Activation of NF-κB signaling regulates ovariectomy-induced bone loss and weight gain.

Postmenopausal women experience bone loss and weight gain. To date, crosstalk between estrogen receptor signals and nuclear factor-κB (NF-κB) has been reported, and estrogen depletion enhances bone resorption by osteoclasts via NF-κB activation. However, it is unclear when and in which tissues NF-κB is activated after menopause, and how NF-κB acts as a common signaling molecule for postmenopausal weight gain and bone loss. Therefore, we examined the role of NF-κB in bone and energy metabolism following menopause. NF-κB reporter mice, which can be used to measure NF-κB activation in vivo, were ovariectomized (OVX) and the luminescence intensity after OVX increased in the metaphyses of the long bones and perigonadal white adipose tissue, but not in the other tissues. OVX was performed on wild-type (WT) and p65 mutant knock-in (S534A) mice, whose mutation enhances the transcriptional activity of NF-κB. Weight gain with worsening glucose tolerance was significant in S534A mice after OVX compared with those of WT mice. The bone density of the sham group in WT or S534A mice did not change, whereas in the S534A-OVX group it significantly decreased due to the suppression of bone formation and increase in bone marrow adipocytes. Disulfiram, an anti-alcoholic drug, suppressed OVX-induced activation of NF-κB in the metaphyses of long bones and white adipose tissue (WAT), as well as weight gain and bone loss. Overall, the activation of NF-κB in the metaphyses of long bones and WAT after OVX regulates post-OVX weight gain and bone loss.

Nuclear factor-κB p65 subunit determines the fate of aging epithelial cells.

Nuclear factor (NF)-κB signaling is not only important for the immune and inflammatory responses but also for the normal development of epithelial cells, such as those in the skin and tooth. Here, we generated epithelial cell-specific p65-deficient (p65Δepi-/-) mice to analyze the roles of NF-κB signaling in epithelial cell developent. Notably, p65Δepi-/- mice exhibited no abnormalities in their appearance compared to the control (p65flox/flox) littermates. Furthermore, no major changes were observed in the skin, hair growth, and shape and color of the incisors and molars. However, 65 % of p65Δepi-/- mice exhibited corneal thickening after 8 weeks of age, and 30 % of p65Δepi-/- mice exhibited hair growth from the mandibular incisors around 24 weeks of age. No hair growth was observed at 36 and 42 weeks of age. However, micro-computed tomography images revealed a large cavity below the mandibular incisors extending to the root of the incisor. Histological analysis revealed that the cavity was occupied by a connective tissue containing hair-like structures with many dark brown granules that disappeared after melanin bleaching, confirming the presence of hair. Although inflammatory cells were also observed near the eruption site of the incisor teeth of p65Δepi-/- mice, no major disturbance was observed in the arrangement of enamel epithelial cells. Overall, these results highlight the role of p65 in the maintenance of epithelial cell homeostasis during aging.

Wnt7b expressed by hypertrophic chondrocytes is a stimulatory factor for endochondral ossification that is regulated by Smad4 activity.

Endochondral ossification contributes to longitudinal skeletal growth. Osteoblasts, which are bone-forming cells, appear close to terminally differentiated hypertrophic chondrocytes during endochondral ossification. We established mice with conditional knockout (cKO) of Smad4, an essential co-activator for transforming growth factor ß family signaling. The mice showed a marked increase in bone volume in the metaphysis as a result of increased bone formation by osteoblasts, in which ß-catenin, an effector of canonical Wnt signaling, accumulated. We identified Wnt7b as a factor with increased expression in growth plate cartilage in Smad4 cKO mice. Wnt7b mRNA was expressed in differentiated chondrocytes and suppressed by BMP4 stimulation. Ablation of Wnt7b blunted the increase in bone in adult Smad4 cKO mice and reduced skeletal growth in juvenile mice. Overall, we conclude that Wnt7b is a crucial factor secreted from hypertrophic chondrocytes to initiate endochondral ossification. These results suggest that Smad4-dependent BMP signaling regulates the Wnt7b-ß-catenin axis during endochondral ossification.

The G protein-coupled receptor GPRC5C is a saccharide sensor with a novel 'off' response.

GPRC5C is an orphan G protein-coupled receptor (GPCR) that belongs to the class C GPCR family. Although GPRC5C is expressed in various organs, its function and ligand are still undetermined. We found that GPRC5C is expressed in mouse taste cells, enterocytes, and pancreatic α-cells. In functional imaging assays, HEK293 cells heterologously expressing GPRC5C and the chimeric G protein α subunit Gα16-gust44 showed robust intracellular Ca2+ increases in response to monosaccharides, disaccharides, and a sugar alcohol, but not an artificial sweetener or sweet-tasting amino acid. Notably, Ca2+ increases occurred after washout, not during stimulation. Our findings suggest that GPRC5C has receptor properties which lead to novel 'off' responses to saccharide detachment and may work as an internal or external chemosensor specifically tuned to natural sugars.

Inhibition of non-canonical NF-κB signaling suppresses periodontal inflammation and bone loss.

Periodontal disease is an infectious disease that affects many people worldwide. Disease progression destroys the alveolar bone and causes tooth loss. We have previously shown that alymphoplasia (aly/aly) mice harboring a loss-of-function mutation in the map3k14 gene, which is involved in p100 to p52 processing of the alternative NF-κB pathway, exhibited mild osteopetrosis due to decreased number of osteoclasts, suggesting the alternative NF-κB pathway as a potential drug target for the amelioration of bone disease. In the present study, wild-type (WT) and aly/aly mice were subjected to silk ligation to establish a periodontitis model. Alveolar bone resorption was suppressed in aly/aly mice by decreased numbers of osteoclasts in the alveolar bone in comparison to WT mice. Furthermore, the expression of receptor activator of NF-κB ligand (RANKL) and TNFα (cytokines involved in osteoclast induction in periligative gingival tissue) was decreased. When primary osteoblasts (POBs) and bone marrow cells (BMCs) derived from WT and aly/aly mice were prepared and co-cultured, osteoclasts were induced from WT-derived BMCs, regardless of the origin of the POBs, but hardly formed from aly/aly mouse-derived BMCs. Furthermore, the local administration of an NIK inhibitor, Cpd33, inhibited osteoclast formation and thereby inhibited alveolar bone resorption in the periodontitis model. Therefore, the NIK-mediated NF-κB alternative pathway can be a therapeutic target for periodontal disease.

p130Cas is required for androgen-dependent postnatal development regulation of submandibular glands.

Salivary glands develop through epithelial-mesenchymal interactions and are formed through repeated branching. The Crk-associated substrate protein (p130Cas) serves as an adapter that forms a complex with various proteins via integrin and growth factor signaling, with important regulatory roles in several essential cellular processes. We found that p130Cas is expressed in ductal epithelial cells of the submandibular gland (SMG). We generated epithelial tissue-specific p130Cas-deficient (p130CasΔepi-) mice and aimed to investigate the physiological role of p130Cas in the postnatal development of salivary glands. Histological analysis showed immature development of granular convoluted tubules (GCT) of the SMG in male p130CasΔepi- mice. Immunofluorescence staining showed that nuclear-localized androgen receptors (AR) were specifically decreased in GCT cells in p130CasΔepi- mice. Furthermore, epidermal growth factor-positive secretory granules contained in GCT cells were significantly reduced in p130CasΔepi- mice with downregulated AR signaling. GCTs lacking p130Cas showed reduced numbers and size of secretory granules, disrupted subcellular localization of the cis-Golgi matrix protein GM130, and sparse endoplasmic reticulum membranes in GCT cells. These results suggest that p130Cas plays a crucial role in androgen-dependent GCT development accompanied with ER-Golgi network formation in SMG by regulating the AR signaling.

Id4 modulates salivary gland homeostasis and its expression is downregulated in IgG4-related disease via miR-486-5p.

Salivary glands are physiologically orchestrated by the coordinated balance between cell differentiation, proliferation, apoptosis, and interactions between epithelial, mesenchymal endothelial, and neuronal cells, and they are frequent sites of manifestations of Sjögren's syndrome (SS) or IgG4-related disease (IgG4-RD). However, little is known about salivary gland homeostasis and its involvement in those diseases. Inhibitor of DNA binding/differentiation 4 (Id4) is an Id protein involved in the transcriptional control of many biological events, including differentiation. Studies of Id4-deficient mice revealed that Id4-deficient submandibular glands were smaller and exhibited accelerated differentiation, compared with those from wild-type littermates. In addition, dry mouth symptoms and Th17 expansion in splenocytes were also observed in the absence of Id4. Furthermore, Id4 levels in the salivary glands of patients with IgG4-RD, but not SS, were significantly decreased compared with those of healthy controls. miRNA-mRNA integrated analysis demonstrated that miR-486-5p was upregulated in IgG4-RD patients and that it might regulate Id4 in the lesion sites. Together, these results provide evidence for the inhibitory role of Id4 in salivary differentiation, and a critical association between Id4 downregulation and IgG4-RD.

Inhibition of the ATG4-LC3 pathway suppressed osteoclast maturation.

Autophagy is a non-selective action in which cells degrade parts of themselves, reusing degraded cellular components. Among autophagy-related gene (ATG) family members, ATG4 proteins play crucial roles in the microtubule-associated protein 1 light chain 3 (LC3) phosphatidylethanolamine (PE) system which is essential for autophagosome maturation. Although autophagy has been shown to be involved in osteoclastic bone resorption, the role of ATG4/LC3 in bone resorption remains unclear. When mouse bone marrow cells were treated with various concentrations of NSC185058 (NSC), a specific inhibitor of ATG4B, 1 h prior to treatment with receptor activator of NF-κB ligand (RANKL) in the presence of macrophage colony stimulating factor (M-CSF), NSC inhibited osteoclastogenesis in a dose-dependent manner. Addition of NSC in the late stages of osteoclast differentiation suppressed multinucleation and reduced the expression of markers for mature osteoclasts such as Dc-stamp, Mmp9, and Ctsk. NSC also suppressed actin ring formation and pit formation in mature osteoclasts. When a periodontitis model involving eight-week-old male mice in which the right maxillary second molar had been ligated with silk thread was injected with or without NSC, alveolar bone resorption was suppressed by a decrease in the number of osteoclasts in the NSC-treated group. These results suggest that LC3 is important for the maturation of osteoclasts and that LC3 inhibition is a new therapeutic strategy for periodontal disease.

Critical Roles of NF-κB Signaling Molecules in Bone Metabolism Revealed by Genetic Mutations in Osteopetrosis.

The nuclear factor-κB (NF-κB) transcription factor family consists of five related proteins, RelA (p65), c-Rel, RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). These proteins are important not only for inflammation and the immune response but also for bone metabolism. Activation of NF-κB occurs via the classic and alternative pathways. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, activate the former, and cytokines involved in lymph node formation, such as receptor activator of NF-κB ligand (RANKL) and CD40L, activate the latter. p50 and p52 double-knockout mice revealed severe osteopetrosis due to the total lack of osteoclasts, which are specialized cells for bone resorption. This finding suggests that the activation of NF-κB is required for osteoclast differentiation. The NF-κB signaling pathway is controlled by various regulators, including NF-κB essential modulator (NEMO), which is encoded by the IKBKG gene. In recent years, mutant forms of the IKBKG gene have been reported as causative genes of osteopetrosis, lymphedema, hypohidrotic ectodermal dysplasia, and immunodeficiency (OL-EDA-ID). In addition, a mutation in the RELA gene, encoding RelA, has been reported for the first time in newborns with high neonatal bone mass. Osteopetrosis is characterized by a diffuse increase in bone mass, ranging from a lethal form observed in newborns to an asymptomatic form that appears in adulthood. This review describes the genetic mutations in NF-κB signaling molecules that have been identified in patients with osteopetrosis.

RANKL elevation activates the NIK/NF-κB pathway, inducing obesity in ovariectomized mice.

Menopausal women are susceptible to visceral obesity, which increases the risk of metabolic disorders. However, the mechanisms of menopause-induced visceral fat accumulation are not fully understood. Circulating levels of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) are elevated in an animal model of menopause. RANKL, a multifunctional cytokine, activates the NF-κB pathway, which serves as a pivotal mediator of inflammatory responses. Here, we investigated whether RANKL-induced non-canonical NF-κB pathway activation induces inflammation and lipid accumulation in adipose tissues. RANKL induced Tnfa expression via the non-canonical NF-κB pathway in bone marrow cells. We therefore analyzed aly/aly mice, in which the non-canonical NF-κB pathway is not activated, owing to an inactive form of NF-κB-inducing kinase. A postmenopausal obesity model was generated by ovariectomy and subsequent high-fat and high-sucrose diet feeding. In aly/aly mice with postmenopausal obesity, serum RANKL levels were elevated, and hepatic lipid accumulation and adipocyte hypertrophy were suppressed, resulting in reduced macrophage infiltration and inflammatory cytokine mRNA expression in visceral adipose tissue. Furthermore, aly/aly mice showed protection from glucose intolerance and insulin resistance, which were observed in ovariectomized WT obese mice. These findings indicate that non-canonical NF-κB pathway activation via serum RANKL elevation contributes to postmenopausal obesity.

Phospholipase C-related but catalytically inactive protein acts as a positive regulator of insulin signalling in adipocytes.

Insulin signalling is tightly controlled by various factors, but the exact molecular mechanism remains incompletely understood. We have previously reported that phospholipase C-related but catalytically inactive protein (PRIP; used here to refer to both PRIP-1 and PRIP-2, also known as PLCL1 and PLCL2, respectively) interacts with Akt1, the central molecule in insulin signalling. Here, we investigated whether PRIP is involved in the regulation of insulin signalling in adipocytes. We found that insulin signalling, including insulin-stimulated phosphorylation of the insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and Akt, and glucose uptake were impaired in adipocytes from PRIP double-knockout (PRIP-KO) mice compared with those from wild-type (WT) mice. The amount of IR expressed on the cell surface was decreased in PRIP-KO adipocytes. Immunoprecipitation assays showed that PRIP interacted with IR. The reduced cell surface IR in PRIP-KO adipocytes was comparable with that in WT cells when Rab5 (Rab5a, -5b and -5c) expression was silenced using specific siRNA. In contrast, the dephosphorylation of IRS-1 at serine residues, some of which have been reported to be involved in the internalisation of IR, was impaired in cells from PRIP-KO mice. These results suggest that PRIP facilitates insulin signalling by modulating the internalisation of IR in adipocytes.

RAF1-MEK/ERK pathway-dependent ARL4C expression promotes ameloblastoma cell proliferation and osteoclast formation.

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/ß-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/ß-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Deletion of epithelial cell-specific p130Cas impairs the maturation stage of amelogenesis.

Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130CasΔepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130CasΔepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130CasΔepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130CasΔepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca2++K+-exchanger in the maturation stage were reduced in p130CasΔepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).

The unique function of p130Cas in regulating the bone metabolism.

p130 Crk-associated substrate (Cas) functions as an adapter protein and plays important roles in certain cell functions, such as cell proliferation, spreading, migration, and invasion. Furthermore, it has recently been reported to have a new function as a mechanosensor. Since bone is a tissue that is constantly under gravity, it is exposed to mechanical stress. Mechanical unloading, such as in a microgravity environment in space or during bed rest, leads to a decrease in bone mass because of the suppression of bone formation and the stimulation of bone resorption. Osteoclasts are multinucleated bone-resorbing giant cells that recognize bone and then form a ruffled border in the resorption lacuna. p130Cas is a molecule located downstream of c-Src that is important for the formation of a ruffled border in osteoclasts. Indeed, osteoclast-specific p130Cas-deficient mice exhibit osteopetrosis due to osteoclast dysfunction, similar to c-Src-deficient mice. Osteoblasts subjected to mechanical stress induce both the phosphorylation of p130Cas and osteoblast differentiation. In osteocytes, mechanical stress regulates bone mass by shuttling p130Cas between the cytoplasm and nucleus. Oral squamous cell carcinoma (OSCC) cells express p130Cas more strongly than epithelial cells in normal tissues. The knockdown of p130Cas in OSCC cells suppressed the cell growth, the expression of receptor activator of NF-κB ligand, which induces osteoclast formation, and bone invasion by OSCC. Taken together, these findings suggest that p130Cas might be a novel therapeutic target molecule in bone diseases, such as osteoporosis, rheumatoid arthritis, bone loss due to bed rest, and bone invasion and metastasis of cancer.

Hepatic glycogenolysis is determined by maternal high-calorie diet via methylation of Pygl and it is modified by oteocalcin administration in mice.

OBJECTIVE: Accumulating evidence indicates that an adverse perinatal environment contributes to a higher risk of metabolic disorders in the later life of the offspring. However, the underlying molecular mechanisms remain largely unknown. Thus, we investigated the contribution of maternal high-calorie diet and osteocalcin to metabolic homeostasis in the offspring. METHODS: Eight-week-old C57Bl/6N female mice were mated with age-matched males and allocated randomly to three groups: a normal-diet (ND) or a high-fat, high-sucrose diet group, which was administered either saline (control) or GluOC (10 ng/g body mass) from the day of mating to that of delivery, and the dams were fed a ND after the delivery. Pups weaned at 24 days after birth were analyzed. RESULTS: A maternal high-fat, high-sucrose diet during pregnancy causes metabolic disorders in the liver of the offspring via hypermethylation of the Pygl gene, encoding glycogen phosphorylase L, which mediates hepatic glycogenolysis. The reduced expression of Pygl induced by the maternal diet causes the hepatic accumulation of glycogen and triglyceride in the offspring, which remains in adulthood. In addition, the administration of uncarboxylated osteocalcin during pregnancy upregulates Pygl expression via both direct CREBH and ATF4 and indirect epigenomic pathways, mitigating the maternal diet-induced obesity and abnormal glucose and lipid metabolism in adulthood. CONCLUSIONS: We propose that maternal energy status is reflected in the hepatic glycogenolysis capacity of the offspring via epigenetic modification of Pygl and uncarboxylated osteocalcin regulates glycogenolysis.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial-mesenchymal transition via the Smad1/5 signaling pathway.

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial-mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.

Volume-regulated chloride channel regulates cell proliferation and is involved in the possible interaction between TMEM16A and LRRC8A in human metastatic oral squamous cell carcinoma cells.

OBJECTIVES: Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line. METHODS: Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays. RESULTS: VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane. CONCLUSIONS: We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.

Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis.

The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands, such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in WT mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat and high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.

A small nuclear acidic protein (MTI-II, Zn2+-binding protein, parathymosin) attenuates TNF-α inhibition of BMP-induced osteogenesis by enhancing accessibility of the Smad4-NF-κB p65 complex to Smad binding element.

Pro-inflammatory cytokines prevent bone regeneration in vivo and activation of nuclear factor-κB (NF-κB) signaling has been proposed to lead to suppression of bone morphogenetic protein (BMP)-induced osteogenesis via direct binding of p65 to Smad4 in vitro. Application of a small nuclear acidic protein (MTI-II) and its delivered peptide, MPAID (MTI-II peptide anti-inflammatory drug) has been described to elicit therapeutic potential via strong anti-inflammatory action following the physical association of MTI-II and MPAID with p65. However, it is unclear whether MTI-II attenuates tumor necrosis factor (TNF)-α inhibition of BMP-induced osteogenesis. Herein, we found that TNF-α-mediated suppression of responses associated with BMP4-induced osteogenesis, including expression of the osteocalcin encoding gene Ocn, Smad binding element (SBE)-dependent luciferase activity, alkaline phosphatase activity, and alizarin red S staining were largely restored by MTI-II and MPAID in MC3T3-E1 cells. Mechanistically, MTI-II and MPAID did not inhibit nuclear translocation of p65 or disassociate Smad4 from p65. Further, results from chromatin immunoprecipitation (ChIP) analyses revealed that Smad4 enrichment in cells over-expressing MTI-II and treated with TNF-α was equivalent to that in cells without TNF-α treatment. Alternatively, Smad4 enrichment was considerably decreased following TNF-α treatment in control cells. Moreover, p65 enrichment in the Id-1 promoter SBE was detected only when cells over-expressing MTI-II were stimulated with TNF-α. Overall, our study concludes that MTI-II restored TNF-α-inhibited suppression of BMP-Smad-induced osteogenic differentiation by enhancing accessibility of the Smad4-p65 complex to the SBE rather than by liberating Smad4 from p65.

A novel inhibitor of NF-κB-inducing kinase prevents bone loss by inhibiting osteoclastic bone resorption in ovariectomized mice.

Musculoskeletal diseases and disorders, including osteoporosis and rheumatoid arthritis are diseases that threaten a healthy life expectancy, and in order to extend the healthy life expectancy of elderly people, it is important to prevent bone and joint diseases and disorders. We previously reported that alymphoplasia (aly/aly) mice, which have a loss-of-function mutation in the Nik gene involved in the processing of p100 to p52 in the alternative NF-κB pathway, show mild osteopetrosis with a decrease in the osteoclast number, suggesting that the alternative NF-κB pathway is a potential drug target for ameliorating bone diseases. Recently, the novel NF-κB-inducing kinase (NIK)-specific inhibitor compound 33 (Cpd33) was developed, and we examined its effect on osteoclastic bone resorption in vitro and in vivo. Cpd33 inhibited the receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis accompanied by a decrease in the expression of nfatc1, dc-stamp, and cathepsin K, markers of osteoclast differentiation, without affecting the cell viability, in a dose-dependent manner. Cdp33 specifically suppressed the RANKL-induced processing of p100 to p52 but not the phosphorylation of p65 or the degradation or resynthesis of IκBα in osteoclast precursors. Cpd33 also suppressed the bone-resorbing activity in mature osteoclasts. Furthermore, Cdp33 treatment prevented bone loss by suppressing the osteoclast formation without affecting the osteoblastic bone formation in ovariectomized mice. Taken together, NIK inhibitors may be a new option for patients with a reduced response to conventional pharmacotherapy or who have serious side effects.