Dracorhodin perchlorate inhibits osteoclastogenesis through repressing RANKL-stimulated NFATc1 activity

Osteolytic skeletal disorders are caused by an imbalance in the osteoclast and osteoblast function. Suppressing the differentiation and resorptive function of osteoclast is a key strategy for treating osteolytic diseases. Dracorhodin perchlorate (D.P), an active component from dragon blood resin, has been used for facilitating wound healing and anti-cancer treatments. In this study, we determined the effect of D.P on osteoclast differentiation and function. We have found that D.P inhibited RANKL-induced osteoclast formation and resorbed pits of hydroxyapatite-coated plate in a dose-dependent manner. D.P also disrupted the formation of intact actin-rich podosome structures in mature osteoclasts and inhibited osteoclast-specific gene and protein expressions. Further, D.P was able to suppress RANKL-activated JNK, NF-κB and Ca²⁺ signalling pathways and reduces the expression level of NFATc1 as well as the nucleus translocation of NFATc1. Overall, these results indicated a potential therapeutic effect of D.P on osteoclast-related conditions.     

Tumour necrosis factor‐α promotes liver ischaemia‐reperfusion injury through the PGC‐1α/Mfn2 pathway

Tumour necrosis factor (TNF)‐α has been considered to induce ischaemia‐reperfusion injury (IRI) of liver which is characterized by energy dysmetabolism. Peroxisome proliferator–activated receptor‐γ co‐activator (PGC)‐1α and mitofusion2 (Mfn2) are reported to be involved in the regulation of mitochondrial function. However, whether PGC‐1α and Mfn2 form a pathway that mediates liver IRI, and if so, what the underlying involvement is in that pathway remain unclear. In this study, L02 cells administered recombinant human TNF‐α had increased TNF‐α levels and resulted in down‐regulation of PGC‐1α and Mfn2 in a rat liver IRI model. This was associated with hepatic mitochondrial swelling, decreased adenosine triphosphate (ATP) production, and increased levels of reactive oxygen species (ROS) and alanine aminotransferase (ALT) activity as well as cell apoptosis. Inhibition of TNF‐α by neutralizing antibody reversed PGC‐1α and Mfn2 expression, and decreased hepatic injury and cell apoptosis both in cell culture and in animals. Treatment by rosiglitazone sustained PGC‐1α and Mfn2 expression both in IR livers, and L02 cells treated with TNF‐α as indicated by increased hepatic mitochondrial integrity and ATP production, reduced ROS and ALT activity as well as decreased cell apoptosis. Overexpression of Mfn2 by lentiviral‐Mfn2 transfection decreased hepatic injury in IR livers and L02 cells treated with TNF‐α. However, there was no up‐regulation of PGC‐1α. These findings suggest that PGC‐1α and Mfn2 constitute a regulatory pathway, and play a critical role in TNF‐α‐induced hepatic IRI. Inhibition of the TNF‐α or PGC‐1α/Mfn2 pathways may represent novel therapeutic interventions for hepatic IRI.     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

KiSS1 suppresses TNFα‐induced breast cancer cell invasion via an inhibition of RhoA‐Mediated NF‐κB activation

Tumor necrosis factor‐alpha (TNFα) induces cancer development and metastasis, which is prominently achieved by nuclear factor‐kappa B (NF‐κB) activation. TNFα‐induced NF‐κB activation enhances cellular mechanisms including proliferation, migration, and invasion. KiSS1, a key regulator of puberty, was initially discovered as a tumor metastasis suppressor. The expression of KiSS1 was lost or down‐regulated in different metastatic tumors. However, it is unclear whether KiSS1 regulates TNFα‐induced NF‐κB activation and further tumor cell migration. In this study, we demonstrate that KiSS1 suppresses the migration of breast cancer cells by inhibiting TNFα‐induced NF‐κB pathway and RhoA activation. Both KiSS1 overexpression and KP10 (kisspeptin‐10) stimulation inhibited TNFα‐induced NF‐κB activity, suppressed TNFα‐induced cell migration and cell attachment to fibronectin in breast cancer cells while KP10 has little effect on cancer cell proliferation. Furthermore, KP10 inhibited TNFα‐induced cell migration and RhoA GTPase activation. Therefore, our data demonstrate that KiSS1 inhibits TNFα‐induced NF‐κB activation via downregulation of RhoA activation and suppression of breast cancer cell migration and invasion. J. Cell. Biochem. 107: 1139–1149, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of PFKFB3 suppresses osteoclastogenesis and prevents ovariectomy‐induced bone loss

Osteoclasts are multinucleated cells derived from the monocyte/macrophage cell lineage under the regulation of receptor activator of nuclear factor‐κB ligand (RANKL). In previous studies, stimulation by RANKL during osteoclastogenesis was shown to induce a metabolic switch to enhanced glycolytic metabolism. Thus, we hypothesized that blockage of glycolysis might serve as a novel strategy to treat osteoclast‐related diseases. In the present study, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase 3 (PFKFB3), an essential regulator of glycolysis, was up‐regulated during osteoclast differentiation. Genetic and pharmacological inhibition of PFKFB3 in bone marrow‐derived macrophages suppressed the differentiation and function of osteoclasts. Moreover, intraperitoneal administration of the PFKFB3 inhibitor PFK15 prevented ovariectomy‐induced bone loss. In addition, glycolytic activity characterized by lactate accumulation and glucose consumption in growth medium was reduced by PFKFB3 inhibition. Further investigation indicated that the administration of L‐lactate partially reversed the repression of osteoclastogenesis caused by PFKFB3 inhibition and abrogated the inhibitory effect of PFK15 on the activation of NF‐κB and MAPK pathways. In conclusion, the results of this study suggest that blockage of glycolysis by targeting PFKFB3 represents a potential therapeutic strategy for osteoclast‐related disorders.     

Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Gα12 or constitutively active RhoA

Gα₁₂–RhoA signaling is a parathyroid hormone (PTH)‐stimulated pathway that mediates effects in bone and may influence genetic susceptibility to osteoporosis. To further elucidate effects of the pathway in osteoblasts, UMR‐106 osteoblastic cells were stably transfected with constitutively active (ca) Gα₁₂ or caRhoA or dominant negative (dn) RhoA and co‐cultured with RAW 264.7 cells to determine effects on hormone‐stimulated osteoclastogenesis. Whereas PTH and calcitriol‐stimulated osteoclastogenesis in co‐cultures with UMR‐106 cells expressing pcDNA or dominant negative RhoA, the osteoclastogenic effects of PTH and calcitriol were significantly attenuated when the UMR‐106 cells expressed either caRhoA or caGα₁₂. These inhibitory effects were partially reversed by the Rho kinase inhibitor Y27632. None of the constructs affected osteoclastogenesis in untreated co‐cultures, and the constructs did not inhibit the osteoclastogenic responses to receptor activator of NFκB ligand (RANKL). To investigate the mechanism of the inhibitory effects of caGα₁₂ and caRhoA, expression of RANKL, osteoprotegerin (OPG), osteopontin (OPN), and intercellular adhesion molecule‐1 (ICAM) in response to PTH or calcitriol was examined in the UMR‐106 cells. In the cells expressing pcDNA or dnRhoA, PTH and calcitriol increased RANKL mRNA and decreased OPG mRNA, whereas these effects were absent in the cells expressing caGα₁₂ or caRhoA. Basal expression of RANKL and OPG was unaffected by the constructs. The results suggest that Gα₁₂–RhoA signaling can inhibit hormone‐stimulated osteoclastogenesis by effects on expression of RANKL and OPG. Since PTH can stimulate the Gα₁₂–RhoA pathway, the current findings could represent a homeostatic mechanism for regulating osteoclastogenic action. J. Cell. Biochem. 111: 1531–1536, 2010. © 2010 Wiley‐Liss, Inc.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Tectorigenin inhibits RANKL‐induced osteoclastogenesis via suppression of NF‐κB signalling and decreases bone loss in ovariectomized C57BL/6

Metabolism of bone is regulated by the balance between osteoblast‐mediated bone formation and osteoclast‐mediated bone resorption. Activation of osteoclasts could lead to osteoporosis. Thus, inhibiting the activity of osteoclasts becomes an available strategy for the treatment of osteoporosis. Tectorigenin is an extract of Belamcanda chinensis In the present study, the anti‐osteoclastogenesis effects of tectorigenin were investigated in vitro and in vivo. The results showed preventive and therapeutic effects of tectorigenin at concentrations of 0, 10, 40, and 80 μmol/L in the maturation and activation of osteoclasts. A signalling study also indicated that tectorigenin treatment reduces activation of NF‐κB signalling in osteoclastogenesis. Animal experiment demonstrated that tectorigenin treatment (1‐10 mg/kg, abdominal injection every 3 days) significantly inhibits bone loss in ovariectomized C57BL/6. Our data suggest that tectorigenin is a potential pharmacological choice for osteoporosis.     

Follistatin‐like protein 1 contributes to dendritic cell and T‐lymphocyte activation in nasopharyngeal carcinoma patients by altering nuclear factor κb and Jun N‐terminal kinase expression

Follistatin‐like protein 1 (FSTL1) is a newly characterized protein that can regulate the immune response in various ways. Dendritic cells (DCs) are central to immune regulation. In this study, we explored the impact of FSTL1 on DC activity in nasopharyngeal carcinoma (NPC) patients. The surface expression of CD40, CD86, and HLA‐DR on DCs was analyzed and showed significantly elevated expression levels, indicating DC maturity. After FSTL1 was added to DCs collected from NPC patients (n = 50), controls (n = 47), and healthy donors (n = 10), interferon γ secretion and T‐cell receptor expression in cytotoxic T lymphocytes were also investigated. In the experimental groups, the expression of the critical immune protein nuclear factor (NF)‐κb was upregulated, whereas Jun N‐terminal kinase (JNK) was downregulated. Our findings demonstrate that FSTL1 plays a critical role in immune regulation, enhancing the antigen presentation ability of DCs by up‐regulating NF‐κb expression and down‐regulating JNK expression.