Activator Protein-1 Activation Research Articles

PPARβ/δ prevents inflammation and fibrosis during diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is a specific type of myocardial disease that often develops in patients suffering from diabetes, which has become the foremost cause of death among them. It is an insidious multifactorial disease caused by complex and partially unknown mechanisms that include metabolic dysregulation, local inflammation, fibrosis, and cardiomyocyte apoptosis. Despite its severity and poor prognosis, it often goes undiagnosed, and there are currently no approved specific drugs to prevent or even treat it. Peroxisome proliferator-activated receptor (PPAR)β/δ is a key metabolic regulator that has been proposed as a potential target for DCM due to its pleiotropic anti-inflammatory properties. Diabetes was induced by multiple low-dose streptozotocin (STZ) administration in wild-type and PPARβ/δ knockout male mice treated with the PPARβ/δ agonist GW0742 or vehicle. Human cardiomyocytes (AC16) and mouse atrial myocytes (HL-1) exposed to hyperglycemia and treated with PPARβ/δ agonists were also used. PPARβ/δ deletion in mice negatively impacted cardiac morphology and function, which was accompanied by interstitial fibrosis and structural remodeling of the heart. This phenotype was further exacerbated in knockout diabetic mice. At the molecular level, PPARβ/δ suppression resulted in increased expression of pro-inflammatory and pro-fibrotic markers. Some of these markers were also induced by diabetes in wild-type mice and were exacerbated in diabetic knockout mice. The activity of the transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) correlated with most of these changes. Remarkably, PPARβ/δ activation partially prevented inflammation and fibrosis in the heart, as well as cardiac atrophy, induced during diabetes in mice, and also in cultured cardiomyocytes exposed to hyperglycemia. Finally, our results suggest that the beneficial effects of PPARβ/δ activation are mediated by the inhibition of mitogen-activated protein kinases (MAPK) activity and subsequent downregulation of the transcriptional activities of NF-κB and AP-1. Overall, the data suggest that PPARβ/δ agonists might be useful in preventing inflammation and fibrosis progression in DCM.

Regulatory Effects of Copper on Ghrelin Secretion in Rat Fundic Glands.

Copper (Cu) is an effective additive in feed for promoting growth. Growth dan axis comprising growth hormone (GH), somatostatin (SS) and GH-releasing hormone (GHRH), with ghrelin regulating their release. The growth-promoting effects of Cu are closely related to ghrelin, but the specific mechanism behind the relationship remains unknown. We investigated the adjustment of ghrelin synthesis and secretion by Cu. Sprague-Dawley rats were fed basal diets with an addition of 0, 120 or 240 mg/kg Cu sulfate for 28 day to establish a growth-promoting model. Signalling molecules relevant to ghrelin synthesis and secretion were detected and mechanistically explored using enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction and Western blot analysis. The 120 mg/kg supplement improved growth performance; significantly increased the serum levels of ghrelin, ghrelin O-acyltransferase (GOAT), acylated ghrelin (AG), GH, and reactive oxygen species (ROS) and decreased those of SS; significantly increased the mRNA and protein expression of ghrelin, GOAT, ghrelin receptor (GHS-R1α), and activator protein 1 (AP-1); increased the phosphorylation ratio of JNK and p38 MAPK; and inhibited the mRNA and protein expression of SS and SS receptor subtype 2 (SSTR2) in gastric fundic gland tissues. Thus, Cu may affect gastric ghrelin synthesis at the transcriptional level by activating the JNK/p38 MAPK pathway through increased ROS levels and regulating the activation of the downstream redox-sensitive transcription factor AP-1. SS plays a crucial determinant role in ghrelin regulation via intragastric Cu. Cu promotes GOAT activity and ghrelin secretion by inhibiting SS secretion, affecting AG levels, and promoting ghrelin acylation through ghrelin/GOAT/GHS-R1α system, modulating ghrelin secretion.

Echinochrome A inhibits HMGB1-induced vascular smooth muscle cell migration by suppressing osteopontin expression.

Echinochrome A (Ech A) isolated from marine organisms is a therapeutic effector for various cardiovascular diseases, but its precise mechanisms are unclear. This study identified the role and mechanisms mediating the effects of Ech A on the migration of vascular smooth muscle cells (VSMCs) induced by high-mobility group box 1 (HMGB1). Compared to the control cells, the migration of VSMCs stimulated with HMGB1 (100 ng/ml) was markedly increased, which was significantly attenuated in cells pretreated with MPIIIB10 (100 ng/ml), a neutralizing monoclonal antibody for osteopontin (OPN). In VSMCs stimulated with HMGB1, the increased expression of OPN mRNA and protein was accompanied by an increased OPN promoter activity. In reporter gene assays using OPN promoter-luciferase constructs, the promoter region 538-234 bp of the transcription start site containing the binding sites for activator protein 1 (AP-1) was shown to be responsible for the increased transcriptional activity by HMGB1. In addition, the binding activity of AP-1 was increased in HMGB1-stimulated cells, highlighting the pivotal role of AP-1 on OPN expression in HMGB1-stimulated VSMCs. An examination of the vascular effects of Ech A showed that the increased AP-1 binding/promoter activities and OPN expression induced by HMGB1 were attenuated in cells pretreated with Ech A (3 or 10 μM). Similarly, Ech A inhibited HMGB1-induced VSMC migration in a concentration-dependent manner. These findings suggest that Ech A inhibits VSMC migration by suppressing OPN expression. Hence, Ech A is suggested as a potential therapeutic strategy for vascular remodeling in the injured vasculatures.

Molecular mechanisms of transmitted endoplasmic reticulum stress mediating immune escape of gastric cancer via PVR overexpression in TAMs

Gastric cancer (GC) is the fourth leading cause of cancer death worldwide. Due to the complex tumor microenvironment (TME), the efficacy of immunotherapy in GC has not met expectations. Malignant changes in the TME induce endoplasmic reticulum stress (ERS). ERS can be transmitted between tumor cells and tumor-associated macrophages (TAMs), promoting tumor immune escape, but the specific mechanism in GC remains unclear. We established a TAM model of transmitted ERS (TERS), and iTRAQ proteomic analysis identified overexpressed proteins. The overexpression of poliovirus receptor (PVR) was screened while flow cytometry and ELISA showed that PVR mediated the immunosuppressive function of TAMs by downregulating the proliferative activity and cytotoxicity of cocultured CD8+ T lymphocytes. With EMSA and dual-luciferase reporter assays, we confirmed that erythropoietin-producing hepatocellular receptor A2 (EphA2) affected PVR expression by increasing the transcriptional activity of activator protein-1 (AP-1). MFC cells were mixed with EphA2 knockdown or control RAW264.7 cells to establish subcutaneous tumor models with or without tunicamycin treatment in vivo. The vivo experiments revealed that ERS promoted subcutaneous xenograft growth, which was reversed by EphA2 knockdown. Clinically, GC patients with high expression of PVR and EphA2 tended to have an immunosuppressive TME, which were determined by immunohistochemical and immunofluorescence analyses. In conclusion, the transcriptional activity of AP-1 is upregulated in ERS-transmitted TAMs through EphA2 to increase PVR expression, which promotes immune escape in GC. Our study provides a new perspective on the role of ERS in tumor immunity.

Blockade of foam cell formation by an AP-1 inhibitor attenuates atherosclerosis in diabetes

Abstract Background Atherosclerosis is a major contributor to cardiovascular disease (CVD) related premature deaths in people with diabetes. Residual increased risk of CVD remains even after treatment of standard risk factors including hyperglycaemia. Therefore, there is an unmet clinical need of novel therapies that can directly target the intrinsic pathobiology of atherosclerotic plaque development. Macrophage associated pro-atherosclerotic processes including impaired cholesterol efflux resulting in foam cell formation are important therapeutic targets in atherosclerosis. Expression of genes linked to impaired cholesterol efflux can be regulated by multiple factors including transcription factors (TFs) such as activator protein 1 (AP-1) complex. Although, previous studies have implicated the role of foam cells in atherosclerosis, the role of AP-1 TF complex in foam cell formation in diabetes induced atherosclerosis is not known. Methods Diabetes was induced with streptozotocin (STZ) in atheroprone Apoe-/- mice and 10 weeks later aortae were subjected to single cell RNA sequencing (scRNA-seq) analysis using 10X Genomics platform. In vitro model mimicking diabetes-induced foam cell formation was established in human monocytes THP-1 derived macrophages using high glucose (25mM) and ox-LDL ± a small molecular inhibitor of AP-1, T-5224. AP-1 activity, gene expression and foam cell formation were assessed with activity assay, RT PCR and Oil Red O staining respectively in this setting. Complementary in vitro AP-1 knockdown experiments using shRNA were also performed. Furthermore, preclinical intervention studies were also performed where Apoe-/- mice made diabetic with STZ were treated 5-weeks post diabetes with T-5224 (30 mg/kg body weight daily for 5 weeks). Results scRNA-seq analysis identified a well-defined cluster of foam cells within macrophages with a diabetes specific proatherogenic transcriptomic profile. Differential gene expression analysis showed increased expression of AP-1 members cFos, Atf3, Atf4 and Junb exclusively in foam cell cluster in diabetes (cut off = FDR<0.05, Log2FC -2/2). Comparison with the ENCODE dataset for AP-1 target genes using Harmonizome version 3 showed that indeed multiple differentially expressed genes in diabetes were targets of AP-1. AP-1 members were also upregulated in the THP-1-based in vitro model of high glucose induced foam cell formation. THP-1 derived foam cells showed increased AP-1 activity and lipid uptake. Interestingly, AP-1 inhibition by T-5224 attenuated foam cell formation. AP-1 knockdown experiments validated experimental findings of AP-1 inhibition studies in THP-1 derived macrophages. Importantly, T-5224 treatment blocked foam cell formation resulting in reduced atherosclerosis in diabetic Apoe-/- mice in our intervention study design. Conclusion This study identified the AP-1 inhibition with T-5224 as a potential treatment for the diabetes induced foam cell formation and associated atherosclerosis.

CRISPR/Cas9-mediated generation of AP-1 activity reporter cell line in human embryonic stem cell (WAe007-A-5)

Activator protein 1 (AP-1) is involved in cell fate determination and function. To monitor the AP-1 activity, we cloned a AP-1 binding sites fragment into the upstream of minimal TATA-box promoter, then a luciferase-GFP reporter (LuciGFP) was designated to the downstream of the promoter. With CRISPR/Cas9, the AP-1-LuciGFP reporter was introduced into AAVS1 locus, a safe harbor for gene editing. Thus, this AP-1-LuciGFP reporter cell line could be subjected to monitor the AP-1 activity during the cell differentiation, cell fate transition and disease modeling.

Ribosomal modification protein rimK-like family member A activates betaine-homocysteine S-methyltransferase 1 to ameliorate hepatic steatosis

Nonalcoholic fatty liver disease (NAFLD) is a serious threat to public health, but its underlying mechanism remains poorly understood. In screening important genes using Gene Importance Calculator (GIC) we developed previously, ribosomal modification protein rimK-like family member A (RIMKLA) was predicted as one essential gene but its functions remained largely unknown. The current study determined the roles of RIMKLA in regulating glucose and lipid metabolism. RIMKLA expression was reduced in livers of human and mouse with NAFLD. Hepatic RIMKLA overexpression ameliorated steatosis and hyperglycemia in obese mice. Hepatocyte-specific RIMKLA knockout aggravated high-fat diet (HFD)-induced dysregulated glucose/lipid metabolism in mice. Mechanistically, RIMKLA is a new protein kinase that phosphorylates betaine-homocysteine S-methyltransferase 1 (BHMT1) at threonine 45 (Thr45) site. Upon phosphorylation at Thr45 and activation, BHMT1 eliminated homocysteine (Hcy) to inhibit the activity of transcription factor activator protein 1 (AP1) and its induction on fatty acid synthase (FASn) and cluster of differentiation 36 (CD36) gene transcriptions, concurrently repressing lipid synthesis and uptake in hepatocytes. Thr45 to alanine (T45A) mutation inactivated BHMT1 to abolish RIMKLA’s repression on Hcy level, AP1 activity, FASn/CD36 expressions, and lipid deposition. BHMT1 overexpression rescued the dysregulated lipid metabolism in RIMKLA-deficient hepatocytes. In summary, RIMKLA is a novel protein kinase that phosphorylates BHMT1 at Thr45 to repress lipid synthesis and uptake. Under obese condition, inhibition of RIMKLA impairs BHMT1 activity to promote hepatic lipid deposition.

Senescent glia link mitochondrial dysfunction and lipid accumulation

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6–11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

MPT0E028, a novel pan-HDAC inhibitor, prevents pulmonary fibrosis through inhibition of TGF-β-induced CTGF expression in human lung fibroblasts: Involvement of MKP-1 activation

Histone deacetylase (HDAC) inhibitors are potential candidates for treating pulmonary fibrosis. MPT0E028, a novel pan-HDAC inhibitor, has been reported to exhibit antitumor activity in several cancer cell lines. In this study, we investigated the mechanism underlying the inhibitory effects of MPT0E028 on the expression of fibrogenic proteins in human lung fibroblasts (WI-38). Our results revealed that MPT0E028 inhibited transforming growth factor-β (TGF-β)-, thrombin-, and endothelin 1-induced connective tissue growth factor (CTGF) expression in a concentration-dependent manner. In addition, MPT0E028 suppressed TGF-β-stimulated expression of fibronectin, collagen I, and α-smooth muscle actin (α-SMA). Furthermore, MPT0E028 inhibited the TGF-β-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). MPT0E028 reduced the increase in SMAD3 and c-Jun phosphorylation, and SMAD3-and activator protein-1 (AP-1)-luciferase activities under TGF-β stimulation. Transfection with mitogen-activated protein kinase phosphatase-1 (MKP-1) siRNA reversed the suppressive effects of MPT0E028 on TGF-β-induced increases in CTGF expression; JNK, p38, and ERK phosphorylation; and SMAD3 and AP-1 activation. Moreover, MPT0E028 increased MKP-1 acetylation and activity in WI-38 cells. Pretreatment with MPT0E028 reduced the fibrosis score and fibronectin, collagen, and α-SMA expression in bleomycin-induced pulmonary fibrosis mice. In conclusion, MPT0E028 induced MKP-1 acetylation and activation, which in turn inhibited TGF-β-stimulated JNK, p38, and ERK phosphorylation; SMAD3 and AP-1 activation; and subsequent CTGF expression in human lung fibroblasts. Thus, MPT0E028 may be a potential drug for treating pulmonary fibrosis.

Antigen Presenting Cell activator protein 1 (AP-1) complex contributes to Salt-sensitive Blood Pressure

Salt-sensitivity of blood pressure (SSBP) affects 50% of the hypertensive and 25% of the normotensive population and is a major independent risk factor for cardiovascular morbidity and mortality. We previously found that SSBP is associated with activation of the NLRP3 inflammasome in antigen presenting cells (APCs) via increased oxidated stress and isolevuglandin (IsoLG) formation, but the underlying mechanisms are unknown. The activator protein 1 (AP-1) (FOS/JUN) has been implicated in activation of the NLRP3 inflammasome but its role in SSBP is not known. We hypothesized that AP-1 transcription factor in APCs senses elevated sodium and contributes to SSBP. We further hypothesize that antioxidant flavonoid, diosmetin prevents AP-1 activation and SSBP. Using bulk RNA-sequencing in human monocytes, we found elevated sodium increases expression of the AP-1 gene family when compared to normal sodium concentration, including cFOS (2378.18 ± 480.7 vs 6494.09 ± 945.55, p= 0.0009), FOSB (53.63 ± 17. 55 vs 131.06 ± 10.30, p= 0.397), cJUN (7313.90 ± 984.93 vs 11370.09 ± 1286.35, p= 0.2563) JUNB (4218.4.36 ± 199.17 vs 570.45 ± 200.91, p= 0.0445) and JUND (3309. 63 ± 270.64 vs 8057.90 ± 1043.05, p= 0.00006). In additional experiments, we enrolled people with hypertension and phenotype them for SSBP using an established in-patient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses in vivo on peripheral blood mononuclear cells (PBMCs). We found that expression of the FOSB and JUNB genes are more sensitive in concert with blood pressure in salt-sensitive (SS) but not salt-resistant (SR) humans. Moreover, diosmetin attenuated high salt-induced hypertension, 123.5 ± 4.1 vs 146.8 ± 2.5 mm Hg in controls, **p< 0.01). In addition, diosmetin also reduced high salt-induced inflammation including APC production of IsoLG-protein adducts, IL-1β, and NLRP3 inflammasome expression. These findings reveal a role for immune cell AP-1 (FOS/JUN) signaling in salt-sensitive hypertension and diosmetin may provide a potential therapeutic target for treatment of SSBP. 1R01HL144941-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Synergistic effect of Toll-like receptor 2 ligands and alendronate on proinflammatory cytokine production in mouse macrophage-like RAW-ASC cells is accompanied by upregulation of MyD88 expression

ObjectivesToll-like receptors (TLRs) recognize whole cells or components of microorganisms. Alendronate (ALN) is an anti-bone-resorptive drug that has inflammatory side effects. The aim in this study was to examine whether ALN augments TLR2 ligand-induced proinflammatory cytokine production using mouse macrophage-like RAW264.7 cells transfected with murine apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) gene (hereafter, referred to as “RAW-ASC cells”). MethodsRAW-ASC cells were pretreated with or without ALN and then incubated with or without TLR2 ligands. The levels of secreted mouse IL-1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in culture supernatants and the activation of activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) were measured using enzyme-linked immunosorbent assay (ELISA). The expressions of myeloid differentiation factor 88 (MyD88), caspase-11, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), ASC, NF-κB p65, and actin were analyzed via Western blotting. TLR2 expression was analyzed using flow cytometry. ResultsALN substantially upregulated the Pam3CSK4-induced release of IL-1α, IL-1β, IL-6, and TNF-α and MyD88 expression in RAW-ASC cells. ST-2825, a MyD88 inhibitor, inhibited the ALN-augmented release of these cytokines. Pretreatment with ALN augmented Pam3CSK4-induced NF-κB activation in RAW-ASC cells and upregulated AP-1 activation. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein and ALN synergically upregulated the release of IL-1α, IL-1β, IL-6 and TNF-α in RAW-ASC cells. ConclusionsOur findings suggest that ALN augments TLR2 ligand-induced proinflammatory cytokine production via the upregulation of MyD88 expression, and this augmentation is accompanied by the activation of NF-κB and AP-1 in RAW-ASC cells.

Integration of ATAC-seq and RNA-seq identifies MX1-mediated AP-1 transcriptional regulation as a therapeutic target for Down syndrome

BackgroundGrowing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation.MethodsWe performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS.ResultsBinding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs.ConclusionsThis study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.

Early Response Transcription Factors in Erythroid Regeneration

Acute anemia activates distinct mechanisms from physiological erythropoiesis to increase red blood cell production. While erythroid regeneration in this context requires some critical cytokines (e.g., BMP-4), little is known about transcription factors (TFs) connecting growth factor stimulation to anemia-specific gene expression and cellular responses. We previously discovered that GATA1 and TAL1 control a cohort of composite cis-elements which are anemia-activated in erythroid progenitors and precursors during regeneration. Intriguingly, cis-regulatory activity and gene activation at these loci are delayed by several days following initial anemia onset by the red blood cell lysing chemical phenylhydrazine. This delay suggested that these elements were utilized for erythroid precursor expansion but not activation. We hypothesize that distinct TF mechanisms are early responders to erythroid regeneration. To identify early anemia response TFs, we compared transcript levels in mouse spleen Kit + erythroid progenitors at days 0 and 1 post-anemia (anemia upregulated genes; p<0.05, fold change>1.5). Among upregulated genes, only 10 were TFs, and 3 of them ( Jun, Junb, Fos) are components of Activator protein 1 (AP-1) transcription factor. All 3 transcripts were subsequently downregulated at 3 days after anemia. In physiological hematopoiesis, AP-1 is highly expressed in most cells but low within the erythroid lineage. These findings suggested transient anemia-activated AP-1 may initiate erythroid regeneration. While AP-1 is a big family, only JUN and FOS family exhibited transient anemia-activation. Genes containing AP-1 binding motifs were cell intrinsically upregulated post-anemia in Jun and Fos-expressing cells. To determine Jun TF activity in anemia, we performed phospho-flow cytometry in Lin- spleen cells from mice after inducing anemia. We discovered that at Day 1 vs Control, the median fluorescence intensity (MFI) of Kit +CD71 +Ter119 + cells stained with total Jun or phospho-Jun (p-Jun) antibodies increased 2.0-fold (p=0.03) and 2.9-fold (p=0.04), respectively. Concurring with transcript levels, both total and p-Jun protein increased between 6- and 24-hours post-anemia and decreased at 72 hours. These changes post-anemia were absent in non-erythroid (CD71 -Ter119 -) and Kit - cells. These data demonstrated a transient increase in JUN TF activity early during the anemia response in erythroid progenitor/precursors. AP-1 activity is commonly elevated in response to a variety of stressors, including hypoxia and inflammation. Given the limited sets of transcripts upregulated at early anemia time points, direct AP-1 targets may be required for establishing an anemia-dependent transcriptome. To identify AP-1 target genes in erythroid regeneration, we selected genetic loci nearby (distance <100kb) AP-1-occupied cis-elements in human erythroleukemia (K562) or mouse erythroid progenitor cells (GEO: GSE31477, 208960, 170378, 172818). 50 potential AP-1 target genes were upregulated in stress erythroid precursors on day 3 vs day 1 post-anemia. Seven anemia-regulated transcription factors (SOX6, E2F2, TAL1, TFDP2, FOXO3, GFI1B, MAFG) reside at loci containing candidate AP-1 cis-elements. The T-cell Acute Lymphoblastic Leukemia-1 (TAL1) TF cooperates with GATA1 at E-box-GATA composite cis-elements to promote transcription of a number of erythroid-specific genes, several of which are needed for effective erythroid regeneration. Knockdown approaches will be used to test whether Jun expression and activity promotes the anemia-dependent transcriptome, stress erythroid precursor differentiation, and cell survival during erythroid regeneration. Given that Jun can be activated by BMP-4 signaling via the Jun kinase (JNK) pathway, and the requirement for BMP-4 in erythroid regeneration, we are investigating whether BMP-4 stimulation (or other cytokines) phosphorylates Jun to establish the regenerative transcriptome at early time points post-anemia. These studies will improve our understanding of transcriptional mechanisms driving erythroid regenerative responses, which can be useful for identifying new therapeutic targets for acute/chronic anemias.

Alendronate augments lipid A‑induced IL‑1β release by ASC‑deficient RAW264 cells via AP‑1 activation.

Alendronate (ALN) is an anti-bone-resorptive drug with inflammatory side effects. ALN upregulates lipid A-induced interleukin (IL)-1α and IL-1β release by J774.1 cells via apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) activation. The present study examined whether ALN augmented lipid A-induced proinflammatory cytokine production using ASC-deficient mouse macrophage-like RAW264 cells. Pretreatment of RAW264 cells with ALN significantly augmented lipid A-induced IL-1β release, although ALN did not upregulate the expression of Toll-like receptor 4, myeloid differentiation factor 88 (MyD88) and caspase-11. Moreover, pretreatment of caspase-11-deficient RAW264.7 cells with ALN significantly augmented lipid A-induced IL-1β release. Notably, ALN upregulated the activation of FosB, c-Jun or JunD, but not c-Fos or NF-κB in RAW264 cells. Furthermore, pretreatment with the activator protein 1 (AP-1) inhibitor SR11302, but not the c-Fos inhibitor T-5224, before addition of ALN inhibited ALN-augmented IL-1β release by lipid A-treated RAW264 cells. SR11302 also reduced ALN-augmented lactate dehydrogenase release by the cells. These findings collectively suggested that ALN augmented lipid A-induced IL-1β release and cell membrane damage in ASC-deficient RAW264 cells via activation of AP-1, but not NF-κB.

Thrombin-Induced COX-2 Expression and PGE2 Synthesis in Human Tracheal Smooth Muscle Cells: Role of PKCδ/Pyk2-Dependent AP-1 Pathway Modulation.

In this study, we confirmed that thrombin significantly increases the production of COX-2 and PGE2 in human tracheal smooth muscle cells (HTSMCs), leading to inflammation in the airways and lungs. These molecules are well-known contributors to various inflammatory diseases. Here, we investigated in detail the involved signaling pathways using specific inhibitors and small interfering RNAs (siRNAs). Our results demonstrated that inhibitors targeting proteins such as protein kinase C (PKC)δ, proline-rich tyrosine kinase 2 (Pyk2), c-Src, epidermal growth factor receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), or activator protein-1 (AP-1) effectively reduced thrombin-induced COX-2 and PGE2 production. Additionally, transfection with siRNAs against PKCδ, Pyk2, c-Src, EGFR, protein kinase B (Akt), or c-Jun mitigated these responses. Furthermore, our observations revealed that thrombin stimulated the phosphorylation of key components of the signaling cascade, including PKCδ, Pyk2, c-Src, EGFR, Akt, and c-Jun. Thrombin activated COX-2 promoter activity through AP-1 activation, a process that was disrupted by a point-mutated AP-1 site within the COX-2 promoter. Finally, resveratrol (one of the most researched natural polyphenols) was found to effectively inhibit thrombin-induced COX-2 expression and PGE2 release in HTSMCs through blocking the activation of Pyk2, c-Src, EGFR, Akt, and c-Jun. In summary, our findings demonstrate that thrombin-induced COX-2 and PGE2 generation involves a PKCδ/Pyk2/c-Src/EGFR/PI3K/Akt-dependent AP-1 activation pathway. This study also suggests the potential use of resveratrol as an intervention for managing airway inflammation.

FRI109 D-Allulose Inhibits NF-kB- And AP1-Mediated Inflammatory Pathways In HepG2 Cells

Abstract Disclosure: N. Gupta: None. J. Batch-Joudi: None. L.D. Graham: None. K. Champagne: None. F. Warda: None. M.J. Haas: None. A. Mooridian: None. Nothing to Disclose: NG, J B-J, LG, KC, FW, MJH, ADM. Source of Research Support: No outside funding was used for this research. Rare sugars such as D-allulose and its derivatives are defined as monosaccharides that are rare in nature. D-allulose is an epimer of D-fructose and is used primarily as a non-nutritive sweetener. D-allulose is also a functional food due to its ability to lower fasting plasma glucose and post-prandial glucose levels in both normoglycemic and diabetics, most likely due to its insulin-mimetic properties as well as its ability to enhance glucagon-like peptide 1. D-alluose has also been shown to possess anti-inflammatory and immunosuppressant activity in allogenic orthotopic liver transportation, neutrophil activation, and monocyte chemoattractant protein-1 expression in endothelial cells. We examined the effects of D-allulose on two well characterized pathways that promote inflammation in HepG2 hepatoblastoma cells, specifically nuclear factor κB (NF-κB) and activator protein 1 (AP1). HepG2 cells were exposed to tumor necrosis factor α (TNFα) (10 ng/ml), lipopolysaccharide (LPS) (10 nM), and high dextrose (27.5 mM). Treatment with 0, 2.8, 5.5, 11, 22, and 27.5 mM D-allulose decreased TNFα-dependent NF-κB and AP1 reporter plasmid activity and 2.8- and 5.5-mM D-alluose decreased NF-κB p50 and p65 subunit expression, measured by Western blot. D-allulose treatment increased IκB expression in TNFα treated cells. D-alluose also suppressed c-jun, phospho-c-jun, and c-fos AP1 subunit expression in TNFα treated cells. Similar effects were observed in D-allulose-treated cells (2.8 and 5.5 mM) exposed to high dextrose and LPS. These results suggest that D-allulose mediates some of its anti-inflammatory effects via suppressing NF-κB and AP1 activity. Presentation: Friday, June 16, 2023

PD-1 instructs a tumor-suppressive metabolic program that restricts glycolysis and restrains AP-1 activity in T cell lymphoma

The PDCD1-encoded immune checkpoint receptor PD-1 is a key tumor suppressor in T cells that is recurrently inactivated in T cell non-Hodgkin lymphomas (T-NHLs). The highest frequencies of PDCD1 deletions are detected in advanced disease, predicting inferior prognosis. However, the tumor-suppressive mechanisms of PD-1 signaling remain unknown. Here, using tractable mouse models for T-NHL and primary patient samples, we demonstrate that PD-1 signaling suppresses T cell malignancy by restricting glycolytic energy and acetyl coenzyme A (CoA) production. In addition, PD-1 inactivation enforces ATP citrate lyase (ACLY) activity, which generates extramitochondrial acetyl-CoA for histone acetylation to enable hyperactivity of activating protein 1 (AP-1) transcription factors. Conversely, pharmacological ACLY inhibition impedes aberrant AP-1 signaling in PD-1-deficient T-NHLs and is toxic to these cancers. Our data uncover genotype-specific vulnerabilities in PDCD1-mutated T-NHL and identify PD-1 as regulator of AP-1 activity.

The potential of activator protein 1 (AP-1) in cancer targeted therapy.

Activator protein-1 (AP-1) is a transcription factor that consists of a diverse group of members including Jun, Fos, Maf, and ATF. AP-1 involves a number of processes such as proliferation, migration, and invasion in cells. Dysfunctional AP-1 activity is associated with cancer initiation, development, invasion, migration and drug resistance. Therefore, AP-1 is a potential target for cancer targeted therapy. Currently, some small molecule inhibitors targeting AP-1 have been developed and tested, showing some anticancer effects. However, AP-1 is complex and diverse in its structure and function, and different dimers may play different roles in different type of cancers. Therefore, more research is needed to reveal the specific mechanisms of AP-1 in cancer, and how to select appropriate inhibitors and treatment strategies. Ultimately, this review summarizes the potential of combination therapy for cancer.

Design of an Herbal Preparation Composed by a Combination of Ruscus aculeatus L. and Vitis vinifera L. Extracts, Magnolol and Diosmetin to Address Chronic Venous Diseases through an Anti-Inflammatory Effect and AP-1 Modulation

Chronic venous disease (CVD) is an often underestimated inflammatory pathological condition that can have a serious impact on quality of life. Many therapies have been proposed to deal with CVD, but unfortunately the symptoms recur with increasing frequency and intensity as soon as treatments are stopped. Previous studies have shown that the common inflammatory transcription factor AP-1 (activator protein-1) and nuclear factor kappa-activated B-cell light chain enhancer (NF-kB) play key roles in the initiation and progression of this vascular dysfunction. The aim of this research was to develop a herbal product that acts simultaneously on different aspects of CVD-related inflammation. Based on the evidence that several natural components of plant origin are used to treat venous insufficiency and that magnolol has been suggested as a putative modulator of AP-1, two herbal preparations based on Ruscus aculeatus root extracts, and Vitis vinifera seed extracts, as well as diosmetin and magnolol, were established. A preliminary MTT-based evaluation of the possible cytotoxic effects of these preparations led to the selection of one of them, named DMRV-2, for further investigation. First, the anti-inflammatory efficacy of DMRV-2 was demonstrated by monitoring its ability to reduce cytokine secretion from endothelial cells subjected to LPS-induced inflammation. Furthermore, using a real-time PCR-based protocol, the effect of DMRV-2 on AP-1 expression and activity was also evaluated; the results obtained demonstrated that the incubation of the endothelial cells with this preparation almost completely nullified the effects exerted by the treatment with LPS on AP-1. Similar results were also obtained for NF-kB, whose activation was evaluated by monitoring its distribution between the cytosol and the nucleus of endothelial cells after the different treatments.

Microtubule Acetylation-Specific Inhibitors Induce Cell Death and Mitotic Arrest via JNK/AP-1 Activation in Triple-Negative Breast Cancer Cells.

Microtubule acetylation has been proposed as a marker of highly heterogeneous and aggressive triple-negative breast cancer (TNBC). The novel microtubule acetylation inhibitors GM-90257 and GM-90631 (GM compounds) cause TNBC cancer cell death but the underlying mechanisms are currently unknown. In this study, we demonstrated that GM compounds function as anti-TNBC agents through activation of the JNK/AP-1 pathway. RNA-seq and biochemical analyses of GM compound-treated cells revealed that c-Jun N-terminal kinase (JNK) and members of its downstream signaling pathway are potential targets for GM compounds. Mechanistically, JNK activation by GM compounds induced an increase in c-Jun phosphorylation and c-Fos protein levels, thereby activating the activator protein-1 (AP-1) transcription factor. Notably, direct suppression of JNK with a pharmacological inhibitor alleviated Bcl2 reduction and cell death caused by GM compounds. TNBC cell death and mitotic arrest were induced by GM compounds through AP-1 activation in vitro. These results were reproduced in vivo, validating the significance of microtubule acetylation/JNK/AP-1 axis activation in the anti-cancer activity of GM compounds. Moreover, GM compounds significantly attenuated tumor growth, metastasis, and cancer-related death in mice, demonstrating strong potential as therapeutic agents for TNBC.