Vitamin K3 attenuates lipopolysaccharide‐induced acute lung injury through inhibition of nuclear factor‐κB activation

Vitamin K is a family of fat‐soluble compounds including phylloquinone (vitamin K₁), menaquinone (vitamin K₂) and menadione (vitamin K₃). Recently, it was reported that vitamin K, especially vitamins K₁ and K₂, exerts a variety of biological effects, and these compounds are expected to be candidates for therapeutic agents against various diseases. In this study, we investigated the anti‐inflammatory effects of vitamin K₃ in in vitro cultured cell experiments and in vivo animal experiments. In human embryonic kidney (HEK)293 cells, vitamin K₃ inhibited the tumour necrosis factor (TNF)‐α‐evoked translocation of nuclear factor (NF)‐κB into the nucleus, although vitamins K₁ and K₂ did not. Vitamin K₃ also suppressed the lipopolysaccharide (LPS)‐induced nuclear translocation of NF‐κB and production of TNF‐α in mouse macrophage RAW264·7 cells. Moreover, the addition of vitamin K₃ before and after LPS administration attenuated the severity of lung injury in an animal model of acute lung injury/acute respiratory distress syndrome (ARDS), which occurs in the setting of acute severe illness complicated by systemic inflammation. In the ARDS model, vitamin K₃ also suppressed the LPS‐induced increase in the serum TNF‐α level and inhibited the LPS‐evoked nuclear translocation of NF‐κB in lung tissue. Despite marked efforts, little therapeutic progress has been made, and the mortality rate of ARDS remains high. Vitamin K₃ may be an effective therapeutic strategy against acute lung injury including ARDS.     

Human mesenchymal stromal cells modulate T‐cell responses through TNF‐α‐mediated activation of NF‐κB

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF‐κB) in human MSCs. This pathway is activated by TNF‐α that is generated following TCR stimulation of T cells. Inhibition of NF‐κB through silencing of IκB kinase β or the TNF‐α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC‐associated NF‐κB activation primarily leads to inhibition of T‐cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF‐α/NF‐κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF‐κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.     

Olfactomedin 1 negatively regulates NF‐κB signalling and suppresses the growth and metastasis of colorectal cancer cells

Uncontrolled growth and distant metastasis are hallmarks of colorectal cancer (CRC), but the mechanisms are poorly understood. Olfactomedin 1 (OLFM1), a member of the olfactomedin domain‐containing protein family, plays an important role in the development of neurogenic tissues. Recently, OLFM1 deregulation was frequently observed in several cancers, and it was induced in colon cell lines after treatment with the demethylating agent 5‐aza‐2'‐deoxycytidine. However, the function of OLFM1 in CRC remains unknown. In this study, we reanalysed published microarray data and found that OLFM1 was significantly down‐regulated in primary CRC samples compared to adjacent non‐cancerous tissues. The results of immunohistochemistry indicated that decreased OLFM1 expression was significantly associated with lymph node status (p = 0.023), distant metastasis (p < 0.001), and AJCC/TNM stage (p = 0.013), and CRC patients with low OLFM1 expression had consistently poor overall survival (OS; p < 0.001) and progression‐free survival (PFS; p < 0.001). Further analysis demonstrated that OLFM1 was epigenetically silenced in CRC tissues and cell lines via promoter hypermethylation. Overexpression and knockdown of OLFM1 attenuated and increased, respectively, CRC cells' proliferation, migration, and invasion in vitro and metastasis to the lung and liver in vivo. Mechanistically, the promotion of growth and metastasis of CRC cells by silencing of OLFM1 was associated with the activation of the non‐canonical NF‐κB signalling pathway. OLFM1 interacted with NF‐κB‐inducing kinase (NIK; MAP3K14) and repressed the phosphorylation of its downstream substrate Ikappa B kinase alpha (IKKα). OLFM1 expression was negatively correlated with the phosphorylation level of IKKα in CRC tissue samples. Knockdown of NIK impaired the ability of OLFM1 to repress NF‐κB signalling, cell growth or migration. Thus, OLFM1 may be a valuable biomarker and therapeutic target for CRC patients. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Expression of classical NF‐κB pathway effectors in human ovarian carcinoma

Darb‐Esfahani S, Sinn B V, Weichert W, Budczies J, Lehmann A, Noske A, Buckendahl A‐C, Müller B M, Sehouli J, Koensgen D, Györffy B, Dietel M & Denkert C
(2010) Histopathology 56. 727–739
Expression of classical NF‐κB pathway effectors in human ovarian carcinoma Aims: Functional studies have demonstrated that nuclear factor (NF)‐κB promotes tumour progression in ovarian cancer cells. However, surprisingly little is known of the expression of effectors of the NF‐κB pathway in human ovarian cancer in vivo. Methods and results: Immunohistochemistry and in situ hybridization revealed that in a cohort of 85 primary ovarian carcinomas, total p65 expression was inversely correlated to nuclear and cytoplasmic phospho‐IκBα (P = 0.002 and P = 0.05, respectively), and IκBα mRNA expression (P = 0.032). In contrast, phospho‐p65 expression was paralleled by the expression of nuclear (P = 0.027) and cytoplasmic phospho‐IκBα (P = 0.01). Total p65 expression was an adverse prognostic factor for overall survival (P = 0.018). In contrast, total IκBα and phosphorylated nuclear and cytoplasmic IκBα expression were favourable prognostic markers (P = 0.001, P = 0.031, P = 0.001, respectively). Cytoplasmic phospho‐IκBα expression remained a significant prognostic factor on multivariate analysis (P = 0.010). In cultured, stimulated OVCAR‐3 ovarian cancer cells the cytoplasmic retranslocation of p65 was delayed by inhibition of the nuclear membrane transporter chromosomal region maintenance/exportin1 protein (CRM1). A positive association of p65 and CRM1 expression was demonstrated in ovarian cancer tissue (P < 0.0001). Conclusions: Total and phosphorylated IκBα protein expression might serve as markers for NF‐κB activation in human ovarian carcinoma. Cytoplasmic localization of p65 may be related to deregulated nucleocytoplasmic transport in carcinomas overexpressing CRM1.     

5,7‐dihydroxy‐3,4,6‐trimethoxyflavone inhibits the inflammatory effects induced by Bacteroides fragilis enterotoxin via dissociating the complex of heat shock protein 90 and IκBα and IκB kinase‐γ in intestinal epithelial cell culture

Enterotoxin produced by enterotoxigenic Bacteroides fragilis (BFT) has been associated with mucosal inflammation and diarrhoeal diseases. In this study, the anti‐inflammatory molecular mechanism of 5,7‐dihydroxy‐3,4,6‐trimethoxyflavone (eupatilin) was characterized in an HT‐29 intestinal epithelial cell line stimulated with BFT. Pre‐treatment of HT‐29 cells with eupatilin decreased the production significantly of both interleukin (IL)‐8 and prostaglandin E₂ induced by BFT in a dose‐dependent manner. BFT‐activated nuclear factor‐kappaB (NF‐κB) signals in HT‐29 cells and pretreatment with eupatilin suppressed NF‐κB activation that resulted in the significant inhibition of IL‐8 and cyclo‐oxygenase‐2 expression. BFT‐induced phosphorylation of both IκBα and IκB kinase (IKK) signals was prevented in eupatilin‐pretreated HT‐29 cells. Transfection of siRNA for IKK‐α and IKK‐β decreased the production of IL‐8 and prostaglandin E₂; however, the transfection of IKK‐β siRNA showed a more significant reduction of BFT‐induced IκBα phosphorylation compared with that of IKK‐α siRNA. In addition, herbimycin A, a specific inhibitor of heat shock protein 90 (Hsp90), decreased the BFT‐induced activation of IKK and NF‐κB, suggesting that Hsp90 is associated with a pathway of IKK‐NF‐κB‐IL‐8/cyclo‐oxygenase‐2 gene signalling. Furthermore, eupatilin dissociated the complex between Hsp90 and IKK‐γ in BFT‐stimulated HT‐29 cells. These results suggest that eupatilin can suppress the NF‐κB signalling pathway by targeting the Hsp90‐IKK‐γ complex in intestinal epithelial cells and may attenuate BFT‐induced inflammatory responses.     

NF‐κB1, NF‐κB2 and c‐Rel differentially regulate susceptibility to colitis‐associated adenoma development in C57BL/6 mice

NF‐κB signalling is an important factor in the development of inflammation‐associated cancers. Mouse models of Helicobacter‐induced gastric cancer and colitis‐associated colorectal cancer have demonstrated that classical NF‐κB signalling is an important regulator of these processes. In the stomach, it has also been demonstrated that signalling involving specific NF‐κB proteins, including NF‐κB1/p50, NF‐κB2/p52, and c‐Rel, differentially regulate the development of gastric pre‐neoplasia. To investigate the effect of NF‐κB subunit loss on colitis‐associated carcinogenesis, we administered azoxymethane followed by pulsed dextran sodium sulphate to C57BL/6, Nfkb1^?/?, Nfkb2^?/?, and c‐Rel^?/?mice. Animals lacking the c‐Rel subunit were more susceptible to colitis‐associated cancer than wild‐type mice, developing 3.5 times more colonic polyps per animal than wild‐type mice. Nfkb2^?/? mice were resistant to colitis‐associated cancer, developing fewer polyps per colon than wild‐type mice (median 1 compared to 4). To investigate the mechanisms underlying these trends, azoxymethane and dextran sodium sulphate were administered separately to mice of each genotype. Nfkb2^?/? mice developed fewer clinical signs of colitis and exhibited less severe colitis and an attenuated cytokine response compared with all other groups following DSS administration. Azoxymethane administration did not fully suppress colonic epithelial mitosis in c‐Rel^?/? mice and less colonic epithelial apoptosis was also observed in this genotype compared to wild‐type counterparts. These observations demonstrate different functions of specific NF‐κB subunits in this model of colitis‐associated carcinogenesis. NF‐κB2/p52 is necessary for the development of colitis, whilst c‐Rel‐mediated signalling regulates colonic epithelial cell turnover following DNA damage. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Paeoniflorin ameliorates collagen‐induced arthritis via suppressing nuclear factor‐κB signalling pathway in osteoclast differentiation

Paeoniflorin (PF), extracted from the root of Paeonia lactiflora Pall, exhibits anti‐inflammatory properties in several autoimmune diseases. Osteoclast, the only somatic cell with bone resorbing capacity, was the direct cause of bone destruction in rheumatoid arthritis (RA) and its mouse model, collagen‐induced arthritis (CIA). The objective of this study was to estimate the effect of PF on CIA mice, and explore the mechanism of PF in bone destruction. We demonstrated that PF treatment significantly ameliorated CIA through inflammatory response inhibition and bone destruction suppression. Furthermore, PF treatment markedly decreased osteoclast number through the altered RANKL/RANK/OPG ratio and inflammatory cytokines profile. Consistently, we found that osteoclast differentiation was significantly inhibited by PF through down‐regulation of nuclear factor‐κB activation in vitro. Moreover, we found that PF suppressed nuclear factor‐κB activation by decreasing its translocation to the nucleus in osteoclast precursor cells. Taken together, our new findings provide insights into a novel function of PF in osteoclastogenesis and demonstrate that PF would be a new therapeutic modality as a natural agent for RA treatment and other autoimmune conditions with bone erosion.     

Hepatitis B virus X protein upregulates expression of calpain small subunit 1 via nuclear facter‐κB/p65 in hepatoma cells

Hepatitis B virus (HBV) infection is closely correlated with the development of hepatocellular carcinoma (HCC), in which hepatitis B virus X protein (HBx) plays crucial roles. HBx is believed to be a multifunctional oncoprotein. It has been reported that the calpain small subunit 1 (Capn4) is upregulated in the HCC tissues and involved in the metastasis of HCC. Therefore, we suppose that HBx may promote hepatoma cell migration through Capn4. In the present study, we investigated the effect of HBx on regulating Capn4 expression in human HCC cells. Our data showed that HBx could increase promoter activity of Capn4 and upregulate the expression of Capn4 at the levels of mRNA and protein in human hepatoma HepG2 (or H7402) cells using luciferase reporter gene assay, real‐time quantitative RT‐PCR assay and Western blot analysis. While, the RNA interference targeting HBx mRNA was able to abolish the upregulation. Interestingly, we found that the inhibition of nuclear factor‐κB (NF‐κB) mediated by siRNA targeting NF‐κB/p65 mRNA or PDTC (an inhibitor of NF‐κB) could attenuate the upregulation of Capn4. While, HBx failed to increase the promoter activity of Capn4 in hepatoma cells when the putative NF‐κB binding site of the Capn4 promoter was mutant, suggesting that NF‐κB is involved in the activation of Capn4 mediated by HBx. In function, wound healing assay showed that HBx could significantly enhance the migration ability of HepG2 cells through upregulating Capn4. Thus, we conclude that HBx upregulate Capn4 through NF‐κB/p65 to promote migration of hepatoma cells. J. Med. Virol. 82:920–928, 2010. © 2010 Wiley‐Liss, Inc.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

Hepatocyte‐specific activation of NF‐κB does not aggravate chemical hepatocarcinogenesis in transgenic mice

The NF‐κB signalling pathway plays important roles in liver organogenesis and carcinogenesis. Mouse embryos deficient in IKKβ die in mid‐gestation, due to excessive apoptosis of hepatoblasts. Although activation of the NF‐κB signalling pathway has been demonstrated in human hepatocellular carcinoma, the role of NF‐κB is controversial. Here, we have generated transgenic mice in which a constitutively active form of IKKβ was expressed in a hepatocyte‐specific manner. Using electrophoretic mobility shift assay, we documented increased NF‐κB activities and up‐regulated levels of NF‐κB downstream target genes, Bcl‐xL and STAT5, in the transgenic mouse livers. These results confirmed that the NF‐κB pathway was activated in the livers of the transgenic mice. However, there was no significant difference in tumour formation between transgenic and wild‐type mice up to an age of 50 weeks. When we treated the transgenic mice with the chemical carcinogen diethylnitrosamine (DEN), we observed no significant differences in the incidence and size of liver tumours formed in these mice with and without DEN treatment at 35 weeks of age, suggesting that the activated NF‐κB pathway in the livers of the transgenic mice did not enhance hepatocarcinogenesis. Interestingly, some of the transient transgenic embryos (E12.5) had abnormal excessive accumulation of nucleated red blood cells in their developing livers. In summary, NF‐κB activation in hepatocytes did not significantly affect chemical hepatocarcinogenesis. In addition, the TTR/IKKCA transgenic mice may serve as a useful model for studying the role of NF‐κB activation in hepatocarcinogenesis as well as inflammatory and metabolic diseases. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.