HTLV-1 infected T cells cause bone loss via small extracellular vesicles.

Adult T cell leukemia (ATL), caused by infection with human T cell leukemia virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T lead to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine RANKL, suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV injected over mouse calvaria in the presence of low dose RANKL caused more osteolysis than RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukemia.

Mer tyrosine kinase regulates bone metabolism, and its deficiency partially ameliorates periodontitis- and ovariectomy-induced bone loss in mice.

Bone homeostasis is maintained by tightly coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. In the present report, the role of Mer tyrosine kinase (MerTK) in bone metabolism was investigated. The expression of MerTK decreased upon BMP2 stimulation of osteoblast precursors. The femurs of Mertk-deficient mice showed significantly increased bone volume with concomitant increase of bone formation and reduction in bone resorption. These bone phenotypes were attributed to the increased osteoblast differentiation and mineralization accounted by the enhanced ß-catenin and Smad signaling in the absence of MerTK in osteoblast precursors. Although the Mertk-deficient bone marrow macrophages were predisposed to enhanced osteoclast differentiation via augmented Ca2+-NFATc1 signaling, the dramatic increase of Tnfsf11b/Tnfsf11 (Opg/Rankl) ratio in Mertk knockout bones and osteoblast precursors corroborated the reduction of osteoclastogenesis in Mertk deficiency. In ligature-induced periodontitis and ovariectomy models, the bone resorption was significantly attenuated in Mertk-deficient mice compared with wild-type control. Taken together, these data indicate novel role of MerTK in bone metabolism and suggest a potential strategy targeting MerTK in treating bone-lytic diseases including periodontitis and osteoporosis.

Conditional loss of IKKα in Osterix + cells has no effect on bone but leads to age-related loss of peripheral fat.

NF-κB has been reported to both promote and inhibit bone formation. To explore its role in osteolineage cells, we conditionally deleted IKKα, an upstream kinase required for non-canonical NF-κB activation, using Osterix (Osx)-Cre. Surprisingly, we found no effect on either cancellous or cortical bone, even following mechanical loading. However, we noted that IKKα conditional knockout (cKO) mice began to lose body weight after 6 months of age with severe reductions in fat mass and lower adipocyte size in geriatric animals. qPCR analysis of adipogenic markers in fat pads of cKO mice indicated no difference in early differentiation, but instead markedly lower leptin with age. We challenged young mice with a high fat diet finding that cKO mice gained less weight and showed improved glucose metabolism. Low levels of recombination at the IKKα locus were detected in fat pads isolated from old cKO mice. To determine whether recombination occurs in adipocytes, we examined fat pads in Osx-Cre;TdT reporter mice; these showed increasing Osx-Cre-mediated expression in peripheral adipocytes from 6 weeks to 18 months. Since Osx-Cre drives recombination in peripheral adipocytes with age, we conclude that fat loss in cKO mice is most likely caused by progressive deficits of IKKα in adipocytes.

Myeloid-Specific PTP1B Deficiency Attenuates Inflammation-Induced and Ovariectomy-Induced Bone Loss in Mice by Inhibiting Osteoclastogenesis.

The differentiation and activity of bone-resorbing osteoclasts are tightly regulated to maintain the homeostasis of healthy bones. In this study, the role of protein tyrosine phosphatase 1B (PTP1B) during osteoclastogenesis was studied in myeloid-specific Ptpn1-deficient (conditional knockout [cKO]) mice. The mRNA and protein expression of PTP1B increased during the formation of mature osteoclasts from mouse bone macrophages on stimulation with macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL). The Ptpn1 cKO mice exhibited increased femoral trabecular bone volume with a decreased number and activity of osteoclasts compared with control mice. The in vitro culture of osteoclast precursors corroborated the inhibition of osteoclastogenesis in cKO cells compared with control, with concomitantly decreased RANKL-dependent proliferation, lower osteoclast marker gene expression, reduced nuclear expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1), diminished intracellular Ca2+ oscillations, and increased phosphorylation of proto-oncogene tyrosine-protein kinase Src on inhibitory tyrosine residue. In a ligature-induced periodontitis model, Ptpn1 cKO mice exhibited attenuated osteoclastogenesis and alveolar bone loss following the induction of inflammation. The Ptpn1-deficient mice were similarly protected from ovariectomy-induced bone loss compared with control mice. These results provide a novel regulatory role of PTP1B in osteoclastogenesis and suggest a potential as a therapeutic target for bone-lytic diseases. © 2021 American Society for Bone and Mineral Research (ASBMR).

Developmental Roles of FUSE Binding Protein 1 (Fubp1) in Tooth Morphogenesis.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

6-Shogaol, an active ingredient of ginger, inhibits osteoclastogenesis and alveolar bone resorption in ligature-induced periodontitis in mice.

BACKGROUND: Periodontitis is an inflammatory disease of the tissues surrounding teeth that causes destruction of connective tissues. During the progress of periodontitis, osteoclasts are solely accountable for the resorption of alveolar bones that leads to the loss of teeth if not properly treated. Thus, the development of effective anti-resorptive therapies will greatly benefit the treatment of periodontitis patients. In the present study, we suggest an inhibitory effect of 6-shogaol, an ingredient of ginger, on osteoclast differentiation and bone resorption. METHODS: Mouse bone marrow cells were cultured in the presence of macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) to investigate the effect of 6-shogaol on osteoclast differentiation and intracellular signaling pathways. 6-shogaol significantly reduced osteoclast differentiation, actin ring formation, and resorption. In the presence of 6-shogaol, osteoclast signaling including the RANKL-induced activation of mitogen-activated protein kinases, Ca2+ oscillation, generation of reactive oxygen species, and nuclear factor of activated T-cells, cytoplasmic 1 nuclear translocation was significantly inhibited in vitro. Furthermore, a ligature-induced periodontitis model in mice was used to determine the role of 6-shogaol in vivo. RESULTS: The administration of 6-shogaol prevented osteoclastogenesis and alveolar bone resorption induced by ligature. Furthermore, the ligature-induced number of macrophages and neutrophils as well as the expression of interleukin-1ß and tumor necrosis factor-α were considerably lower in the periodontal tissues following shogaol injection. CONCLUSION: These results confirm the anti-osteoclastogenic effect of 6-shogaol and suggest the possibility of application as an anti-resorptive strategy in periodontitis.

A novel Bruton's tyrosine kinase inhibitor, acalabrutinib, suppresses osteoclast differentiation and Porphyromonas gingivalis lipopolysaccharide-induced alveolar bone resorption.

BACKGROUND: Periodontitis is not only one of the most prevalent inflammatory diseases among adults, but also commonly linked to numerous systemic conditions including cardiovascular diseases, stroke, and diabetes. Although osteoclasts are responsible for the alveolar bone resorption during periodontitis pathogenesis, the development of pharmacologic strategies targeting these cells has not been vastly fruitful. METHODS: Bone marrow macrophages were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) to examine the direct effect of acalabrutinib on osteoclastogenesis. Ca2+ oscillation and nuclear localization of NFATc1 in osteoclast precursors were examined to determine the precise molecular mechanism. LPS-induced alveolar bone loss model was employed for studying effect in in vivo bone resorption. RESULTS: Acalabrutinib directly inhibited RANKL and LPS-induced in vitro osteoclast differentiation. In addition, acalabrutinib inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases and reduced the expression of NF-κB. The inhibitory mechanism involved suppression of Ca2+ oscillation in osteoclast precursors resulting in the decreased NFATc1 expression and nuclear localization, which is a crucial prerequisite for osteoclastogenesis. The administration of acalabrutinib significantly reduced P. gingivalis lipopolysaccharide-induced alveolar bone erosion in mice. CONCLUSION: These data indicate that acalabrutinib is an effective inhibitor of osteoclastogenesis both in vitro and in vivo, with a potential for a novel strategy against bone destruction by periodontitis.

Fluvastatin Inhibits Osteoclast Differentiation and Porphyromonas gingivalis Lipopolysaccharide-Induced Alveolar Bone Erosion in Mice.

BACKGROUND: Statins have been widely used to treat hypercholesterolemia. In addition to inhibition of cholesterol synthesis, recent reports suggest a bone anabolic property of statins. However, little notice has been paid to the direct effect of statins on osteoclastogenesis and bone resorption. METHODS: The effect of fluvastatin on osteoclast differentiation was determined using in vitro culture of mouse bone marrow macrophages (BMMs) in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL). The role of fluvastatin on bone erosion was examined in the Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS)-induced alveolar bone loss model in mice. RESULTS: Fluvastatin significantly inhibited both RANKL- and LPS-induced osteoclast differentiation in mouse BMMs. Fluvastatin also markedly reduced expression of osteoclast differentiation marker genes Acp5, Calcr, and Ctsk as well as fusion markers Atp6v0d2 and Dcstamp. These were accompanied by decreased expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1 transcription factors. Fluvastatin reduced generation of reactive oxygen species upon the addition of RANKL and LPS, suggesting an antioxidant role. Finally, administration of fluvastatin in mice conspicuously reduced Pg LPS-induced osteoclastogenesis and alveolar bone erosion in vivo. CONCLUSION: Combined, these results suggest fluvastatin directly inhibited osteoclastogenesis and efficiently blocked bone erosion.