The transcription factor BBX regulates phosphate homeostasis through the modulation of FGF23.

Fibroblast growth factor 23 (FGF23) plays an important role in phosphate homeostasis, and increased FGF23 levels result in hypophosphatemia; however, the molecular mechanism underlying increased FGF23 expression has not been fully elucidated. In this study, we found that mice lacking the bobby sox homolog (Bbx-/-) presented increased FGF23 expression and low phosphate levels in the serum and skeletal abnormalities such as a low bone mineral density (BMD) and bone volume (BV), as well as short and weak bones associated with low bone formation. Osteocyte-specific deletion of Bbx using Dmp-1-Cre resulted in similar skeletal abnormalities, elevated serum FGF23 levels, and reduced serum phosphate levels. In Bbx-/- mice, the expression of sodium phosphate cotransporter 2a (Npt2a) and Npt2c in the kidney and Npt2b in the small intestine, which are negatively regulated by FGF23, was downregulated, leading to phosphate excretion/wasting and malabsorption. An in vitro Fgf23 promoter analysis revealed that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced transactivation of the Fgf23 promoter was significantly inhibited by BBX overexpression, whereas it was increased following Bbx knockdown. Interestingly, 1,25(OH)2D3 induced an interaction of the 1,25(OH)2D3 receptor (VDR) with BBX and downregulated BBX protein levels. Cycloheximide (CHX) only partially downregulated BBX protein levels, indicating that 1,25(OH)2D3 regulates BBX protein stability. Furthermore, the ubiquitination of BBX followed by proteasomal degradation was required for the increase in Fgf23 expression induced by 1,25(OH)2D3. Collectively, our data demonstrate that BBX negatively regulates Fgf23 expression, and consequently, the ubiquitin-dependent proteasomal degradation of BBX is required for FGF23 expression, thereby regulating phosphate homeostasis and bone development in mice.

The MCP-3/Ccr3 axis contributes to increased bone mass by affecting osteoblast and osteoclast differentiation.

Several CC subfamily chemokines have been reported to regulate bone metabolism by affecting osteoblast or osteoclast differentiation. However, the role of monocyte chemotactic protein 3 (MCP-3), a CC chemokine, in bone remodeling is not well understood. Here, we show that MCP-3 regulates bone remodeling by promoting osteoblast differentiation and inhibiting osteoclast differentiation. In a Ccr3-dependent manner, MCP-3 promoted osteoblast differentiation by stimulating p38 phosphorylation and suppressed osteoclast differentiation by upregulating interferon beta. MCP-3 increased bone morphogenetic protein 2-induced ectopic bone formation, and mice with MCP-3-overexpressing osteoblast precursor cells presented increased bone mass. Moreover, MCP-3 exhibited therapeutic effects by abrogating receptor activator of nuclear factor kappa-B ligand-induced bone loss. Therefore, MCP-3 has therapeutic potential for diseases involving bone loss due to its positive role in osteoblast differentiation and negative role in osteoclast differentiation.

Therapeutic Potential of 4-Hexylresorcinol in Preserving Testicular Function in Streptozotocin-Induced Diabetic Rats.

It is known that many diabetic patients experience testicular atrophy. This study sought to investigate the effect of 4-hexylresorcinol (4HR) on testicular function in rats with streptozotocin (STZ)-induced diabetes, focusing on testicular weight, sperm motility, histological alterations, and serum testosterone levels to understand the efficacy of 4HR on testes. Our findings reveal that 4HR treatment significantly improves testicular health in diabetic rats. Notably, the STZ group exhibited a testicular weight of 1.22 ± 0.48 g, whereas the STZ/4HR group showed a significantly enhanced weight of 1.91 ± 0.26 g (p < 0.001), aligning closely with the control group's weight of 1.99 ± 0.17 g and the 4HR group's weight of 2.05 ± 0.24 g, indicating no significant difference between control and 4HR groups (p > 0.05). Furthermore, the STZ/4HR group demonstrated significantly improved sperm motility compared to the STZ group, with apoptotic indicators notably reduced in the STZ/4HR group relative to the STZ group (p < 0.05). These results underscore the therapeutic potential of 4HR for maintaining testicular function under diabetic conditions.

Effects of 4-hexylresorcinol on facial skeletal development in growing rats: Considerations for diabetes.

Objective: : To investigate the long-term effects of 4-hexylresorcinol (4HR) on facial skeletal growth in growing male rats, with a focus on diabetic animal models. Methods: : Forty male rats were used. Of them, type 1 diabetes mellitus was induced in 20 animals by administering 40 mg/kg streptozotocin (STZ), and they were assigned to either the STZ or 4HR-injected group (STZ/4HR group). The remaining 20 healthy rats were divided into control and 4HR groups. We administered 4HR subcutaneously at a weekly dose of 10 mg/kg until the rats were euthanized. At 16 weeks of age, whole blood was collected, and micro-computed tomography of the skull and femur was performed. Results: : All craniofacial linear measurements were smaller in the STZ group than in the control group. The mandibular molar width was significantly smaller in the 4HR group than in the control group (P = 0.031) but larger in the STZ/4HR group than in the STZ group (P = 0.011). Among the diabetic animals, the STZ/4HR group exhibited significantly greater cortical bone thickness, bone mineral density, and bone volume than the STZ group. Serum testosterone levels were also significantly higher in the STZ/4HR group than in the STZ group. Conclusions: : 4HR administration may have divergent effects on mandibular growth and bone mass in healthy and diabetic rats. In the context of diabetes, 4HR appears to have beneficial effects, potentially through the modulation of mitochondrial respiration.

Peptidylarginine deiminase 2 plays a key role in osteogenesis by enhancing RUNX2 stability through citrullination.

Peptidylarginine deiminase (PADI) 2 catalyzes the post-translational conversion of peptidyl-arginine to peptidyl-citrulline in a process called citrullination. However, the precise functions of PADI2 in bone formation and homeostasis remain unknown. In this study, our objective was to elucidate the function and regulatory mechanisms of PADI2 in bone formation employing global and osteoblast-specific Padi2 knockout mice. Our findings demonstrate that Padi2 deficiency leads to the loss of bone mass and results in a cleidocranial dysplasia (CCD) phenotype with delayed calvarial ossification and clavicular hypoplasia, due to impaired osteoblast differentiation. Mechanistically, Padi2 depletion significantly reduces RUNX2 levels, as PADI2-dependent stabilization of RUNX2 protected it from ubiquitin-proteasomal degradation. Furthermore, we discovered that PADI2 binds to RUNX2 and citrullinates it, and identified ten PADI2-induced citrullination sites on RUNX2 through high-resolution LC-MS/MS analysis. Among these ten citrullination sites, the R381 mutation in mouse RUNX2 isoform 1 considerably reduces RUNX2 levels, underscoring the critical role of citrullination at this residue in maintaining RUNX2 protein stability. In conclusion, these results indicate that PADI2 plays a distinct role in bone formation and osteoblast differentiation by safeguarding RUNX2 against proteasomal degradation. In addition, we demonstrate that the loss-of-function of PADI2 is associated with CCD, thereby providing a new target for the treatment of bone diseases.

Functional Role of Phospholipase D in Bone Metabolism.

Phospholipase D (PLD) proteins are major enzymes that regulate various cellular functions, such as cell growth, cell migration, membrane trafficking, and cytoskeletal dynamics. As they are responsible for such important biological functions, PLD proteins have been considered promising therapeutic targets for various diseases, including cancer and vascular and neurological diseases. Intriguingly, emerging evidence indicates that PLD1 and PLD2, 2 major mammalian PLD isoenzymes, are the key regulators of bone remodeling; this suggests that these isozymes could be used as potential therapeutic targets for bone diseases, such as osteoporosis and rheumatoid arthritis. PLD1 or PLD2 deficiency in mice can lead to decreased bone mass and dysregulated bone homeostasis. Although both mutant mice exhibit similar skeletal phenotypes, PLD1 and PLD2 play distinct and nonredundant roles in bone cell function. This review summarizes the physiological roles of PLD1 and PLD2 in bone metabolism, focusing on recent findings of the biological functions and action mechanisms of PLD1 and PLD2 in bone cells.

Cbfß Is a Novel Modulator against Osteoarthritis by Maintaining Articular Cartilage Homeostasis through TGF-ß Signaling.

TGF-ß signaling is a vital regulator for maintaining articular cartilage homeostasis. Runx transcription factors, downstream targets of TGF-ß signaling, have been studied in the context of osteoarthritis (OA). Although Runx partner core binding factor ß (Cbfß) is known to play a pivotal role in chondrocyte and osteoblast differentiation, the role of Cbfß in maintaining articular cartilage integrity remains obscure. This study investigated Cbfß as a novel anabolic modulator of TGF-ß signaling and determined its role in articular cartilage homeostasis. Cbfß significantly decreased in aged mouse articular cartilage and human OA cartilage. Articular chondrocyte-specific Cbfb-deficient mice (Cbfb△ac/△ac) exhibited early cartilage degeneration at 20 weeks of age and developed OA at 12 months. Cbfb△ac/△ac mice showed enhanced OA progression under the surgically induced OA model in mice. Mechanistically, forced expression of Cbfß rescued Type II collagen (Col2α1) and Runx1 expression in Cbfß-deficient chondrocytes. TGF-ß1-mediated Col2α1 expression failed despite the p-Smad3 activation under TGF-ß1 treatment in Cbfß-deficient chondrocytes. Cbfß protected Runx1 from proteasomal degradation through Cbfß/Runx1 complex formation. These results indicate that Cbfß is a novel anabolic regulator for cartilage homeostasis, suggesting that Cbfß could protect OA development by maintaining the integrity of the TGF-ß signaling pathway in articular cartilage.

Peroxiredoxin 5 regulates osteogenic differentiation through interaction with hnRNPK during bone regeneration.

Peroxiredoxin 5 (Prdx5) is involved in pathophysiological regulation via the stress-induced cellular response. However, its function in the bone remains largely unknown. Here, we show that Prdx5 is involved in osteoclast and osteoblast differentiation, resulting in osteoporotic phenotypes in Prdx5 knockout (Prdx5Ko) male mice. To investigate the function of Prdx5 in the bone, osteoblasts were analyzed through immunoprecipitation (IP) and liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) methods, while osteoclasts were analyzed through RNA-sequencing. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) was identified as a potential binding partner of Prdx5 during osteoblast differentiation in vitro. Prdx5 acts as a negative regulator of hnRNPK-mediated osteocalcin (Bglap) expression. In addition, transcriptomic analysis revealed that in vitro differentiated osteoclasts from the bone marrow-derived macrophages of Prdx5Ko mice showed enhanced expression of several osteoclast-related genes. These findings indicate that Prdx5 might contribute to the maintenance of bone homeostasis by regulating osteoblast differentiation. This study proposes a new function of Prdx5 in bone remodeling that may be used in developing therapeutic strategies for bone diseases.

Characterization of a conjugated polysuccinimide-carboplatin compound.

Carboplatin, an advanced anticancer drug with excellent efficacy against ovarian cancer, was developed to alleviate the side effects that often occur with cisplatin and other platinum-based compounds. Our study reports the in vitro characteristics, viability, and activity of cells expressing the inducible nitric oxide synthase (iNOS) gene after carboplatin was conjugated with polysuccinimide (PSI) and administered in combination with other widely used anticancer drugs. PSI, which has promising properties as a drug delivery material, could provide a platform for prolonging carboplatin release, regulating its dosage, and improving its side effects. The iNOS gene has been shown to play an important role in both cancer cell survival and inhibition. Herein, we synthesized a PSI-carboplatin conjugate to create a modified anticancer agent and confirmed its successful conjugation. To ensure its solubility in water, we further modified the structure of the PSI-carboplatin conjugate with 2-aminoethanol groups. To validate its biological characteristics, the ovarian cancer cell line SKOV-3 and normal ovarian Chinese hamster ovary cells were treated with the PSI-carboplatin conjugate alone and in combination with paclitaxel and topotecan, both of which are used in conventional chemotherapy. Notably, PSI-carboplatin conjugation can be used to predict changes in the genes involved in cancer growth and inhibition. In conclusion, combination treatment with the newly synthesized polymer-carboplatin conjugate and paclitaxel displayed anticancer activity against ovarian cancer cells but was not toxic to normal ovarian cancer cells, resulting in the development of an effective candidate anticancer drug without severe side effects.

Effect of teriparatide on drug treatment of tuberculous spondylitis: an experimental study.

Tuberculous spondylitis often develops catastrophic bone destruction with uncontrolled inflammation. Because anti-tuberculous drugs do not have a role in bone formation, a combination drug therapy with a bone anabolic agent could help in fracture prevention and promote bone reconstruction. This study aimed to investigate the influence of teriparatide on the effect of anti-tuberculous drugs in tuberculous spondylitis treatment. We used the virulent Mycobacterium tuberculosis (Mtb) H37Rv strain. First, we investigated the interaction between teriparatide and anti-tuberculosis drugs (isoniazid and rifampin) by measuring the minimal inhibitory concentration (MIC) against H37Rv. Second, we evaluated the therapeutic effect of anti-tuberculosis drugs and teriparatide on our previously developed in vitro tuberculous spondylitis model of an Mtb-infected MG-63 osteoblastic cell line using acid-fast bacilli staining and colony-forming unit counts. Selected chemokines (interleukin [IL]-8, interferon γ-induced protein 10 kDa [IP-10], monocyte chemoattractant protein [MCP]-1, and regulated upon activation, normal T cell expressed and presumably secreted [RANTES]) and osteoblast proliferation (alkaline phosphatase [ALP] and alizarin red S [ARS] staining) were measured. Teriparatide did not affect the MIC of isoniazid and rifampin. In the Mtb-infected MG-63 spondylitis model, isoniazid and rifampin treatment significantly reduced Mtb growth, and cotreatment with teriparatide did not change the anti-tuberculosis effect of isoniazid (INH) and rifampin (RFP). IP-10 and RANTES levels were significantly increased by Mtb infection, whereas teriparatide did not affect all chemokine levels as inflammatory markers. ALP and ARS staining indicated that teriparatide promoted osteoblastic function even with Mtb infection. Cotreatment with teriparatide and the anti-tuberculosis drugs activated bone formation (ALP-positive area increased by 705%, P = 0.0031). Teriparatide was effective against Mtb-infected MG63 cells without the anti-tuberculosis drugs (ARS-positive area increased by 326%, P = 0.0037). Teriparatide had no effect on the efficacy of anti-tuberculosis drugs and no adverse effect on the activity of Mtb infection in osteoblasts. Furthermore, regulation of representative osteoblastic inflammatory chemokines was not changed by teriparatide treatment. In the in vitro Mtb-infected MG-63 cell model of tuberculous spondylitis, cotreatment with the anti-tuberculosis drugs and teriparatide increased osteoblastic function.

Phospholipase D2 controls bone homeostasis by modulating M-CSF-dependent osteoclastic cell migration and microtubule stability.

Phospholipase D2 (PLD2), a signaling protein, plays a central role in cellular communication and various biological processes. Here, we show that PLD2 contributes to bone homeostasis by regulating bone resorption through osteoclastic cell migration and microtubule-dependent cytoskeletal organization. Pld2-deficient mice exhibited a low bone mass attributed to increased osteoclast function without altered osteoblast activity. While Pld2 deficiency did not affect osteoclast differentiation, its absence promoted the migration of osteoclast lineage cells through a mechanism involving M-CSF-induced activation of the PI3K-Akt-GSK3ß signaling pathway. The absence of Pld2 also boosted osteoclast spreading and actin ring formation, resulting in elevated bone resorption. Furthermore, Pld2 deletion increased microtubule acetylation and stability, which were later restored by treatment with a specific inhibitor of Akt, an essential molecule for microtubule stabilization and osteoclast bone resorption activity. Interestingly, PLD2 interacted with the M-CSF receptor (c-Fms) and PI3K, and the association between PLD2 and c-Fms was reduced in response to M-CSF. Altogether, our findings indicate that PLD2 regulates bone homeostasis by modulating osteoclastic cell migration and microtubule stability via the M-CSF-dependent PI3K-Akt-GSK3ß axis.

Skeletal muscle mitoribosomal defects are linked to low bone mass caused by bone marrow inflammation in male mice.

BACKGROUND: Mitochondrial oxidative phosphorylation (OxPhos) is a critical regulator of skeletal muscle mass and function. Although muscle atrophy due to mitochondrial dysfunction is closely associated with bone loss, the biological characteristics of the relationship between muscle and bone remain obscure. We showed that muscle atrophy caused by skeletal muscle-specific CR6-interacting factor 1 knockout (MKO) modulates the bone marrow (BM) inflammatory response, leading to low bone mass. METHODS: MKO mice with lower muscle OxPhos were fed a normal chow or high-fat diet and then evaluated for muscle mass and function, and bone mineral density. Immunophenotyping of BM immune cells was also performed. BM transcriptomic analysis was used to identify key factors regulating bone mass in MKO mice. To determine the effects of BM-derived CXCL12 (C-X-C motif chemokine ligand 12) on regulation of bone homeostasis, a variety of BM niche-resident cells were treated with recombinant CXCL12. Vastus lateralis muscle and BM immune cell samples from 14 patients with hip fracture were investigated to examine the association between muscle function and BM inflammation. RESULTS: MKO mice exhibited significant reductions in both muscle mass and expression of OxPhos subunits but increased transcription of mitochondrial stress response-related genes in the extensor digitorum longus (P < 0.01). MKO mice showed a decline in grip strength and a higher drop rate in the wire hanging test (P < 0.01). Micro-computed tomography and von Kossa staining revealed that MKO mice developed a low mass phenotype in cortical and trabecular bone (P < 0.01). Transcriptomic analysis of the BM revealed that mitochondrial stress responses in skeletal muscles induce an inflammatory response and adipogenesis in the BM and that the CXCL12-CXCR4 (C-X-C chemokine receptor 4) axis is important for T-cell homing to the BM. Antagonism of CXCR4 attenuated BM inflammation and increased bone mass in MKO mice. In humans, patients with low body mass index (BMI = 17.2 ± 0.42 kg/m2 ) harboured a larger population of proinflammatory and cytotoxic senescent T-cells in the BMI (P < 0.05) and showed reduced expression of OxPhos subunits in the vastus lateralis, compared with controls with a normal BMI (23.7 ± 0.88 kg/m2 ) (P < 0.01). CONCLUSIONS: Defects in muscle mitochondrial OxPhos promote BM inflammation in mice, leading to decreased bone mass. Muscle mitochondrial dysfunction is linked to BM inflammatory cytokine secretion via the CXCL12-CXCR4 signalling axis, which is critical for inducing low bone mass.

Excessive osteoclast activation by osteoblast paracrine factor RANKL is a major cause of the abnormal long bone phenotype in Apert syndrome model mice.

The fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling pathway plays important roles in the development and growth of the skeleton. Apert syndrome caused by gain-of-function mutations of FGFR2 results in aberrant phenotypes of the skull, midface, and limbs. Although short limbs are representative features in patients with Apert syndrome, the causative mechanism for this limb defect has not been elucidated. Here we quantitatively confirmed decreases in the bone length, bone mineral density, and bone thickness in the Apert syndrome model of gene knock-in Fgfr2S252W/+ (EIIA-Fgfr2S252W/+ ) mice. Interestingly, despite these bone defects, histological analysis showed that the endochondral ossification process in the mutant mice was similar to that in wild-type mice. Tartrate-resistant acid phosphatase staining revealed that trabecular bone loss in mutant mice was associated with excessive osteoclast activity despite accelerated osteogenic differentiation. We investigated the osteoblast-osteoclast interaction and found that the increase in osteoclast activity was due to an increase in the Rankl level of osteoblasts in mutant mice and not enhanced osteoclastogenesis driven by the activation of FGFR2 signaling in bone marrow-derived macrophages. Consistently, Col1a1-Fgfr2S252W/+ mice, which had osteoblast-specific expression of Fgfr2 S252W, showed significant bone loss with a reduction of the bone length and excessive activity of osteoclasts was observed in the mutant mice. Taken together, the present study demonstrates that the imbalance in osteoblast and osteoclast coupling by abnormally increased Rankl expression in Fgfr2S252W/+ mutant osteoblasts is a major causative mechanism for bone loss and short long bones in Fgfr2S252W/+ mice.

Effects of 4-Hexylresorcinol on Craniofacial Growth in Rats.

4-Hexylresorcinol (4HR) has been used as a food additive, however, it has been recently demonstrated as a Class I histone deacetylase inhibitor (HDACi). Unlike other HDACi, 4HR can be taken through foods. Unfortunately, some HDACi have an influence on craniofacial growth, therefore, the purpose of this study was to evaluate the effects of 4HR on craniofacial growth. Saos-2 cells (osteoblast-like cells) were used for the evaluation of HDACi and its associated activities after 4HR administration. For the evaluation of craniofacial growth, 12.8 mg/kg of 4HR was administered weekly to 4 week old rats (male: 10, female: 10) for 12 weeks. Ten rats were used for untreated control (males: 5, females: 5). Body weight was recorded every week. Serum and head samples were collected at 12 weeks after initial administration. Craniofacial growth was evaluated by micro-computerized tomography. Serum was used for ELISA (testosterone and estrogen) and immunoprecipitation high-performance liquid chromatography (IP-HPLC). The administration of 4HR (1-100 µM) showed significant HDACi activity (p < 0.05). Body weight was significantly different in male rats (p < 0.05), and mandibular size was significantly smaller in 4HR-treated male rats with reduced testosterone levels. However, the mandibular size was significantly higher in 4HR treated female rats with increased growth hormone levels. In conclusion, 4HR had HDACi activity in Saos-2 cells. The administration of 4HR on growing rats showed different responses in body weight and mandibular size between sexes.

Septal chondrocyte hypertrophy contributes to midface deformity in a mouse model of Apert syndrome.

Midface hypoplasia is a major manifestation of Apert syndrome. However, the tissue component responsible for midface hypoplasia has not been elucidated. We studied mice with a chondrocyte-specific Fgfr2S252W mutation (Col2a1-cre; Fgfr2S252W/+) to investigate the effect of cartilaginous components in midface hypoplasia of Apert syndrome. In Col2a1-cre; Fgfr2S252W/+ mice, skull shape was normal at birth, but hypoplastic phenotypes became evident with age. General dimensional changes of mutant mice were comparable with those of mice with mutations in EIIa-cre; Fgfr2S252W/+, a classic model of Apert syndrome in mice. Col2a1-cre; Fgfr2S252W/+ mice showed some unique facial phenotypes, such as elevated nasion, abnormal fusion of the suture between the premaxilla and the vomer, and decreased perpendicular plate of the ethmoid bone volume, which are related to the development of the nasal septal cartilage. Morphological and histological examination revealed that the presence of increased septal chondrocyte hypertrophy and abnormal thickening of nasal septum is causally related to midface deformities in nasal septum-associated structures. Our results suggest that careful examination and surgical correction of the nasal septal cartilage may improve the prognosis in the surgical treatment of midface hypoplasia and respiratory problems in patients with Apert syndrome.

Hypoxia-inducible factor 2α is a novel inhibitor of chondrocyte maturation.

Hypoxic environment is essential for chondrocyte maturation and longitudinal bone growth. Although hypoxia-inducible factor 1 alpha (Hif-1α) has been known as a key player for chondrocyte survival and function, the function of Hif-2α in cartilage is mechanistically and clinically relevant but remains unknown. Here we demonstrated that Hif-2α was a novel inhibitor of chondrocyte maturation through downregulation of Runx2 stability. Mechanistically, Hif-2α binding to Runx2 inhibited chondrocyte maturation by Runx2 degradation through disrupting Runx2/Cbfß complex formation. The Hif-2α-mediated-Runx2 degradation could be rescued by Cbfß transfection due to the increase of Runx2/Cbfß complex formation. Consistently, mesenchymal cells derived from Hif-2α heterozygous mice were more rapidly differentiated into hypertrophic chondrocytes than those of wild-type mice in a micromass culture system. Collectively, these findings demonstrate that Hif-2α is a novel inhibitor for chondrocyte maturation by disrupting Runx2/Cbfß complex formation and consequential regulatory activity.

The estrogen-related receptor γ modulator, GSK5182, inhibits osteoclast differentiation and accelerates osteoclast apoptosis.

Estrogen-related receptor γ (ERRγ), a member of the orphan nuclear receptor family, is a key mediator in cellular metabolic processes and energy homeostasis. Therefore, ERRγ has become an attractive target for treating diverse metabolic disorders. We recently reported that ERRγ acts as a negative regulator of osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In the present study, we explored the effects of an ERRγ-specific modulator, GSK5182, on ERRγ-regulated osteoclast differentiation and survival. Interestingly, GSK5182 increased ERRγ protein levels much as does GSK4716, which is an ERRγ agonist. GSK5182 inhibited osteoclast generation from bone-marrow-derived macrophages without affecting cytotoxicity. GSK5182 also attenuated RANKL-mediated expression of c-Fos and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), pivotal transcription factors for osteoclastogenesis. Arrested osteoclast differentiation was associated with reduced RANK expression, but not with the M-CSF receptor, c-Fms. GSK5182 strongly blocked the phosphorylation of IκBα, c-Jun N-terminal kinase, and extracellular signal-regulated kinase in response to RANKL. GSK5182 also suppressed NF-κB promoter activity in a dose-dependent manner. In addition to osteoclastogenesis, GSK5182 accelerated osteoclast apoptosis by caspase-3 activation. Together, these results suggest that GSK5182, a synthetic ERRγ modulator, may have potential in treating disorders related to bone resorption. [BMB Reports 2021; 54(5): 266-271].

Deletion of phospholipase D1 decreases bone mass and increases fat mass via modulation of Runx2, ß-catenin-osteoprotegerin, PPAR-γ and C/EBPα signaling axis.

In osteoporosis, mesenchymal stem cells (MSCs) prefer to differentiate into adipocytes at the expense of osteoblasts. Although the balance between adipogenesis and osteogenesis has been closely examined, the mechanism of commitment determination switch is unknown. Here we demonstrate that phospholipase D1 (PLD1) plays a key switch in determining the balance between bone and fat mass. Ablation of Pld1 reduced bone mass but increased fat in mice. Mechanistically, Pld1/- MSCs inhibited osteoblast differentiaion with diminished Runx2 expression, while osteoclast differentiation was accelerated in Pld1-/- bone marrow-derived macrophages. Pld1-/- osteoblasts showed decreased expression of osteogenic makers. Increased number and resorption activity of osteoclasts in Pld1-/- mice were corroborated with upregulation of osteoclastogenic markers. Moreover, Pld1-/- osteoblasts reduced ß-catenin mediated-osteoprotegerin (OPG) with increased RANKL/OPG ratio which resulted in accelerated osteoclast differentiation. Thus, low bone mass with upregulated osteoclasts could be due to the contribution of both osteoblasts and osteoclasts during bone remodeling. Moreover, ablation of Pld1 further increased bone loss in ovariectomized mice, suggesting that PLD1 is a negative regulator of osteoclastogenesis. Furthermore, loss of PLD1 increased adipogenesis, body fat mass, and hepatic steatosis along with upregulation of PPAR-γ and C/EBPα. Interestingly, adipocyte-specific Pld1 transgenic mice rescued the compromised phenotypes of fat mass and adipogenesis in Pld1 knockout mice. Collectively, PLD1 regulated the bifurcating pathways of mesenchymal cell lineage into increased osteogenesis and decreased adipogenesis, which uncovered a previously unrecognized role of PLD1 in homeostasis between bone and fat mass.

PDK2 Deficiency Prevents Ovariectomy-Induced Bone Loss in Mice by Regulating the RANKL-NFATc1 Pathway During Osteoclastogenesis.

Estrogen deficiency leads to osteoporosis as a result of an imbalance in bone remodeling due to greater bone resorption. Estrogen deficiency increases the osteoclastic resorption of bone, and many of the FDA-approved therapies for osteoporosis are antiresorptive drugs that mainly act by reducing osteoclast activity. The mitochondrial enzyme pyruvate dehydrogenase kinase (PDK) is a critical regulator of aerobic glycolysis that exerts its effects by phosphorylating the pyruvate dehydrogenase complex (PDC), which is responsible for oxidative phosphorylation. In the present study, we found that during osteoclast differentiation, PDK2 expression increased more than that of the other PDK isoenzymes. Bone loss was delayed and the number of osteoclasts was lower in ovariectomized (OVX) Pdk2-/- mice than in OVX wild-type mice. The differentiation of osteoclasts was suppressed in Pdk2-/- bone marrow-derived monocyte/macrophage lineage cells, which was associated with lower phosphorylation of cAMP response element-binding protein (CREB) and c-FOS, and a consequent reduction in NFATc1 transcription. Administration of AZD7545, a specific inhibitor of PDK2, prevented the OVX-induced bone loss and reduced the phosphorylation of CREB and c-FOS, and the protein expression of NFATc1, in osteoclasts. Collectively, these results indicate that the inhibition of PDK2 prevents osteoporosis in estrogen-deficient mice by reducing aberrant osteoclast activation, probably via inhibition of the RANKL-CREB-cFOS-NFATc1 pathway. These findings imply that PDK2 inhibitors might be repurposed for the therapy of estrogen deficiency-induced osteoporosis. © 2020 American Society for Bone and Mineral Research (ASBMR).

N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide is a novel inhibitor of resorptive bone loss in mice.

The dynamic balance between bone formation and bone resorption is vital for the retention of bone mass. The abnormal activation of osteoclasts, unique cells that degrade the bone matrix, may result in many bone diseases such as osteoporosis. Osteoporosis, a bone metabolism disease, occurs when extreme osteoclast-mediated bone resorption outstrips osteoblast-related bone synthesis. Therefore, it is of great interest to identify agents that can regulate the activity of osteoclasts and prevent bone loss-induced bone diseases. In this study, we found that N-[2-(4-benzoyl-1-piperazinyl)phenyl]-2-(4-chlorophenoxy) acetamide (PPOAC-Bz) exerted a strong inhibitory effect on osteoclastogenesis. PPOAC-Bz altered the mRNA expressions of several osteoclast-specific marker genes and blocked the formation of mature osteoclasts, suppressing F-actin belt formation and bone resorption activity in vitro. In addition, PPOAC-Bz prevented OVX-induced bone loss in vivo. These findings highlighted the potential of PPOAC-Bz as a prospective drug for the treatment of osteolytic disorders.