IL-8 Promoter Activity Research Articles

Reciprocal regulation of c-Maf tyrosine phosphorylation by TEC and PTPN22 in TH2 cells (CCR3P.210)

Abstract c-Maf plays an important role in regulating cytokine production in TH cells. It contains an N-terminal transactivation domain, which is connected to the C-terminal DNA binding domain through a hinge domain. c-Maf’s transactivation of IL-4 is optimized by phosphorylation at Tyr21, Tyr92, and Tyr131. However, the molecular mechanism regulating its tyrosine phosphorylation remains unknown. Here, we demonstrate that Tec kinase family member TEC, but not RLK or ITK, is a tyrosine kinase of c-Maf and that TEC enhances c-Maf-dependent IL-4 promoter activity. This effect of TEC is counteracted by PTPN22, which physically interacts with and facilitates tyrosine dephosphorylation of c-Maf thereby attenuating its transcriptional activity. We further show that phosphorylation of Tyr21/92/131 of c-Maf is also critical for its recruitment to the IL-21 promoter and optimal production of this cytokine by TH17 cells. Thus, manipulating tyrosine phosphorylation of c-Maf through its kinases and phosphatases can have significant impact on TH cell-mediated immune responses.

Lack of TNFR2 expression by CD4+ T cells limits IL-2 production and promotes Th17 differentiation (BA11P.138)

Abstract Tumor necrosis factor (TNF) plays dualistic pro-inflammatory and immune suppressive roles that lead to unpredictable outcomes of TNF blockade in autoimmune disease. Recent evidence has identified contrasting co-stimulatory effects of TNF on effector T cells and regulatory T cells in controlling autoimmunity. The objective of this study was to determine whether CD4+ T cell autonomous TNFR1 and TNFR2 differentially regulate IL-2 production. We utilize B10.A 5C.C7 TCR Tg Rag2-/- IL-2-GFP reporter mice that are deficient for TNFR1 and/or TNFR2 and memTNFΔ1-12 FoxP3-GFP reporter mice that encode an uncleavable tmTNF to demonstrate that TNFR2, but not TNFR1, and membrane, but not soluble, TNF promote Il2 promoter activity and decrease Il2 mRNA decay to increase IL-2 production in response to CD4+ T cell stimulation. We further demonstrate differential DNA binding of NF-κB in TNFR1- and TNFR2-deficient CD4+ T cells following activation. Adoptive transfer of naive CD4+ T cells from TNFR2-deficient IL-2-GFP heterozygous (IL-2+/-), but not IL-2-GFP homozygous (IL-2-/-), 5C.C7 Rag2-/- TCR Tg mice into B10.A Rag2-/- hosts and subsequent immunization with MCC88-103 + LPS yielded greater IL-17 production upon CD4+ T cell re-challenge in comparison to transfer of TNFR2-sufficient 5C.C7 TCR CD4+ T cells. Taken together, these results suggest that selective blockade of TNFR2 on CD4+ T cells is sufficient to inhibit IL-2 production and promote TH17 differentiation.

Olive oil phenolic extract regulates interleukin-8 expression by transcriptional and posttranscriptional mechanisms in Caco-2 cells.

In this study, we investigated the ability of a phenolic extract from extra virgin olive oil (OPE) to modulate the inflammatory response in intestinal epithelial cells. Undifferentiated and differentiated Caco-2 cells were challenged with LPS (50 μg/mL) or IL-1β (5 ng/mL) to mimic the early and intermediate phase of intestinal inflammation, respectively. The effects of OPE on nuclear factor-κB-driven transcription and IL-8 promoter activity were evaluated in transfection assays, coupled to p65 nuclear translocation. Modulation of IL-8 mRNA levels by OPE was measured by quantitative RT-PCR while effects on protein levels by ELISA. Specific mitogen activated protein kinases inhibitors were used to investigate mRNA stability and the involvement of related signaling pathways. OPE prevented IL-8 expression and secretion in LPS-treated Caco-2 cells. In the presence of IL-1β OPE exhibited opposing effects on IL-8 gene transcription and mRNA/protein levels. While in IL-1β-treated cells IL-8 promoter activity was inhibited by treatment with OPE, IL-8 mRNA stability was strongly enhanced, leading to increased protein expression. Inhibitors of p38 and extracellular signal-regulated kinases partly prevented OPE effect on IL-8 mRNA levels. Intestinal epithelial cells represent a direct target of the action of olive oil phenols where they regulate IL-8 expression by transcriptional and posttranscriptional mechanisms.

15-Lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway.

The IL-6/signal transducer and activator of transcription 3 (STAT3) pathway is a critical signaling pathway for colitis-associated colorectal cancer (CAC). Peroxisome proliferator-activated receptor (PPAR)-δ, a lipid nuclear receptor, up-regulates IL-6. 15-Lipoxygenase-1 (15-LOX-1), which is crucial to production of lipid signaling mediators to terminate inflammation, down-regulates PPAR-δ. 15-LOX-1 effects on IL-6/STAT3 signaling and CAC tumorigenesis have not been determined. We report that intestinally targeted transgenic 15-LOX-1 expression in mice inhibited azoxymethane- and dextran sodium sulfate-induced CAC, IL-6 expression, STAT3 phosphorylation, and IL-6/STAT3 downstream target (Notch3 and MUC1) expression. 15-LOX-1 down-regulation was associated with IL-6 up-regulation in human colon cancer mucosa. Reexpression of 15-LOX-1 in human colon cancer cells suppressed IL-6 mRNA expression, STAT3 phosphorylation, IL-6 promoter activity, and PPAR-δ mRNA and protein expression. PPAR-δ overexpression in colonic epithelial cells promoted CAC tumorigenesis in mice and increased IL-6 expression and STAT3 phosphorylation, whereas concomitant 15-LOX-1 expression in colonic epithelial cells (15-LOX-1-PPAR-δ-Gut mice) suppressed these effects: the number of tumors per mouse (mean ± sem) was 4.22 ± 0.68 in wild-type littermates, 6.67 ± 0.83 in PPAR-δ-Gut mice (P = 0.026), and 2.25 ± 0.25 in 15-LOX-1-PPAR-δ-Gut mice (P = 0.0006). Identification of 15-LOX-1 suppression of PPAR-δ to inhibit IL-6/STAT3 signaling-driven CAC tumorigenesis provides mechanistic insights that can be used to molecularly target CAC.

FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3.

Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4-6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs.

The N-N non-covalent domain of the nucleocapsid protein of type 2 porcine reproductive and respiratory syndrome virus enhances induction of IL-10 expression.

Porcine reproductive and respiratory syndrome virus (PRRSV) usually establishes a prolonged infection and causes an immunosuppressive state. It has been proposed that IL-10 plays an important role in PRRSV-induced immunosuppression. However, this mechanism has not been completely elucidated. In this study, we found that transfection of 3D4/2 macrophages with the N protein gene of type 2 PRRSV significantly upregulated IL-10 expression at the transcriptional level. Moreover, alanine substitution mutation analysis revealed that the N protein residues 33-37, 65-68 and 112-123 were related to the upregulation of IL-10 promoter activity. Recombinant PRRSV with mutations at residues 33-37 in the N protein (rQ33-5A and rS36A) recovered from corresponding infectious cDNA clones and induced significantly lower levels of IL-10 production in infected monocyte-derived dendritic cells, as compared with their revertants rQ33-5A(R) and rS36A(R), and the wild-type recombinant PRRSV strain rNT/wt. These data indicate that the type 2 PRRSV N protein plays an important role in IL-10 induction and the N-N non-covalent domain is associated with this activity.

Involvement of intracellular calcium mobilization in IL-8 activation in human retinal pigment epithelial cells.

Calcium signaling is an important intracellular pathway. Increased intracellular calcium is associated with cytokine regulation and inflammatory signals secretion. The purpose of this study is to understand the molecular mechanisms by which calcium signaling controls IL-8 activation in human RPE cells. Fluorescence-based calcium imaging and different mutants of IL-8 plasmids were used in this study. The IL-8 promoter activation, gene expression, and secretion were detected by using luciferase reporter assay, quantitative real-time PCR (Q-PCR), and ELISA, respectively. In addition, pharmacological inhibitors and small interfering RNA (siRNA) were applied to clarify the mechanisms of IL-8 activation. Our study reported that intracellular calcium mobilization activated IL-8 gene expression and secretion. Application of pharmacological inhibitor BAY 11-7082, siRNA, and plasmids of the nuclear factor κ light chain enhancer of activated B cells (NF-κB) binding site, we identified that NF-κB is the main transcription factor involved in intracellular calcium mobilization-mediated IL-8 activation in human RPE cells. Collectively, our findings highlight the important role of intracellular calcium mobilization in the activation of IL-8. These findings may be helpful for the clinical applications in the age-related macular degeneration (AMD) prevention and treatment.

The effect of in vitro gastrointestinal digestion on the anti-inflammatory activity of Vitis vinifera L. leaves.

Botanicals are widely consumed all over the world for health purposes, with increased usage in the general population, in many different types of products, including foods and plant food supplements. Several reports support for the beneficial effects of botanicals against gastrointestinal inflammation. However, no studies regarding the anti-inflammatory activity in the gastrointestinal tract of red vine leaves have been reported so far. The present work investigates the biological activity of Vitis vinifera L. water extract (VVWE) from dried leaves in two in vitro models of gastric and intestinal inflammation. The extract was characterized by a validated HPLC-DAD method, and tested on human epithelial gastric (AGS) and intestinal (Caco-2) cells with the aim to investigate the inhibitory effect on IL-8 secretion and promoter activity, before and after in vitro gastric or gastrointestinal digestion. Our results show that the water extract from red vine leaves inhibits TNFα-induced IL-8 secretion and expression in human gastric epithelial cells; the effect should be maintained, although to a lesser extent, after gastric digestion. In contrast, the effect after intestinal digestion is dramatically decreased since degradation of the active components in the gut does not allow the extract to efficiently counteract TNFα or IL-1β induced IL-8 expression and the NF-κB pathway. The main molecular target of VVWE at the gastric level includes TNFα-induced activation of NF-κB and occurs at concentrations easily reachable after PFS consumption based on red vine leaf water extract as the ingredient. Our findings suggest that PFS containing water extracts from Vitis vinifera L. leaves could be useful to inhibit/attenuate gastric inflammation inhibiting IL-8 secretion and expression through impairment of the NF-κB pathway.

IL-1β Induces IL-6 production in retinal Müller cells predominantly through the activation of P38 MAPK/NF-κB signaling pathway

IL-6 plays an important role in various inflammatory ocular diseases, including diabetic retinopathy. Müller cells are the major source of inflammatory mediators, including IL-6, in the retina. However, the mechanism of regulating IL-6 production in these cells remains unclear. Examination of signaling pathways in human retinal Müller cells (MIO-M1 cell line) cultured with IL-1β, TNF-α, IL-6, IL-8, VEGF, IFN-γ, glucose or mannitol showed that IL-1β was the most potent stimulator of IL-6 production. In addition, IL-1 β also increased NF-κB p50 protein level and phosphorylation of p38 MAPK, ERK1/2 and c-Jun. Induction of IL-6 production by IL-1β was significantly reduced by addition of p38 MAPK (SB203580), MEK1/2 (U0126) or NF-κB (BAY11-7082) inhibitors, with the highest effect being observed with SB203580. To explore the specific elements in IL-6 promoter responsible for IL-1β-induction of IL-6 expression, a series of plasmids bearing various IL-6 promoter mutations were transiently expressed in MIO-MI cells cultured in the presence or absence of IL-1β (10ng/ml) and/or SB203580 (10µM). Results showed that IL-6 promoter activity of the parent pIL-6-Luc651 was significantly enhanced by IL-1β, but the level was significantly attenuated by SB203580. Furthermore, the IL-6 promoter activity was also reduced upon deletion of NF-κB, AP-1 or C/EBP binding sites, with NF-κB deletion being the greatest. These results are the first demonstration that IL-1β induces IL-6 production in Müller cells by activation of IL-6 promoter activity predominantly through the p38 MAPK/NF-κB pathway.

Regulation of IL-8 Promoter Activity by Verrucarin A in Human Monocytic THP-1 Cells

Macrocyclic trichothecenes have been frequently detected in fungi in water-damaged buildings and exhibited higher toxicity than the well-studied trichothecenes; however, the mechanism underlying their toxicity has been poorly understood. In this study, transcriptional regulation of the cytokine interleukin (IL)-8 by a macrocyclic trichothecene, verrucarin A (VA), in human monocytic THP-1 cells is reported. Consistent with previous findings, VA was 100-fold more cytotoxic than deoxynivalenol (DON), while ochratoxin A (OA) was not cytotoxic. In cells transduced with the wild-type IL-8 promoter luciferase construct, VA induced a biphasic dose response composed of an upregulation of luciferase expression at low concentrations of 0.01−1 ng/ml and a downregulation at high levels of 10 ng/ml and higher. In contrast, DON induced a sigmoid-shaped dose response with the EC50 of 11.6 ng/ml, while OA did not markedly affect the IL-8 expression. When cells were transduced with IL-8 promoter with a mutation of transcription factor nuclear factor-κB (NF-κB)-binding site, VA (1 ng/ml), DON (1000 ng/ml), and tumor necrosis factor (TNF) α (20 ng/ml)-induced luciferase expression were impaired. In addition, the NF-κB inhibitor caffeic acid phenethyl ester inhibited VA-, DON-, and TNFα-induced luciferase expression. Mutation of the CCAAT/enhancer-binding protein (CEBP) β binding site of the IL-8 promoter affected only DON-, but not VA- and TNFα-induced luciferase expression. Taken together, these results suggested that VA activated IL-8 promoter via an NF-κB-dependent, but not CEBPβ-dependent, pathway in human monocytes.

Biochemical properties of the recurrent LMX1b truncated mutant carried in a Taiwanese family with nail-patella syndrome

Loss of the DNA-binding activity of a transcription factor is detrimental to its function in responsive gene regulation. We diagnosed a Taiwanese family with nail-patella syndrome (NPS) whose members inherited the mutated LMX1b transcription factor with no DNA-binding homeodomain. The loss-of-function variants cause haploinsufficiency of LMX1b, leading to the clinical manifestation of NPS. The underlying molecular mechanism is unclear. To test whether the recurrent pathogenic truncated LMX1b-R198X reported in our patients might be a functional protein. Its biochemical properties were explored. The luciferase reporter driven by the human interleukin (IL)-6 gene promoter was assayed to measure the transcriptional activity of LMX1b. The nuclear localization of different enhanced green fluorescent protein-tagged LMX1b proteins was observed using fluorescence microscopy. Western blotting was employed to evaluate the expression of various transfected LMX1b constructs. LMX1b-R198X enhanced the IL-6 promoter activity activated by the wild-type LMX1b and diminished the promoter activity induced by phorbol 12-myristate 13-acetate. LMX1b-R198X carried out its effect differentially in the expression of various human genes. The nuclear localization of the wild-type LMX1b was disrupted by the C-terminus truncation. The protein stability exhibited by LMX1b-R198X appears to be much higher than that of the wild-type protein. We demonstrated that loss of function might not be the only way for mutated LMX1b to cause haploinsufficiency as the main pathogenic mechanism for NPS. LMX1b-R198X has less nuclear localization and higher stability than the wild-type protein; consequently, it might function as a competitor to sequester other effectors by protein-protein interaction to interfere with downstream transcriptional events.

CD4 T Cell-autonomous TNF receptor type 2 promotes interleukin-2 transcription (IRM6P.723)

Abstract Interleukin (IL)-2 plays a key role in adjusting the dynamic state of equilibrium between effector T cells (Teffs) and regulatory T cells (Tregs) as well as channeling the outcome of immune responses. IL-2 is mainly produced by activated CD4 T cells and the absence of IL-2 signaling results in lethal autoimmunity. In this study we investigated the role of tumor necrosis factor alpha (TNF) receptors type 1 (TNFR1) and type 2 (TNFR2) on the regulation of IL-2 at the level of transcription during the primary activation of CD4 T cells. We generated TNFR1- and/or TNFR2-deficient-IL-2-GFF knock-in reporter mice to examine Il2 promoter activity at the single cell level. We found that the deficiency of TNFR2, but not TNFR1, markedly diminished the number of CD4 T cells that produced IL-2. We further correlated decreased Il2 transcription with impaired intracellular c-Rel expression in TNFR2 KO CD4 T cells. IL-2 production was augmented in response to the addition of soluble TNF implicating a role for transmembrane TNF (tmTNF). Collectively, these data suggest that CD4 T cell autonomous tmTNF/TNFR2 signaling contributes to the number of IL-2 producers by providing a critical third signal necessary for threshold Il2 promoter activity. These results have important implications for understanding the contribution of IL-2 signaling on the dynamic equilibrium between Teff cells and Treg cells and the outcome of inflammatory immune responses.

MiR-200c regulates IL8 expression by targeting IKBKB: a potential mediator of inflammation in leiomyoma pathogenesis.

We have previously reported that leiomyoma expressed lower levels of miR-200c and elevated IL8 as compared to paired myometrium. Here we addressed the regulatory functions of miR-200c on the expression of inflammatory mediators and cellular viability using leiomyomas and paired myometrium and their isolated primary smooth muscle cells. Our results indicated that gain-of function or knockdown of miR-200c in leiomyoma smooth muscle cells (LSMC) regulated IL8 mRNA and protein expression through direct targeting of IKBKB and alteration of NF-kB activity. Additionally, leiomyoma expressed higher levels of phosphorylated IKBKB with no significant difference in the level of IKBKB mRNA and protein as compared to matched myometrium. Gain-of function of miR-200c in LSMC resulted in decreased IkBαphosphorylation and p65 nuclear translocation, which led to decreased p65 transcriptional activity of IL8 promoter, and increased caspase 3/7 activity which was not reversible following IL8 restoration. Collectively, our results suggest that NF-κB signaling pathway is a target of miR-200c regulatory function, and low level of miR-200c expression in leiomyoma by transcriptional regulation of inflammatory mediators such as IL8, in part account for development of leiomyomas.

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice

Polymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis.

Areca nut extracts exert different effects in oral cancer cells depending on serum concentration: A clue to the various oral alterations in betel quid chewers

Betel quid chewing is associated with various pathologic alterations in oral mucosa. However, the molecular mechanism behind so many contradictory alterations remains unclear. Here we aimed to build a model to facilitate the related studies in cultured cells. In our results, areca nut extract (ANE) was found to exert different effects in oral cells depending on the supplemented serum level. ANE strongly induced DNA damage, necrotic ballooning, and inflammatory cytokines under lower serum concentration while might convert to facilitate deregulated growth of serum-supplemented cells via modulating the activity/expression of factors such as E-cadherin and Snail. Despite ANE significantly activated NF-κB, a mediator critical for inflammation, inhibition of NF-κB did not prevent the activation of IL8 promoter. We further discovered Y705-dephosphorylated STAT3 might enhance IL8 transcription. Since necrosis and the inflammatory cytokines could cause massive inflammation, infiltration of interstitial fluid might potentiate cellular resistance against the acute cytotoxicity of ANE and further support the proliferation of transforming cells. Induction of VEGF and angiogenesis under lower serum condition also paved the way for cell growth and subsequent metastasis. Accordingly, we concluded that in correlation with serum infiltration ANE caused particular effects in oral cells and possibly the various clinicopathological alterations in vivo.

Sublytic C5b‐9 induces IL‐6 and TGF‐β1 production by glomerular mesangial cells in rat Thy‐1 nephritis through p300‐mediated C/EBPβ acetylation

CCAAT/enhancer-binding protein (C/EBPβ)-enhanced IL-6 and TGF-β1 promoter activity and p300-mediated C/EBPβ acetylation were involved in up-regulation of IL-6 and TGF-β1 expression in GMCs attacked by sublytic C5b-9. In detail, the elements of C/EBPβ binding to rat IL-6 and TGF-β1 promoter and 3 acetylated sites of rat C/EBPβ protein were first revealed. Furthermore, silencing the p300 or C/EBPβ gene in rat kidney significantly reduced the production of IL-6 and TGF-β1 and renal lesions in Thy-1N rats. Together, these data indicate that the mechanism of IL-6 and TGF-β1 production in renal tissue of Thy-1N rats is associated with sublytic C5b-9 up-regulated p300 and p300-mediated C/EBPβ acetylation as well as C/EBPβ-activated IL-6 and TGF-β1 genes.

Nuclear factor 45 of tongue sole (Cynoglossus semilaevis): Evidence for functional differentiation between two isoforms in immune defense against viral and bacterial pathogens

Nuclear factor 45 (NF45) is known to play an important role in regulating interleukin-2 expression in mammals. The function of fish NF45 is largely unknown. In a previous study, we reported the identification of a NF45 (named CsNF45) from half smooth tongue sole (Cynoglossus semilaevis). In the present study, we identified an isoform of CsNF45 (named CsNF45i) from half smooth tongue sole and examined its biological properties in comparison with CsNF45. We found that CsNF45i is a truncated version of CsNF45 and lacks the N-terminal 38 residues of CsNF45. Genetic analysis showed that the CsNF45 gene consists of 14 exons and 13 introns, and that CsNF45 and CsNF45i are the products of alternative splicing. Constitutive expression of CsNF45 and CsNF45i occurred in multiple tissues but differed in patterns. Experimental infection with viral and bacterial pathogens upregulated the expression of both isoforms but to different degrees, with potent induction of CsNF45 being induced by bacterial pathogen, while dramatic induction of CsNF45i being induced by viral pathogen. Transient transfection analysis showed that both isoforms were localized in the nucleus and able to stimulate the activity of IL-2 promoter to comparable extents. To examine their in vivo effects, the two isoforms were overexpressed in tongue sole. Subsequent analysis showed that following viral and bacterial infection, the viral loads in CsNF45i-overexpressing fish were significantly lower than those in CsNF45-overexpressing fish, whereas the bacterial loads in CsNF45-overexpressing fish were significantly lower than those in CsNF45i-overexpressing fish. These results indicate that both CsNF45 and CsNF45i possess immunoregulatory properties, however, the two isoforms most likely participate in different aspects of host immune defense that target different pathogens.

In oesophageal squamous cells exposed to acidic bile salt medium, omeprazole inhibits IL-8 expression through effects on nuclear factor-κB and activator protein-1

ObjectiveOesophagitis might result from the effects of chemokines produced by oesophageal cells in response to gastro-oesophageal reflux, and not solely from the direct, caustic effects of refluxed gastric juice. Proton...

Specific Inhibition of the Redox Activity of Ape1/Ref-1 by E3330 Blocks Tnf-Α-Induced Activation of Il-8 Production in Liver Cancer Cell Lines

APE1/Ref-1 is a main regulator of cellular response to oxidative stress via DNA-repair function and co-activating activity on the NF-κB transcription factor. APE1 is central in controlling the oxidative stress-based inflammatory processes through modulation of cytokines expression and its overexpression is responsible for the onset of chemoresistance in different tumors including hepatic cancer. We examined the functional role of APE1 overexpression during hepatic cell damage related to fatty acid accumulation and the role of the redox function of APE1 in the inflammatory process. HepG2 cells were stably transfected with functional and non-functional APE1 encoding plasmids and the protective effect of APE1 overexpression toward genotoxic compounds or FAs accumulation, was tested. JHH6 cells were stimulated with TNF-α in the presence or absence of E3330, an APE1 redox inhibitor. IL-8 promoter activity was assessed by a luciferase reporter assay, gene expression by Real-Time PCR and cytokines (IL-6, IL-8, IL-12) levels measured by ELISA. APE1 over-expression did not prevent cytotoxicity induced by lipid accumulation. E3330 treatment prevented the functional activation of NF-κB via the alteration of APE1 subcellular trafficking and reduced IL-6 and IL-8 expression induced by TNF-α and FAs accumulation through blockage of the redox-mediated activation of NF-κB. APE1 overexpression observed in hepatic cancer cells may reflect an adaptive response to cell damage and may be responsible for further cell resistance to chemotherapy and for the onset of inflammatory response. The efficacy of the inhibition of APE1 redox activity in blocking TNF-α and FAs induced inflammatory response opens new perspectives for treatment of inflammatory-based liver diseases.

Interleukin (IL)-32β-mediated CCAAT/Enhancer-binding Protein α (C/EBPα) Phosphorylation by Protein Kinase Cδ (PKCδ) Abrogates the Inhibitory Effect of C/EBPα on IL-10 Production

We previously reported that IL-32β promotes IL-10 production in myeloid cells. However, the underlying mechanism remains elusive. In this study, we demonstrated that IL-32β abrogated the inhibitory effect of CCAAT/enhancer-binding protein α (C/EBPα) on IL-10 expression in U937 cells. We observed that the phosphorylation of C/EBPα Ser-21 was inhibited by a PKCδ-specific inhibitor, rottlerin, or IL-32β knockdown by siRNA and that IL-32β shifted to the membrane from the cytosol upon phorbol 12-myristate 13-acetate treatment. We revealed that IL-32β suppressed the binding of C/EBPα to IL-10 promoter by using ChIP assay. These data suggest that PKCδ and IL-32β may modulate the effect of C/EBPα on IL-10 expression. We next demonstrated by immunoprecipitation that IL-32β interacted with PKCδ and C/EBPα, thereby mediating C/EBPα Ser-21 phosphorylation by PKCδ. We showed that IL-32β suppressed the inhibitory effect of C/EBPα on IL-10 promoter activity. However, the IL-10 promoter activity was reduced to the basal level by rottlerin treatment. When C/EBPα serine 21 was mutated to glycine (S21G), the inhibitory effect of C/EBPα S21G on IL-10 promoter activity was not modulated by IL-32β. Taken together, our results show that IL-32β-mediated C/EBPα Ser-21 phosphorylation by PKCδ suppressed C/EBPα binding to IL-10 promoter, which promoted IL-10 production in U937 cells.