Proepithelin stimulates growth plate chondrogenesis via nuclear factor-kappaB-p65-dependent mechanisms

Proepithelin, a previously unrecognized growth factor in cartilage, has recently emerged as an important regulator for cartilage formation and function. In the present study, we provide several lines of evidences in proepithelin-mediated induction of cell proliferation, differentiation, and apoptosis in the metatarsal growth plate. Proepithelin-mediated stimulation of metatarsal growth and growth plate chondrogenesis was neutralized by pyrrolidine dithiocarbamate, a known NF-κB inhibitor. In rat growth plate chondrocytes, proepithelin induced NF-κB-p65 nuclear translocation, and nuclear NF-κB-p65 initiated its target gene cyclin D1 to regulate chondrocyte functions. The inhibition of NF-κB-p65 expression and activity (by p65 short interfering RNA (siRNA) and pyrrolidine dithiocarbamate, respectively) in chondrocytes reversed the proepithelin-mediated induction of cell proliferation and differentiation and the proepithelin-mediated prevention of cell apoptosis. Moreover, the inhibition of the phosphatidylinositol 3-kinase and Akt abolished the effects of proepithelin on NF-κB activation. Finally, using siRNA and antisense strategies, we demonstrated that endogenously produced proepithelin by chondrocytes is important for chondrocyte growth in serum-deprived conditions. These results support the hypothesis that the induction of NF-κB activity of in growth plate chondrocytes is critical in proepithelin-mediated growth plate chondrogenesis and longitudinal bone growth.     

The molecular mechanisms of the effect of Dexamethasone and Cyclosporin A on TLR4 /NF-κB signaling pathway activation in oral lichen planus

Toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-κB) signaling transduction pathway play important roles in the pathogenesis of several chronic inflammatory diseases, but its function in oral lichen planus (OLP) remains unclear. In this study, we examined the expression of TLR4 and NF-κB-p65 and inflammatory cytokines TNF-α and IL-1β by immunohistochemistry in OLP tissues, and found that TLR4 and NF-κB-p65 were significantly upregulated in OLP compared to normal oral mucosa (P<0.05). We used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to simulate the local OLP immune environment to some extent. RT-PCR and immunoblotting analyses showed significant activation of TLR4 and NF-κB-p65 in the circumstance of LPS-induced inflammatory response. The high expression of TLR4 and NF-κB-p65 are correlated with expression of cytokines TNF-α and IL-1β (P<0.05). We further showed that NF-κB could act as an anti-apoptotic molecule in OLP. We conclude that TLR4 and the NF-κB signaling pathway may interact with the perpetuation of OLP. Steroids and cyclosporine are effective in the treatment of symptomatic OLP. However, there was some weak evidence for the mechanism over Dexamethasone (DeX) and Cyclosporine A (CsA) for the palliation of symptomatic OLP. In the present study, we found that Dexamethasone and Cyclosporine A negatively regulated NF-κB signaling pathway under LPS simulation in HaCaT cells by inhibiting TLR4 expression, on the other hand, Cyclosporine A could inhibit HaCaT cell proliferation by the induction of the apoptosis of HaCaT cells to protect OLP from the destruction of epidermal cells effectively.     

CO-releasing molecules and increased heme oxygenase-1 induce protein S-glutathionylation to modulate NF-κB activity in endothelial cells

Protein glutathionylation is a protective mechanism that functions in response to mild oxidative stress. Carbon monoxide (CO) can increase the reactive oxygen species concentration from a low level via the inhibition of cytochrome c oxidase. We therefore hypothesized that CO would induce NF-κB-p65 glutathionylation and then show anti-inflammatory effects. In this study, we found that CO-releasing molecules suppress TNFα-induced monocyte adhesion to endothelial cells (ECs) and reduce ICAM-1 expression. Moreover, CO donors were further found to exert their inhibitory effects by blocking NF-κB-p65 nuclear translocation, but do so independent of IκBα degradation, in TNFα-treated ECs. In addition, p65 protein glutathionylation represents the response signal to CO donors and is reversed by the reducing agent dithiothreitol. Thiol modification of the cysteine residue in the p65 RHD region was required for the CO-modulated NF-κB activation. The suppression of p65 glutathionylation by a GSH synthesis inhibitor, BSO, and by catalase could also attenuate TNFα-induced p65 nuclear translocation and ICAM-1 expression. CO donors induce Nrf2 activation and Nrf2 siRNA suppresses CO-induced p65 glutathionylation and inhibition. Furthermore, we found that the CO donors induce heme oxygenase-1 (HO-1) expression, which increases p65 glutathionylation. In contrast, HO-1 siRNA attenuates CO donor- and hemin-induced p65 glutathionylation. Our results thus indicate that the glutathionylation of p65 is likely to be responsible for CO-mediated NF-κB inactivation and that the HO-1-dependent pathway may prolong the inhibitory effects of CO donors upon TNFα treatment of ECs.     

血管活性肠肽和NF-κB信号通路在骨关节炎中的作用机制

为了考察血管活性肠肽和NF-κB信号通路在骨关节炎中的作用机制,本研究以5周龄SD雄性大鼠为研究材料,通过改良Hulth法建立骨关节炎大鼠模型,然后向大鼠关节内注射血管活性肠肽质粒进行治疗。研究显示,骨关节炎大鼠软骨细胞数量明显减少,细胞弥漫性增加,出现大量细胞簇。在用血管活性肠肽质粒处理后,大鼠膝关节的病理改变显著改善,并且OARSI评分显著降低(p<0.05)。血管活性肠肽质粒处理可显著降低大鼠血清IL-2和TNF-α水平,并升高IL-4水平(p<0.05)。血管活性肠肽质粒处理可显著降低滑膜细胞的增殖能力(p<0.05)。血管活性肠肽质粒处理可显著上调滑膜细胞中的CollagenⅡ和Osteoprotegerin蛋白表达,并下调ADAMTS-5和MMP-13蛋白表达。血管活性肠肽质粒处理可显著下调p-p65表达,并上调p-IκBα表达。综上所述,本研究表明血管活性肠肽可通过抑制滑膜细胞增殖、炎症反应、软骨退变等作用来治疗骨关节炎,其作用机制与抑制NF-κB信号通路的激活有关。     

Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Astragalus polysaccharides alleviates LPS-induced inflammation via the NF-κB/MAPK signaling pathway

Early weaning usually causes intestinal disorders, enteritis, and diarrhea in young animals and human infants. Astragalus polysaccharides (APS) possesses anti-inflammatory activity. To study the anti-inflammatory mechanisms of APS and its potential effects on intestinal health, we performed an RNA sequencing (RNA-seq) study in lipopolysaccharide (LPS)-stimulated porcine intestinal epithelial cells (IPEC-J2) in vitro. In addition, LPS-stimulated BALB/c mice were used to study the effects of APS on intestinal inflammation in vivo. The results from the RNA-seq analysis show that there were 107, 756, and 5 differentially expressed genes in the control versus LPS, LPS versus LPS+APS, and control versus LPS+APS comparison groups, respectively. The results of Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways play significant roles in the regulation of inflammatory factors and chemokine expression by APS. Further verification of the above two pathways by using western blot and immunofluorescence analysis revealed that the gene expression levels of the phosphorylated p38 MAPK, ERK1/2, and NF-κB p65 were inhibited by APS, while the expression of IκB-α protein was significantly increased (p < .05), indicating that APS inhibits the production of inflammatory factors and chemokines by the inhibition of activation of the MAPK and NF-κB inflammatory pathways induced by LPS stimulation. Animal experiments further demonstrated that prefeeding APS in BALB/c mice can alleviate the expression of the jejunal inflammatory factors interleukin 6 (IL-6), IL-Iβ, and tumor necrosis factor-α induced by LPS stimulation and improve jejunal villus morphology.     

Hepatocyte growth factor preferentially activates the anti-inflammatory arm of NF-κB signaling to induce A20 and protect renal proximal tubular epithelial cells from inflammation

Inflammation induces the NF-κB dependent protein A20 in human renal proximal tubular epithelial cells (RPTEC), which secondarily contains inflammation by shutting down NF-κB activation. We surmised that inducing A20 without engaging the pro-inflammatory arm of NF-κB could improve outcomes in kidney disease. We showed that hepatocyte growth factor (HGF) increases A20 mRNA and protein levels in RPTEC without causing inflammation. Upregulation of A20 by HGF was NF-κB/RelA dependent as it was abolished by overexpressing IκBα or silencing p65/RelA. Unlike TNFα, HGF caused minimal IκBα and p65/RelA phosphorylation, with moderate IκBα degradation. Upstream, HGF led to robust and sustained AKT activation, which was required for p65 phosphorylation and A20 upregulation. While HGF treatment of RPTEC significantly increased A20 mRNA, it failed to induce NF-κB dependent, pro-inflammatory MCP-1, VCAM-1, and ICAM-1 mRNA. This indicates that HGF preferentially upregulates protective (A20) over pro-inflammatory NF-κB dependent genes. Upregulation of A20 supported the anti-inflammatory effects of HGF in RPTEC. HGF pretreatment significantly attenuated TNFα-mediated increase of ICAM-1, a finding partially reversed by silencing A20. In conclusion, this is the first demonstration that HGF activates an AKT-p65/RelA pathway to preferentially induce A20 but not inflammatory molecules. This could be highly desirable in acute and chronic renal injury where A20-based anti-inflammatory therapies are beneficial.     

Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles

Intestinal organoids were established as an ex vivo model of the intestinal epithelium. We investigated whether organoids resemble the intestinal epithelium in their microRNA (miRNA) profiles. Total RNA samples were obtained from crypt and villus fractions in murine intestine and from cultured organoids. Microarray analysis showed that organoids largely resembled intestinal epithelial cells in their miRNA profiles. In silico prediction followed by qRT-PCR suggested that six genes are regulated by corresponding miRNAs along the crypt-villus axis, suggesting miRNA regulation of epithelial cell renewal in the intestine. However, such expression patterns of miRNAs and their target mRNAs were not reproduced during organoids maturation. This might be due to lack of luminal factors and endocrine, nervous, and immune systems in organoids and different cell populations between in vivo epithelium and organoids. Nevertheless, we propose that intestinal organoids provide a useful in vitro model to investigate miRNA expression in intestinal epithelial cells.     

HE4、NF-κBp65和MMP-9在卵巢上皮性肿瘤中的表达及其临床意义

目的探讨HE4、NF-κBp65以及MMP-9的表达与上皮性卵巢肿瘤临床病理生物学行为的关系以及对患者预后的影响。方法应用免疫组化对80例卵巢上皮性癌、10例交界性上皮性肿瘤及10例良性上皮性肿瘤组织进行HE4、NF-κB p65及MMP-9蛋白的检测。结果 HE4、NF-κBp65及MMP-9蛋白的阳性表达率在卵巢癌组均高于交界性及良性肿瘤组（P〈0.05）。三个指标的表达与EOC的组织学分级、腹腔脏器及淋巴结转移以及PTNM临床分期有关（P〈0.05）;在EOC中,NF-κBp65分别与HE4、MMP-9的表达呈正相关（r1=0.673,P〈0.05;r2=0.775,P〈0.05）。多因素分析,PTNM分期、MMP-9的表达是影响卵巢癌术后患者预后的独立因素（P〈0.05）;HE4阳性组与阴性组5年生存率分别为12.7%和84.0%,MMP-9阳性组与阴性组5年生存率分别为8.7%和70.6%,差异均有统计学意义（P〈0.05）。结论 NF-κBp65可能通过上调HE4、MMP-9的表达促进EOC的浸润和转移,联合检测HE4、NF-κBp65以及MMP-9的表达可能作为预测和评价患者预后的生物学指标。     

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.     

Expression of TNFAIP3 in intestinal epithelial cells protects from DSS- but not TNBS-induced colitis

Intestinal epithelial cells (IEC) maintain gastrointestinal homeostasis by providing a physical and functional barrier between the intestinal lumen and underlying mucosal immune system. The activation of NF-κB and prevention of apoptosis in IEC are required to maintain the intestinal barrier and prevent colitis. How NF-κB activation in IEC prevents colitis is not fully understood. TNFα-induced protein 3 (TNFAIP3) is a NF-κB-induced gene that acts in a negative-feedback loop to inhibit NF-κB activation and also to inhibit apoptosis; therefore, we investigated whether TNFAIP3 expression in the intestinal epithelium impacts susceptibility of mice to colitis. Transgenic mice expressing TNFAIP3 in IEC (villin-TNFAIP3 Tg mice) were exposed to dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the severity and characteristics of mucosal inflammation and barrier function were compared with wild-type mice. Villin-TNFAIP3 Tg mice were protected from DSS-induced colitis and displayed reduced production of NF-κB-dependent inflammatory cytokines. Villin-TNFAIP3 Tg mice were also protected from DSS-induced increases in intestinal permeability and induction of IEC death. Villin-TNFAIP3 Tg mice were not protected from colitis induced by TNBS. These results indicate that TNFAIP3 expression in IEC prevents colitis involving DSS-induced IEC death, but not colitis driven by T cell-mediated inflammation. As TNFAIP3 inhibits NF-κB activation and IEC death, expression of TNFAIP3 in IEC may provide an avenue to inhibit IEC NF-κB activation without inducing IEC death and inflammation.     

Electrolytically generated acid functional water inhibits NF-κB activity by attenuating nuclear-cytoplasmic shuttling of p65 and p50 subunits

Background: Human β-defensin 2 (hBD2) gene expression is dependent on nuclear factor kappa B (NF-κB) activity. We have previously demonstrated that electrolytically generated acid functional water (FW) induces the expression of hBD2 in the human oral squamous cell carcinoma (OSCC) cell line Ca9-22. However, the induction was not dependent on NF-κB activity; in fact, FW inhibited NF-κB activity. Therefore, we hypothesized that FW might reduce spontaneous interleukin 8 (IL-8) secretion by Ca9-22 cells, which is heavily dependent on NF-κB activity. This study aimed at demonstrating the inhibitory effect of FW on NF-κB activity. Methods: Ca9-22 cells were incubated with FW, and spontaneous IL-8 secretion was observed by enzyme-linked immunosorbent assay. Luciferase assay was performed using the 5′-untranslated region of the IL-8 gene. The steps of NF-κB activation blocked by FW were evaluated by localization of the NF-κB subunits p65 and p50 by immunofluorescence staining. Western blotting was further performed to confirm the changes in NF-κB subunit localization. Results: The Ca9-22 cells spontaneously secreted IL-8, which was rapidly and drastically inhibited by FW treatment. The luciferase assay demonstrated the inhibitory action of FW, which was diminished by deletion of the NF-κB binding site from this construct. FW treatment altered the distribution of both the p65 and p50 subunits. P65, which was localized in the nucleus during the resting state, moved to the cytoplasm after FW treatment, whereas, p50, localized in the cytoplasm during the resting state, moved to the nucleus subsequent to FW treatment. Conclusions: The results from this study indicate that FW might inhibit spontaneous IL-8 secretion by redistribution of the NF-κB subunits within the cells.     

The α2AR/Caveolin-1/p38MAPK/NF-κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia-reperfusion

Intestinal ischaemia-reperfusion (I/R) injury can result in acute lung injury due to ischaemia and hypoxia. Dexmedetomidine (Dex), a highly selective alpha2-noradrenergic receptor (α2AR) agonist used in anaesthesia, is reported to regulate inflammation in organs. This study aimed to investigate the role and mechanism of Dex in lung injury caused by intestinal I/R. After establishing a rat model of intestinal I/R, we measured the wet-to-dry specific gravity of rat lungs upon treatments with Dex, SB239063 and the α2AR antagonist Atipamezole. Moreover, injury scoring and histopathological studies of lung tissues were performed, followed by ELISA detection on tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 expression. Correlation of Caveolin-1 (Cav-1) protein expression with p38, p-p38, p-p65 and p65 in rat lung tissues was analysed, and the degree of cell apoptosis in lung tissues after intestinal I/R injury was detected by TUNEL assay. The lung injury induced by intestinal I/R was a dynamic process. Moreover, Dex had protective effects against lung injury by mediating the expression of Cal-1 and α_2A-AR. Specifically, Dex promoted Cav-1 expression via α_2A-AR activation and mitigated intestinal I/R-induced lung injury, even in the presence of Atipamezole. The protective effect of Dex on intestinal I/R-induced lung injury was also closely related to α_2A-AR/p38 mitogen-activated protein kinases/nuclear factor-kappaB (MAPK/NF-κB) pathway. Dex can alleviate pulmonary inflammation after in intestinal I/R by promoting Cav-1 to inhibit the activation of p38 and NF-κB. In conclusion, Dex can reduce pulmonary inflammatory response even after receiving threats from both intestinal I/R injury and Atipamezole.     

The glutathionylation of p65 modulates NF-κB activity in 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂-treated endothelial cells

Protein glutathionylation is a posttranslational modification of cysteine residues with glutathione in response to mild oxidative stress. Because 15-deoxy-Δ12,14-prostaglandin J(2) (15d-PGJ(2)) is an electrophilic prostaglandin that can increase glutathione (GSH) levels and augment reactive oxygen species (ROS) production, we hypothesized that it induces NF-κB-p65 glutathionylation and would exert anti-inflammatory effects. Herein, we show that 15d-PGJ(2) suppresses the expression of ICAM-1 and NF-κB-p65 nuclear translocation. 15d-PGJ(2) upregulates the Nrf2-related glutathione synthase gene and thereby increases the GSH levels. Consistent with this, Nrf2 siRNA molecules abolish the inhibition of p65 nuclear translocation in 15d-PGJ(2)-induced endothelial cells (ECs). ECs treated with GSSG show increased thiol modifications of p65 and also a block in TNFα-induced p65 nuclear translocation and ICAM-1 expression, but not in IκBα degradation. However, the overexpression of glutaredoxin 1 was found to be accompanied by a modest increase in NF-κB activity. Furthermore, we found that multiple cysteine residues in p65 are responsible for glutathionylation. 15d-PGJ(2) was observed to induce p65 glutathionylation and is suppressed by a GSH synthesis inhibitor, buthionine sulfoximine, by catalase, and by Nrf2 siRNA molecules. Our results thus indicate that the GSH/ROS-dependent glutathionylation of p65 is likely to be responsible for 15d-PGJ(2)-mediated NF-κB inactivation and for the enhanced inhibitory effects of 15d-PGJ(2) on TNFα-treated ECs.