Analyzing the impact of ATF3 in tumorigenesis and immune cell infiltration of ovarian tumor: a bioinformatics study.

ATF3 is an essential transcription activator in regulating cancer-related genetic expression. To identify the role of ATF3 in ovarian tumor, we investigated the correlation between ATF3 expression and the clinicopathological properties using multiple database. The cBioPortal and GEPIA database displayed the clinical information of ovarian patients harboring or without harboring ATF3 mutation. Furthermore, we assessed the relationship between survival and ATF3 expression level using Kaplan-Meier plotter, which reveals that the ovarian patients with higher expression of ATF3 suffered the worse overall survival and progression-free survival. The differentially expressed genes were analyzed using gene ontology, protein-protein interaction network, and gene set enrichment analysis to identify the hub gene and critical pathways, significantly affecting the tumorigenesis of ovarian tumor. Finally, we assessed the correlation between ATF3 and immune cell infiltration using Tumor Immunoassay Resource (TIMER) database. The results demonstrated that higher expression has a positive correlation with macrophage infiltration, expression for M1- and M2-type macrophages. Our study suggests that ATF3 can regulate the cell cycle and heme-related oxidative phosphorylation process, and it may be a critical factor to regulate the macrophage cell to be infiltrated into ovarian cancer. ATF3 can be used as a biomarker for diagnosis and therapy of ovarian tumor.

Critical Role of Intestinal Microbiota in ATF3-Mediated Gut Immune Homeostasis.

Secretory Ig A (sIgA) plays an important role in the maintenance of intestinal homeostasis via cross-talk with gut microbiota. The defects in sIgA production could elicit dysbiosis of commensal microbiota and subsequently facilitate the development of inflammatory bowel disease. Our previous study revealed activating transcription factor 3 (ATF3) as an important regulator of follicular helper T (TFH) cells in gut. ATF3 deficiency in CD4+ T cells impaired the development of gut TFH cells, and therefore diminished sIgA production, which increased the susceptibility to murine colitis. However, the potential role of microbiota in ATF3-mediated gut homeostasis remains incompletely understood. In this study, we report that both Atf3-/- and CD4creAtf3fl/fl mice displayed profound dysbiosis of gut microbiota when compared with their littermate controls. The proinflammatory Prevotella taxa, especially Prevotella copri, were more abundant in ATF3-deficient mice when compared with littermate controls. This phenotype was obviously abrogated by adoptive transfer of either TFH cells or IgA+ B cells. Importantly, depletion of gut microbiota dramatically alleviated the severity of colitis in Atf3-/- mice, whereas transfer of microbiota from Atf3-/- mice to wild-type recipients increased their susceptibility to colitis. Collectively, these observations indicate the importance of IgA-microbiota interaction in ATF3-mediated gut homeostasis.

Activating transcription factor 3 modulates the macrophage immune response to Mycobacterium tuberculosis infection via reciprocal regulation of inflammatory genes and lipid body formation.

Infection of macrophages by Mycobacterium tuberculosis elicits an immune response that clears the bacterium. However, the bacterium is able to subvert the innate immune response. Differential expression of transcription factors (TFs) is central to the dynamic balance of this interaction. Among other functions, TFs regulate the production of antibacterial agents such as nitric oxide, pro-inflammatory cytokines and neutral lipids which are stored in lipid bodies (LBs) and favour bacterial survival. Here, we demonstrate that the TF activating transcription factor 3 (ATF3) is upregulated early during infection of macrophages or mice. Depletion of ATF3 enhances mycobacterial survival in macrophages suggesting its host-protective role. ATF3 interacts with chromatin remodelling protein brahma-related gene 1 and both associate with the promoters of interleukin-12p40, interleukin-6 and nitric oxide synthase 2, to activate expression of these genes. Strikingly, ATF3 downregulates LB formation by associating at the promoters of positive regulators of LB formation such as cholesterol 25 hydroxylase and the microRNA-33 locus. ATF3 represses the association of the activating mark, acetyl histone H4 lysine 8 at the promoter of cholesterol 25 hydroxylase. Our study suggests opposing roles of ATF3 in regulation of distinct sets of macrophage genes during infection, converging on a host-protective immune response.

Transcriptional factor ATF3 protects against colitis by regulating follicular helper T cells in Peyer's patches.

Disruption of mucosal immunity plays a critical role in the pathogenesis of inflammatory bowel disease, yet its mechanism remains not fully elucidated. Here, we found that activating transcription factor 3 (ATF3) protects against colitis by regulating follicular helper T (TFH) cells in the gut. The expression of ATF3 in CD4+ T cells was negatively correlated with the severity of ulcerative colitis in clinical patients. Mice with ATF3 deficiency in CD4+ T cells (CD4creAtf3fl/fl ) were much more susceptible to dextran sulfate sodium-induced colitis. The frequencies of TFH cells, not other T cell subsets, were dramatically decreased in Peyer's patches from CD4creAtf3fl/fl mice compared with Atf3fl/fl littermate controls. The defective TFH cells significantly diminished germinal center formation and IgA production in the gut. Importantly, adoptive transfer of TFH or IgA+ B cells caused significant remission of colitis in CD4creAtf3fl/fl mice, indicating the TFH-IgA axis mediated the effect of ATF3 on gut homeostasis. Mechanistically, B cell lymphoma 6 was identified as a direct transcriptional target of ATF3 in CD4+ T cells. In summary, we demonstrated ATF3 as a regulator of TFH cells in the gut, which may represent a potential immunotherapeutic target in colitis.

ATF3 Stimulates IL-17A by Regulating Intracellular Ca2+/ROS-Dependent IL-1ß Activation During Streptococcus pneumoniae Infection.

Activating transcription factor-3 (ATF3) in the ER stress pathway induces cytokine production and promotes survival during gram-positive bacterial infection. IL-17A is a critical cytokine that is essential for clearance of Streptococcus pneumoniae. However, the mechanism by which ATF3 induces IL-17A production remains unknown. Here, we show that ATF3 induces IL-17A production via NLRP3 inflammasome-dependent IL-1ß secretion. Survival rates were comparable in IL-17A-depleted and ATF3 KO mice but were lower than in WT mice treated with isotype control, indicating that ATF3 positively regulated IL-17A production. Indeed, ATF3 KO mice showed a marked reduction in IL-17A protein and mRNA expression compared to levels in WT mice. Moreover, mitochondrial IL-1ß production by bone marrow-derived macrophages was significantly reduced in ATF3 KO mice as a result of the disruption of cellular ROS and Ca2+ homeostasis. Accordingly, ATF3 KO mice displayed diminished survival and bacterial clearance following S. pneumoniae infection. Taken together, these data suggest a mechanism in which macrophage ATF3 promotes IL-17A production in γδ T cells to rapidly induce host defenses during early S. pneumoniae infection.

Overall Downregulation of mRNAs and Enrichment of H3K4me3 Change Near Genome-Wide Association Study Signals in Systemic Lupus Erythematosus: Cell-Specific Effects.

This study was designed to define gene expression and H3K4me3 histone modifications in T cells, B cells, and monocytes in systemic lupus erythematosus (SLE). Array studies of total peripheral blood mononuclear cells have demonstrated gene expression signatures related to neutrophils, interferon, and other inflammatory pathways. It is not clear how consistent these effects are across different cell types. In this study, RNA-seq and chromatin immunoprecipitation-seq were utilized to identify gene expression patterns and H3K4me3 histone modifications related to promoter activation in SLE. Across the three cell types, there was 55% concordance for gene expression changes related to SLE. Key conserved pathways were ribosome biogenesis among upregulated genes and heat shock response among downregulated genes. ETS family transcription factors (TFs) and STAT1 were revealed as common regulators by position weight matrices. When epigenetic changes were leveraged with gene expression, the pivotal TFs ATF3 and FOS were defined with ATF3 also cross-referencing with gene expression-identified TFs. Genome-wide association study (GWAS) single nucleotide polymorphisms associated with SLE were cross-referenced with both mRNA and H3K4me3 changes in SLE. Baseline mRNA expression and H3K4me3 peak height was higher at sites that cross-referenced with GWAS signals, however, all three cell types exhibited an overall decrease in expression of GWAS-associated RNAs differentially expressed in SLE. H3K4me3 changes in SLE were also enriched in GWAS-associated sites. In summary, the SLE disease process is associated with both shared and cell-specific changes in gene expression and epigenetics. Surprisingly, GWAS-associated RNAs were overall markedly decreased across all three cell types. TF analysis identified ATF3, FOS, STAT1, and ETS family members as critical, all pathways with a recognized relationship to the SLE disease process. GWAS signals clearly mark both cell-type specific changes in SLE as well as concordant changes across all three cell types. Interpretation of single nucleotide polymorphism effects in SLE will require tissue-specific mechanistic studies and therapeutics will require mechanistic studies in multiple cell types.

Neuroinflammation after Traumatic Brain Injury Is Enhanced in Activating Transcription Factor 3 Mutant Mice.

Traumatic brain injury (TBI) induces a neuroinflammatory response resulting in astrocyte and microglia activation at the lesion site. This involves upregulation of neuroinflammatory genes, including chemokines and interleukins. However, so far, there is lack of knowledge on transcription factors (TFs) modulating this TBI-associated gene expression response. Herein, we analyzed activating transcription factor 3 (ATF3), a TF encoding a regeneration-associated gene (RAG) predominantly studied in peripheral nervous system (PNS) injury. ATF3 contributes to PNS axon regeneration and was shown before to regulate inflammatory processes in other injury models. In contrast to PNS injury, data on ATF3 in central nervous system (CNS) injury are sparse. We used Atf3 mouse mutants and a closed-head weight-drop-based TBI model in adult mice to target the rostrolateral cortex resulting in moderate injury severity. Post-TBI, ATF3 was upregulated already at early time points (i.e,. 1-4 h) post-injury in the brain. Mortality and weight loss upon TBI were slightly elevated in Atf3 mutants. ATF3 deficiency enhanced TBI-induced paresis and hematoma formation, suggesting that ATF3 limits these injury outcomes in wild-type mice. Next, we analyzed TBI-associated RAG and inflammatory gene expression in the cortical impact area. In contrast to the PNS, only some RAGs (Atf3, Timp1, and Sprr1a) were induced by TBI, and, surprisingly, some RAG encoding neuropeptides were downregulated. Notably, we identified ATF3 as TF-regulating proneuroinflammatory gene expression, including CCL and CXCL chemokines (Ccl2, Ccl3, Ccl4, and Cxcl1) and lipocalin. In Atf3 mutant mice, mRNA abundance was further enhanced upon TBI compared to wild-type mice, suggesting immune gene repression by wild-type ATF3. In accord, more immune cells were present in the lesion area of ATF3-deficient mice. Overall, we identified ATF3 as a new TF-mediating TBI-associated CNS inflammatory responses.

Marginal zone B cells control the response of follicular helper T cells to a high-cholesterol diet.

Splenic marginal zone B (MZB) cells, positioned at the interface between circulating blood and lymphoid tissue, detect and respond to blood-borne antigens. Here we show that MZB cells in mice activate a homeostatic program in response to a high-cholesterol diet (HCD) and regulate both the differentiation and accumulation of T follicular helper (TFH) cells. Feeding mice an HCD resulted in upregulated MZB cell surface expression of the immunoregulatory ligand PDL1 in an ATF3-dependent manner and increased the interaction between MZB cells and pre-TFH cells, leading to PDL1-mediated suppression of TFH cell motility, alteration of TFH cell differentiation, reduced TFH abundance and suppression of the proatherogenic TFH response. Our findings reveal a previously unsuspected role for MZB cells in controlling the TFH-germinal center response to a cholesterol-rich diet and uncover a PDL1-dependent mechanism through which MZB cells use their innate immune properties to limit an exaggerated adaptive immune response.

Characterisation of Immune and Neuroinflammatory Changes Associated with Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) and associated neuropathic pain is a debilitating adverse effect of cancer treatment. Current understanding of the mechanisms underpinning CIPN is limited and there are no effective treatment strategies. In this study, we treated male C57BL/6J mice with 4 cycles of either Paclitaxel (PTX) or Oxaliplatin (OXA) over a week and tested pain hypersensitivity and changes in peripheral immune responses and neuroinflammation on days 7 and 13 post 1st injection. We found that both PTX and OXA caused significant mechanical allodynia. In the periphery, PTX and OXA significantly increased circulating CD4+ and CD8+ T-cell populations. OXA caused a significant increase in the percentage of interleukin-4+ lymphocytes in the spleen and significant down-regulation of regulatory T (T-reg) cells in the inguinal lymph nodes. However, conditional depletion of T-reg cells in OXA-treated transgenic DEREG mice had no additional effect on pain sensitivity. Furthermore, there was no leukocyte infiltration into the nervous system of OXA- or PTX-treated mice. In the peripheral nervous system, PTX induced expression of the neuronal injury marker activating transcription factor-3 in IB4+ and NF200+ sensory neurons as well as an increase in the chemokines CCL2 and CCL3 in the lumbar dorsal root ganglion. In the central nervous system, PTX induced significant astrocyte activation in the spinal cord dorsal horn, and both PTX and OXA caused reduction of P2ry12+ homeostatic microglia, with no measurable changes in IBA-1+ microglia/macrophages in the dorsal and ventral horns. We also found that PTX induced up-regulation of several inflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL11, CCL4, CCL3, IL-12p70 and GM-CSF) in the spinal cord. Overall, these findings suggest that PTX and OXA cause distinct pathological changes in the periphery and nervous system, which may contribute to chemotherapy-induced neuropathic pain.

Effect of lipid-bound apolipoprotein A-I cysteine mutant on ATF3 in RAW264.7 cells.

Activating transcription factor 3 (ATF3) is a TLR-induced repressor that plays an important role in the inhibition of specific inflammatory signals. We previously constructed recombinant high density lipoproteins (rHDL) (including rHDLWT, rHDLM, rHDL228 and rHDL74) and found that rHDL74 had a strong anti-inflammatory ability. In the present study, we investigate the roles of recombinant apolipoprotein A-I (ApoA-I) (rHDLWT) and its cysteine mutant HDLs (rHDLM, rHDL228 and rHDL74) on ATF3 function in RAW264.7 cells stimulated by lipopolysaccharide. Our results showed that compared with the LPS group, rHDL74 can decrease the level of TNF-α and IL-6, whereas rHDL228 increases their expression levels. RT-PCR and Western blotting results showed that compared with the LPS group, rHDL74, rHDLWT and rHDLM can markedly increase the expression level of ATF3, whereas the level of ATF3 decreases in the rHDL228 group. In summary, the different anti-inflammatory mechanisms of the ApoA-I cysteine mutants might be associated with the regulation of ATF3 level.

The citrus flavonoid naringenin confers protection in a murine endotoxaemia model through AMPK-ATF3-dependent negative regulation of the TLR4 signalling pathway.

Excessive activation of the TLR4 signalling pathway is critical for inflammation-associated disorders, while negative regulators play key roles in restraining TLR4 from over-activation. Naringenin is a citrus flavonoid with remarkable anti-inflammatory activity, but the mechanisms underlying its inhibition of LPS/TLR4 signalling are less clear. This study investigated the molecular targets and therapeutic effects of naringenin in vitro and in vivo. In LPS-stimulated murine macrophages, naringenin suppressed the expression of TNF-α, IL-6, TLR4, inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2) and NADPH oxidase-2 (NOX2). Naringenin also inhibited NF-κB and mitogen-activated protein kinase (MAPK) activation. However, it did not affect the IRF3 signalling pathway or interferon production, which upregulate activating transcription factor 3 (ATF3), an inducible negative regulator of TLR4 signalling. Naringenin was demonstrated to directly increase ATF3 expression. Inhibition of AMPK and its upstream calcium-dependent signalling reduced ATF3 expression and dampened the anti-inflammatory activity of naringenin. In murine endotoxaemia models, naringenin ameliorated pro-inflammatory reactions and improved survival. Furthermore, it induced AMPK activation in lung tissues, which was required for ATF3 upregulation and the enhanced anti-inflammatory activity. Overall, this study reveals a novel mechanism of naringenin through AMPK-ATF3-dependent negative regulation of the LPS/TLR4 signalling pathway, which thereby confers protection against murine endotoxaemia.

Identification and characterization of latency-associated peptide-expressing γδ T cells.

γδ T cells are a subset of lymphocytes specialized in protecting the host against pathogens and tumours. Here we describe a subset of regulatory γδ T cells that express the latency-associated peptide (LAP), a membrane-bound TGF-ß1. Thymic CD27+IFN-γ+CCR9+α4ß7+TCRγδ+ cells migrate to the periphery, particularly to Peyer's patches and small intestine lamina propria, where they upregulate LAP, downregulate IFN-γ via ATF-3 expression and acquire a regulatory phenotype. TCRγδ+LAP+ cells express antigen presentation molecules and function as antigen presenting cells that induce CD4+Foxp3+ regulatory T cells, although TCRγδ+LAP+ cells do not themselves express Foxp3. Identification of TCRγδ+LAP+ regulatory cells provides an avenue for understanding immune regulation and biologic processes linked to intestinal function and disease.

Toll-like receptors: Activation, signalling and transcriptional modulation.

Families of innate immune receptors serve as the bodies primary defence system by recognising and rapidly responding to infection by microorganisms or to endogenous danger signals and initiating inflammatory processes. Whilst Toll-like receptors (TLRs) were the first family to be discovered, important and exciting discoveries continue to emerge into the molecular mechanisms that control their activation and regulation. Herein, I will provide an overview of TLR activation and their downstream signalling cascades, and discuss some of the recent findings concerning the assembly of a TLR oligomeric signalling platform, known as the Myddosome. Further, a brief examination of the importance of crosstalk between multiple TLRs or between TLRs and other innate immune receptors for appropriate and coordinated immune responses will be presented. Finally, I will discuss the importance of mechanisms that regulate TLRs with a focus on the role of activating transcription factor 3 (ATF3) in modulating transcriptional responses downstream of TLRs.

PIKfyve, a class III lipid kinase, is required for TLR-induced type I IFN production via modulation of ATF3.

Type I IFN plays a key role in antiviral responses. It also has been shown that deregulation of type I IFN expression following abnormal activation of TLRs contributes to the pathogenesis of systemic lupus erythematosus. In this study, we find that PIKfyve, a class III lipid kinase, is required for endolysosomal TLR-induced expression of type I IFN in mouse and human cells. PIKfyve binds to phosphatidylinositol 3-phosphate and synthesizes phosphatidylinositol 3,5-bisphosphate, and plays a critical role in endolysosomal trafficking. However, PIKfyve modulates type I IFN production via mechanisms independent of receptor and ligand trafficking in endolysosomes. Instead, pharmacological or genetic inactivation of PIKfyve rapidly induces expression of the transcription repressor ATF3, which is necessary and sufficient for suppression of type I IFN expression by binding to its promoter and blocking its transcription. Thus, we have uncovered a novel phosphoinositide-mediated regulatory mechanism that controls TLR-mediated induction of type I IFN, which may provide a new therapeutic indication for the PIKfyve inhibitor.

Early growth response protein-1 mediates lipotoxicity-associated placental inflammation: role in maternal obesity.

Obesity is associated with low-grade chronic inflammation, which contributes to cellular dysfunction promoting metabolic disease. Obesity during pregnancy leads to a proinflammatory milieu in the placenta; however, the underlying causes for obesity-induced placental inflammation remain unclear. Here, we examine the mechanisms by which saturated fatty acids and inflammatory cytokines induce inflammation in placental trophoblasts. We conducted global transcriptomic profiling in BeWo cells following palmitate and/or TNFα treatment and gene/protein expression analyses of MAPK pathways and characterized downstream transcription factors directly regulating inflammatory cytokines. Microarray analysis revealed increased expression of genes regulating inflammation, stress response, and immediate early response in cytotrophoblasts in response to palmitic acid (PA), TNFα, or a combination of both (PA + TNFα). Both gene ontology and gene set enrichment analysis revealed MAPK and EGR-1 signaling to be upregulated in BeWo cells, which was confirmed via immunoblotting. Importantly, activation of JNK signaling was necessary for increased proinflammatory cytokine (IL-6, TNFα, and IL-8) and EGR1 mRNA. Consistent with the requirement of JNK signaling, ChIP analysis confirmed the recruitment of c-Jun and other MAPK-responsive immediate early factors on the EGR1 promoter. Moreover, recruitment of EGR-1 on cytokine promoters (IL-6, TNFα, and IL-8) and an impaired proinflammatory response following knockdown of EGR-1 suggested it as a central component of the mechanism facilitating inflammatory gene expression. Finally, akin to in vitro findings, term placenta from obese women also had both increased JNK and p38 signaling and greater EGR-1 protein relative to lean women. Our results demonstrate that lipotoxic insults induce inflammation in placental cells via activation of JNK/EGR-1 signaling.

Activating transcription factor 3 regulates immune and metabolic homeostasis.

Integration of metabolic and immune responses during animal development ensures energy balance, permitting both growth and defense. Disturbed homeostasis causes organ failure, growth retardation, and metabolic disorders. Here, we show that the Drosophila melanogaster activating transcription factor 3 (Atf3) safeguards metabolic and immune system homeostasis. Loss of Atf3 results in chronic inflammation and starvation responses mounted primarily by the larval gut epithelium, while the fat body suffers lipid overload, causing energy imbalance and death. Hyperactive proinflammatory and stress signaling through NF-κB/Relish, Jun N-terminal kinase, and FOXO in atf3 mutants deregulates genes important for immune defense, digestion, and lipid metabolism. Reducing the dose of either FOXO or Relish normalizes both lipid metabolism and gene expression in atf3 mutants. The function of Atf3 is conserved, as human ATF3 averts some of the Drosophila mutant phenotypes, improving their survival. The single Drosophila Atf3 may incorporate the diversified roles of two related mammalian proteins.

Ectopic expression of human MutS homologue 2 on renal carcinoma cells is induced by oxidative stress with interleukin-18 promotion via p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) signaling pathways.

Human MutS homologue 2 (hMSH2), a crucial element of the highly conserved DNA mismatch repair system, maintains genetic stability in the nucleus of normal cells. Our previous studies indicate that hMSH2 is ectopically expressed on the surface of epithelial tumor cells and recognized by both T cell receptor γδ (TCRγδ) and natural killer group 2 member D (NKG2D) on Vδ2 T cells. Ectopically expressed hMSH2 could trigger a γδ T cell-mediated cytolysis. In this study, we showed that oxidative stress induced ectopic expression of hMSH2 on human renal carcinoma cells. Under oxidative stress, both p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways have been confirmed to mediate the ectopic expression of hMSH2 through the apoptosis-signaling kinase 1 (ASK1) upstream and activating transcription factor 3 (ATF3) downstream of both pathways. Moreover, renal carcinoma cell-derived interleukin (IL)-18 in oxidative stress was a prominent stimulator for ectopically induced expression of hMSH2, which was promoted by interferon (IFN)-γ as well. Finally, oxidative stress or pretreatment with IL-18 and IFN-γ enhanced γδ T cell-mediated cytolysis of renal carcinoma cells. Our results not only establish a mechanism of ectopic hMSH2 expression in tumor cells but also find a biological linkage between ectopic expression of hMSH2 and activation of γδ T cells in stressful conditions. Because γδ T cells play an important role in the early stage of innate anti-tumor response, γδ T cell activation triggered by ectopically expressed hMSH2 may be an important event in immunosurveillance for carcinogenesis.

JNK inhibition by SP600125 attenuates trans-10, cis-12 conjugated linoleic acid-mediated regulation of inflammatory and lipogenic gene expression.

Supplementation with a mixture of trans-10, cis-12 (t10,c12) and cis-9, trans-11 (c9,t11) isomers of conjugated linoleic acid (CLA), or t10,c12 CLA alone, reduces body weight and fat deposition in animals and some humans. However, these anti-obesity actions of t10,c12 CLA are routinely accompanied by increased markers of inflammation and insulin resistance. Thus, we examined the extent to which blocking c-Jun NH2-terminal kinase (JNK) signaling using the JNK inhibitor SP600125 attenuated markers of inflammation and insulin resistance in primary human adipocytes treated with t10,c12 CLA. SP600125 attenuated t10,c12 CLA-mediated phosphorylation of cJun and increased protein levels of activating transcription factor (ATF) 3, two downstream targets of JNK. SP600125 attenuated t10,c12 CLA-mediated induction of inflammatory genes, including interleukin (IL)-6, IL-8, IL-1ß, ATF3, monocyte chemoattractant protein (MCP)-1, and cyclooxygenase-2. Consistent with these data, SP600125 prevented t10,c12 CLA-mediated secretion of IL-8, IL-6, and MCP-1. SP600125 prevented t10,c12 CLA suppression of lipogenic genes including peroxisome proliferator activated receptor gamma, liver X receptor, sterol regulatory element binding protein, acetyl-CoA carboxylase, and stearoyl-CoA desaturase. Additionally, SP600125 blocked t10,c12 CLA-mediated induction of suppressor of cytokine synthesis-3 and suppression of adiponectin and insulin-dependent glucose transporter 4 mRNA levels. Collectively, these data suggest that JNK signaling plays an important role in t10,c12 CLA-mediated regulation of inflammatory and lipogenic gene expression in primary cultures of human adipocytes.

Activating transcription factor 3 (ATF3) promotes sublytic C5b-9-induced glomerular mesangial cells apoptosis through up-regulation of Gadd45α and KLF6 gene expression.

The sublytic C5b-9 complexes can result in glomerular mesangial cells (GMCs) apoptosis, which involved in the initiation and development of rat Thy-1 nephritis. Activating transcription factor 3 (ATF3) is an immediate early gene for cells to cope with a variety of stress signals, and our previous study revealed that ATF3 could promote GMCs apoptosis attacked by sublytic C5b-9. But the mechanism of ATF3 promoting GMCs apoptosis triggered by sublytic C5b-9 attack has not been elucidated. In this study, the data showed that the expression of ATF3, growth arrest and DNA damage-45 alpha (Gadd45α), Krüppel-like factor 6 (KLF6) and proliferating cell nuclear antigen (PCNA) in the GMCs in response to sublytic C5b-9 stimulation for the indicated time was significantly increased, and ATF3 expression could lead to GMCs apoptosis through up-regulation of Gadd45α and KLF6, but not up-regulation of PCNA. Furthermore, Gadd45α was identified as a downstream target gene regulated by ATF3 directly, and KLF6 might be regulated by ATF3 in an indirect manner.

Determination of complementary antibody pairs using protein A capture with the AlphaScreen assay format.

The utility of antibody reagents for the detection of specific cellular targets for both research and diagnostic applications is widespread and continually expanding. Often it is useful to develop specific antibodies as reagent pairs that distinguish different epitopes of the target such that sandwich enzyme-linked immunosorbent assay can be used for selective and specific detection. However, the identification of pairing antibodies is often cumbersome and labor-intensive even with the use of designed peptide-specific epitopes as antigens. We have developed a robust and high-throughput method for identifying pairing complementary antibodies derived either from commercial sources or during a rabbit hybridoma monoclonal screening and selection process using protein A capture with the AlphaScreen bead-based assay format. We demonstrate the value and effectiveness of this assay with three protein targets: Akt2, ATF3, and NAEß (the ß-subunit of the neddylation activation enzyme).