The PDE4 Inhibitors Roflumilast and Rolipram Rescue ADO2 Osteoclast Resorption Dysfunction.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption caused by heterozygous missense mutations in the chloride channel 7 (CLCN7). Adenylate cyclase, which catalyzes the formation of cAMP, is critical for lysosomal acidification in osteoclasts. We found reduced cAMP levels in ADO2 osteoclasts compared to wild-type (WT) osteoclasts, leading us to examine whether regulating cAMP would improve ADO2 osteoclast activity. Although forskolin, a known activator of adenylate cyclase and cAMP levels, negatively affected osteoclast number, it led to an overall increase in ADO2 and WT osteoclast resorption activity in vitro. Next, we examined cAMP hydrolysis by the phosphodiesterase 4 (PDE4) proteins in ADO2 versus WT osteoclasts. QPCR analysis revealed higher expression of the three major PDE4 subtypes (4a, 4b, 4d) in ADO2 osteoclasts compared in WT, consistent with reduced cAMP levels in ADO2 osteoclasts. In addition, we found that the PDE4 antagonists, rolipram and roflumilast, stimulated ADO2 and WT osteoclast formation in a dose-dependent manner. Importantly, roflumilast and rolipram displayed a concentration-dependent increase in osteoclast resorption activity which was greater in ADO2 than WT osteoclasts. Moreover, treatment with roflumilast rescued cAMP levels in ADO2 OCLs. The key findings from our studies demonstrate that osteoclasts from ADO2 mice exhibit reduced cAMP levels and PDE4 inhibition rescues cAMP levels and ADO2 osteoclast activity dysfunction in vitro. The mechanism of action of PDE4 inhibitors and their ability to reduce the high bone mass of ADO2 mice in vivo are currently under investigation. Importantly, these studies advance the understanding of the mechanisms underlying the ADO2 osteoclast dysfunction which is critical for the development of therapeutic approaches to treat clinically affected ADO2 patients.

Effect of Roflumilast, a Selective PDE4 Inhibitor, on Bone Phenotypes in ADO2 Mice.

Autosomal Dominant Osteopetrosis type II (ADO2) is a rare bone disease of impaired osteoclastic bone resorption that usually results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene. We previously created mouse models of ADO2 (p.G213R) with one of the most common mutations (G215R) as found in humans and demonstrated that this mutation in mice phenocopies the human disease of ADO2. Previous studies have shown that roflumilast (RF), a selective phosphodiesterase 4 (PDE4) inhibitor that regulates the cAMP pathway, can increase osteoclast activity. We also observed that RF increased bone resorption in both wild-type and ADO2 heterozygous osteoclasts in vitro, suggesting it might rescue bone phenotypes in ADO2 mice. To test this hypothesis, we administered RF-treated diets (0, 20 and 100 mg/kg) to 8-week-old ADO2 mice for 6 months. We evaluated bone mineral density and bone micro-architecture using longitudinal in-vivo DXA and micro-CT at baseline, and 6-, 12-, 18-, and 24-week post-baseline time points. Additionally, we analyzed serum bone biomarkers (CTX, TRAP, and P1NP) at baseline, 12-, and 24-week post-baseline. Our findings revealed that RF treatment did not improve aBMD (whole body, femur, and spine) and trabecular BV/TV (distal femur) in ADO2 mice compared to the control group treated with a normal diet. Furthermore, we did not observe any significant changes in serum levels of bone biomarkers due to RF treatment in these mice. Overall, our results indicate that RF does not rescue the osteopetrotic bone phenotypes in ADO2 heterozygous mice.

Megakaryocyte Secreted Factors Regulate Bone Marrow Niche Cells During Skeletal Homeostasis, Aging, and Disease.

The bone marrow microenvironment contains a diverse array of cell types under extensive regulatory control and provides for a novel and complex mechanism for bone regulation. Megakaryocytes (MKs) are one such cell type that potentially acts as a master regulator of the bone marrow microenvironment due to its effects on hematopoiesis, osteoblastogenesis, and osteoclastogenesis. While several of these processes are induced/inhibited through MK secreted factors, others are primarily regulated by direct cell-cell contact. Notably, the regulatory effects that MKs exert on these different cell populations has been found to change with aging and disease states. Overall, MKs are a critical component of the bone marrow that should be considered when examining regulation of the skeletal microenvironment. An increased understanding of the role of MKs in these physiological processes may provide insight into novel therapies that can be used to target specific pathways important in hematopoietic and skeletal disorders.

Osteocyte-Derived CaMKK2 Regulates Osteoclasts and Bone Mass in a Sex-Dependent Manner through Secreted Calpastatin.

Calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) regulates bone remodeling through its effects on osteoblasts and osteoclasts. However, its role in osteocytes, the most abundant bone cell type and the master regulator of bone remodeling, remains unknown. Here we report that the conditional deletion of CaMKK2 from osteocytes using Dentine matrix protein 1 (Dmp1)-8kb-Cre mice led to enhanced bone mass only in female mice owing to a suppression of osteoclasts. Conditioned media isolated from female CaMKK2-deficient osteocytes inhibited osteoclast formation and function in in vitro assays, indicating a role for osteocyte-secreted factors. Proteomics analysis revealed significantly higher levels of extracellular calpastatin, a specific inhibitor of calcium-dependent cysteine proteases calpains, in female CaMKK2 null osteocyte conditioned media, compared to media from female control osteocytes. Further, exogenously added non-cell permeable recombinant calpastatin domain I elicited a marked, dose-dependent inhibition of female wild-type osteoclasts and depletion of calpastatin from female CaMKK2-deficient osteocyte conditioned media reversed the inhibition of matrix resorption by osteoclasts. Our findings reveal a novel role for extracellular calpastatin in regulating female osteoclast function and unravel a novel CaMKK2-mediated paracrine mechanism of osteoclast regulation by female osteocytes.

Pyk2 deficiency enhances bone mass during midpalatal suture expansion.

OBJECTIVE: To determine if Pyk2 deficiency increases midpalatal suture bone mass and preserves sutural integrity after maxillary expansion. SETTING AND SAMPLE: Thirty-six male Pyk2 knockout (KO) and control (WT) mice at 6 weeks of age. MATERIALS AND METHODS: Mice received nickel-titanium spring expanders delivering 0 g (no intervention control), 10 or 20 g force for 14 days. High-resolution micro-CT was used to determine bone volume/tissue volume (BV/TV), sutural width and intermolar width. Effects on osteoclasts, chondrocytes and suture morphology were determined by histomorphometry. RESULTS: Pyk2-KO controls (0 g) had 7% higher BV/TV compared with WT controls. Expanded Pyk2-KO maxillae also exhibited 12% (10 g) and 18% (20 g) higher BV/TV than WT mice. Although bone loss following expansion occurred in both genotypes, BV/TV was decreased to a greater extent in WT maxillae (-10% at 10g; -22% at 20 g) compared with Pyk2-KO maxillae (-11% only at 20 g). Expanded WT maxillae also showed a greater increase in sutural width, intermolar width and fibrous connective tissue width compared with expanded Pyk2-KO maxillae. Moreover, osteoclast number was increased 77% (10 g) and 132% (20 g) in expanded WT maxillae, but remained unchanged in expanded Pyk2-KO, compared to their respective controls. Cartilage area and chondrocyte number were increased to the same extent in expanded WT and Pyk2-KO sutures. CONCLUSIONS: These findings suggest that midpalatal suture expansion increases osteoclast formation in WT but not Pyk2-KO mice, leading to higher BV/TV in expanded Pyk2-KO maxillae. These studies suggest Pyk2-targeted strategies may be beneficial to increase bone density and preserve sutural integrity during maxillary expansion.

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro.

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43def ), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.

MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.

Parathyroid hormone (PTH) affects the skeleton by acting on osteocytes (Ots) in bone through yet unclear mechanisms. We report that matrix metalloproteinase 14 (MMP14) expression/activity are increased in bones from mice with genetic constitutive activation (ca) of the PTH receptor 1 (PTH1R) in Ots (caPTH1ROt) and in bones from mice exposed to elevated PTH levels but not in mice lacking [conditional knockout (cKO)] the PTH1R in Ots (cKOPTH1ROt). Furthermore, PTH upregulates MMP14 in human bone cultures and in Ot-enriched bones from floxed control mice but not from cKOPTH1ROt mice. MMP14 activity increases soluble receptor activator of NF-κΒ ligand production, which in turn, stimulates osteoclast differentiation and resorption. Pharmacologic inhibition of MMP14 activity reduced the high bone remodeling exhibited by caPTH1ROt mice or induced by chronic PTH elevation and decreased bone resorption but allowed full stimulation of bone formation induced by PTH injections, thereby potentiating bone gain. Thus, MMP14 is a new member of the intricate gene network activated in Ots by PTH1R signaling that can be targeted to adjust the skeletal responses to PTH in favor of bone preservation.-Delgado-Calle, J., Hancock, B., Likine, E. F., Sato, A. Y., McAndrews, K., Sanudo, C., Bruzzaniti, A., Riancho, J. A., Tonra, J. R., Bellido, T. MMP14 is a novel target of PTH signaling in osteocytes that controls resorption by regulating soluble RANKL production.

The antimicrobial properties, cytotoxicity, and differentiation potential of double antibiotic intracanal medicaments loaded into hydrogel system.

OBJECTIVE: This study evaluated the antimicrobial properties, cytotoxicity, and mineralization potential of methylcellulose hydrogels loaded with low concentrations of double antibiotic pastes (DAP). MATERIALS AND METHODS: The direct and residual antibacterial effects of 1, 5, and 10 mg/mL of DAP loaded into hydrogels as well as calcium hydroxide (Ca(OH)2) were tested against single-species biofilms of Enterococcus faecalis and dual-species biofilms (Enterococcus faecalis and Prevotella intermedia). The effects of DAP hydrogels on proliferation and mineralization of dental pulp stem cells (DPSC) were tested using MTT assays, alkaline phosphate activity (ALP), and alizarin red staining. Fisher's exact tests, Wilcoxon rank sum tests, and one-way ANOVA were used for statistical analyses (α = 0.05). RESULTS: All tested concentrations of DAP hydrogels as well as Ca(OH)2 demonstrated significant direct antibacterial effects against single- and dual-species biofilms. However, only 5 and 10 mg/mL of DAP hydrogels exhibited significant residual antibacterial effects against both types of tested biofilms. Only 1 mg/mL of DAP hydrogels did not have significant negative effects on DPSC viability, ALP activity, and mineralization nodule formation. However, 5 and 10 mg/mL of DAP hydrogels caused significant negative effects on cytotoxicity and mineralization nodule formation of DPSC. CONCLUSIONS: Hydrogels containing 1 mg/mL DAP offered significant direct antibacterial effects against single- and dual-species biofilms without causing significant negative effects on viability, ALP activity, and mineralization nodule formation of DPSC. CLINICAL RELEVANCE: The methylcellulose-based hydrogel proposed in this study can be used clinically as a biocompatible system to deliver controlled low concentrations of DAP.

The proto-oncogene function of Mdm2 in bone.

Mouse double minute 2 (Mdm2) is a multifaceted oncoprotein that is highly regulated with distinct domains capable of cellular transformation. Loss of Mdm2 is embryonically lethal, making it difficult to study in a mouse model without additional genetic alterations. Global overexpression through increased Mdm2 gene copy number (Mdm2Tg ) results in the development of hematopoietic neoplasms and sarcomas in adult animals. In these mice, we found an increase in osteoblastogenesis, differentiation, and a high bone mass phenotype. Since it was difficult to discern the cell lineage that generated this phenotype, we generated osteoblast-specific Mdm2 overexpressing (Mdm2TgOb ) mice in 2 different strains, C57BL/6 and DBA. These mice did not develop malignancies; however, these animals and the MG63 human osteosarcoma cell line with high levels of Mdm2 showed an increase in bone mineralization. Importantly, overexpression of Mdm2 corrected age-related bone loss in mice, providing a role for the proto-oncogenic activity of Mdm2 in bone health of adult animals.

Lnk Deficiency Leads to TPO-Mediated Osteoclastogenesis and Increased Bone Mass Phenotype.

The Lnk adapter protein negatively regulates the signaling of thrombopoietin (TPO), the main megakaryocyte (MK) growth factor. Lnk-deficient (-/-) mice have increased TPO signaling and increased MK number. Interestingly, several mouse models exist in which increased MK number leads to a high bone mass phenotype. Here we report the bone phenotype of these mice. MicroCT and static histomorphometric analyses at 20 weeks showed the distal femur of Lnk-/- mice to have significantly higher bone volume fraction and trabecular number compared to wild-type (WT) mice. Notably, despite a significant increase in the number of osteoclasts (OC), and decreased bone formation rate in Lnk-/- mice compared to WT mice, Lnk-/- mice demonstrated a 2.5-fold greater BV/TV suggesting impaired OC function in vivo. Additionally, Lnk-/- mouse femurs exhibited non-significant increases in mid-shaft cross-sectional area, yet increased periosteal BFR compared to WT femurs was observed. Lnk-/- femurs also had non-significant increases in polar moment of inertia and decreased cortical bone area and thickness, resulting in reduced bone stiffness, modulus, and strength compared to WT femurs. Of note, Lnk is expressed by OC lineage cells and when Lnk-/- OC progenitors are cultured in the presence of TPO, significantly more OC are observed than in WT cultures. Lnk is also expressed in osteoblast (OB) cells and in vitro reduced alkaline phosphatase activity was observed in Lnk-/- cultures. These data suggest that both direct effects on OB and OC as well as indirect effects of MK in regulating OB contributes to the observed high bone mass. J. Cell. Biochem. 118: 2231-2240, 2017. © 2017 Wiley Periodicals, Inc.

Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms.

Osteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.

C-Mpl Is Expressed on Osteoblasts and Osteoclasts and Is Important in Regulating Skeletal Homeostasis.

C-Mpl is the receptor for thrombopoietin (TPO), the main megakaryocyte (MK) growth factor, and c-Mpl is believed to be expressed on cells of the hematopoietic lineage. As MKs have been shown to enhance bone formation, it may be expected that mice in which c-Mpl was globally knocked out (c-Mpl(-/-) mice) would have decreased bone mass because they have fewer MKs. Instead, c-Mpl(-/-) mice have a higher bone mass than WT controls. Using c-Mpl(-/-) mice we investigated the basis for this discrepancy and discovered that c-Mpl is expressed on both osteoblasts (OBs) and osteoclasts (OCs), an unexpected finding that prompted us to examine further how c-Mpl regulates bone. Static and dynamic bone histomorphometry parameters suggest that c-Mpl deficiency results in a net gain in bone volume with increases in OBs and OCs. In vitro, a higher percentage of c-Mpl(-/-) OBs were in active phases of the cell cycle, leading to an increased number of OBs. No difference in OB differentiation was observed in vitro as examined by real-time PCR and functional assays. In co-culture systems, which allow for the interaction between OBs and OC progenitors, c-Mpl(-/-) OBs enhanced osteoclastogenesis. Two of the major signaling pathways by which OBs regulate osteoclastogenesis, MCSF/OPG/RANKL and EphrinB2-EphB2/B4, were unaffected in c-Mpl(-/-) OBs. These data provide new findings for the role of MKs and c-Mpl expression in bone and may provide insight into the homeostatic regulation of bone mass as well as bone loss diseases such as osteoporosis.

High bone mass in mice lacking Cx37 because of defective osteoclast differentiation.

Connexin (Cx) proteins are essential for cell differentiation, function, and survival in all tissues with Cx43 being the most studied in bone. We now report that Cx37, another member of the connexin family of proteins, is expressed in osteoclasts, osteoblasts, and osteocytes. Mice with global deletion of Cx37 (Cx37(-/-)) exhibit higher bone mineral density, cancellous bone volume, and mechanical strength compared with wild type littermates. Osteoclast number and surface are significantly lower in bone of Cx37(-/-) mice. In contrast, osteoblast number and surface and bone formation rate in bones from Cx37(-/-) mice are unchanged. Moreover, markers of osteoblast activity ex vivo and in vivo are similar to those of Cx37(+/+) littermates. sRANKL/M-CSF treatment of nonadherent Cx37(-/-) bone marrow cells rendered a 5-fold lower level of osteoclast differentiation compared with Cx37(+/+) cell cultures. Further, Cx37(-/-) osteoclasts are smaller and have fewer nuclei per cell. Expression of RANK, TRAP, cathepsin K, calcitonin receptor, matrix metalloproteinase 9, NFATc1, DC-STAMP, ATP6v0d1, and CD44, markers of osteoclast number, fusion, or activity, is lower in Cx37(-/-) osteoclasts compared with controls. In addition, nonadherent bone marrow cells from Cx37(-/-) mice exhibit higher levels of markers for osteoclast precursors, suggesting altered osteoclast differentiation. The reduction of osteoclast differentiation is associated with activation of Notch signaling. We conclude that Cx37 is required for osteoclast differentiation and fusion, and its absence leads to arrested osteoclast maturation and high bone mass in mice. These findings demonstrate a previously unrecognized role of Cx37 in bone homeostasis that is not compensated for by Cx43 in vivo.

A novel role for thrombopoietin in regulating osteoclast development in humans and mice.

Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor-kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders.

Ascorbic acid as a modulator of inflammatory response against Candida albicans.

Aim: To evaluate the behavior of oral keratinocytes in the presence of Vitamin C (Vit C) and its anti-inflammatory potential. Materials & methods: Oral keratinocytes were initially exposed to 0.1-2.5 mM of Vit C and the metabolic activity and cell migration were evaluated using MTS assay and Ibidi culture inserts, respectively. After, the cells were challenged with Candida albicans and inflammatory markers were analyzed by qPCR. Results: The treatment was not cytotoxic, and the highest concentrations increased the metabolic activity at 24 h. Vit C delayed the cell migration at 48 and 72 h. Interestingly, it downregulated the genes IL-8 and IL-1ß. Conclusion: Vit C could be an interesting adjuvant to anti-fungal treatment due to its anti-inflammatory potential.

Vitamin C, also known as ascorbic acid, is a vitamin commonly found in fruits and vegetables. It is popular for supporting our immune system, so is commonly taken as a supplement. We looked at the action of vitamin C on cells from the mouth and its potential to reduce inflammation in a fungal disease of the mouth ­ oral candidiasis. We showed that vitamin C is not toxic to cells of the mouth and may reduce inflammation in cells infected by the fungus. This suggests that vitamin C could be used as a complementary therapy for oral candidiasis.

Megakaryocytes regulate expression of Pyk2 isoforms and caspase-mediated cleavage of actin in osteoblasts.

The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton.

Defective microtubule-dependent podosome organization in osteoclasts leads to increased bone density in Pyk2(-/-) mice.

The protein tyrosine kinase Pyk2 is highly expressed in osteoclasts, where it is primarily localized in podosomes. Deletion of Pyk2 in mice leads to mild osteopetrosis due to impairment in osteoclast function. Pyk2-null osteoclasts were unable to transform podosome clusters into a podosome belt at the cell periphery; instead of a sealing zone only small actin rings were formed, resulting in impaired bone resorption. Furthermore, in Pyk2-null osteoclasts, Rho activity was enhanced while microtubule acetylation and stability were significantly reduced. Rescue experiments by ectopic expression of wild-type or a variety of Pyk2 mutants in osteoclasts from Pyk2(-/-) mice have shown that the FAT domain of Pyk2 is essential for podosome belt and sealing zone formation as well as for bone resorption. These experiments underscore an important role of Pyk2 in microtubule-dependent podosome organization, bone resorption, and other osteoclast functions.

Sex-specific differences in direct osteoclastic versus indirect osteoblastic effects underlay the low bone mass of Pannexin1 deletion in TRAP-expressing cells in mice.

Pannexin1 (Panx1) is a hemichannel-forming protein that participates in the communication of cells with the extracellular space. To characterize the role of osteoclastic Panx1 on bone, Panx1fl/fl;TRAP-Cre (Panx1ΔOc) mice were generated, and compared to Panx1fl/fl littermates at 6 weeks of age. Total and femoral BMD was ~20% lower in females and males whereas spinal BMD was lower only in female Panx1ΔOc mice. µCT analyses showed that cortical bone of the femoral mid-diaphysis was not altered in Panx1ΔOc mice. In contrast, cancellous bone in the distal femur and lumbar vertebra was significantly decreased in both female and male Panx1ΔOc mice compared to Panx1fl/fl controls and was associated with higher osteoclast activity in female Panx1ΔOc mice, with no changes in the males. On the other hand, vertebral bone formation was decreased for both sexes, resulting from lower mineral apposition rate in the females and lower mineralizing surface in the males. Consistent with an osteoclastic effect in female Panx1ΔOc mice, osteoclast differentiation with RANKL/M-CSF and osteoclast bone resorbing activity in vitro were higher in female, but not male, Panx1ΔOc mice, compared to Panx1fl/fl littermates. Surprisingly, although Panx1 expression was normal in bone marrow stromal-derived osteoblasts from male and female Panx1ΔOc mice, mineral deposition by male (but not female) Panx1ΔOc osteoblasts was lower than controls, and it was reduced in male Panx1fl/fl osteoblasts when conditioned media prepared from male Panx1ΔOc osteoclast cultures was added to the cell culture media. Thus, deletion of Panx1 in TRAP-expressing cells in female mice leads to low bone mass primarily through a cell autonomous effect in osteoclast activity. In contrast, our evidence suggests that changes in the osteoclast secretome drive reduced osteoblast function in male Panx1ΔOc mice, resulting in low bone mass.

Osteoclast-mediated bone loss observed in a COVID-19 mouse model.

The consequences of SARS-CoV-2 infection on the musculoskeletal system represent a dangerous knowledge gap. Aging patients are at added risk for SARS-CoV-2 infection; therefore, a greater understanding of the resulting musculoskeletal sequelae of SARS-CoV-2 infection may help guide clinical strategies. This study examined fundamental bone parameters among mice treated with escalating viral loads. Male C57BL/6J (WT, n = 17) and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (K18-hACE2 transgenic mice, n = 21) expressing human ACE2 (TG) were divided into eight groups (n = 4-6/group) and subjected to intranasal dosing of 0, 1 × 103, 1 × 104, and 1 × 105 PFU (plaque forming units) of human SARS-CoV-2. Animal health was assessed daily by veterinary staff using established and validated scoring criteria (activity, posture, body condition scores and body weight). We report here that mock and WT infected mice were healthy and completed the study, surviving until 12-14 days post infection (dpi). In contrast, the TG mice infected with 1 × 105 PFU all experienced severe health declines that necessitated early euthanasia (6-7 dpi). For TG mice infected with 1 × 104 PFU, 2 mice were also euthanized after 7 dpi, while 3 mice showed signs of moderate disease at day 6 dpi, but recovered fully by day 11 dpi. Four of the 5 TG mice that were infected with 1 × 103 PFU remained healthy throughout the study. This suggests that our study mimics what is seen during human disease, where some patients develop severe disease resulting in death, while others have moderate to severe disease but recover, and others are asymptomatic. At necropsy, femurs were extracted and analyzed by µCT. No difference was found in µCT determined bone parameters among the WT groups. There was, however, a significant 24.4% decrease in trabecular bone volume fraction (p = 0.0009), 19.0% decrease in trabecular number (p = 0.004), 6.2% decrease in trabecular thickness (p = 0.04), and a 9.8% increase in trabecular separation (p = 0.04) among surviving TG mice receiving any viral load compared to non-infected controls. No differences in cortical bone parameters were detected. TRAP staining revealed surviving infected mice had a significant 64% increase in osteoclast number, a 27% increase in osteoclast surface, and a 38% increase in osteoclasts per bone surface. While more studies are needed to investigate the long-term consequences of SARS-CoV-2 infection on skeletal health, this study demonstrates a significant reduction in several bone parameters and corresponding robust increases in osteoclast number observed within 2 weeks post-infection in surviving asymptomatic and moderately affected mice.

Nmp4/CIZ suppresses the response of bone to anabolic parathyroid hormone by regulating both osteoblasts and osteoclasts.

How parathyroid hormone (PTH) increases bone mass is unclear, but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth, we treated 10-week-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH(1-34) at 30 µg/kg daily or vehicle, 7 days/week, for 2, 3, or 7 weeks. Null mice treated with hormone (7 weeks) gained more vertebral and tibial cancellous bone than WT animals, paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2 weeks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone, these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3 weeks into treatment. Unexpectedly, the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptide, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts was elevated in Nmp4-KO mice at 2 weeks, but not 3 weeks, of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity and number of both osteoblasts and osteoclasts.