCs. These results may contribute to optimizing bone regeneration in diabetic patients.

EFFECTS OF COLLAGEN HYDROLYSATE DIGESTS ON CELLS INVOLVED IN BONE REMODELING

Purpose: Although classically a “wear and tear” disease associated with decreased cartilage, osteoarthritis (OA) is now considered a disease of the whole joint. Thus, bone health has become particularly important in OA. Bone remodeling is tightly regulated by bone-forming osteoblasts (OBs) and bone-resorbing osteoclasts (OCs). An imbalance between bone formation and resorption adversely impacts joint integrity and health. To manage OA symptoms, supplements such as collagen hydrolysates (CHs) are often used, which exert their effects via bioactive peptides (BAPs).

Biomaterial Scaffolds Made of Chemically Cross-Linked Gelatin Microsphere Aggregates (C-GMSs) Promote Vascularized Bone Regeneration.

Various scaffolding systems have been attempted to facilitate vascularization in tissue engineering by optimizing biophysical properties (e.g., vascular-like structures, porous architectures, surface topographies) or loading biochemical factors (e.g., growth factors, hormones). However, vascularization during ossification remains an unmet challenge that hampers the repair of large bone defects. In this study, reconstructing vascularized bones in situ against critical-sized bone defects is endeavored using newly developed scaffolds made of chemically cross-linked gelatin microsphere aggregates (C-GMSs). The rationale of this design lies in the creation and optimization of cell-material interfaces to enhance focal adhesion, proliferation, and function of anchorage-dependent functional cells. In vitro trials are carried out by coculturing human aortic endothelial cells (HAECs) and murine osteoblast precursor cells (MC3T3-E1) within C-GMS scaffolds, in which endothelialized bone-like constructs are yielded. Angiogenesis and osteogenesis induced by C-GMSs scaffold are further confirmed via subcutaneous-embedding trials in nude mice. In situ trials for the repair of critical-sized femoral defects are subsequently performed in rats. The acellular C-GMSs with interconnected macropores, exhibit the capability to recruit the endogenous cells (e.g., bone-forming cells, vascular forming cells, immunocytes) and then promote vascularized bone regeneration as well as integration with host bone.

Chitosan regulated electrochemistry for dense hydroxyapatite/MgO nanocomposite coating with antibiosis and osteogenesis on titanium alloy

Preparation of hydroxyapatite (HA) coating on the titanium surface through the electrochemical method can improve its osteoinductivity.However, simple HA coating is of low density and easy to shed off, which may result in decreased osteogenesis and antibiosis. Here, chitosan (CS) and Ca2+, mg2+ were co-hybridized by pulse electrodeposition (PED) in situ to obtain nanocomposite coating with omogeneously dispersed HA nanoparticles (HA-NPs) and mg(OH)2 nanoparticles (Mg(OH)2-NPs), and dense composite HA/MgO coatings were then prepared after high-temperature sintering. The HA / MgO coating is nano spherical，showed steady and biocompatible Ca2+, Mg2+ release due to the regulation of CS and high-temperature sintering, and exhibited superior apatite-forming bioactivity. The antibacterial rate of HA/MgO coating against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were both over 96%. Osteoblasts (OBs) and vascular endothelial cells (VECs) showed good adhesion and proliferation on the nanocomposite coating, indicating that the nanocomposite coating had favorable angiogenic and osteogenesis.

Tanshinol suppresses osteosarcoma by specifically inducing apoptosis of U2-OS cells through p53-mediated mechanism

Ethnopharmacological relevanceRadix Salviae miltiorrhizae (also called Danshen in traditional Chinese medicine) is a famous herbal medicine, which has been frequently used to treat blood stasis syndrome including osteosarcoma (OS) in traditional Chinese medicine. Main components of Danshen have been assumed to exhibit anti-OS capacity. Nevertheless, tanshinol (TS, main component of Danshen)'s efficacy and mechanism in OS hasn't been clearly described ever since. This drew our attention, since OS is the most frequent primary bone carcinomas in children and adolescents, with a high incidence and fatality rate. Unfortunately, chemotherapy for OS has faced many clinical challenges due to the increasing chemoresistance and recurrence. This study was then designed to deeply explore TS's role in OS therapy. Aim of the studyTo explore the anti-OS efficacy and mechanism of TS, we conducted in vivo and in vitro experiments by using a zebrafish xenograft model and U2-OS cells. Materials and methodsCCK-8 assay, DAPI and γ-H2A.X immunofluorescence staining, and flow cytometry (apoptosis verification) were employed to determine the anti-proliferative and pro-apoptotic effects of TS. qPCR and Western blot were used to examine TSʹs molecular actions and mechanism on apoptosis of U2-OS cells. ResultsThe in vivo data showed that TS significantly inhibited U2-OS tumor growth in larval zebrafish from 2 to 20 ng/mL. In vitro data indicated that TS exerted significant anti-proliferative and pro-apoptotic effects on U2-OS cells in a dose-dependent manner. Moreover, TS has no inhibitory effect on bMSCs, suggesting its safety on normal bone-forming cells. Molecular data illustrated that TS obviously activated the p53 signaling-related proteins (p-p53, Bax, CASP3, CASP9) and its upstream JNK (p-JNK, p-c-JUN) and ATM (p-ATM) signaling molecules through phosphorylation and cleavage, followed by up-regulation of the pro-apoptotic genes, NOXA, PUMA, TP53, BAX, and BIM, and down-regulation of Bcl-2 protein. ConclusionIn sum, TS specifically induced apoptosis of U2-OS cells by activating p53 signaling pathways, indicating TS as a promising candidate for OS treatment.

Effects of Vitamin A (Retinol) Release from Calcium Phosphate Matrices and Porous 3D Printed Scaffolds on Bone Cell Proliferation and Maturation

Vitamin A is a fat-soluble compound widely known for vision health. Highly variable reports on its effects on bone health have necessitated further research to truly understand its role on bone cell proliferation. Retinol, one bioactive form of vitamin A, is incorporated into synthetic bone graft scaffolds for low load-bearing clinical bone treatment. The objective of this work is to understand the effects of retinol on osteoblast and osteoclast cells when embedded within calcium phosphate matrices, including interconnected porous 3D printed tricalcium phosphate scaffolds. Results show that hydrophobic retinol can be released from bone scaffolds when a combination of biodegradable polymers, polycaprolactone and polyethylene glycol, are employed as drug carriers. The release of retinol in vitro can support a 20 ± 1% increase in osteoblast (bone-forming) cell proliferation with proper cell adhesion and filopodial extensions. Osteoclast cell morphology is necrosed and torn with a reduction in proliferation at approximately 6 ± 1% when retinol is present. In addition, inhibition of osteoclastic resorption pit bays is noted using scanning electron microscopy. With the scaffolds' round pore interconnectivity facilitating retinol release, this system can provide an alternative to traditional bone grafts while additionally supporting bone healing through enhanced osteoblast cell proliferation and inhibition of osteoclast resorption activity.

DNMT1 is a negative regulator of osteogenesis.

ABSTRACTThe role and underlying mechanisms of DNA methylation in osteogenesis/chondrogenesis remain poorly understood. We here reveal DNA methyltransferase 1 (DNMT1), which is responsible for copying DNA methylation onto the newly synthesized DNA strand after DNA replication, is overexpressed in sponge bone of people and mice with senile osteoporosis and required for suppression of osteoblast (OB) differentiation of mesenchymal stem cells (MSCs) and osteoprogenitors. Depletion of DNMT1 results in demethylation at the promoters of key osteogenic genes such as RORA and Fgfr2, and consequent upregulation of their transcription in vitro. Mechanistically, DNMT1 binds exactly to the promoters of these genes and are responsible for their 5-mc methylation. Conversely, simultaneous depletion of RORA or Fgfr2 blunts the effects of DNMT1 silencing on OB differentiation, suggesting RORA or Fgfr2 may be crucial for modulating osteogenic differentiation downstream of DNMT1. Collectively, these results reveal DNMT1 as a key repressor of OB differentiation and bone formation while providing us a new rationale for specific inhibition of DNMT1 as a potential therapeutic strategy to treat age-related bone loss.

Natural killer cells activity against multiple myeloma cells is modulated by osteoblast-induced IL-6 and IL-10 production

BackgroundNatural killer (NK) cells are part of the innate arm of the immune system; as such NK cells can be activated rapidly to target virus-infected cells and tumor cells without prior sensitization. The human NK-92MI cell line is among the most widely used NK cell in preclinical research studies and has also been approved for clinical applications. Previous studies have shown that osteoblasts (OSB) confer drug resistance in multiple myeloma (MM) and other cancers that metastasize to the bone marrow. AimWe evaluated here how OSB, which are bone forming cells and a key cellular component of the bone marrow microenvironment, modulate the cytotoxic activity of NK-92MI cells against the MM.1S multiple myeloma cell line. MethodsThe osteoblastic niche was recapitulated with either the osteoblastic cell line hFOB 1.19 (hFOB) or primary osteoblasts (P-OSB) derived from surgical resections. Time-lapse imaging was utilized to quantify changes in MM.1S cell viability under different conditions, including: (1) Co-culture of MM.1S with NK92MI cells, (2) triple-culture of hFOB or P-OSB with MM.1S and NK-92MI, and (3) MM.1S or NK-92MI cells primed with OSB-derived supernatant. Cytokine analysis was conducted to quantify potential secreted factors associated with the protective effects of OSB. ResultsThe physical presence of OSB hindered the activity of NK-92MI cells, resulting in the increased viability of MM.1S compared to co-cultures which lacked OSB. This observation was accompanied by reduced perforin and granzyme A secretion from NK-92MI cells. Contact of OSB and NK-92MI cells also induced interleukin 6 (IL-6) and interleukin 10 (IL-10) production; two cytokines which are known to impair the NK cell immunity against MM and other cancers. OSB supernatant also conferred cytoprotection to MM.1S, suggesting a dual mechanism by which OSB may modulate both NK and MM cells. ConclusionsWe demonstrated here that OSB can negatively impact the activity of NK cells against MM. As NK cells and their chimeric antigen receptor-modified versions become more widely used in the clinic, our results suggest that understanding the role of OSB as potential immunoregulators of the NK cell-mediated cytotoxic response in the bone marrow tumor microenvironment may provide new opportunities for enhancing the effectiveness of this potent immunotherapeutic approach.

Plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects that prevents menopausal-related osteoporosis in mice

Osteoporosis is caused by enhanced bone resorption and relatively reduced bone formation. There is an unmet need to develop new agents with both antiresorptive and anabolic effects to treat osteoporosis, although drugs with either effect alone are available. A small molecular compound, plumbagin, was reported to inhibit receptor activator of nuclear factor kappa-B ligand–induced osteoclast (OC) differentiation by inhibiting IκBα phosphorylation–mediated canonical NF-κB activation. However, the key transcriptional factor RelA/p65 in canonical NF-κB pathway functions to promote OC precursor survival but not terminal OC differentiation. Here, we found that plumbagin inhibited the activity of NF-κB inducing kinase, the key molecule that controls noncanonical NF-κB signaling, in an ATP/ADP-based kinase assay. Consistent with this, plumbagin inhibited processing of NF-κB2 p100 to p52 in the progenitor cells of both OCs and osteoblasts (OBs). Interestingly, plumbagin not only inhibited OC but also stimulated OB differentiation in vitro. Importantly, plumbagin prevented trabecular bone loss in ovariectomized mice. This was associated with decreased OC surfaces on trabecular surface and increased parameters of OBs, including OB surface on trabecular surface, bone formation rate, and level of serum osteocalcin, compared to vehicle-treated mice. In summary, we conclude that plumbagin is a NF-κB-inducing kinase inhibitor with dual anabolic and antiresorptive effects on bone and could represent a new class of agent for the prevention and treatment of osteoporosis.

Biological Effects of Cyclin-Dependent Kinase Inhibitors Ribociclib, Palbociclib and Abemaciclib on Breast Cancer Bone Microenvironment.

The CDK4/6 inhibitors (CDKi) palbociclib, ribociclib, and abemaciclib are currently approved in combination with anti-estrogen therapy for the treatment of advanced and/or metastatic hormone receptor-positive/HER2-neu-negative breast cancer patients. Given the high incidence of bone metastases in this population, we investigated and compared the potential effects of palbociclib, ribociclib, and abemaciclib on the breast cancer bone microenvironment. Primary osteoclasts (OCs) and osteoblasts (OBs) were obtained from human monocyte and mesenchymal stem cells, respectively. OC function was evaluated by tartrate-resistant acid phosphatase assay and real-time PCR; OB activity was assessed by an alizarin red assay. OB/breast cancer co-culture models were generated via the seeding of MCF-7 cells on a layer of OBs, and tumor cell proliferation was analyzed using flow cytometry. Here, we showed that ribociclib, palbociclib, and abemaciclib exerted similar inhibitory effects on the OC differentiation and expression of bone resorption markers without affecting OC viability. On the other hand, the three CDKi did not affect the ability of OB to produce bone matrix, even if the higher doses of palbociclib and abemaciclib reduced the OB viability. In OB/MCF-7 co-culture models, palbociclib demonstrated a lower anti-tumor effect than ribociclib and abemaciclib. Overall, our results revealed the direct effects of CDKi on the tumor bone microenvironment, highlighting differences potentially relevant for clinical practice.

Interleukin-17 promotes osteoclastogenesis and periodontal damage via autophagy in vitro and in vivo

ObjectiveBecause of its potent pro-inflammatory properties, interleukin-17 (IL-17) contributes to the pathogenesis of various inflammatory diseases. This study explored the effects of IL-17 on osteoclastogenesis in an osteoclast monoculture and osteoblast-osteoclast co-culture system, as tools to investigate the molecular mechanisms underlying the interactions between osteoclastogenesis and autophagy. MethodsVarious ratios of calvarial osteoblasts (OB) and osteoclast precursor cells (mouse macrophage cell line RAW264.7, hereinafter referred to as OC) were tested. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the optimum osteoblasts:osteoclasts ratio. IL-17 was added to the co-culture system to test its effects on multinucleated osteoclast formation and osteoclast-related proteins. We assessed the effects of IL-17 on receptor activator of nuclear factor-kappa B ligand (RANKL) expression in osteoblasts, and determined if IL-17 alone could modulate osteoclast formation in an osteoclast monoculture. Administration of exogenous RANKL combined with IL-17 was employed to stimulate RAW264.7 cells osteoclastogenesis and to determine production of osteoclasts and autophagy-related proteins. We knocked down Beclin1 expression in RAW264.7 cells and examined the expression of autophagy-related and osteoclast-related proteins in RAW264.7 cells and the co-culture system, and the TAK1-binding protein 3 (TAB3)/ extracellular signal regulated kinase (ERK) pathway. ResultsA ratio of 20 OB : 1 OC yielded the highest rate of osteoclast formation. Low IL-17 concentrations increased osteoclastogenesis in co-cultures significantly, but high levels of IL-17 had the opposite effect. IL-17 alone could not induce formation of TRAP+ multinucleated cells in RAW264.7 cells. Low IL-17 concentrations promoted osteoclast differentiation and autophagy in RAW264.7 cells induced by exogenous RANKL, but high IL-17 concentrations inhibited this process. Knockdown of Beclin1 reversed the enhanced effects of 0.1 ng/mL IL-17 on osteoclastogenesis and autophagy in RAW264.7 cells. The TAB3/ERK pathway was also blocked after autophagy inhibition. ConclusionIn the co-culture model used in this study, a ratio of 20 OB:1 OC proved to be the optimal ratio to facilitate osteoclast formation. IL-17 regulated RANKL-induced osteoclastogenesis via autophagy. The Beclin1/TAB3/ERK pathway was involved in osteoclast autophagy.