YTHDF1 promotes the osteolytic bone metastasis of breast cancer via inducing EZH2 and CDH11 translation.

Bone metastasis is common in breast cancer and more effective therapies are required, however, its molecular mechanism is poorly understood. Additionally, the role of the m6A reader YTHDF1 in bone metastasis of breast cancer has not been reported. Here, we reveal that the increased expression of YTHDF1 is clinically correlated with breast cancer bone metastases. YTHDF1 promotes migration, invasion, and osteoblast adhesion and induces osteoclast differentiation of cancer cells in vitro and vivo. Mechanically, RNA-seq, MeRIP-seq and RIP-seq analysis, and molecular biology experiments demonstrate that YTHDF1 translationally enhances EZH2 and CDH11 expression by reading m6A-enriched sites of their transcripts. Moreover, adeno-associated virus (AAV) was used to deliver shYTHDF1 (shYTHDF1-AAV) in intratibial injection models, eliciting a significant suppressive effect on breast cancer bone metastatic formation and osteolytic destruction. Overall, we uncovered that YTHDF1 promotes osteolytic bone metastases of breast cancer by inducing EZH2 and CDH11 translation.

A case report of multicentric carpotarsal osteolysis syndrome: Depiction of a debilitating disease course.

Multicentric carpotarsal osteolysis syndrome (MCTO) is a rare skeletal disorder characterized by progressive osteolysis involving the carpal and tarsal bones, and often associated with nephropathy. It is caused by heterozygous mutation in the MAF bZIP transcription factor B (MAFB) gene. Heterogeneous clinical manifestation and wide spectrum of disease severity have been observed in patients with MCTO. Here, we report a case of a male patient who presented with kidney failure in childhood with progressive disabling skeletal deformity. He was diagnosed with MCTO at 31-years-old, where a de novo pathogenic heterozygous variant in NM_005461.5:c.212C>A: p.(Pro71His) of the MAFB gene was identified. While there has been little data on the long-term prognosis and life expectancy of this disease, this case report sheds light on the debilitating disease course with multiple significant morbidities of a patient with MCTO throughout his lifetime of 33 years.

Multicentric carpotarsal osteolysis syndrome with variants of MAFB gene: a case report and literature review.

BACKGROUND: Multicentric carpotarsal osteolysis (MCTO) is a rare genetic disorder characterized by the progressive loss of bone in the hands, feet, and other skeletal structures. It presents with symptoms that may resemble those of juvenile idiopathic arthritis, making diagnosis challenging for clinicians. The identification of MAF BZIP Transcription Factor B (MAFB) mutations as significant contributors to MCTO represents a major breakthrough in our understanding of the pathogenesis of this rare skeletal disorder. CASE PRESENTATION: Our objective was to present the phenotype, treatment, and outcome of a patient with a variant of MAFB-induced MCTO to broaden the range of clinical features associated with MCTO and share our clinical experience for improved diagnosis and treatment. In our case, early MRI examination of the bones and whole exome sequencing enabled an early and accurate MCTO diagnosis, and timely Denosumab administration resulted in no deterioration. CONCLUSION: This suggests that MRI examination and whole exome sequencing should be considered when MCTO is suspected, and Denosumab might be an option in the treatment of MCTO.

Elucidation of molecular basis of osteolytic bone lesions in advanced multiple myeloma.

Osteolytic bone lesion is a major cause of lower quality of life and poor prognosis in patients with multiple myeloma (MM), but molecular pathogenesis of the osteolytic process in MM remains elusive. Fms-like tyrosine kinase 3 ligand (FLT3L) was reported to be elevated in bone marrow (BM) and blood of patients with advanced MM who often show osteolysis. Here, we investigated a functional link of FLT3L to osteolytic process in MM. We recruited 86, 306, and 52 patients with MM, acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), respectively. FLT3L levels of patients with hematologic malignancies were measured in BM-derived plasma and found to be significantly higher in MM than in AML or ALL, which rarely show osteolysis. FLT3L levels were further elevated in MM patients with bone lesion compared with patients without bone lesion. In vitro cell-based assays showed that the administration of FLT3L to HEK293T, HeLa, and U2OS cells led to an increase in the DKK1 transcript level through STAT3 phosphorylation at tyrosine 705. WNT reporter assay showed that FLT3L treatment reduced WNT signaling and nuclear translocation of ß-catenin. These results collectively show that the FLT3L-STAT3-DKK1 pathway inhibits WNT signaling-mediated bone formation in MM, which can cause osteolytic bone lesion. Finally, transcriptomic profiles revealed that FLT3L and DKK1 were predominantly elevated in the hyperdiploidy subtype of MM. Taken together, FLT3L can serve as a promising biomarker for predicting osteolytic bone lesion and also a potential therapeutic target to prohibit the progression of the osteolytic process in MM with hyperdiploidy.

Mettl3­mediated m6A RNA methylation regulates osteolysis induced by titanium particles.

Peri­prosthetic osteolysis (PPO) induced by wear particles is considered the primary cause of titanium prosthesis failure and revision surgery. The specific molecular mechanisms involve titanium particles inducing multiple intracellular pathways, which impact disease prevention and the targeted therapy of PPO. Notably, N6­methyladenosine (m6A) serves critical roles in epigenetic regulation, particularly in bone metabolism and inflammatory responses. Thus, the present study aimed to determine the role of RNA methylation in titanium particle­induced osteolysis. Results of reverse transcription­quantitative PCR (RT­qPCR), western blotting, ELISA and RNA dot blot assays revealed that titanium particles induced osteogenic inhibition and proinflammatory responses, accompanied by the reduced expression of methyltransferase­like (Mettl) 3, a key component of m6A methyltransferase. Specific lentiviruses vectors were employed for Mettl3 knockdown and overexpression experiments. RT­qPCR, western blotting and ELISA revealed that the knockdown of Mettl3 induced osteogenic inhibition and proinflammatory responses comparable with that induced by titanium particle, while Mettl3 overexpression attenuated titanium particle­induced cellular reactions. Methylated RNA immunoprecipitation­qPCR results revealed that titanium particles mediated the methylation of two inhibitory molecules, namely Smad7 and SMAD specific E3 ubiquitin protein ligase 1, via Mettl3 in bone morphogenetic protein signaling, leading to osteogenic inhibition. Furthermore, titanium particles induced activation of the nucleotide binding oligomerization domain 1 signaling pathway through methylation regulation, and the subsequent activation of the MAPK and NF­κB pathways. Collectively, the results of the present study indicated that titanium particles utilized Mettl3 as an upstream regulatory molecule to induce osteogenic inhibition and inflammatory responses. Thus, the present study may provide novel insights into potential therapeutic targets for aseptic loosening in titanium prostheses.

Aseptic loosening around total joint replacement in humans is regulated by miR-1246 and miR-6089 via the Wnt signalling pathway.

BACKGROUND: Total joint replacement for osteoarthritis is one of the most successful surgical procedures in modern medicine. However, aseptic loosening continues to be a leading cause of revision arthroplasty. The diagnosis of aseptic loosening remains a challenge as patients are often asymptomatic until the late stages. MicroRNA (miRNA) has been demonstrated to be a useful diagnostic tool and has been successfully used in the diagnosis of other diseases. We aimed to identify differentially expressed miRNA in the plasma of patients with aseptic loosening. METHODS: Adult patients undergoing revision arthroplasty for aseptic loosening and age- and gender-matched controls were recruited. Samples of bone, tissue and blood were collected, and RNA sequencing was performed in 24 patients with aseptic loosening and 26 controls. Differentially expressed miRNA in plasma was matched to differentially expressed mRNA in periprosthetic bone and tissue. Western blot was used to validate protein expression. RESULTS: Seven miRNA was differentially expressed in the plasma of patients with osteolysis (logFC >|2|, adj-P < 0.05). Three thousand six hundred and eighty mRNA genes in bone and 427 mRNA genes in tissue samples of osteolysis patients were differentially expressed (logFC >|2|, adj-P < 0.05). Gene enrichment analysis and pathway analysis revealed two miRNA (miR-1246 and miR-6089) had multiple gene targets in the Wnt signalling pathway in the local bone and tissues which regulate bone metabolism. CONCLUSION: These results suggest that aseptic loosening may be regulated by miR-1246 and miR-6089 via the Wnt signalling pathway.

Conditional knockout of PDK1 in osteoclasts suppressed osteoclastogenesis and ameliorated prostate cancer-induced osteolysis in murine model.

BACKGROUND: The development and maintenance of normal bone tissue is maintained by balanced communication between osteoblasts and osteoclasts. The invasion of cancer cells disrupts this balance, leading to osteolysis. As the only bone resorbing cells in vivo, osteoclasts play important roles in cancer-induced osteolysis. However, the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in osteoclast resorption remains unclear. METHODS: In our study, we used a receptor activator of nuclear factor-kappa B (RANK) promoter-driven Cre-LoxP system to conditionally delete the PDK1 gene in osteoclasts in mice. We observed the effect of osteoclast-specific knockout of PDK1 on prostate cancer-induced osteolysis. Bone marrow-derived macrophage cells (BMMs) were extracted and induced to differentiate osteoclasts in vitro to explore the role of PDK1 in osteoclasts. RESULTS: In this study, we found that PDK1 conditional knockout (cKO) mice exhibited smaller body sizes when compared to the wild-type (WT) mice. Moreover, deletion of PDK1 in osteoclasts ameliorated osteolysis and rPDK1educed bone resorption markers in the murine model of prostate cancer-induced osteolysis. In vivo, we discovered that osteoclast-specific knockout of suppressed RANKL-induced osteoclastogenesis, bone resorption function, and osteoclast-specific gene expression (Ctsk, TRAP, MMP-9, NFATc1). Western blot analyses of RANKL-induced signaling pathways showed that conditional knockout of PDK1 in osteoclasts inhibited the early nuclear factor κB (NF-κB) activation, which consequently suppressed the downstream induction of NFATc1. CONCLUSION: These findings demonstrated that PDK1 performs an important role in osteoclastogenesis and prostate cancer-induced osteolysis by modulating the PDK1/AKT/NF-κB signaling pathway.

Clinical, radiographic and molecular characterization of two unrelated families with multicentric osteolysis, nodulosis, and arthropathy.

BACKGROUND: Multicentric osteolysis nodulosis and arthropathy (MONA) is a rare autosomal recessive disorder characterized by marked progressive bone loss and joint destruction resulting in skeletal deformities. MONA is caused by MMP2 deficiency. Here we report clinical and molecular analyses of four patients in two families from Pakistan and Finland. METHODS: Clinical analyses including radiography were completed and blood samples were collected. The extracted DNA was subjected to whole-exome analysis or target gene sequencing. Segregation analyses were performed in the nuclear pedigree. Pathogenicity prediction scores for the selected variants and conservation analyses of affected amino acids were observed. RESULTS: The phenotype in the four affected individuals was consistent with multicentric osteolysis or MONA, as the patients had multiple affected joints, osteolysis of hands and feet, immobility of knee joint and progressive bone loss. Long-term follow up of the patients revealed the progression of the disease. We found a novel MMP2 c.1336 + 2T > G homozygous splice donor variant segregating with the phenotype in the Pakistani family while a MMP2 missense variant c.1188 C > A, p.(Ser396Arg) was homozygous in both Finnish patients. In-silico analysis predicted that the splicing variant may eventually introduce a premature stop codon in MMP2. Molecular modeling for the p.(Ser396Arg) variant suggested that the change may disturb MMP2 collagen-binding region. CONCLUSION: Our findings expand the genetic spectrum of Multicentric osteolysis nodulosis and arthropathy. We also suggest that the age of onset of this disorder may vary from childhood up to late adolescence and that a significant degree of intrafamilial variability may be present.

A report of 5 Indian families with multicentric carpotarsal osteolysis syndrome.

Multicentric carpotarsal osteolysis syndrome (MCTO) is a rare autosomal dominant skeletal dysplasia characterised by swelling and restriction of movement in the wrist and ankle joints, as well as osteolysis of the carpal and tarsal bones, that can be misdiagnosed as juvenile idiopathic arthritis. We describe five Indian families with heterozygous nonrecurrent missense pathogenic variants in exon 1 of MAF bZIP transcription factor B (MAFB).

The checkpoint inhibitor PD-1H/VISTA controls osteoclast-mediated multiple myeloma bone disease.

Multiple myeloma bone disease is characterized by the development of osteolytic bone lesions. Recent work identified matrix metalloproteinase 13 as a myeloma-derived fusogen that induces osteoclast activation independent of its proteolytic activity. We now identify programmed death-1 homolog, PD-1H, as the bona fide MMP-13 receptor on osteoclasts. Silencing PD-1H or using Pd-1h-/- bone marrow cells abrogates the MMP-13-enhanced osteoclast fusion and bone-resorptive activity. Further, PD-1H interacts with the actin cytoskeleton and plays a necessary role in supporting c-Src activation and sealing zone formation. The critical role of PD-1H in myeloma lytic bone lesions was confirmed using a Pd-1h-/- myeloma bone disease mouse model wherein myeloma cells injected into Pd-1h-/-Rag2-/- results in attenuated bone destruction. Our findings identify a role of PD-1H in bone biology independent of its known immunoregulatory functions and suggest that targeting the MMP-13/PD-1H axis may represent a potential approach for the treatment of myeloma associated osteolysis.

Abnormal expression pattern of lncRNA H19 participates in multiple myeloma bone disease by unbalancing osteogenesis and osteolysis.

BACKGROUND: Accumulating genetic and epigenetic alterations in multiple myeloma (MM) have been demonstrated to be closely associated with osteolytic bone disease, generally characterized as increased osteoclast formation and decreased osteoblast activity. Previously, serum long non-coding RNA (lncRNA) H19 has been proved to be a biomarker for the diagnosis of MM. Whereas, its role in MM-associated bone homeostasis remains largely elusive. METHODS: A cohort of 42 MM patients and 40 healthy volunteers were enrolled for evaluating differential expressions of H19 and its downstream effectors. The proliferative capacity of MM cells was monitored by CCK-8 assay. Alkaline phosphatase (ALP) staining and activity detection, either with Alizarin red staining (ARS) were employed to assess osteoblast formation. Osteoblast- or osteoclast-associated gene were detected using qRT-PCR and western blot analysis. Bioinformatics analysis, RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) were subjected to verify H19/miR-532-3p/E2F7/EZH2 axis, which was accounted for epigenetic suppression of PTEN. The functional role of H19 on MM development through unbalancing osteolysis and osteogenesis was also confirmed in the murine MM model. RESULTS: Upregulation of serum H19 was observed in MM patients, suggesting its positive correlation with the poor prognosis of MM patients. Loss of H19 dramatically weakened cell proliferation of MM cells, promoted osteoblastic differentiation, and impaired osteoclast activity. While reinforced H19 exhibited the opposite effects. Akt/mTOR signaling plays an indispensable role in H19-mediated osteoblast formation and osteoclastgenesis. Mechanistically, H19 served as a sponge for miR-532-3p to upregulate E2F7, a transcriptional activator of EZH2, thereby accounting for modulating epigenetic suppression of PTEN. The in vivo experiments further validated that H19 exerted important impacts on tumor growth through breaking the balance between osteogenesis and osteolysis via Akt/mTOR signaling. CONCLUSION: Collectively, increased enrichment of H19 in MM cells exhibits an essential role in MM development by disturbing bone homeostasis.

Multicentric Carpotarsal Osteolysis: a Contemporary Perspective on the Unique Skeletal Phenotype.

PURPOSE OF REVIEW: Multicentric carpotarsal osteolysis (MCTO) is an ultra-rare disorder characterized by osteolysis of the carpal and tarsal bones, subtle craniofacial deformities, and nephropathy. The molecular pathways underlying the pathophysiology are not well understood. RECENT FINDINGS: MCTO is caused by heterozygous mutations in MAFB, which encodes the widely expressed transcription factor MafB. All MAFB mutations in patients with MCTO result in replacement of amino acids that cluster in a phosphorylation region of the MafB transactivation domain and account for a presumed gain-of-function for the variant protein. Since 2012, fewer than 60 patients with MCTO have been described with 20 missense mutations in MAFB. The clinical presentations are variable, and a genotype-phenotype correlation is lacking. Osteolysis, via excessive osteoclast activity, has been regarded as the primary mechanism, although anti-resorptive agents demonstrate little therapeutic benefit. This paper appraises current perspectives of MafB protein action, inflammation, and dysfunctional bone formation on the pathogenesis of the skeletal phenotype in MCTO. More research is needed to understand the pathogenesis of MCTO to develop rational therapies.

Circular RNA CircSLC8A1 contributes to osteogenic differentiation in hBMSCs via CircSLC8A1/miR-144-3p/RUNX1 in periprosthetic osteolysis.

Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real-time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain- and loss-of-function methods. The association of CircSLC8A1 and miR-144-3p, along with miR-144-3p and RUNX1, was predicted using bioinformatics. RNA pull-down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL-mouse model was also investigated using adeno-associated virus. CircSLC8A1 was found to be downregulated in PPOL patients' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR-144-3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR-144-3p and participated in PPOL through the miR-144-3p/RUNX1 pathway in hBMSCs. Micro-CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR-144-3p/RUNX1 pathway may provide a potential target for prevention of PPOL.

CST6 suppresses osteolytic bone disease in multiple myeloma by blocking osteoclast differentiation.

Osteolytic bone disease is a hallmark of multiple myeloma (MM). A significant fraction (~20%) of MM patients do not develop osteolytic lesions (OLs). The molecular basis for the absence of bone disease in MM is not understood. We combined PET-CT and gene expression profiling (GEP) of purified BM CD138+ MM cells from 512 newly diagnosed MM patients to reveal that elevated expression of cystatin M/E (CST6) was significantly associated with the absence of OL in MM. An enzyme-linked immunosorbent assay revealed a strong correlation between CST6 levels in BM serum/plasma and CST6 mRNA expression. Both recombinant CST6 protein and BM serum from patients with high CST6 significantly inhibited the activity of the osteoclast-specific protease cathepsin K and blocked osteoclast differentiation and function. Recombinant CST6 inhibited bone destruction in ex vivo and in vivo myeloma models. Single-cell RNA-Seq showed that CST6 attenuates polarization of monocytes to osteoclast precursors. Furthermore, CST6 protein blocks osteoclast differentiation by suppressing cathepsin-mediated cleavage of NF-κB/p100 and TRAF3 following RANKL stimulation. Secretion by MM cells of CST6, an inhibitor of osteoclast differentiation and function, suppresses osteolytic bone disease in MM and probably other diseases associated with osteoclast-mediated bone loss.

A review on the role of PRNCR1 in human disorders with an especial focus on cancer.

Prostate Cancer Associated Non-Coding RNA 1 (PRNCR1) is a long non-coding RNA (lncRNA) which is transcribed from chromosome 8, plus strand. This lncRNA has been reported to be an oncogenic transcript participating in the pathogenesis of several kinds of cancers. Some single nucleotide polymorphisms within this lncRNA affect cancer risk. Moreover, few studies have revealed its possible roles in some non-neoplastic conditions, such as cisplatin-induced acute kidney injury, osteolysis after hip replacement, preeclampsia and pulmonary disorders. In the present narrative review, we explain diverse roles of PRNCR1 in human disorders.

LINC01534/miR-135b-5p/PTPRT axis regulates inflammatory response in loosening total hip replacement via modulating NF-κB signaling pathway.

Aseptic loosening after total hip replacement brings adverse health outcomes and increased risk for complications. The resorptive activity of inflammatory cells activated by the presence of wear-generated debris plays a critical role in debris-induced osteolysis. Previous studies indicate that the abnormally expressed LINC01534 plays a critical role in inflammatory responses. In this study, we aimed to elucidate the functional role and underlying mechanism of LINC01534 in debris-induced osteolysis. We first confirmed that LINC01534 was highly expressed in hip cartilage tissues from aseptic loosening patients. By using an IL-1ß-induced inflammation model mimicking debris-induced osteolysis, we demonstrated that LINC01534 promoted IL-1ß-induced inflammatory response in hip chondrocytes. Knockdown of LINC01534 inhibited the expression of inflammatory IL-6, IL-8, and TNF-α in hip chondrocytes. Our results showed that LINC01534 functioned as a competing endogenous RNA (ceRNA) by sponging miR-135b-5p in hip chondrocytes. Moreover, bioinformatics analysis and luciferase reporter assay demonstrated that CCHC-Type Zinc Finger Nucleic Acid Binding Protein (PTPRT) is a downstream target of miR-135b-5p. Knockdown of PTPRT attenuated the IL-1ß-induced inflammatory responses in hip chondrocytes. In addition, we revealed that inhibition of miR-135b-5p or overexpression of PTPRT could antagonize the effects of LINC01534 knockdown on inflammation attenuation in hip chondrocytes. Mechanistically, we demonstrated that LINC01534/miR-135b-5p/PTPRT axis regulated the NF-κB signaling pathway in hip chondrocytes. Taken together, our findings suggest that LINC01534/miR-135b-5p/PTPRT axis might be a valuable therapeutic target for the treatment of debris-induced osteolysis.

Dental and Orthopaedic Implant Loosening: Overlap in Gene Expression Regulation.

Objectives: Endoprosthetic loosening still plays a major role in orthopaedic and dental surgery and includes various cellular immune processes within peri-implant tissues. Although the dental and orthopaedic processes vary in certain parts, the clinical question arises whether there are common immune regulators of implant loosening. Analyzing the key gene expressions common to both processes reveals the mechanisms of osteoclastogenesis within periprosthetic tissues of orthopaedic and dental origin. Methods: Donor peripheral blood mononuclear cells (PBMCs) and intraoperatively obtained periprosthetic fibroblast-like cells (PPFs) were (co-)cultured with [± macrophage-colony stimulating factor (MCSF) and Receptor Activator of NF-κB ligand (RANKL)] in transwell and monolayer culture systems and examined for osteoclastogenic regulations [MCSF, RANKL, osteoprotegerin (OPG), and tumor necrosis factor alpha (TNFα)] as well as the ability of bone resorption. Sequencing analysis compared dental and orthopaedic (co-)cultures. Results: Monolayer co-cultures of both origins expressed high levels of OPG, resulting in inhibition of osteolysis shown by resorption assay on dentin. The high OPG-expression, low RANKL/OPG ratios and a resulting inhibition of osteolysis were displayed by dental and orthopaedic PPFs in monolayer even in the presence of MCSF and RANKL, acting as osteoprotective and immunoregulatory cells. The osteoprotective function was only observed in monolayer cultures of dental and orthopaedic periprosthetic cells and downregulated in the transwell system. In transwell co-cultures of PBMCs/PPFs profound changes of gene expression, with a significant decrease of OPG (20-fold dental versus 100 fold orthopaedic), were identified. Within transwell cultures, which offer more in vivo like conditions, RANKL/OPG ratios displayed similar high levels to the original periprosthetic tissue. For dental and orthopaedic implant loosening, overlapping findings in principal component and heatmap analysis were identified. Conclusions: Thus, periprosthetic osteoclastogenesis may be a correlating immune process in orthopaedic and dental implant failure leading to comparable reactions with regard to osteoclast formation. The transwell cultures system may provide an in vivo like model for the exploration of orthopaedic and dental implant loosening.

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

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

Regulatory roles of miRNAs 16, 133a, and 223 on osteoclastic bone destruction caused by breast cancer metastasis.

Osteolytic bone metastasis leads to skeletal­related events, resulting in a decline in the patient activities and survival; therefore, it is important to understand the mechanism underlying bone metastasis. Recent studies have suggested that microRNAs (miRNAs or miRs) are involved in osteoclast differentiation and/or osteolytic bone metastasis; however, the roles of miRNAs have not been elucidated. In the present study, the roles of miRNAs in bone destruction caused by breast cancer metastasis were investigated in vitro and in vivo. miR­16, miR­133a and miR­223 were transfected into a human breast cancer cell line, MDA­MB­231. The expression of osteolytic factors in conditioned medium (miR­CM) collected from the culture of transfected cells was assessed. To evaluate the effects of miRNAs on osteoclast differentiation and activities, tartrate­resistant acid phosphatase (TRAP) staining and bone resorptive assays were performed in osteoclasts following miR­CM treatment. To create in vivo bone metastasis models for histological and morphometric evaluation, miRNA­transfected MDA­MB­231 cells were transplanted into the proximal tibia of nude mice. Expression of osteolytic factors, including receptor activator for nuclear factor­κB ligand (RANKL), interleukin (IL)­1ß, IL­6, parathyroid hormone­related protein (PTHrP), and tumor necrosis factor (TNF), was increased in miR­16­CM, whereas it was decreased in both miR­133a­CM and miR­223­CM. TRAP staining and bone resorptive assays revealed that osteoclast function and activities were promoted by miR­16­CM treatment, whereas they were suppressed by miR­133a­CM and miR­223­CM. Consistent with in vitro findings, in vivo experiments revealed that the overexpression of miR­16 increased osteoclast activities and bone destruction in MDA­MB­231 cells, whereas the opposite results were observed in both miR­133a­ and miR­223­transfected MDA­MB­231 cells. Our results indicated that miR­16 promoted osteoclast activities and bone destruction caused by breast cancer metastasis in the bone microenvironment, whereas miR­133a and miR­223 suppressed them. These miRNAs could be potential biomarkers and therapeutic targets for breast cancer bone metastasis.

Biphasic α2ß1 Integrin Expression in Breast Cancer Metastasis to Bone.

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2ß1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2ß1 in the context of bone metastasis. In this study, we aimed to understand the role of α2ß1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2ß1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2ß1 expression and bone-metastatic potential. Inhibiting α2ß1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.