Impaired Osteoblast Differentiation Research Articles

HIF1 activation safeguards cortical bone formation against impaired oxidative phosphorylation.

Energy metabolism, through pathways such as oxidative phosphorylation (OxPhos) and glycolysis, plays a pivotal role in cellular differentiation and function. Our study investigates the impact of OxPhos disruption in cortical bone development by deleting Mitochondrial Transcription Factor A (TFAM). TFAM controls OxPhos by regulating the transcription of mitochondrial genes. The cortical bone, constituting the long bones' rigid shell, is sheathed by the periosteum, a connective tissue layer populated with skeletal progenitors that spawn osteoblasts, the bone-forming cells. TFAM-deficient mice presented with thinner cortical bone, spontaneous midshaft fractures, and compromised periosteal cell bioenergetics, characterized by reduced ATP levels. Additionally, they exhibited an enlarged periosteal progenitor cell pool with impaired osteoblast differentiation. Increasing Hypoxia-Inducible Factor 1a (HIF1) activity within periosteal cells significantly mitigated the detrimental effects induced by TFAM deletion. HIF1 is known to promote glycolysis in all cell types. Our findings underscore the indispensability of OxPhos for the proper accrual of cortical bone mass and indicate a compensatory mechanism between OxPhos and glycolysis in periosteal cells. The study opens new avenues for understanding the relationship between energy metabolism and skeletal health and suggests that modulating bioenergetic pathways may provide a therapeutic avenue for conditions characterized by bone fragility.

Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis.

Mitofusin 2 plays a critical role in maintaining the functional integrity of the neuromuscular-skeletal axis

PurposeMitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. MethodsMfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). ResultsMice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. ConclusionDespite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.

Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination

Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.

Glutathione limits RUNX2 oxidation and degradation to regulate bone formation.

Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled, since elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underlie many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential in neutralizing ROS and establish a proosteogenic reduction and oxidation reaction (REDOX) environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis led to acute degradation of RUNX2, impaired osteoblast differentiation, and reduced bone formation. Conversely, reducing ROS using catalase enhanced RUNX2 stability and promoted osteoblast differentiation and bone formation when GSH biosynthesis was limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilized RUNX2 and improved bone development in the Runx2+/- haplo-insufficient mouse model of human cleidocranial dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarify how ROS negatively impacts osteoblast differentiation and bone formation.

Cell differentiation and matrix organization are differentially affected during bone formation in osteogenesis imperfecta zebrafish models with different genetic defects impacting collagen type I structure

Osteogenesis imperfecta (OI) is a family of rare heritable skeletal disorders associated with dominant mutations in the collagen type I encoding genes and recessive defects in proteins involved in collagen type I synthesis and processing and in osteoblast differentiation and activity. Historically, it was believed that the OI bone phenotype was only caused by abnormal collagen type I fibrils in the extracellular matrix, but more recently it became clear that the altered bone cell homeostasis, due to mutant collagen retention, plays a relevant role in modulating disease severity in most of the OI forms and it is correlated to impaired bone cell differentiation. Despite in vitro evidence, in vivo data are missing. To better understand the physiopathology of OI, we used two zebrafish models: Chihuahua (Chi/+), carrying a dominant p.G736D substitution in the α1 chain of collagen type I, and the recessive p3h1−/−, lacking prolyl 3-hydroxylase (P3h1) enzyme. Both models share the delay of collagen type I folding, resulting in its overmodification and partial intracellular retention. The regeneration of the bony caudal fin of Chi/+ and p3h1−/− was employed to investigate the impact of abnormal collagen synthesis on bone cell differentiation. Reduced regenerative ability was evident in both models, but it was associated to impaired osteoblast differentiation and osteoblastogenesis/adipogenesis switch only in Chi/+. On the contrary, reduced osteoclast number and activity were found in both models during regeneration. The dominant OI model showed a more detrimental effect in the extracellular matrix organization. Interestingly, the chemical chaperone 4-phenylbutyrate (4-PBA), known to reduce cellular stress and increase collagen secretion, improved bone formation only in p3h1−/− by favoring caudal fin growth without affecting bone cell markers expression. Taken together, our in vivo data proved the negative impact of structurally abnormal collagen type I on bone formation but revealed a gene mutation-specific effect on bone cell differentiation and matrix organization in OI. These, together with the distinct ability to respond to the chaperone treatment, underline the need for precision medicine approaches to properly treat the disease.

Transient downregulation of NR4A1 leads to impaired osteoblast differentiation through the TGF-ß pathway, and Elesclomol (STA-4783) rescues this phenotype.

Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway.

A single-center study of bone mineral density in adult patients with severe hemophilia A in correlation with markers of bone metabolism

Osteopenia and osteoporosis are well-known hemophilia A comorbidities. The pathogenesis of bone turnover alteration resulting in reduced bone mass includes impaired osteoblastic differentiation and disinhibition of RANKL-induced osteoclastogenesis as a result of a low FVIII level. To evaluate the bone mineral density (BMD) in adult patients with severe hemophilia A and assess a possible correlation with the bone remodeling biomarkers OPG/RANKL, CTX-1, osteocalcin, and Vit D. 28 male subjects with severe hemophilia A and 33 age-matched controls were recruited. The biomarkers were tested with the ELISA assay and BMD with DEXA of the lumbar spine (LS) and total hip (TH). The patients had lower LS-BMD (-0.955±0.145 vs. 1.118±0.079, p=0.05) and TH-BMD (-0.840±0.147 vs. 0.951±0.075, p=0.05) than those of the controls. The TH T-scores were -1.41±0.91 vs. 0.4±0.49 (p=0.05) and the LS T-scores -1.16±1.046 vs. 0.14±0.72 (p=0.05). 66.6% of patients under 50 years had osteopenia and 8.3% had osteoporosis. Fifty percent of those over 50 years old had osteopenia and 20% had osteoporosis. We found significantly higher OPG levels (123.69±107.05 vs. 41.98±18.95, p=0.05) than that in controls and lower sRANKL levels (23.49±29.39 vs. 131.32±201.27, p=0.05) and sRANKL/OPG ratio (0.27±0.35 vs. 5.28±10.01, p=0.05) than those in controls. A positive correlation was found between sRANKL and the BMD T-score of lumbar spine (p=0.001) in the patient group. sRANKL level and ratio can be used as predictors of low BMD.

Atf7ip Inhibits Osteoblast Differentiation via Negative Regulation of the Sp7 Transcription Factor

Epigenetic modifications are critical for cell differentiation and growth. As a regulator of H3K9 methylation, Setdb1 is implicated in osteoblast proliferation and differentiation. The activity and nucleus localization of Setdb1 are regulated by its binding partner, Atf7ip. However, whether Atf7ip is involved in the regulation of osteoblast differentiation remains largely unclear. In the present study, we found that Atf7ip expression was upregulated during the osteogenesis of primary bone marrow stromal cells and MC3T3-E1 cells, and was induced in PTH-treated cells. The overexpression of Atf7ip impaired osteoblast differentiation in MC3T3-E1 cells regardless of PTH treatment, as measured by the expression of osteoblast differentiation markers, Alp-positive cells, Alp activity, and calcium deposition. Conversely, the depletion of Atf7ip in MC3T3-E1 cells promoted osteoblast differentiation. Compared with the control mice, animals with Atf7ip deletion in the osteoblasts (Oc-Cre;Atf7ipf/f) showed more bone formation and a significant increase in the bone trabeculae microarchitecture, as reflected by μ-CT and bone histomorphometry. Mechanistically, Atf7ip contributed to the nucleus localization of Setdb1 in MC3T3-E1, but did not affect Setdb1 expression. Atf7ip negatively regulated Sp7 expression, and through specific siRNA, Sp7 knockdown attenuated the enhancing role of Atf7ip deletion in osteoblast differentiation. Through these data, we identified Atf7ip as a novel negative regulator of osteogenesis, possibly via its epigenetic regulation of Sp7 expression, and demonstrated that Atf7ip inhibition is a potential therapeutic measure for enhancing bone formation.

A High Percentage of CD16+ Monocytes Correlates with the Extent of Bone Erosion in Chronic Lymphocytic Leukemia Patients: The Impact of Leukemic B Cells in Monocyte Differentiation and Osteoclast Maturation

Significant skeletal alterations are present in Chronic Lymphocytic Leukemia (CLL) patients; bone erosion, particularly evident in the long bone shaft, appeared increased in the progressive disease stage. Moreover, the partial colonization of the bone with reactive bone marrow we documented via PET-FDG imaging suggests that neoplastic cell overgrowth contributes to bone derangement. Indeed, cytokines released by leukemic B cells impair osteoblast differentiation and enhance osteoclast formation in vitro. CD16, Fcγ-RIIIa, has been previously indicated as a marker of osteoclast precursors. We demonstrate, here, that the percentage of circulating monocytes, CD16+, is significantly higher in CLL patients than in normal controls and directly correlated with the extent of bone erosion. When we assessed if healthy monocytes, treated with a CLL-conditioned medium, modulated RANK, RANKL and CD16, we observed that all these molecules were up-regulated and CD16 to a greater extent. Altogether, these findings suggest that leukemic cells facilitate osteoclast differentiation. Interestingly, the evidence that monocytes, polarized toward the M2 phenotype, were characterized by high CD16 expression and showed a striking propensity to differentiate toward osteoclasts may provide further explanations for the enhanced levels of bone erosion detected, in agreement with the high number of immunosuppressive-M2 cells present in these patients.

Sickle Cell Disease Compromises the Hematopoietic Stem Cells Bone Marrow Niche with Age

Sickle cell disease (SCD) is characterized by chronic inflammation caused by red blood cell hemolysis, which can trigger oxidative stress and tissue injury that contribute to disease. Recent studies have shown that SCD pathophysiology reconstituted post-transplant in mouse models disrupts the architecture of bone marrow (BM) sinusoidal endothelium and causes a defect of mesenchymal stromal cells (MSCs) function. However, the impact of SCD on the ability MSCs and endothelial cells (ECs) to support of human and mouse hematopoiesis remains largely unknown. Using a mouse model of SCD, we now report that both sinusoidal (LepR+) and arteriolar MSCs (PαS) are more abundant in SCD BM, where they also display increased proliferation at 2-, 6- and 12-months of age. Additionally, 12-month-old SCD mice display a massive loss of sinusoidal ECs. Young SCD patient (n=9, age 2-18) BM also displayed increased MSC numbers relative to age-matched healthy controls (n=13). SCD MSCs isolated from 6- and 12-month old mice, maintained increased cell cycle when cultured ex vivo, showed decreased CFU-F potential, and impaired osteoblast differentiation. In ex vivo co-cultures of primary murine BM MSCs, ECs and HSCs, we found that 2-, 6-, and 12-month old SCD stroma (MSCs and ECs) failed to support non-SCD HSCs relative to controls. Phenotypic HSCs recovered from these co-cultures displayed a loss of CFU potential and transplantation activity. In tri-cultures, SCD MSCs, but not SCD ECs, drove increased HSC proliferation, apoptosis, and lymphoid-biased transplantation. In addition, MSCs isolated from BM of young SCD patients also displayed increased cell cycle, increased pro-inflammatory cytokine secretion, and decreased ability to support the expansion and CFU potential of co-cultured healthy human CD34+ cells, relative to MSCs isolated from young non-SCD donors. Bulk RNA-sequencing of 2-, 6-, and 12-month old murine SCD MSCs revealed gene signatures consistent with accelerated aging, increased proliferation, loss of extracellular matrix (ECM), impaired immune modulation, and reduced secretion of factors known to support HSCs. Genetic restoration of ECM rescued the ability of SCD MSCs to expand HSCs in ex vivo co-cultures, and support HSCs with robust CFU and transplantation potential. In sum, our data reveals a loss of HSPC supportive potential in BM MSCs in young individuals with SCD and that loss of ECM production by MSCs contributes to dysfunction in the HSC BM niche in both young and aged SCD mice.

Glutamate Metabolism Shapes Osteoclast Differentiation: New Potential Therapeutic Target to Block Osteoclastic Bone Resorption in Multiple Myeloma Patients

Multiple Myeloma (MM) cells are characterized by an impaired Glutamine (Gln) metabolism and Gln-addiction feature. As a result of this, MM patients have lower levels of Gln and higher levels of its main metabolite Glutamate (Glu) in the bone marrow (BM) microenvironment as compared with Smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS) patients. Moreover, we have recently shown that MM-dependent Gln-exhausted microenvironment impairs osteoblast differentiation. Based on this evidence, we have hypothesized that alterations of Gln/Glu axis may have a significant impact on the activation of osteoclasts (OCLs) and in the development of osteolytic lesions. We purified BM CD14+ monocytes from a cohort of 30 patients with monoclonal gammopathies (5 MGUS, 12 SMM and 13 newly diagnosed MM (ND-MM)). Cells have been incubated in osteoclastogenic medium in the presence or absence of Glu. The samples were subsequently analyzed by real-time PCR, Western blot and TRAP staining after 3 or 14 days. Amino acid uptake and amino acid content have been assessed under the same conditions. Glu uptake inhibitors have been tested throughout osteoclastogenesis. Our data showed that the presence of Glu significantly increases the number of OCLs generated from MGUS/SMM CD14+ compared with cells differentiated in the absence of the amino acid (p<0.01). In these patients, Glu increases the expression of RANK as well as NF-κB and NFATc1 protein level pointing to a stimulatory effect on osteoclastogenic signaling pathway. On the contrary, CD14+ obtained from MM patients have a lower response to Glu probably as a consequence of the high Glu microenvironment imposed by MM cells. Consistently, MM CD14+ cells are prone to generate more OCLs also in the absence of Glu compared with CD14+ cells obtained from premalignant samples (median number of OCLs: MGUS/SMM 33, MM 123, p=0.02). The analysis of intracellular amino acids (Gln, Glu, and α-ketoglutarate (2-OG)) indicated that Glu content significantly increases after 8 days of differentiation while decreasing after 14 days pointing to a fast accumulation of Glu upon differentiation. The content of both Gln and 2-OG do not vary appreciably throughout the differentiation. These data suggest that OCLs differentiation is associated with an increased uptake of extracellular Glu. Consistently, the activity of the sodium-dependent Glu transporters EAATs, increases already after 3 days of incubation in differentiating medium without Glu, with a further increase in the presence of the amino acid. Expression analysis revealed that, among EAATs, EAAT1 (encoded by SLC1A3 gene) is responsible for the uptake of extracellular Glu upon differentiation both in the presence and in the absence of the amino acid. The expression of other Glu transporters do not change during differentiation. Selective inhibitors of EAAT1, UCPH101 and TFB-TBOA completely block the formation of multinucleated OCLs in the presence of Glu (p<0.01). In parallel to EAAT1 induction, Glu treatment increases the expression of Calcineurin, the serine/threonine phosphatase activated by increased intracellular calcium levels indicating the possible modulation of calcium signaling by Glu. In conclusion, our results indicate that high BM Glu level sustains OCLs formation by stimulating of NF-κB/NFATc1/calcineurin pathway. EAAT1 transporter is responsible for the uptake of extracellular Glu during osteoclastogenesis while its inhibition markedly blocks OCL formation. Therefore, targeting glutamate metabolism could be a therapeutic strategy to block OCL formation in MM patients.

MiR-539-3p impairs osteogenesis by suppressing Wnt interaction with LRP-6 co-receptor and subsequent inhibition of Akap-3 signaling pathway.

X-linked hypophosphatemia (XLH), an inheritable form of rickets is caused due to mutation in Phex gene. Several factors are linked to the disease’s aetiology, including non-coding RNA molecules (miRNAs), which are key post-transcriptional regulators of gene expression and play a significant role in osteoblast functions. MicroRNAs sequence analysis showed differentially regulated miRNAs in phex silenced osteoblast cells. In this article, we report miR-539-3p, an unidentified novel miRNA, in the functional regulation of osteoblast. MiR-539-3p overexpression impaired osteoblast differentiation. Target prediction algorithm and experimental confirmation by luciferase 3’ UTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over expression of miR-539-3p in osteoblasts down regulated Wnt/beta catenin signaling components and deteriorated trabecular microarchitecture leading to decreased bone formation in ovariectomized (Ovx) mice. Additionally, biochemical bone resorption markers like CTx and Trap-5b were elevated in serum samples of mimic treated group, while, reverse effect was observed in anti-miR treated animals along with increased bone formation marker P1NP. Moreover, transcriptome analysis with miR-539-3p identified a novel uncharacterized Akap-3 gene in osteoblast cells, knock down of which resulted in downregulation of osteoblast differentiation markers at both transcriptional and translational level. Overall, our study for the first time reported the role of miR-539-3p in osteoblast functions and its downstream Akap-3 signalling in regulation of osteoblastogenesis.

Chi3L1 is a therapeutic target in bone metabolism and a potential clinical marker in patients with osteoporosis

BMP2 is clinically used as an ectopic bone inducer and plays a significant role in bone development, formation, and diseases. Chitinase 3-like 1 protein (Chi3L1) is found in the skeletal system. However, Chi3L1-mediated bone metabolism and aging-related bone erosion via BMP2 signaling have not yet been demonstrated. Herein, Chi3L1 increased BMP2-induced osteoblast differentiation in mesenchymal precursor cells and human primary osteoblasts. Chi3L1KO(-/-) showed abnormal bone development, and primary osteoblasts isolated from Chi3L1KO(-/-) exhibited impaired osteoblast differentiation and maturation. Chi3L1 also potentiated BMP2 signaling and RUNX2 expression in primary osteoblasts. Chi3L1 interacted with BMPRIa, which increased the surface expression of BMPRIa and promoted BMP2 signaling to induce osteoblast differentiation. Chi3L1KO(-/-) mice showed bone formation reduced with a decrease in RUNX2 expression in calvarial defects. Chi3L1KO(-/-) mice exhibited aging-related osteoporotic bone loss with decreases in the levels of RUNX2 and OPG, while serum PYD level and osteoclast number increased. Chi3L1 increased OPG via non-canonical BMP2 signaling in osteoblasts, which suppressed osteoclastogenesis in BMMs. Furthermore, ROC analysis showed that serum Chi3L1 level clinically decreased in osteoporosis patients. Our findings demonstrate that Chi3L1 promotes bone formation, suppresses osteoclastogenesis, and prevents aging-related osteoporosis.

Systems biology approach delineates critical pathways associated with disease progression in rheumatoid arthritis

Rheumatoid Arthritis (RA) is a chronic systemic autoimmune disease leading to inflammation, cartilage cell death, synoviocyte proliferation, and increased and impaired differentiation of osteoclasts and osteoblasts leading to joint erosions and deformities. Transcriptomics, proteomics, and metabolomics datasets were analyzed to identify the critical pathways that drive the RA pathophysiology. Single nucleotide polymorphisms (SNPs) associated with RA were analyzed for the functional implications, clinical outcomes, and blood parameters later validated by literature. SNPs associated with RA were grouped into pathways that drive the immune response and cytokine production. Further gene set enrichment analysis (GSEA) was performed on gene expression omnibus (GEO) data sets of peripheral blood mononuclear cells (PBMCs), synovial macrophages, and synovial biopsies from RA patients showed enrichment of Th1, Th2, Th17 differentiation, viral and bacterial infections, metabolic signalling and immunological pathways with potential implications for RA. The proteomics data analysis presented pathways with genes involved in immunological signaling and metabolic pathways, including vitamin B12 and folate metabolism. Metabolomics datasets analysis showed significant pathways like amino-acyl tRNA biosynthesis, metabolism of amino acids (arginine, alanine aspartate, glutamate, glutamine, phenylalanine, and tryptophan), and nucleotide metabolism. Furthermore, our commonality analysis of multi-omics datasets identified common pathways with potential implications for joint remodeling in RA. Disease-modifying anti-rheumatic drugs (DMARDs) and biologics treatments were found to modulate many of the pathways that were deregulated in RA. Overall, our analysis identified molecular signatures associated with the observed symptoms, joint erosions, potential biomarkers, and therapeutic targets in RA. Communicated by Ramaswamy H. Sarma

Defective bone repletion in aged Balb/cBy mice was caused by impaired osteoblastic differentiation.

This study was undertaken to gain mechanistic information about bone repair using the bone repletion model in aged Balb/cBy mice. onemonth-old (young) mice were fed a calcium-deficient diet for 2weeks and 8month-old (adult) and 21-25month-old (aged) female mice for 4weeks during depletion, which was followed by feeding a calcium-sufficient diet for 16days during repletion. To determine if prolonged repletion would improve bone repair, an additional group of aged mice were repleted for 4 additional weeks. Control mice were fed calcium-sufficient diet throughout. In vivo bone repletion response was assessed by bone mineral density gain and histomorphometry. In vitro response was monitored by osteoblastic proliferation, differentiation, and senescence. There was no significant bone repletion in aged mice even with an extended repletion period, indicating an impaired bone repletion. This was not due to an increase in bone cell senescence or reduction in osteoblast proliferation, but to dysfunctional osteoblastic differentiation in aged bone cells. Osteoblasts of aged mice had elevated levels of cytosolic and ER calcium, which were associated with increased Cav1.2 and CaSR (extracellular calcium channels) expression but reduced expression of Orai1 and Stim1, key components of Stored Operated Ca2+ Entry (SOCE). Activation of Cav1.2 and CaSR leads to increased osteoblastic proliferation, but activation of SOCE is associated with osteoblastic differentiation. The bone repletion mechanism in aged Balb/cBy mice is defective that is caused by an impaired osteoblast differentiation through reducedactivationof SOCE.

Deletion of Tfam in Prx1-Cre expressing limb mesenchyme results in spontaneous bone fractures.

Osteoblasts require substantial amounts of energy to synthesize the bone matrix and coordinate skeleton mineralization. This study analyzed the effects of mitochondrial dysfunction on bone formation, nano-organization of collagen and apatite, and the resultant mechanical function in mouse limbs. Limb mesenchyme-specific Tfam knockout (Tfamf/f;Prx1-Cre: Tfam-cKO) mice were analyzed morphologically and histologically, and gene expressions in the limb bones were assessed by in situ hybridization, qPCR, and RNA sequencing (RNA-seq). Moreover, we analyzed the mitochondrial function of osteoblasts in Tfam-cKO mice using mitochondrial membrane potential assay and transmission electron microscopy (TEM). We investigated the pathogenesis of spontaneous bone fractures using immunohistochemical analysis, TEM, birefringence analyzer, microbeam X-ray diffractometer and nanoindentation. Forelimbs in Tfam-cKO mice were significantly shortened from birth, and spontaneous fractures occurred after birth, resulting in severe limb deformities. Histological and RNA-seq analyses showed that bone hypoplasia with a decrease in matrix mineralization was apparent, and the expression of type I collagen and osteocalcin was decreased in osteoblasts of Tfam-cKO mice, although Runx2 expression was unchanged. Decreased type I collagen deposition and mineralization in the matrix of limb bones in Tfam-cKO mice were associated with marked mitochondrial dysfunction. Tfam-cKO mice bone showed a significantly lower Young's modulus and hardness due to poor apatite orientation which is resulted from decreased osteocalcin expression. Mice with limb mesenchyme-specific Tfam deletions exhibited spontaneous limb bone fractures, resulting in severe limb deformities. Bone fragility was caused by poor apatite orientation owing to impaired osteoblast differentiation and maturation.

Canonical Wnt signaling works downstream of iron overload to prevent ferroptosis from damaging osteoblast differentiation

Excessive iron has emerged in a large population of patients suffering from degenerative or hematological diseases with a common outcome, osteoporosis. However, its underlying mechanism remains to be clarified in order to formulate effective prevention and intervention against the loss of bone-forming osteoblasts. We show herein that increased intracellular iron by ferric ammonium citrate (FAC) mimicking the so-called non-transferrin bound iron concentrations leads to ferroptosis and impaired osteoblast differentiation. FAC upregulates the expression of Trfr and DMT1 genes to increase iron uptake, accumulating intracellular labile ferrous iron for iron overload status. Then, the excessive ferrous iron generates reactive oxygen species (ROS) and lipid peroxidation products (LPO), causing ferroptosis with its typical mitochondrial morphological changes, such as shrinkaged and condensed membrane with diminution and loss of crista and outer membrane rupture. We further examined that ferroptosis is the main cause responsible for FAC-disrupted osteoblast differentiation, although apoptosis and senescence are concurrently induced as well. Mechanistically, we revealed that iron dose-dependently down-regulates the expression of Wnt target genes and inhibits the transcription of Wnt reporter TopFlash construct, so as to inhibit the canonical Wnt signaling. Wnt agonist, ferroptosis inhibitor, or antioxidant melatonin reverses iron-inhibited canonical Wnt signaling to restore osteoblast differentiation by reducing ROS and LPO production to prevent ferroptosis notably without reducing iron overload. This study proposes a working model against excessive iron-induced osteoporosis: iron chelator deferoxamine or the above three drugs prevent ferroptosis, restore traditional Wnt signaling to maintain osteoblast differentiation no matter whether iron overload is removed or not. Additionally, iron chelator should be used to a suitable extent because iron itself is necessary for osteogenic differentiation.

Expression and Regulatory Network Analysis of BICC1 for Aged Sca-1-Positive Bone Narrow Mesenchymal Stem Cells.

Background The impaired osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs) is a major cause of bone remodeling imbalance and osteoporosis. The bicaudal C homologue 1 (BICC1) gene is a genetic regulator of bone mineral density (BMD) and promotes osteoblast differentiation. The purpose of this study is to explore the probable function of BICC1 in osteoporosis and osteogenic differentiation of aged BMSCs. Methods We examined the GSE116925 microarray dataset obtained from the Gene Expression Omnibus (GEO) database. The GEO2R algorithm identified differentially expressed genes (DEGs) in Sca-1+ BMSCs from young (3 months old) and old (18 months old) mice. Then, to identify the most crucial genes, we used pathway enrichment analysis and a protein-protein interaction (PPI) network. Furthermore, starBase v2.0 was used to generate the regulatory networks between BICC1 and related competing endogenous RNAs (ceRNAs). NetworkAnalyst was used to construct TF-gene networks and TF-miRNA-gene networks of BICC1 and ceRNA. Furthermore, we investigated the Bicc1 expression in aged Sca-1-positive BMSCs. Result We detected 923 DEGs and discovered that epidermal growth factor receptor (EGFR) was the top hub gene with a high degree of linkage. According to the findings of the PPI module analysis, EGFR was mostly engaged in cytokine signaling in immune system and inflammation-related signaling pathways. 282 ceRNAs were found to interact with the BICC1 gene. EGFR was not only identified as a hub gene but also as a BICC1-related ceRNA. Then, we predicted 11 common TF-genes and 7 miRNAs between BICC1 and EGFR. Finally, we found that BICC1 mRNA and EGFR mRNA were significantly overexpressed in aged Sca-1-positive BMSCs. Conclusion As a genetic gene that affects bone mineral density, BICC1 may be a new target for clinical treatment of senile osteoporosis by influencing osteogenic differentiation of BMSCs through EGFR-related signaling. However, the application of the results requires support from more experimental data.

Deletion of Rheb1 in Osteocytes Leads to Osteopenia Characterized by Reduced Bone Formation and Enhanced Bone Resorption.

Ras homologue enriched in brain 1 (Rheb1), an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), is known to modulate various cellular processes. However, its impact on bone metabolism in vivo remains unknown. The study aimed at understanding the role of Rheb1 on bone homeostasis. We measured the serum parameters and performed histomorphometry, quantitative real-time polymerase chain reaction, and Western blotting, along with the generation of mouse gene knockout (KO) model, and conducted a microcomputed tomography analysis and tartrate-resistant acid phosphatase staining, to delineate the impacts of Rheb1 on bone homeostasis. In the Rheb1 KO mice, the results showed that Rheb1 KO caused significant damage to the bone microarchitecture, indicating that mTORC1 activity was essential for the regulation of bone homeostasis. Specifically, suppressed mineralization activity in primary osteoblasts and a decreased osteoblast number were observed in the Rheb1 KO mice, demonstrating that loss of Rheb1 led to impaired osteoblastic differentiation. Furthermore, the higher apoptotic ratio in Rheb1-null osteocytes could promote Tnfsf11 expression and lead to an increase in osteoclasts, indicating increased bone resorption activity in the KO mice. The findings confirmed that Rheb1 deletion in osteoblasts/osteocytes led to osteopenia due to impaired bone formation and enhanced bone resorption.