The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts.

Schizophyllan (SPG), a ß-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1ß/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Cytosolic malate dehydrogenase regulates RANKL-mediated osteoclastogenesis via AMPK/c-Fos/NFATc1 signaling.

Cytosolic malate dehydrogenase (malate dehydrogenase 1, MDH1) plays pivotal roles in the malate/aspartate shuttle that might modulate metabolism between the cytosol and mitochondria. In this study, we investigated the role of MDH1 in osteoclast differentiation and formation. MDH1 expression was induced by receptor activator of nuclear factor kappa-B ligand (RANKL) treatment. Knockdown of MDH1 by infection with retrovirus containing MDH1-specific shRNA (shMDH1) reduced mature osteoclast formation and bone resorption activity. Moreover, the expression of marker genes associated with osteoclast differentiation was downregulated by shMDH1 treatment, suggesting a role of MDH1 in osteoclast differentiation. In addition, intracellular ATP production was reduced following the activation of adenosine 5' monophosphate-activated protein kinase (AMPK), a cellular energy sensor and negative regulator of RANKL-induced osteoclast differentiation, in shMDH1-infected osteoclasts compared to control cells. In addition, the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a critical transcription factor of osteoclastogenesis, was decreased with MDH1 knockdown during RANKL-mediated osteoclast differentiation. These findings provide strong evidence that MDH1 plays a critical role in osteoclast differentiation and function via modulation of the intracellular energy status, which might affect AMPK activity and NFATc1 expression.

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Small molecular weight PEGylation of diosgenin in an in vivo animal study for diabetic auditory impairment treatment.

Diosgenin was modified to control its in vivo bioavailability by conjugating a hydrophilic unit, tetraethylene glycol. The diosgenin-tetraethylene glycol conjugate (TE) was orally administered in streptozotocin induced diabetic mice for this auditory protection study. The bioactivity improvement of TE for in vivo diabetic auditory impairment treatment was clearly observed in three different auditory tests and compared with that of diosgenin. The improvement in in vivo efficacy suggests that the small molecular weight PEGylation of diosgenin is a synthetically robust and systematically applicable strategy to reform the poor pharmacokinetics of a hydrophobic aglycone.

Inhibitory Effect of Rosae Multiflorae Fructus Extracts on the Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis through Modulation of P38- and Ca2+-Mediated Nuclear Factor of Activated T-Cells Cytoplasmic 1 Expression.

BACKGROUND: Rosae Multiflorae fructus (RMF), known to have anti-inflammatory and antioxidant properties, has been used as a traditional remedy for inflammatory diseases such as arthritis in Eastern Asia. However, its effect on osteoclasts, which play a crucial role in resorptive inflammatory bone diseases, is yet to be elucidated. METHODS: The effect of extract of RMF (RMF-E) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated osteoclastogenesis was examined by tartrate-resistant acid phosphatase (TRAP) staining, real-time polymerase chain reaction and western blot analysis. In addition, RANKL-induced Ca2+-oscillation was also investigated. RESULTS: RMF-E remarkably inhibited TRAP+-osteoclast and resorptive pit formation in a dose-dependent manner. In addition, the expression of c-Fos and nuclear factor of activated T-cells cytoplasmic, known as pivotal transcription factors for osteoclast formation in vitro and in vivo, and that of the osteoclast differentiation markers such as Acp5, Oscar, CtsK, Atp6v0d2, Tm7sf4, and Nfatc1 were significantly decreased by RMF-E treatment during osteoclastogenesis. The inhibitory effect of RMF-E on RANKL-induced osteoclastogenesis was caused by the suppression of p38 mitogen-activated protein kinase activation, and RANKL-induced Ca2+-oscillation removal via inactivation of Bruton's tyrosine kinase (BTK), and subsequently phospholipase C-γ2. CONCLUSIONS: RMF-E negatively regulates osteoclast differentiation and formation. These findings suggest the possibility of RMF-E as a traditional therapeutic agent against osteoclast-related bone disorders such as osteoporosis, rheumatoid arthritis, and periodontitis.

Actin-binding LIM protein 1 regulates receptor activator of NF-κB ligand-mediated osteoclast differentiation and motility.

Actin-binding LIM protein 1 (ABLIM1), a member of the LIM-domain protein family, mediates interactions between actin filaments and cytoplasmic targets. However, the role of ABLIM1 in osteoclast and bone metabolism has not been reported. In the present study, we investigated the role of ABLIM1 in the receptor activator of NF-κB ligand (RANKL)- mediated osteoclastogenesis. ABLIM1 expression was induced by RANKL treatment and knockdown of ABLIM1 by retrovirus infection containing Ablim1-specific short hairpin RNA (shAblim1) decreased mature osteoclast formation and bone resorption activity in a RANKL-dose dependent manner. Coincident with the downregulated expression of osteoclast differentiation marker genes, the expression levels of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), critical transcription factors of osteoclastogenesis, were also decreased in shAblim1-infected osteoclasts during RANKLmediated osteoclast differentiation. In addition, the motility of preosteoclast was reduced by ABLIM1 knockdown via modulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/Rac1 signaling pathway, suggesting another regulatory mechanism of ABLIM1 in osteoclast formation. These data demonstrated that ABLIM1 is a positive regulator of RANKLmediated osteoclast formation via the modulation of the differentiation and PI3K/Akt/Rac1-dependent motility. [BMB Reports 2018; 51(7): 356-361].

Pueraria lobate Inhibits RANKL-Mediated Osteoclastogenesis Via Downregulation of CREB/PGC1ß/c-Fos/NFATc1 Signaling.

Puerariae radix, the dried root of Pueraria lobate Ohwi, is known to prevent bone loss in ovariectomized mice; however, the precise molecular mechanisms are not understood. In this study, we investigated the effects and underlying mechanisms of action of Puerariae radix extract (PRE) on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. PRE dose-dependently inhibited osteoclast differentiation and formation, decreased the bone-resorbing activity of osteoclasts, and downregulated the expression of osteoclast differentiation marker genes. The expression of osteoclastogenic factors produced by PRE-treated osteoblasts such as RANKL, macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) was comparable to that of untreated (control) cells. However, the formation of osteoclasts via bone marrow cell and calvaria-derived osteoblast co-cultures was suppressed by PRE treatment. Therefore, the inhibitory effects of PRE on osteoclastogenesis clearly targeted osteoclasts, but not osteoblasts. PRE treatment considerably reduced RANKL-induced mitogen-activated protein kinases (MAPKs) activity, especially c-Jun N-terminal kinase, in osteoclast precursor cells. In addition, PRE markedly suppressed cAMP response element-binding protein (CREB) activation and the induction of peroxisome proliferator-activated receptor gamma coactivator 1ß (PGC1ß), which stimulate osteoclastogenesis - an effect that was not observed for puerarin and 17-ß estradiol. Finally, PRE treatment significantly repressed the expression of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), which is a master transcription factor for osteoclastogenesis in vitro and in vivo. Overall, these results strongly suggest that PRE is an effective inhibitor of RANKL-induced osteoclastogenesis and may be a potent therapeutic agent for bone-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.