miR‐29a modulates tumor necrosis factor‐α‐induced osteogenic inhibition by targeting Wnt antagonists

We previously found that miR‐29a was significantly downregulated in Ankylosing spondylitis (AS) patients, a chronic inflammatory disease associated with bone metabolic disorder, however, the underlying mechanism remains unclear. In this study, we demonstrated that miR‐29a regulates tumor necrosis factor‐α (TNF‐α) mediated bone loss mainly by targeting DKK1 and GSK3β, thus activating the Wnt/β‐catenin pathway. Our findings may provide new insight into the pathogenesis of the bone metabolism disorder in inflammation environment and provide promising therapeutic target.     

Yes‐associated protein promotes tumour necrosis factor α–treated cementoblast mineralization partly by inactivating NF‐κB pathway

Cementum regeneration, as one of the most difficult challenges of periodontal regeneration, is influenced by inflammatory factors. Inflammation may hamper or promote periodontal tissue repair under different circumstances, as it is found to do in dentin‐pulp complex and bone tissue. Our team demonstrated that YAP promotes mineralization of OCCM, a cementoblast cell line. However, the effect of YAP on its mineralization under inflammatory microenvironment is unclear. In this study, cementogenesis in vitro was up‐regulated after transient TNF‐α treatment for 30 minutes. YAP expression also was increased by TNF‐α treatment. YAP overexpression promoted OCCM mineralization after the cells were transiently treated with TNF‐α because YAP overexpression inhibited NF‐κB pathway activity, while YAP knockdown elevated it. The inhibited mineralization potential and activated NF‐κB pathway activity by YAP knockdown also were partly rescued by the application of the NF‐κB inhibitor Bay 11‐7082. These results demonstrated that YAP plays a positive role in the mineralization of TNF‐α transiently treated cementoblast, partly by inhibiting the NF‐κB pathway activity.     

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin promotes astrocyte activation and the secretion of tumor necrosis factor‐α via PKC/SSeCKS‐dependent mechanisms

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a ubiquitous environmental pollutant that could induce significant toxic effects in the human nervous system. However, the underlying molecular mechanism has not been entirely elucidated. Reactive astrogliosis has implicated in various neurological diseases via the production of a variety of pro‐inflammatory mediators. Herein, we investigated the potential role of TCDD in facilitating astrocyte activation and the underlying molecular mechanisms. We showed that TCDD induced rapid astrocyte activation following TCDD exposure, which was accompanied by significantly elevated expression of Src‐Suppressed‐C Kinase Substrate (SSeCKS), a protein involved in protein kinase C (PKC)‐mediated Nuclear Factor kappa B signaling, suggesting a possible involvement of PKC‐induced SSeCKS activation in TCDD‐triggered reactive astroglia. In keeping with the finding, we found that the level of phosphorylated Nuclear Factor kappa B p65 was remarkably increased after TCDD treatment. Furthermore, interference of SSeCKS attenuated TCDD‐induced inducible nitric oxide synthase, glial fibrillary acidic protein, phospho‐p65 expression, and tumor necrosis factor‐α secretion in astrocytes. In addition, pre‐treatment with PKC inhibitor also attenuated TCDD‐induced astrocyte activation, as well as SSeCKS expression. Interestingly, we found that TCDD treatment could lead to SSeCKS perinuclear localization, which could be abolished after treatment with PKC inhibitor. Finally, we showed that inhibition of PKC activity or SSeCKS expression would impair TCDD‐triggered tumor necrosis factor‐α secretion. Our results suggested that TCDD exposure could lead to astrocyte activation through PKC/SSeCKS‐dependent mechanisms, highlighting that astrocytes might be important target of TCDD‐induced neurotoxicity.

     

Identification of ciliary neurotrophic factor receptor α as a mediator of neurotoxicity induced by α‐synuclein

Accumulating evidence suggests that extracellular α‐synuclein (eSNCA) plays an important role in the pathogenesis of Parkinson's disease or related synucleinopathies by inducing neurotoxicity directly or indirectly via microglial or astroglial activation. However, the mechanisms by which this occurs remain to be characterized. To explore these mechanisms, we combined three biochemical techniques – stable isotope labeling of amino acid in cell cultures (SILAC), biotin labeling of plasma membrane proteins followed by affinity purification, and analysis of unique proteins binding to SNCA peptides on membrane arrays. The SILAC proteomic analysis identified 457 proteins, of which, 245 or 172 proteins belonged to membrane or membrane associated proteins, depending on the various bioinformatics tools used for interpretation. In dopamine neuronal cells treated with eSNCA, the levels of 86 membrane proteins were increased and 35 were decreased compared with untreated cells. In peptide array analysis, 127 proteins were identified as possibly interacting with eSNCA. Of those, seven proteins were overlapped with the membrane proteins that displayed alterations in relative abundance after eSNCA treatment. One was ciliary neurotrophic factor receptor, which appeared to modulate eSNCA‐mediated neurotoxicity via mechanisms related to JAK1/STAT3 signaling but independent of eSNCA endocytosis.     

Solution structure of the calmodulin‐like C‐terminal domain of Entamoeba α‐actinin2

Cell motility is dependent on a dynamic meshwork of actin filaments that is remodelled continuously. A large number of associated proteins that are severs, cross‐links, or caps the filament ends have been identified and the actin cross‐linker α‐actinin has been implied in several important cellular processes. In Entamoeba histolytica, the etiological agent of human amoebiasis, α‐actinin is believed to be required for infection. To better understand the role of α‐actinin in the infectious process we have determined the solution structure of the C‐terminal calmodulin‐like domain using NMR. The final structure ensemble of the apo form shows two lobes, that both resemble other pairs of calcium‐binding EF‐hand motifs, connected with a mobile linker. Proteins 2016; 84:461–466. © 2016 Wiley Periodicals, Inc.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102     

Endogenous CSE/H2S system mediates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes

Tumour necrosis factor‐α (TNF‐ α)is a major contributor to the pathogenesis of insulin resistance associated with obesity and type 2 diabetes. It has been found that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. We have hypothesized that TNF‐α‐induced insulin resistance is involved in endogenous H₂S generation. The aim of the present study is to investigate the role of endogenous H₂S in TNF‐α‐induced insulin resistance by studying 3T3‐L1 adipocytes. We found that treatment of 3T3‐L1 adipocytes with TNF‐α leads to deficiency in insulin‐stimulated glucose consumption and uptake and increase in endogenous H₂S generation. We show that cystathionine γ‐lyase (CSE) is catalysed in 3T3‐L1 adipocytes to generate H₂S and that CSE expression and activity are upregulated by TNF‐α treatment. Inhibited CSE by its potent inhibitors significantly attenuates TNF‐α‐induced insulin resistance in 3T3‐L1 adipocytes, whereas H₂S treatment of 3T3‐L1 adipocytes impairs insulin‐stimulated glucose consumption and uptake. These data indicate that endogenous CSE/H₂S system contributes to TNF‐α‐caused insulin resistance in 3T3‐L1 adipocytes. Our findings suggest that modulation of CSE/H₂S system is a potential therapeutic avenue for insulin resistance. Copyright © 2012 John Wiley & Sons, Ltd.     

Pulsatilla decoction and its active ingredients inhibit secretion of NO,ET‐1, TNF‐α, and IL‐1α in LPS‐induced rat intestinal microvascular endothelial cells

To investigate the pharmacological mechanism of the traditional Chinese medicine, Pulsatilla decoction (PD), the levels of nitric oxide (NO), endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNF‐α), and interleukin‐1α (IL‐1α) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with PD and its seven active ingredients, namely anemoside B₄, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with lipopolysaccharide (LPS) at 1 µg ml^?1 for 3 h and then treated with PD at 1, 5, and 10 mg ml^?1 and its seven ingredients at 1, 5, and 10 µg ml^?1 for 21 h, respectively. The results revealed that PD, anemonin, berberine, and esculetin inhibited the production of NO; PD, anemonin, and esculetin inhibited the secretion of ET‐1; PD, anemoside B₄, berberine, jatrorrhizine, and aesculin downregulated TNF‐α expression; PD, anemoside B₄, berberine, and palmatine decreased the content of IL‐1α. It showed that PD and its active ingredients could significantly inhibit the secretion of NO, ET‐1, TNF‐α, and IL‐1α in LPS‐induced RIMECs and suggested they would reduce inflammatory response via these cytokines. Copyright © 2009 John Wiley & Sons, Ltd.     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

Sirt1–hypoxia‐inducible factor‐1α interaction is a key mediator of tubulointerstitial damage in the aged kidney

Although it is known that the expression and activity of sirtuin 1 (Sirt1) decrease in the aged kidney, the role of interaction between Sirt1 and hypoxia‐inducible factor (HIF)‐1α is largely unknown. In this study, we investigated whether HIF‐1α could be a deacetylation target of Sirt1 and the effect of their interaction on age‐associated renal injury. Five‐week‐old (young) and 24‐month‐old (old) C57Bl/6J mice were assessed for their age‐associated changes. Kidneys from aged mice showed increased infiltration of CD68‐positive macrophages, higher expression of extracellular matrix (ECM) proteins, and more apoptosis than young controls. They also showed decreased Sirt1 expression along with increased acetylated HIF‐1α. The level of Bcl‐2/adenovirus E1B‐interacting protein 3, carbonic anhydrase 9, Snail, and transforming growth factor‐β1, which are regulated by HIF‐1α, was significantly higher in aged mice suggesting that HIF‐1α activity was increased. In HK‐2 cells, Sirt1 inhibitor sirtinol and siRNA‐mediated knockdown of Sirt1 enhanced apoptosis and ECM accumulation. During hypoxia, Sirt1 was down‐regulated, which allowed the acetylation and activation of HIF‐1α. Resveratrol, a Sirt1 activator, effectively prevented hypoxia‐induced production of ECM proteins, mitochondrial damage, reactive oxygen species generation, and apoptosis. The inhibition of HIF‐1α activity by Sirt1‐induced deacetylation of HIF‐1α was confirmed by Sirt1 overexpression under hypoxic conditions and by resveratrol treatment or Sirt1 overexpression in HIF‐1α‐transfected HK‐2 cells. Finally, we confirmed that chronic activation of HIF‐1α promoted apoptosis and fibrosis, using tubular cell‐specific HIF‐1α transgenic mice. Taken together, our data suggest that Sirt1‐induced deacetylation of HIF‐1α may have protective effects against tubulointerstitial damage in aged kidney.     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.