Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

RANKL triggers resistance to TRAIL-induced cell death in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) occurs as a malignancy of the oral cavity. RANK ligand (RANKL) is essential for osteoclast formation/bone resorption. Recently, we showed autoregulation of receptor activator of nuclear factor-κB ligand (RANKL) stimulates OSCC cell proliferation. OSCC cells show resistance to tumor necrosis factor related apoptosis inducing ligand (TRAIL) treatment. Therefore, we hypothesize that RANKL promotes resistance for TRAIL induction of OSCC apoptotic cell death. In this study, SCC14A and SCC74A cells cultured with TRAIL revealed high-level expression of RANKL which increased resistance to TRAIL inhibition of tumor cell proliferation. RANKL stimulation inhibited terminal deoxynucleotidyl transferase dUTP nick end labeling positive staining in TRAIL-treated cells. CRISPR/Cas-9 knockout of RANKL (RANKL-KO) increased caspase-9, caspase-3 activity and cytochrome c release in OSCC cells. RANKL inhibited proapoptotic proteins BAD and BAX expression. TRAIL treatment suppressed the SQSTM1/p62 and RANKL restored the expression. Interestingly, RANKL alone significantly increased proteasome activity. RANKL-KO in OSCC cells inhibited autophagic activity as evidenced by decreased light chain 3B-II and beclin-1 expression. Thus, RANKL stimulation of OSCC tumor cells triggered resistance for TRAIL-induced OSCC cell death. Taken together, blockade of RANKL may inhibit OSCC tumor progression and enhance the potential of TRAIL induced OSCC tumor cell apoptosis.

Proteasome inhibition suppress microgravity elevated RANK signaling during osteoclast differentiation.

Microgravity (µXg) induces bone loss in astronauts during space missions. Therefore, it is necessary to delineate the underlying mechanisms which leads to bone loss for developing countermeasures. Osteoclasts (OCLs) are multinucleated cells, which resorb bone. Previously, we have demonstrated that simulated µXg enhances OCL formation. However, control of osteoclast bone resorption activity under µXg remains unclear. The OCL formation has been shown to be regulated by ubiquitin-proteasome pathway. Hence, we hypothesized that proteasome inhibition could regulate osteoclast differentiation under µXg. In this study, we identified that RAW264.7 preosteoclast cells treated with proteasome inhibitor (MG-132) suppress RANK receptor expression essential for OCL differentiation, but no effect on TRAF-6. We identified that MG-132 treatment abolished K48-linked poly-ubiquitination under µXg. Immunostaining confirms inhibition of protein ubiquitination and RANK expression in preosteoclast cells. Furthermore, proteasome inhibition suppresses the expression of SQSTM1/p62 under both the ground based Xg and µXg conditions. Also, confocal microscopy using Lyso-Tracker demonstrated that proteasomal inhibition suppress the co-localization of p62 and lysosomes. MG-132 inhibited RANKL induced proteasome activity. RAW264.7 cells treated with the proteasome inhibitor showed an increased level of p-c-Jun activity in control cultures, however decreased under µXg. In contrast, c-Fos and NFATc1 expression was decreased. In-addition, mouse bone marrow cultures treated with MG-132 suppress OCL formation and bone resorption activity. Thus, our findings suggest that proteasome inhibition represents a novel therapeutic approach for bone loss under µXg in space environment.

Inhibition of acid ceramidase regulates MHC class II antigen presentation and suppression of autoimmune arthritis.

The bioactive sphingolipid ceramide affects immune responses although its effect on antigen (Ag) processing and delivery by HLA class II to CD4+T-cells remains unclear. Therefore, we examined the actions of a novel cell-permeable acid ceramidase (AC) inhibitor [(1R,2R) N myristoylamino-(4'-nitrophenyl)-propandiol-1,3] on antigen presentation and inflammatory cytokine production by Ag-presenting cells (APCs) such as B-cells, macrophages, and dendritic cells. We found that AC inhibition in APCs perturbed Ag-processing and presentation via HLA-DR4 (MHC class II) proteins as measured by coculture assay and T-cell production of IL-2. Mass spectral analyses showed that B13 treatment significantly raised levels of four types of ceramides in human B-cells. B13 treatment did not alter Ag internalization and class II protein expression, but significantly inhibited lysosomal cysteinyl cathepsins (B, S and L) and thiol-reductase (GILT), HLA class II Ag-processing, and generation of functional class II-peptide complexes. Ex vivo Ag presentation assays showed that inhibition of AC impaired primary and recall CD4+T-cell responses and cytokine production in response against type II collagen. Further, B13 delayed onset and reduced severity of inflamed joints and cytokine production in the collagen-induced arthritis mouse model in vivo. These findings suggest that inhibition of AC in APCs may dysregulate endolysosomal proteases and HLA class II-associated self-antigen presentation to CD4+T-cells, attenuating inflammatory cytokine production and suppressing host autoimmune responses.

Kynurenine Promotes RANKL-Induced Osteoclastogenesis In Vitro by Activating the Aryl Hydrocarbon Receptor Pathway.

There is increasing evidence of the involvement of the tryptophan metabolite kynurenine (KYN) in disrupting osteogenesis and contributing to aging-related bone loss. Here, we show that KYN has an effect on bone resorption by increasing osteoclastogenesis. We have previously reported that in vivo treatment with KYN significantly increased osteoclast number lining bone surfaces. Here, we report the direct effect of KYN on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in Raw 264.7 macrophage cells, and we propose a potential mechanism for these KYN-mediated effects. We show that KYN/RANKL treatment results in enhancement of RANKL-induced osteoclast differentiation. KYN drives upregulation and activation of the key osteoclast transcription factors, c-fos and NFATc1 resulting in an increase in the number of multinucleated TRAP+ osteoclasts, and in hydroxyapatite bone resorptive activity. Mechanistically, the KYN receptor, aryl hydrocarbon receptor (AhR), plays an important role in the induction of osteoclastogenesis. We show that blocking AhR signaling using an AhR antagonist, or AhR siRNA, downregulates the KYN/RANKL-mediated increase in c-fos and NFATc1 and inhibits the formation of multinucleated TRAP + osteoclasts. Altogether, this work highlights that the novelty of the KYN and AhR pathways might have a potential role in helping to regulate osteoclast function with age and supports pursuing additional research to determine if they are potential therapeutic targets for the prevention or treatment of osteoporosis.

Endoplasmic reticulum stress, autophagic and apoptotic cell death, and immune activation by a natural triterpenoid in human prostate cancer cells.

Though the current therapies are effective at clearing an early stage prostate cancer, they often fail to treat late-stage metastatic disease. We aimed to investigate the molecular mechanisms underlying the anticancer effects of a natural triterpenoid, ganoderic acid DM (GA-DM), on two human prostate cancer cell lines: the androgen-independent prostate carcinoma (PC-3), and androgen-sensitive prostate adenocarcinoma (LNCaP). Cell viability assay showed that GA-DM was relatively more toxic to LNCaP cells than to PC-3 cells (IC50 s ranged 45-55 µM for PC-3, and 20-25 µM for LNCaP), which may have occurred due to differential expression of p53. Hoechst DNA staining confirmed detectable nuclear fragmentation in both cell lines irrespective of the p53 status. GA-DM treatment decreased Bcl-2 proteins while it upregulated apoptotic Bax and autophagic Beclin-1, Atg5, and LC-3 molecules, and caused an induction of both early and late events of apoptotic cell death. Biochemical analyses of GA-DM-treated prostate cancer cells demonstrated that caspase-3 cleavage was notable in GA-DM-treated PC-3 cells. Interestingly, GA-DM treatment altered cell cycle progression in the S phase with a significant growth arrest in the G2 checkpoint and enhanced CD4 + T cell recognition of prostate tumor cells. Mechanistic study of GA-DM-treated prostate cancer cells further demonstrated that calpain activation and endoplasmic reticulum stress contributed to cell death. These findings suggest that GA-DM is a candidate for future drug design for prostate cancer as it activates multiple pathways of cell death and immune recognition.

Autoregulation of RANK ligand in oral squamous cell carcinoma tumor cells.

Oral squamous cell carcinoma (OSCC) is the most common malignancy among oral cancers and shows potent activity for local bone invasion. Receptor activator of nuclear factor κB (RANK) ligand (RANKL) is critical for bone-resorbing osteoclast formation. We previously demonstrated that OSCC tumor cells express high levels of RANKL. In this study, confocal microscopy demonstrated RANKL specific receptor, RANK expression in OSCC tumor cell lines (SCC1, SCC12, and SCC14a). We also confirmed the expression of RANK and RANKL in primary human OSCC tumor specimens. However, regulatory mechanisms of RANKL expression and a functional role in OSCC tumor progression are unclear. Interestingly, we identified that RANKL expression is autoregulated in OSCC tumor cells. The RANKL specific inhibitor osteoprotegerin (OPG) treatment to OSCC cells inhibits autoregulation of RANKL expression. Further, we showed conditioned media from RANKL CRISPR-Cas9 knockout OSCC cells significantly decreased osteoclast formation and bone resorption activity. In addition, RANKL increases OSCC tumor cell proliferation. RANKL treatment to OSCC cells demonstrated a dose-dependent increase in RANK intracellular adaptor protein, TRAF6 expression, and activation of IKK and IκB signaling molecules. We further identified that transcription factor NFATc2 mediates autoregulation of RANKL expression in OSCC cells. Thus, our results implicate RANKL autoregulation as a novel mechanism that facilitates OSCC tumor cell growth and osteoclast differentiation/bone destruction.

Microgravity modulation of syncytin-A expression enhance osteoclast formation.

Microgravity (µXg) experienced by astronauts during space flights causes accelerated bone loss. However, the molecular basis of µXg induced bone loss in space is unclear. Osteoclast (OCL) is the primary bone-resorbing cell. We previously demonstrated that simulated µXg promotes OCL formation. In this study, we identified that µXg induces syncytin-A expression in RAW264.7 preosteoclast cells without RANKL stimulation. We further tested the effect of osteotropic factors such as CXCL5 and 1,25(OH)2 D3 to regulate the syncytin-A expression in preosteoclast cells subjected to µXg compared to ground based (Xg) cultures. CXCL5 (25 ng/mL) and 1,25(OH)2 D3 (10 ng/mL) increased syncytin-A expression under Xg conditions. However, µXg alone upregulates syncytin-A expression compared to Xg control preosteoclast cells. Confocal microscopy using Lyso-Tracker identified syncytin-A expression co-localized with lysosomes in preosteoclast cells. Acridine orange staining showed RANKL elevated autophagy activity in these cells. Further, siRNA suppression of syncytin-A significantly inhibits autophagy activity in RAW264.7 cells. In addition, knockdown of syncytin-A expression inhibits µXg increased OCL formation in mouse bone marrow cultures. Thus, our findings suggest that targeting syncytin-A expression may be an effective countermeasure to control bone loss under microgravity conditions.

SOCS-1/3 participation in FGF-2 signaling to modulate RANK ligand expression in paget's disease of bone.

Paget's disease of bone (PDB) is a chronic focal skeletal disorder characterized by excessive bone resorption followed by disorganized new bone formation. Measles virus nucleocapsid (MVNP) is implicated in pathogenesis of PDB. RANK ligand (RANKL), a critical osteoclastogenic factor expressed on bone marrow stromal/preosteoblast cells is upregulated in PDB. We recently demonstrated that fibroblast growth factor-2 (FGF-2) which induces RANKL expression is elevated in PDB. In this study, we hypothesized that FGF-2 modulates suppressors of cytokine signaling (SOCS) to induce RANKL expression in PDB. We identified increased levels of SOCS-1/3 mRNA expression in bone marrow mononuclear cells derived from patients with PDB compared to normal subjects. Interestingly, conditioned media obtained from MVNP transduced osteoclast progenitor cells significantly increased SOCS-1/3 mRNA expression in stromal/preosteoblast cells. We next examined if SOCS participates in FGF-2 signaling to modulate RANKL gene expression. We showed that FGF-2 stimulation significantly increased SOCS-1/3 expression in human bone marrow stromal/preosteoblast cells. In addition, co-expression of SOCS-1/3 with hRANKL gene promoter-luciferase reporter plasmid in marrow stromal cells demonstrated a significant increase in promoter activity without FGF-2 stimulation. Furthermore, siRNA inhibition of STAT-1 suppresses FGF-2 increased SOCS-1/3 expression in these cells. Thus, our results suggest that SOCS participates in FGF-2 modulation of RANKL expression in PDB.

CXCL5 stimulation of RANK ligand expression in Paget's disease of bone.

Paget's disease of bone (PDB) is a chronic focal skeletal disorder that affects 2-3% of the population over 55 years of age. PDB is marked by highly localized areas of bone turnover with increased osteoclast activity. Evidence suggests a functional role for measles virus nucleocapsid protein (MVNP) in the pathogenesis of PDB. In the present study, we identified elevated levels (≈ 180-fold) of CXCL5 mRNA expression in bone marrow cells from patients with PDB compared with that in normal subjects. In addition, CXCL5 levels are increased (five-fold) in serum samples from patients with PDB. Furthermore, MVNP transduction in human bone marrow monocytes significantly increased CXCL5 mRNA expression. Real-time PCR analysis showed that CXCL5 stimulation increased (6.8-fold) RANKL mRNA expression in normal human bone marrow-derived stromal (SAKA-T) cells. Moreover, CXCL5 increased (5.2-fold) CXCR1 receptor expression in these cells. We further showed that CXCL5 treatment elevated the expression levels of phospho-ERK1/2 and phospho-p38. CXCL5 also significantly increased phosphorylation of CREB (cAMP response element-binding) in bone marrow stromal/preosteoblast cells. Chromatin immuneprecipitation (ChIP) assay confirmed phospho-CREB binding to RANKL gene promoter region. Further, the suppression of p-CREB expression by the inhibitors of ERK1/2, p38 and PKA significantly decreased CXCL5 stimulation of hRANKL gene promoter activity. Thus, our results suggest that CREB is a downstream effector of CXCL5 signaling and that increased levels of CXCL5 contribute to enhanced levels of RANKL expression in PDB.

NIP45 negatively regulates RANK ligand induced osteoclast differentiation.

Receptor activator of NF-κB ligand (RANKL)-RANK receptor signaling to induce NFATc1 transcription factor is critical for osteoclast differentiation and bone resorption. RANK adaptor proteins, tumor necrosis factor receptor-associated factors (TRAFs) play an essential role in RANKL signaling. Evidence indicates that NIP45 (NFAT interacting protein) binds with TRAFs and NFATc2. We therefore hypothesized that NIP45 regulates RANKL induced osteoclast differentiation. In this study, we demonstrate that RANKL treatment down regulates NIP45 expression in mouse bone marrow derived pre-osteoclast cells. Lentiviral (pGIPZ) mediated shRNA knock-down of NIP45 expression in RANKL stimulated pre-osteoclast cells resulted in increased levels of NFATc1, NFATc2, and TRAF6 but not TRAF2 expression compared to control shRNA transduced cells. Also, NIP45 suppression elevated p-IκB-α levels and NF-κB-luciferase reporter activity. Confocal microscopy demonstrated NIP45 colocalized with TRAF6 in the cytosol of osteoclast progenitor cells. In contrast, RANKL stimulation induced NIP45 nuclear translocation and colocalization with NFATc2 in these cells. Coimmuneprecipitation assay demonstrated NIP45 binding with NFATc2 but not NFATc1. We further show that shRNA knock-down of NIP45 expression in pre-osteoclast cells significantly increased RANKL induced osteoclast differentiation and bone resorption activity. Taken together, our results indicate that RANKL signaling down regulates NIP45 expression and that NIP45 is a negative regulator of osteoclast differentiation.

PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

Cytokine signaling via various transcription factors regulates receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from monocyte/macrophage lineage cells involved in propagation and resolution of inflammatory bone destruction. Protein inhibitor of activated STAT3 (PIAS3) was initially identified as a molecule that inhibits DNA binding of STAT3 and regulates many transcription factors through distinct mechanisms. To analyze PIAS3 function in osteoclasts in vivo, we have generated transgenic mice in which PIAS3 is specifically expressed in the osteoclast lineage using the tartrate-resistant acid phosphatase (TRAP) gene promoter. PIAS3 transgenic mice showed an osteopetrotic phenotype due to impairment of osteoclast differentiation. Overexpression of PIAS3 in RAW264.7 cells suppressed RANKL-induced osteoclastogenesis by inhibiting the expression of c-Fos and NFATc1. Interestingly, PIAS3 inhibits the transcriptional activity of microphthalmia-associated transcription factor (MITF) independent of sumoylation. Down-regulation of PIAS3 markedly enhances RANKL-mediated osteoclastogenesis in RAW264.7 cells. Furthermore, overexpression of PIAS3 in mouse primary osteoblast (POB), down-regulates RANKL expression induced by interleukin-6 (IL-6) cytokine family, and inhibits osteoclast formation from bone marrow macrophages (BMMs) in vitro coculture system. Down-regulation of PIAS3 leads to the accelerated expression of RANKL in POB stimulated with IL-6 and soluble IL-6 receptor (sIL-6R). Taken together, our results clearly indicate that PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

Microarray profile of gene expression during osteoclast differentiation in modelled microgravity.

Microgravity (µXg) leads to a 10-15% loss of bone mass in astronauts during space flight. Osteoclast (OCL) is the multinucleated bone-resorbing cell. In this study, we used the NASA developed ground-based rotating wall vessel bioreactor (RWV), rotary cell culture system (RCCS) to simulate µXg conditions and demonstrated a significant increase (2-fold) in osteoclastogenesis compared to normal gravity control (Xg). Gene expression profiling of RAW 264.7 OCL progenitor cells in modelled µXg by Agilent microarray analysis revealed significantly increased expression of critical molecules such as cytokines/growth factors, proteases and signalling proteins, which play an important role in enhanced OCL differentiation/function. Transcription factors such as c-Jun, MITF and CREB implicated in OCL differentiation are upregulated; however no significant change in the levels of NFATc1 expression in preosteoclast cells subjected to modelled µXg. We also identified high-level expression of calcium-binding protein, S100A8 (calcium-binding protein molecule A8/calgranulin A) in preosteoclast cells under µXg. Furthermore, modelled µXg stimulated RAW 264.7 cells showed elevated cytosolic calcium (Ca(2+)) levels/oscillations compared to Xg cells. siRNA knock-down of S100A8 expression in RAW 264.7 cells resulted in a significant decrease in modelled µXg stimulated OCL differentiation. We also identified elevated levels of phospho-CREB in preosteoclast cells subjected to modelled µXg compared to Xg. Thus, modelled µXg regulated gene expression profiling in preosteoclast cells provide new insights into molecular mechanisms and therapeutic targets of enhanced OCL differentiation/activation to prevent bone loss and fracture risk in astronauts during space flight missions.

Role of CXC chemokine ligand 13 in oral squamous cell carcinoma associated osteolysis in athymic mice.

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion; however, the molecular mechanisms of tumor osteolysis are unclear. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in OSCC cells (SCC1, SCC12 and SCC14a). OSCC cell-conditioned media (20%) induced osteoclast differentiation which was inhibited by OPG in peripheral blood monocyte cultures indicating that OSCC cells produce soluble RANKL. Recombinant hCXCL13 (10 ng/ml) significantly enhanced RANKL-stimulated osteoclast differentiation in these cultures. Trans-well migration assay identified that CXCL13 induces chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti-CXCR5 receptor antibody. Zymogram analysis of conditioned media from OSCC cells revealed matrix metalloproteinase-9 (MMP-9) activity. Interestingly, CXCL13 treatment to OSCC cells induced CXCR5 and MMP-9 expression suggesting an autocrine regulatory function in OSCC cells. To examine the OSCC tumor cell bone invasion/osteolysis, we established an in vivo model for OSCC by subcutaneous injection of OSCC cells onto the surface of calvaria in NCr-nu/nu athymic mice, which developed tumors in 4-5 weeks. muCT analysis revealed numerous osteolytic lesions in calvaria from OSCC tumor-bearing mice. Histochemical staining of calvarial sections from these mice revealed a significant increase in the numbers of TRAP-positive osteoclasts at the tumor-bone interface. Immunohistochemical analysis confirmed CXCL13 and MMP-9 expression in tumor cells. Thus, our data implicate a functional role for CXCL13 in bone invasion and may be a potential therapeutic target to prevent osteolysis associated with OSCC tumors in vivo.

Mutation of the sequestosome 1 (p62) gene increases osteoclastogenesis but does not induce Paget disease.

Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62(P392L) mutation contributes to the development of this disease. To determine the effects of p62(P392L) expression on osteoclasts in vitro and in vivo, we introduced either the p62(P392L) or WT p62 gene into normal osteoclast precursors and targeted p62(P392L) expression to the osteoclast lineage in transgenic mice. p62(P392L)-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-kappaB ligand (RANKL) and TNF-alpha and showed increased NF-kappaB signaling but did not demonstrate increased 1,25-(OH)(2)D(3) responsivity, TAF(II)-17 expression, or nuclear number per osteoclast. Mice expressing p62(P392L) developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62(P392L) expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.

Bone loss in survival motor neuron (Smn(-/-) SMN2) genetic mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is characterized by degenerating lower motor neurons and an increased incidence of congenital bone fractures. Survival motor neuron (SMN) levels are significantly reduced due to deletions/mutations in the telomeric SMN1 gene in these patients. We utilized the Smn(-/-) SMN2 mouse model of SMA to determine the functional role for SMN in bone remodelling. microCT analysis of lumber vertebrae, tibia and femur bones from SMA mice revealed an osteoporotic bone phenotype. Histological analysis demonstrated a thin porous cortex of cortical bone and thin trabeculae at the proximal end of the growth plate in the vertebrae of SMA mice compared to wild-type mice. Histochemical staining of the vertebrae showed the presence of abundant activated osteoclasts on the sparse trabeculae and on the endosteal surface of the thin cortex in SMA mice. Histomorphometric analysis of vertebrae from SMA mice showed an increased number of osteoclasts. Serum TRAcP5b and urinary NTx levels were elevated, consistent with increased bone resorption in these mice. SMA mice showed a significant decrease in the levels of osteoblast differentiation markers, osteocalcin, osteopontin and osterix mRNA expression; however, there were no change in the levels of alkaline phosphatase expression compared to WT mice. SMA mouse bone marrow cultures revealed an increased rate of osteoclast formation (54%) and bone resorption capacity (46%) compared to WT mice. Pre-osteoclast cells from SMA mice showed constitutive up-regulation of RANK receptor signalling molecules critical for osteoclast differentiation. Our results implicate SMN function in bone remodelling and skeletal pathogenesis in SMA. Understanding basic mechanisms of SMN action in bone remodelling may uncover new therapeutic targets for preventing bone loss/fracture risk in SMA.

Specific Commensal Bacterium Critically Regulates Gut Microbiota Osteoimmunomodulatory Actions During Normal Postpubertal Skeletal Growth and Maturation.

The commensal gut microbiota critically regulates immunomodulatory processes that influence normal skeletal growth and maturation. However, the influence of specific microbes on commensal gut microbiota osteoimmunoregulatory actions is unknown. We have shown previously that the commensal gut microbiota enhances TH17/IL17A immune response effects in marrow and liver that have procatabolic/antianabolic actions in the skeleton. Segmented filamentous bacteria (SFB), a specific commensal gut bacterium within phylum Firmicutes, potently induces TH17/IL17A-mediated immunity. The study purpose was to delineate the influence of SFB on commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal development. Two murine models were utilized: SFB-monoassociated mice versus germ-free (GF) mice and specific-pathogen-free (SPF) mice +/- SFB. SFB colonization was validated by 16S rDNA analysis, and SFB-induced TH17/IL17A immunity was confirmed by upregulation of Il17a in ileum and IL17A in serum. SFB-colonized mice had an osteopenic trabecular bone phenotype, which was attributed to SFB actions suppressing osteoblastogenesis and enhancing osteoclastogenesis. Intriguingly, SFB-colonized mice had increased expression of proinflammatory chemokines and acute-phase reactants in the liver. Lipocalin-2 (LCN2), an acute-phase reactant and antimicrobial peptide, was substantially elevated in the liver and serum of SFB-colonized mice, which supports the notion that SFB regulation of commensal gut microbiota osteoimmunomodulatory actions are mediated in part through a gut-liver-bone axis. Proinflammatory TH17 and TH1 cells were increased in liver-draining lymph nodes of SFB-colonized mice, which further substantiates that SFB osteoimmune-response effects may be mediated through the liver. SFB-induction of Il17a in the gut and Lcn2 in the liver resulted in increased circulating levels of IL17A and LCN2. Recognizing that IL17A and LCN2 support osteoclastogenesis/suppress osteoblastogenesis, SFB actions impairing postpubertal skeletal development appear to be mediated through immunomodulatory effects in both the gut and liver. This research reveals that specific microbes critically impact commensal gut microbiota immunomodulatory actions regulating normal postpubertal skeletal growth and maturation. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

IL-12 stimulates the osteoclast inhibitory peptide-1 (OIP-1/hSca) gene expression in CD4+ T cells.

Immune cell products such as interferon (IFN)-gamma and interleukin (IL)-12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide-1 (OIP-1/hSca), a Ly-6 gene family member and showed IFN-gamma modulation of OIP-1 expression in bone marrow cells. Whether, IL-12 regulates OIP-1 expression in the bone microenvironment is unclear. Real-time PCR analysis revealed that IL-12 treatment significantly enhanced OIP-1 mRNA expression in human bone marrow mononuclear cells. Because IL-12 induces IFN-gamma production by T cells, we tested whether IFN-gamma participates in IL-12 stimulation of OIP-1 gene expression in these cells. IL-12 treatment in the presence of IFN-gamma neutralizing antibody significantly increased OIP-1 mRNA expression, suggesting that IL-12 directly regulates OIP-1 gene expression. Interestingly, real-time PCR analysis demonstrated that IL-12 induces OIP-1 expression (3.2-fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL-12 (10 ng/ml) treatment of Jurkat cells transfected with OIP-1 gene (-1 to -1,988 bp) promoter-luciferase reporter plasmid demonstrated a 5-fold and 2.7-fold increase in OIP-1 gene promoter activity in the presence and absence of antibody against IFN-gamma, respectively. We showed that STAT-1,3 inhibitors treatment significantly decreased IL-12 stimulated OIP-1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT-3, but not STAT-1 binding to the OIP-1 gene promoter in response to IL-12 stimulation. These results suggest that IL-12 stimulates the OIP-1 gene expression through STAT-3 activation in CD4+ T cells.

Sphingosine-1-Phosphate Receptor 2 Controls Podosome Components Induced by RANKL Affecting Osteoclastogenesis and Bone Resorption.

Proinflammatory cytokine production, cell chemotaxis, and osteoclastogenesis can lead to inflammatory bone loss. Previously, we showed that sphingosine-1-phosphate receptor 2 (S1PR2), a G protein coupled receptor, regulates inflammatory cytokine production and osteoclastogenesis. However, the signaling pathways regulated by S1PR2 in modulating inflammatory bone loss have not been elucidated. Herein, we demonstrated that inhibition of S1PR2 by a specific S1PR2 antagonist (JTE013) suppressed phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinases (MAPKs), and nuclear factor kappa-B (NF-κB) induced by an oral bacterial pathogen, Aggregatibacter actinomycetemcomitans, and inhibited the release of IL-1ß, IL-6, TNF-α, and S1P in murine bone marrow cells. In addition, shRNA knockdown of S1PR2 or treatment by JTE013 suppressed cell chemotaxis induced by bacteria-stimulated cell culture media. Furthermore, JTE013 suppressed osteoclastogenesis and bone resorption induced by RANKL in murine bone marrow cultures. ShRNA knockdown of S1PR2 or inhibition of S1PR2 by JTE013 suppressed podosome components, including PI3K, Src, Pyk2, integrin ß3, filamentous actin (F-actin), and paxillin levels induced by RANKL in murine bone marrow cells. We conclude that S1PR2 plays an essential role in modulating proinflammatory cytokine production, cell chemotaxis, osteoclastogenesis, and bone resorption. Inhibition of S1PR2 signaling could be a novel therapeutic strategy for bone loss associated with skeletal diseases.

Osteoclast inhibitory peptide-1 (OIP-1) inhibits measles virus nucleocapsid protein stimulated osteoclast formation/activity.

Paget's disease (PD) of bone is characterized by increased activity of large abnormal osteoclasts (OCLs) which contain paramyxoviral nuclear and cytoplasmic inclusions. MVNP gene expression has been shown to induce pagetic phenotype in OCLs. We previously characterized the osteoclast inhibitory peptide-1 (OIP-1/hSca) which inhibits OCL formation/bone resorption. OIP-1 is a glycophosphatidylinositol (GPI)-linked membrane protein containing a 79 amino acid extra cellular peptide and a 32 amino acid carboxy terminal GPI-linked peptide (c-peptide) which is critical for OCL inhibition. In this study, we demonstrate that OIP-1 c-peptide significantly decreased (43%) osteoclast differentiation of peripheral blood mononuclear cells from patients with PD. Also, OIP-1 treatment to normal human bone marrow mononuclear cells transduced with the MVNP inhibited (41%) osteoclast precursor (CFU-GM) growth in methyl-cellulose cultures. We further tested if OIP-1 overexpression in the OCL lineage in transgenic mice inhibits MVNP stimulated OCL formation. MVNP transduction and RANKL stimulation of OIP-1 mouse bone marrow cells showed a significant decrease (43%) in OCL formation and inhibition (38%) of bone resorption area compared to wild-type mice. Western blot analysis identified that OIP-1 decreased (3.5-fold) MVNP induced TRAF2 expression during OCL differentiation. MVNP or OIP-1 expression did not affect TRAF6 levels. Furthermore, OIP-1 expression resulted in a significant inhibition of MVNP stimulated ASK1, Rac1, c-Fos, p-JNK, and NFATc1 expression during OCL differentiation. These results suggest that OIP-1 inhibits MVNP induced pagetic OCL formation/activity through suppression of RANK signaling. Thus, OIP-1 may have therapeutic utility against excess bone resorption in patients with PD.