Inhibits Osteoblast Differentiation Research Articles

Wnt3a-induced LRP6 phosphorylation enhances osteoblast differentiation to alleviate osteoporosis through activation of mTORC1/β-catenin signaling

ObjectiveOsteoporosis (OP) is a common cause of morbidity and mortality in older individuals. The importance of Wnt3a in osteogenic activity and bone tissue homeostasis is well known. Here, we explored the possible molecular mechanism by which Wnt3a mediates the LRP6/mTORC1/β-catenin axis to regulate osteoblast differentiation in OP. MethodsOP-related key genes were identified through a bioinformatics analysis. A ROS17/2.8 cell differentiation system for rat osteogenic progenitors and a rat model of senile OP were constructed for in vitro and in vivo mechanism verification. ResultsBioinformatics analysis revealed that LRP6 was poorly expressed in OP and may play a key role in the occurrence of OP by affecting osteoblast differentiation. LRP6 knockdown inhibited osteoblast differentiation in an in vitro model. In addition, Wnt3a promoted osteoblast differentiation by inducing LRP6 phosphorylation. Moreover, LRP6 promoted mTORC1 expression, which indirectly promoted β-catenin expression, thus promoting osteoblast differentiation. Finally, an in vivo assay revealed that LRP6 inhibition improved OP. ConclusionOur study provides evidence that Wnt3a induces phosphorylation of LRP6 to activate the mTORC1/β-catenin axis, thus promoting osteoblast differentiation and ultimately improving OP in aged rats.

Therapeutic Potential of Stearoyl-CoA Desaturase1 (SCD1) in Modulating the Effects of Fatty Acids on Osteoporosis.

Osteoporosis is a common skeletal disease, primarily associated with aging, that results from decreased bone density and bone volume. This reduction significantly increases the risk of fractures in osteoporosis patients compared to individuals with normal bone density. Additionally, the bone regeneration process in these patients is slow, making complete healing difficult. Along with the decline in bone volume and density, osteoporosis is characterized by an increase in marrow adipose tissue (MAT), which is fat within the bone. In this altered bone microenvironment, osteoblasts are influenced by various factors secreted by adipocytes. Notably, saturated fatty acids promote osteoclast activity, inhibit osteoblast differentiation, and induce apoptosis, further reducing osteoblast formation. In contrast, monounsaturated fatty acids inhibit osteoclast formation and mitigate the apoptosis caused by saturated fatty acids. Leveraging these properties, we aimed to investigate the effects of overexpressing stearoyl-CoA desaturase 1 (SCD1), an enzyme that converts saturated fatty acids into monounsaturated fatty acids, on osteogenic differentiation and bone regeneration in both in vivo and in vitro models. Through this novel approach, we seek to develop a stem cell-based therapeutic strategy that harnesses SCD1 to improve bone regeneration in the adipocyte-rich osteoporotic environment.

TMEM175 plays a crucial role in osteoblast differentiation by regulating lysosomal function and autophagy

Bone provides structural support, enables movement, protects internal organs, regulates calcium and phosphorus levels, and contains bone marrow essential for hematopoiesis. Osteoblasts are specialized cells responsible for bone formation through the secretion of extracellular matrix components. Transmembrane protein 175 (TMEM175), which functions as an endosomal/lysosomal K+ channel and a lysosomal H+ channel, regulates lysosomal function and autophagy. Despite the recognized importance of lysosomes and autophagy in osteoblast differentiation, the specific role of TMEM175 in osteoblast differentiation has not been revealed. In this study, we investigated whether TMEM175 is associated with human bone mineral densityand fracture and examined the role of TMEM175 in osteoblast differentiation. In analyses of single nucleotide polymorphismsof pore ion channel genes using the mouse2human database, a significant correlation between TMEM175 single nucleotide polymorphisms and human bone mineral density and fracture was identified. TMEM175 expression levels were found to increase during osteoblast differentiation from bone chip-derived mesenchymal stem cells (BMSCs). Knockdown of TMEM175 in BMSCs suppressed osteoblast differentiation, as evidenced by decreased matrix mineralization and lower expression levels of osteoblast marker genes. Further analysis indicated that TMEM175 deficiency leads to lysosomal dysfunction and partially impairs autophagic clearance during osteoblast differentiation. Moreover, the TMEM175 inhibitor 4-aminopyridinedecreased osteoblast differentiation of BMSCs. Taken together, this study reveals that TMEM175 plays an important role in osteoblast differentiation by regulating lysosomal function and autophagic clearance.

7050 Effect of Spironolactone On Reducing Sugar-Induced Cellular Damage In MC3T3-E1 Osteoblastic Cells

Abstract Disclosure: S. Yun: None. H. Sang: None. S. Park: None. K. Suh: None. H. Kim: None. S. Chin: None. Background: In diabetes, prolonged hyperglycemia causes oxidative stress, resulting in pancreatic beta cells' dysfunction and diabetic complications. Oxidative stress can inhibit osteoblast differentiation and induce dysfunction and apoptosis. Spironolactone is an aldosterone antagonist used to treat hypertension and heart failure. In this study, we observed whether spironolactone can reduce osteoblast cytotoxicity and differentiation caused by oxidative stress induced by 2-deoxy-d-ribose (dRib). Methods: MC3T3-E1 cells were treated with spironolactone (0-100 uM) in the presence of 15 mM dRib. The cytotoxicity (lactate dehydrogenase, LDH) and differentiation (collagen content, alkaline phosphatase [ALP] activity, and mineralization) of osteoblasts, inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin [IL]-6), oxidative stress (reactive oxygen species [ROS], mitochondrial superoxide), endoplasmic reticulum (ER) stress (activating transcription factor 6 [ATF-6], inositol-requiring 1 [IRE1]), mitochondrial function (mitochondrial membrane potential [MMP], adenosine triphosphate [ATP]), glyoxalase I activity, and glutathione (GSH) were measured. Results: Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented the dRib-induced cytotoxicity of osteoblasts and promoted differentiation of osteoblasts suppressed by dRib. Spironolactone significantly reduced dRib-induced inflammatory cytokines, ROS, mitochondrial superoxide, and ER stress. dRib-induced mitochondrial dysfunction was significantly improved by treatment with spironolactone. dRib is detoxified by the glyoxalase system. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I in dRib. Conclusion: Spironolactone could reduce dRib-induced cytotoxicity and promote differentiation in MC3T3-E1 osteoblastic cells by reducing oxidative stress and inflammatory cytokines, increasing mitochondrial biogenesis, and detoxifying dRib. These results suggest that spironolactone can be useful in the prevention and treatment of diabetic bone disease. Presentation: 6/1/2024

Melatonin Inhibits ET-1 Production to Break Crosstalk Between Prostate Cancer and Bone Cells: Implication for Osteoblastic Bone Metastasis Treatment.

Bone metastasis is the primary cause of death among patients with advanced prostate cancer (PCa). PCa tends to spread to bones and acquire the bone-like phenotype, causing osteoblastic bone metastasis. Unfortunately, there is no effective treatment for this condition. However, melatonin, which regulates our circadian rhythm, has been found to have anti-tumor properties. It has yet to be established whether it is effective in treating osteoblastic PCa metastasis. Our findings show that melatonin inhibits the production of endothelin-1 (ET-1) in osteoblastic PCa cells, suppressing osteoblast differentiation. Clinical results indicate that bone metastatic PCa patients have higher levels of ET-1 compared to nonmetastatic PCa patients. Furthermore, melatonin-induced miR-let-7f-5p inhibits ET-1-promoted osteoblast differentiation in osteoblastic PCa. Melatonin also suppresses the property of osteomimicry in osteoblastic PCa cells. Importantly, in the intratibia injection PCa metastasis model, melatonin decreased osteoblastic PCa tumor growth, inhibiting ET-1 production and osteoblast differentiation in vivo. Taken together, melatonin inhibits osteoblastic PCa-regulated osteoblastogenesis by reducing ET-1 production through upregulation of miR-let-7f-5p, while suppressing the property of osteomimicry in osteoblastic PCa. Melatonin therapy could be a promising approach to treating bone metastasis in osteoblastic PCa.

BMP3b regulates bone mass by inhibiting BMP signaling

Bone morphogenetic protein 3b (BMP3b), also known as growth differentiation factor 10 (GDF10), is a non-osteogenic BMP highly expressed in the skeleton. Although in vitro studies have shown that BMP3b suppresses osteoblast differentiation, the physiological role of BMP3b in regulating bone mass in vivo remains unknown. Here, we show that BMP3b deletion in mice leads to a high bone mass phenotype via an unexpected novel mechanism involving de-repression of canonical BMP/Smad signaling. BMP3b null mice were viable, and exhibited no significant difference in body size compared to wildtype control. Trabecular bone parameters assessed by histomorphometry and μCT, revealed a significant increase in bone volume and bone mineral density. Expression of osteoblast-differentiation genes were elevated in bone tissue of BMP3b null mice, whereas expression of osteoclast-related genes remained unchanged. Consistent with this, Bmp3b was highly expressed in osteoblasts relative to osteoclast cells. Ex-vivo culture of primary bone marrow mesenchymal stem cells (BMSCs) and primary bone marrow-derived osteoclasts revealed that inactivation of BMP3b enhances osteogenesis without affecting osteoclastogenesis. Mechanistically, we found that BMP3b suppressed BMP4-induced Smad1/5 phosphorylation and inhibited the activity of a BMP4-driven Id-1 luciferase reporter. Protein-protein interaction assays revealed that BMP3b competitively interfered with the association of BMP4 and BMP type I receptors. These findings suggest that BMP3b regulates bone mass by acting as a BMP receptor antagonist. Thus, maintenance of bone mass involves antagonism of canonical BMP/Smad signaling by a member of the BMP family.

GDF10 is a negative regulator of vascular calcification

Cardiovascular mortality is particularly high and increasing in patients with chronic kidney disease, with vascular calcification (VC) as a major pathophysiologic feature. VC is a highly regulated biological process similar to bone formation involving osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). We have previously demonstrated that loss of T-cell death-associated gene 51 (TDAG51) expression leads to an attenuation of medial VC. We now show a significant induction of circulating levels of growth differentiation factor 10 (GDF10) in TDAG51-/- mice, which was of interest due to its established role as an inhibitor of osteoblast differentiation. The objective of this study was to examine the role of GDF10 in the osteogenic transdifferentiation of VSMCs. Using primary mouse and human VSMCs, as well as exvivo aortic ring cultures, we demonstrated that treatment with recombinant human (rh) GDF10 mitigated phosphate-mediated hydroxyapatite (HA) mineral deposition. Furthermore, exvivo aortic rings from GDF10-/- mice exhibited increased HA deposition compared to C57BL/6J controls. To explain our observations, we identified that rhGDF10 treatment reduced protein expression of runt-related transcription factor 2, a key driver of osteogenic transdifferentiation of VSMCs and VC. In support of these findings, invivo treatment with rhGDF10 attenuated VD3-induced VC. Furthermore, we demonstrated an increase in circulating GDF10 in patients with chronic kidney disease with clinically defined severe VC, as assessed by coronary artery calcium score. Thus, our studies identify GDF10 as a novel inhibitor of mineral deposition and as such, may represent a potential novel biomarker and therapeutic target for the detection and management of VC.

Stanniocalcin 1 and 1,25-dihydroxyvitamin D3 cooperatively regulate bone mineralization by osteoblasts

Stanniocalcin 1 (STC1) is a calcium- and phosphate-regulating hormone that is expressed in all tissues, including bone tissues, and is involved in calcium and phosphate homeostasis. Previously, STC1 expression was found to be increased by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administration in renal proximal tubular cells. In this study, we investigated whether STC1 directly regulates osteoblast differentiation or reciprocally controls the effects of 1,25(OH)2D3 on osteoblasts to contribute to bone homeostasis. We found that STC1 inhibited osteoblast differentiation in vitro and bone morphogenetic protein 2 (BMP2)-induced ectopic bone formation in vivo. Moreover, 1,25(OH)2D3 increased STC1 expression through direct binding to the Stc1 promoter of the vitamin D receptor (VDR). STC1 activated the 1,25(OH)2D3–VDR signaling pathway through the upregulation of VDR expression mediated by the inhibition of Akt phosphorylation in osteoblasts. STC1 further increased the effects of 1,25(OH)2D3 on receptor activator of nuclear factor-κB ligand (RANKL) secretion and inhibited osteoblast differentiation by exhibiting a positive correlation with 1,25(OH)2D3. The long-bone phenotype of transgenic mice overexpressing STC1 specifically in osteoblasts was not significantly different from that of wild-type mice. However, compared with that in the wild-type mice, 1,25(OH)2D3 administration significantly decreased bone mass in the STC1 transgenic mice. Collectively, these results suggest that STC1 negatively regulates osteoblast differentiation and bone formation; however, the inhibitory effect of STC1 on osteoblasts is transient and can be reversed under normal conditions. Nevertheless, the synergistic effect of STC1 and 1,25(OH)2D3 through 1,25(OH)2D3 administration may reduce bone mass by inhibiting osteoblast differentiation.

KIAA0753 enhances osteoblast differentiation suppressed by diabetes.

Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation invitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.

Analysis of PANoptosis-related ceRNA network reveals lncRNA MIR17HG involved in osteogenic differentiation inhibition impaired by tumor necrosis factor-α

BackgroundInflammatory cytokines such as Interleukin 1β(IL1β), IL6,Tumor Necrosis Factor-α (TNF-α) can inhibit osteoblast differentiation and induce osteoblast apoptosis. PANoptosis, a newly identified type of programmed cell death (PCD), may be influenced by long noncoding RNA (lncRNAs) which play important roles in regulating inflammation. However, the potential role of lncRNAs in inflammation and PANoptosis during osteogenic differentiation remains unclear. This study aimed to investigate the regulatory functions of lncRNAs in inflammation and apoptosis during osteogenic differentiation.Methods and resultsHigh-throughput sequencing was used to identify differentially expressed genes involved in osteoblast differentiation under inflammatory conditions. Two lncRNAs associated with inflammation and PANoptosis during osteogenic differentiation were identified from sequencing data and Gene Expression Omnibus (GEO) databases. Their functionalities were analyzed using diverse bioinformatics methodologies, resulting in the construction of the lncRNA-miRNA-mRNA network. Among these, lncRNA (MIR17HG) showed a high correlation with PANoptosis. Bibliometric methods were employed to collect literature data on PANoptosis, and its components were inferred. PCR and Western Blotting experiments confirmed that lncRNA MIR17HG is related to PANoptosis in osteoblasts during inflammation.ConclusionsOur data suggest that TNF-α-induced inhibition of osteogenic differentiation and PANoptosis in MC3T3-E1 osteoblasts is associated with MIR17HG. These findings highlight the critical role of MIR17HG in the interplay between inflammation, PANoptosis, and osteogenic differentiation, suggesting potential therapeutic targets for conditions involving impaired bone formation and inflammatory responses.

Osteoclast-derived exosomes influence osteoblast differentiation in osteoporosis progression via the lncRNA AW011738/ miR-24-2-5p/ TREM1 axis

AimsTo investigate the molecular mechanism of osteoclast-derived exosomes in osteoporosis. Main methodsRANKL induced osteoclast model was screened for significantly differentially expressed lncRNAs and mRNAs by whole RNA sequencing. Exosomes were characterized using electron microscopy, western blotting and nanosight. Overexpression or knockdown of AW011738 was performed to explore its function. The degree of osteoporosis in an osteoporosis model was assessed by mirco-CT. The osteoclast model, osteoblast differentiation ability and the molecular mechanism of lncRNA AW011738/miR-24-2-5p/TREM1 axis in osteoporosis were assessed by dual luciferase reporter gene assay, Western blotting (WB), immunofluorescence and ALP staining. Bioinformatics was used to predict interactions of key osteoporosis-related genes with miRNAs, transcription factors, and potential drugs after upregulation of AW011738. To predict the protein-protein interaction (PPI) network associated with key genes, GO and KEGG analyses were performed on the key genes. The ssGSVA was used to predict changes in the immune microenvironment. Key findingsOsteoclast-derived exosomes containing lncRNA AW011738 decreased the osteogenesis-related markers and accelerated bone loss in OVX mice. Osteoclast (si-AW011738)-derived exosomes showed a significant increase in biomarkers of osteoblast differentiation in vitro compared to the si-NC group. As analyzed by mirco-CT, tail vein injected si-AW011738 OVX mice were less osteoporotic than the control group. AW011738 inhibited osteoblast differentiation by regulating TREM1 expression through microRNA. Meanwhile, overexpression of miR-24-2-5p inhibited TREM1 expression to promote osteoblast differentiation. SignificanceOsteoclast-derived exosomes containing lncRNA AW011738 inhibit osteogenesis in MC3T3-E1 cells through the lncRNA AW011738/miR-24-2-5p/TREM1 axis and exacerbate osteoporosis in OVX mice.

Differential but complementary roles of HIF-1α and HIF-2α in the regulation of bone homeostasis

Bone is a highly dynamic tissue undergoing continuous formation and resorption. Here, we investigated differential but complementary roles of hypoxia-inducible factor (HIF)-1α and HIF-2α in regulating bone remodeling. Using RNA-seq analysis, we identified that specific genes involved in regulating osteoblast differentiation were similarly but slightly differently governed by HIF-1α and HIF-2α. We found that increased HIF-1α expression inhibited osteoblast differentiation via inhibiting RUNX2 function by upregulation of Twist2, confirmed using Hif1a conditional knockout (KO) mouse. Ectopic expression of HIF-1α via adenovirus transduction resulted in the increased expression and activity of RANKL, while knockdown of Hif1a expression via siRNA or osteoblast-specific depletion of Hif1a in conditional KO mice had no discernible effect on osteoblast-mediated osteoclast activation. The unexpected outcome was elucidated by the upregulation of HIF-2α upon Hif1a overexpression, providing evidence that Hif2a is a transcriptional target of HIF-1α in regulating RANKL expression, verified through an experiment of HIF-2α knockdown after HIF-1α overexpression. The above results were validated in an ovariectomized- and aging-induced osteoporosis model using Hif1a conditional KO mice. Our findings conclude that HIF-1α plays an important role in regulating bone homeostasis by controlling osteoblast differentiation, and in influencing osteoclast formation through the regulation of RANKL secretion via HIF-2α modulation.

PM2.5 exposure inhibits osteoblast differentiation by increasing the ubiquitination and degradation of Smad4

Increasing epidemiological evidence has shown that PM2.5 exposure is significantly associated with the occurrence of osteoporosis. It has been well demonstrated that PM2.5 exposure enhanced the differentiation and function of osteoclasts by indirectly causing chronic inflammation, while the mechanism in osteoblasts remains unclear. In our study, toxic effects were evaluated by direct exposure of 20–80 μg/ml PM2.5 to MC3T3-E1 cells and BMSCs. The results showed that PM2.5 exposure did not affect cell viability via proliferation and apoptosis, but significantly inhibited osteoblast differentiation in a dose-dependent manner. Osteogenic transcription factors Runx2 and Sp7 and other biomarkers Alp and Ocn decreased after PM2.5 exposure. RNA-seq revealed TGF-β signaling was involved in PM2.5 exposure inhibited osteoblast differentiation, which led to P-Smad1/5 and P-Smad2 reduction in the nucleus by increasing the ubiquitination and degradation of Smad4. At last, the inflammation response increased in MC3T3-E1 cells with PM2.5 exposure. Moreover, the mRNA levels of Mmp9 increased in bone marrow-derived macrophage cells treated with the conditional medium collected from MC3T3-E1 cells exposed to PM2.5. Overall, these results indicated that PM2.5 exposure inhibits osteoblast differentiation and concurrently increases the maturation of osteoclasts. Our study provides in-depth mechanistic insights into the direct impact of PM2.5 exposure on osteoblast, which would indicate the unrecognized role of PM2.5 on osteoporosis.

Phosvitin phosphopeptides alleviate the inhibitory effect of oxidative stress on osteoblast function through FoxO/Wnt signaling

Oxidative stress inhibits osteoblast differentiation and function, leading to the development of osteoporosis. The effects of Phosvitin phosphopeptides (PPPs) on the osteogenic function of osteoblast were determined based on oxidative stress MC3T3-E1 cell model induced by hydrogen peroxide. The results showed that 50 μg/mL of PPPs significantly increased cell viability from 56.00% (H2O2) to 106.80% for 72 h. DCFH-DA fluorescence assay demonstrated that PPPs could remarkably reduce reactive oxygen species levels induced by H2O2. The alkaline phosphatase activity of oxidative stress-treated osteoblast increased from 2.36 Kim'unit/100 mL (H2O2 group) to 8.16 Kim'unit/100 mL, along with an increase in the activity of total superoxide dismutase, glutathione peroxidase, catalase, and the number of mineralized nodules in response to PPPs treatment. Overall, the results of this work indicated that PPPs had a stimulating impact on the Wnt/β-catenin signaling pathway while simultaneously inhibiting the forkhead box class O (FoxO) signaling pathway in MC3T3-E1 osteoblast induced by H2O2. Consequently, these findings demonstrate that PPPs have the ability to protect the osteogenic function of MC3T3-E1 osteoblasts against oxidative damage through the activation of the FoxO/Wnt signaling pathway.

BIGH3 mediates apoptosis and gap junction failure in osteocytes during renal cell carcinoma bone metastasis progression

Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.

Titanium dioxide nanoparticles oral exposure induce osteoblast apoptosis, inhibit osteogenic ability and increase lipogenesis in mouse

Titanium dioxide nanoparticles (TiO2-NPs) are widely used in food, paint, coating, cosmetic, and composite orthodontic material. As a common food additive, TiO2-NPs can accumulate in various organs of human body, but the effect and underlying mechanism of bone remain unclear. Here mice were exposed to TiO2-NPs by oral gavage, and histological staining of femoral sections showed that TiO2-NPs reduced bone formation and enhanced osteoclast activity and lipogenesis, contributing to decreased trabecula bone. Transmission electron microscope (TEM) as well as biochemical and flow cytometry analysis of osteoblast exhibited that TiO2-NPs accumulated in osteoblast cytoplasm and impaired mitochondria ultrastructure with increased reactive oxygen species (ROS) and lipid hyperoxide, resulting in osteoblast apoptosis. In terms of mechanism, TiO2-NPs treatment inhibited expression of AKT and then increased pro-apoptotic protein Bax expression which was failure to form heterodimers with decreased anti-apoptotic Bcl-2, activating downstream Caspase-9 and Caspase-3 and inducing apoptosis. Additionally, TiO2-NPs suppressed Wnt3a level and then activated anti-Glycogen synthesis kinase (GSK-3β) phosphorylation, and ultimately resulted in degradation of β-catenin which down-regulated Runt-related transcription factor 2 (Runx2) and Osterix, inhibiting expression of osteogenic related proteins. Together, these results revealed that exposure of TiO2-NPs induced apoptosis and inhibited osteoblast differentiation through suppressing PI3K/AKT and Wnt/β-catenin signaling pathways, resulting in reduction of trabecula bone.

Dietary restriction plus exercise change gene expression of Cxcl12 abundant reticular cells in female mice.

Low energy availability due to excessive exercise lowers bone mass and impairs various physiological functions, including immunity and hematopoiesis. We focused on Cxcl12 abundant reticular (CAR) cells, which are bone marrow mesenchymal stem cells and are essential for the maintenance of hematopoietic and immune cells in bone marrow. We examine the functional changes in CAR cells resulting from dietary restriction combined with exercise. Five-week-old wild-type female mice were divided into an ad libitum group (CON), a 60% dietary restriction group (DR), an ad libitum with exercise group (CON + ex), and a 60% dietary restriction with exercise group (DR + ex). Blood parameters, bone structure parameters, and bone marrow fat volume were evaluated after 5weeks. In addition, bone marrow CAR cells were isolated by cell sorting and analyzed for gene expression by RT-qPCR. Bone mineral density (BMD) was significantly decreased in DR and DR + ex compared to CON and CON + ex. Especially, cortical bone mass and thickness were significantly decreased in DR and DR + ex groups, whereas trabecular bone mass was significantly increased. Bone marrow fat volume was significantly increased in DR and DR + ex groups compared to CON and CON + ex. The number of leukocytes in the blood was significantly decreased in the DR + ex group compared to the other three groups. RT-qPCR showed a significant decrease in gene expression of both Foxc1 and Runx2 in CAR cells of the DR + ex group compared to CON. Dietary restriction combined with exercise promotes CAR cell differentiation into bone marrow adipocyte and suppresses osteoblast differentiation.

Abstract 2761: Activating RET mutations promotes osteoblastic bone metastasis by inhibiting osteoclastogenesis and stimulating osteoblast activity via regulation of osteoprotegerin

Abstract Bone metastases (BM) are a source of increased mortality and significant morbidity in patients with medullary thyroid cancer (MTC), with ~50% survival at five years post-diagnosis. MTC causes osteoblastic, osteolytic, or mixed lesions, yet the underlying mechanisms are unknown. The RET proto-oncogene encodes a transmembrane tyrosine kinase receptor and is the driver oncogene in ~60% of MTC cases where a germline or somatic activating mutation results in ligand-independent constitutive activation of the receptor. However, the role of RET mutations in the genesis of bone metastasis lesions is unknown. We show that activating RET mutations promotes an osteoblastic phenotype in mice implanted with patient-derived MTC cells by increasing bone formation and decreasing bone resorption. We found that the radiographic appearance of osteoblastic lesions differed between the types of RET mutations (C634W and M918T). MTC cells with RETC634W mutation showed a four-fold increase in bone mineral density, trabecular bone volume, number, and thickness with a decreased number of osteoblasts compared to the non-tumor-bearing femur. TRAP staining revealed that osteoclast numbers per long bone were reduced in the injected femur compared to the control. In contrast, the MTC-RETM918T mutation showed an increased cortical thickness and porosity with reduced medullary area and increased bone formation rate compared to the non-tumor bearing femur. MTC cells formed tumors with extensive mineralized tissue, composed of bone matrix surrounded by osteoblast-like cells and osteoid. The knockdown of RET in the MTC-RETM918T model significantly reduced cortical thickness and increased the number of osteoclasts per bone surface. We observed increased osteoprotegerin (OPG) expression in MTC-bearing femurs compared to controls, suggesting decreased bone resorption. The factors secreted by MTC cells, including OPG, inhibited osteoclast formation from primary bone marrow cells and Raw264 cells. In addition, RET knockdown by shRNA in MTC cells decreased OPG expression. Moreover, the MTC-conditioned media (CM) increased osteoblast differentiation and mineralization of the primary culture of pre-osteoblasts isolated from calvarial bone, long bones, and MC3T3 cells. The conditioned media of RET-depleted cells inhibits osteoblast differentiation and mineralization. The expression of alkaline phosphatase, osteocalcin, OPG, and osterix increased in pre-osteoblast incubated with MTC-CM. Compared to primary non-metastatic tumors, the OPG expression increases in tissues of sporadic MTC bone metastases harboring RETM918T mutation. These results suggest that MTC with RET-activating mutations induces osteoblastic bone metastasis due to decreased bone resorption and increased bone formation. Citation Format: Rozita Bagheri-Yarmand, Joseph L. Kidd, Gabriel M. Pagnotti, Trupti Trivedi, Leah Guerra, Xinhai Wan, Jian Song, Sue-Hwa Lin, Theresa A Guise. Activating RET mutations promotes osteoblastic bone metastasis by inhibiting osteoclastogenesis and stimulating osteoblast activity via regulation of osteoprotegerin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2761.

LSD1 modulates the bone metastasis of breast cancer cells through hnRNPA2B1-mediated sorting of exosomal miRNAs

Bone metastasis is a key contributor to morbidity and mortality of breast cancer patients. We have previously shown that exosomal miRNAs derived from LSD1 knockdown (KD) breast cancer cells inhibit osteoblast differentiation and promote osteoclast differentiation. However, how LSD1 regulates exosomal miRNAs and whether miRNAs promote bone metastasis through the formation of pre-metastatic niches remains unclear. In vivo experiments demonstrates that exosomes derived from LSD1 KD breast cancer cells significantly promoted bone metastasis. To explore the mechanism underlying the effect of LSD1 on exosomes in breast cancer cells, exosomal and cellular miRNAs from control, LSD1 KD, and rescue cells were sequenced. Interestingly, approximately 80% of LSD1-associated miRNAs were downregulated in exosomes from LSD1 KD cells. The consensus sequence UAGGGC, was identified in many miRNAs downregulated in LSD1 KD exosomes. We found that hnRNPA2B1 regulated the exosomal sorting of miR-6881-3p and some other miRNAs. LSD1 deficiency reduced hnRNPA2B1 expression in breast cancer cells by decreasing the level of H3K9me2 demethylation in the promoter region of the hnRNPA2B1 gene. Our study revealed that LSD1 plays a crucial role in the regulation of exosomal sorting of miRNA.

DDIT3 deficiency accelerates bone remodeling during bone healing by enhancing osteoblast and osteoclast differentiation through ULK1-mediated autophagy

The coordination of osteoblasts and osteoclasts is essential for bone remodeling. DNA damage inducible script 3 (DDIT3) is an important regulator of bone and participates in cell differentiation, proliferation, autophagy, and apoptosis. However, its role in bone remodeling remains unexplored. Here, we found that Ddit3 knockout (Ddit3-KO) enhanced both bone formation and resorption. The increased new bone formation and woven bone resorption, i.e., enhanced bone remodeling capacity, was found to accelerate bone defect healing in Ddit3-KO mice. In vitro experiments showed that DDIT3 inhibited both osteoblast differentiation and Raw264.7 cell differentiation by regulating autophagy. Cell coculture assay showed that Ddit3-KO decreased the ratio of receptor activator of nuclear factor-κβ ligand (RANKL) to osteoprotegerin (OPG) in osteoblasts, and Ddit3-KO osteoblasts inhibited osteoclast differentiation. Meanwhile, DDIT3 knockdown (DDIT3-sh) increased receptor activator of nuclear factor-κβ (RANK) expression in Raw264.7 cells, and DDIT3-sh Raw264.7 cells promoted osteoblast differentiation, whereas, DDIT3 overexpression had the opposite effect. Mechanistically, DDIT3 promoted autophagy partly by increasing ULK1 phosphorylation at serine555 (pULK1-S555) and decreasing ULK1 phosphorylation at serine757 (pULK1-S757) in osteoblasts, thereby inhibiting osteoblast differentiation. DDIT3 inhibited autophagy partly by decreasing pULK1-S555 in Raw264.7 cells, thereby suppressing osteoclastic differentiation. Taken together, our data indicate that DDIT3 is one of the elements regulating bone remodeling and bone healing, which may become a potential target in bone defect treatment.