AP-1 Subunits Research Articles

Spatial proteomics and transcriptomics of the maternal-fetal interface in placenta accreta spectrum

In severe Placenta Accreta Spectrum (PAS), trophoblasts gain deep access in the myometrium (placenta increta). This study investigated alterations at the fetal-maternal interface in PAS cases using a systems biology approach consisting of immunohistochemistry, spatial transcriptomics and proteomics. We identified spatial variation in the distribution of CD4+, CD3+ and CD8+ T-cells at the maternal-interface in placenta increta cases. Spatial transcriptomics identified transcription factors involved in promotion of trophoblast invasion such as AP-1 subunits ATF-3 and JUN, and NFKB were upregulated in regions with deep myometrial invasion. Pathway analysis of differentially expressed genes demonstrated that degradation of extracellular matrix (ECM) and class 1 MHC protein were increased in increta regions, suggesting local tissue injury and immune suppression. Spatial proteomics demonstrated that increta regions were characterised by excessive trophoblastic proliferation in an immunosuppressive environment. Expression of inhibitors of apoptosis such as BCL-2 and fibronectin were increased, while CTLA-4 was decreased and increased expression of PD-L1, PD-L2 and CD14 macrophages. Additionally, CD44, which is a ligand of fibronectin that promotes trophoblast invasion and cell adhesion was also increased in increta regions. We subsequently examined ligand receptor interactions enriched in increta regions, with interactions with ITGβ1, including with fibronectin and ADAMS, emerging as central in increta. These ITGβ1 ligand interactions are involved in activation of epithelial–mesenchymal transition and remodelling of ECM suggesting a more invasive trophoblast phenotype. In PAS, we suggest this is driven by fibronectin via AP-1 signalling, likely as a secondary response to myometrial scarring.

An AAGAB-to-CCDC32 handover mechanism controls the assembly of the AP2 adaptor complex

Vesicular transport relies on multimeric trafficking complexes to capture cargo and drive vesicle budding and fusion. Faithful assembly of the trafficking complexes is essential to their functions but remains largely unexplored. Assembly of AP2 adaptor, a heterotetrameric protein complex regulating clathrin-mediated endocytosis, is assisted by the chaperone AAGAB. Here, we found that AAGAB initiates AP2 assembly by stabilizing its α and σ2 subunits, but the AAGAB:α:σ2 complex cannot recruit additional AP2 subunits. We identified CCDC32 as another chaperone regulating AP2 assembly. CCDC32 recognizes the AAGAB:α:σ2 complex, and its binding leads to the formation of an α:σ2:CCDC32 ternary complex. The α:σ2:CCDC32 complex serves as a template that sequentially recruits the µ2 and β2 subunits of AP2 to complete AP2 assembly, accompanied by CCDC32 release. The AP2-regulating function of CCDC32 is disrupted by a disease-causing mutation. These findings demonstrate that AP2 is assembled by a handover mechanism switching from AAGAB-based initiation complexes to CCDC32-based template complexes. A similar mechanism may govern the assembly of other trafficking complexes exhibiting the same configuration as AP2.

Anti-Metastatic Effects of Standardized Polysaccharide Fraction from Diospyros kaki Leaves via GSK3β/β-Catenin and JNK Inactivation in Human Colon Cancer Cells.

A polysaccharide fraction from Diospyros kaki (PLE0) leaves was previously reported to possess immunostimulatory, anti-osteoporotic, and TGF-β1-induced epithelial-mesenchymal transition inhibitory activities. Although a few beneficial effects against colon cancer metastasis have been reported, we aimed to investigate the anti-metastatic activity of PLE0 and its underlying molecular mechanisms in HT-29 and HCT-116 human colon cancer cells. We conducted a wound-healing assay, invasion assay, qRT-PCR analysis, western blot analysis, gelatin zymography, luciferase assay, and small interfering RNA gene silencing in colon cancer cells. PLE0 concentration-dependently inhibited metastasis by suppressing cell migration and invasion. The suppression of N-cadherin and vimentin expression as well as upregulation of E-cadherin through the reduction of p-GSK3β and β-catenin levels resulted in the outcome of this effect. PLE0 also suppressed the expression and enzymatic activity of matrix metalloproteinases (MMP)-2 and MMP-9, while simultaneously increasing the protein and mRNA levels of the tissue inhibitor of metalloproteinases (TIMP-1). Furthermore, signaling data disclosed that PLE0 suppressed the transcriptional activity and phosphorylation of p65 (a subunit of NF-κB), as well as the phosphorylation of c-Jun and c-Fos (subunits of AP-1) pathway. PLE0 markedly suppressed JNK phosphorylation, and JNK knockdown significantly restored PLE0-regulated MMP-2/-9 and TIMP-1 expression. Collectively, our data indicate that PLE0 exerts an anti-metastatic effect in human colon cancer cells by inhibiting epithelial-mesenchymal transition and MMP-2/9 via downregulation of GSK3β/β-catenin and JNK signaling.

Abstract 6522: Epigenetic modulation decreases myeloid suppression to improve response to immune checkpoint inhibition in breast cancer

Abstract Immune checkpoint inhibitors (ICIs) promote a durable anti-tumor immune response. Unfortunately, many patients with breast cancer are thought to be intrinsically resistant. One potential mechanism of resistance is a myeloid suppressed tumor microenvironment (TME). Targeting myeloid suppression is under investigation as a therapeutic strategy to sensitize tumors to ICI. In our trial, NCI-9844, we demonstrated that the combination of the epigenetic modulator, entinostat with ICIs—nivolumab (anti-PD-1) + ipilimumab (anti-CTLA-4), leads to a 25% objective response rate in patients with advanced breast cancer. Work in our preclinical models showed entinostat decreases suppressive function by myeloid derived suppressor cells (MDSCs). We hypothesized that entinostat decreases intra-tumoral MDSC suppression via the STAT3/AP-1 axis and drives cellular shifts within the TME to improve response to ICI. Here, we examined expression of upstream, downstream, and interacting factors within the STAT3 pathway in MDSCs treated with entinostat. Macro-dissected lung metastases from entinostat treated NeuN mice revealed differential gene expression of AP-1 subunits JunB and FOSL1 in MDSCs identified by single cell RNA sequencing (scRNAseq). Western blot of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. In an entinostat treated MDSC-like cell line, J774M, JunB phosphorylation and FOSL1 were both decreased. scRNAseq also revealed decreased gene expression of STAT3 upstream receptors IL4RA and IFNGR1 and increased expression of STAT3 negative regulator SOCS3. Chromatin immunoprecipitation sequencing is planned to further narrow the molecular mechanism of action. Cell-cell communication analysis of scRNAseq data was performed to evaluate the contribution of entinostat treated macrophages, dendritic cells (DCs), and natural killer (NK) cells on MDSC function. We observed increased frequency of DC populations (classic DC2s and Ccr7 DCs). Following combined treatment with entinostat + ICIs, we also found increased DC to T cell signaling strength for receptor-ligand pairs involved in T cell activation, including Tnfsf9__Tnfrsf9, Cd70_Cd27, H2-dmb2_Cd4, and Cd80_Cd28. Analysis of macrophage-T cell interactions revealed a synergistic decrease in signaling associated with immunosuppression after combined treatment, such as Cd80_Ctla4, Tnf_Tnfrsf1b, and Entpd1_Adora2a. Suppressive function of treated sub-populations of macrophages is planned to determine functional effect of findings. Finally, we are using imaging mass cytometry and bulk RNA sequencing to evaluate proposed mechanisms in patient specimens (NCI-9844). The goal of this work is to identify the molecular and cellular mechanisms driving myeloid suppression of the TME thereby identifying potential therapeutic targets. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Jesse Kreger, Yingtong Liu, Batul Al-Zubeidy, Ludmila Danilova, Sarah M. Shin, Valerie H. Narumi, Sofi Castanon, Julie Jang, Elana J. Fertig, Ashley Cimino-Mathews, Elizabeth M. Jaffee, Vered Stearns, Roisin M. Connolly, Won Jin Ho, Adam MacLean, Evanthia T. Roussos Torres. Epigenetic modulation decreases myeloid suppression to improve response to immune checkpoint inhibition in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6522.

AP-1/C-FOS and AP-1/FRA2 differentially regulate early and late adipogenic differentiation of mesenchymal stem cells.

Obesity is defined as an abnormal accumulation of adipose tissue in the body and is a major global health problem due to increased morbidity and mortality. Adipose tissue is made up of adipocytes, which are fat-storing cells, and the differentiation of these fat cells is known as adipogenesis. Several transcription factors (TFs)such as CEBPβ, CEBPα, PPARγ, GATA, and KLFhave been reported to play a key role in adipogenesis. In this study, we report one more TFAP-1, which is found to be involved in adipogenesis. Human mesenchymal stem cells were differentiated into adipocytes, and the expression pattern of different subunits of AP-1 was examined during adipogenesis. It was observed that C-FOS was predominantly expressed at an early stage (Day 2), whereas FRA2 expression peaked at later stages (Days 6 and 8) of adipogenesis. Chromatin immunoprecipitation-sequencing analysis revealed that C-FOS binds mainly to the promoters of WNT1, miR-30a, and ANAPC7and regulates their expression during mitotic clonal expansion. In contrast, FRA2 binds to the promoters of CIDEA, NOTCH1, ARAF, and MYLK, regulating their expression and lipid metabolism. Data obtained clearly indicatethat the differential expression of C-FOS and FRA2 is crucial for different stages of adipogenesis. This also raises the possibility of considering AP-1 as a therapeutic target for treating obesity and related disorders.

JUN upregulation drives aberrant transposable element mobilization, associated innate immune response, and impaired neurogenesis in Alzheimer’s disease

Adult neurogenic decline, inflammation, and neurodegeneration are phenotypic hallmarks of Alzheimer’s disease (AD). Mobilization of transposable elements (TEs) in heterochromatic regions was recently reported in AD, but the underlying mechanisms are still underappreciated. Combining functional genomics with the differentiation of familial and sporadic AD patient derived-iPSCs into hippocampal progenitors, CA3 neurons, and cerebral organoids, we found that the upregulation of the AP-1 subunit, c-Jun, triggers decondensation of genomic regions containing TEs. This leads to the cytoplasmic accumulation of HERVK-derived RNA-DNA hybrids, the activation of the cGAS-STING cascade, and increased levels of cleaved caspase-3, suggesting the initiation of programmed cell death in AD progenitors and neurons. Notably, inhibiting c-Jun effectively blocks all these downstream molecular processes and rescues neuronal death and the impaired neurogenesis phenotype in AD progenitors. Our findings open new avenues for identifying therapeutic strategies and biomarkers to counteract disease progression and diagnose AD in the early, pre-symptomatic stages.

Early divergent responses to virulent and attenuated vaccine isolates of Flavobacterium covae sp. nov. In channel catfish, Ictalurus punctatus

Columnaris disease continues to inflict substantial losses among freshwater cultured species since its first description one hundred years ago. The experimental and anecdotal evidence suggests an expanded range and rising virulence of columnaris worldwide due to the warming global climate. The channel catfish (Ictalurus punctatus) are particularly vulnerable to columnaris. A recently developed live attenuated vaccine (17–23) for Flavobacterium columnare (now Flavobacterium covae sp. nov.) demonstrated superior protection for vaccinated catfish against genetically diverse columnaris isolates. In this study, we aimed to elucidate the molecular mechanisms and patterns of immune evasion and host manipulation linked to virulence by comparing gene expression changes in the host after the challenge with a virulent (BGSF-27) or live attenuated F. covae sp. nov. vaccine (17–23). Thirty-day-old fry were accordingly challenged with either virulent or vaccine isolates. Gill tissues were collected at 0 h (control), 1 h, and 2 h post-infection, which are two critical time points in early host-pathogen interactions. Transcriptome profiling of the gill tissues revealed a larger number (518) of differentially expressed genes (DEGs) in vaccine-exposed fish than those exposed to the virulent pathogen (321). Pathway analyses suggested potent suppression of early host immune responses by the virulent isolate through a higher expression of nuclear receptor corepressors (NCoR) responsible for antagonizing macrophage and T-cell signaling. Conversely, in vaccinated fry, we observed induction of Ca2+/calmodulin-dependent protein kinase II (CAMKII), responsible for clearing NCoR, and commensurate up-regulation of transcription factor AP-1 subunits, c-Fos, and c-Jun. As in mammalian systems, AP-1 expression was connected with a broad immune activation in vaccinated fry, including induction of CC chemokines, proteinases, iNOS, and IL-12b. Relatedly, divergent expression patterns of Src tyrosine kinase Lck, CD44, and CD28 indicated a delay or suppression of T-cell adhesion and activation in fry exposed to the virulent isolate. Broader implications of these findings will be discussed. The transcriptomic differences between virulent and attenuated bacteria may offer insights into how the host responds to the vaccination or infection and provide valuable knowledge to understand the early immune mechanisms of columnaris disease in aquaculture.

Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis.

Radiation pneumonitis is a critical complication that constrains the use of radiation therapy for thoracic malignancies, leading to substantial morbidity via respiratory distress and lung function impairment. The role of Natural killer (NK) cells in inflammatory diseases is well-documented; however, their involvement in radiation pneumonitis is not fully understood. To explore the involvement of NK cells in radiation pneumonitis, we analyzed tissue samples for NK cell presence and function. The study utilized immunofluorescence staining, western blotting, and immunoprecipitation to investigate CXCL10 and ROS levels, autophagy activity, and NKG2D receptor dynamics in NK cells derived from patients and animal models subjected to radiation. In this study, we observed an augmented infiltration of NK cells in tissues affected by radiation pneumonitis, although their function was markedly diminished. In animal models, enhancing NK cell activity appeared to decelerate the disease progression. Concomitant with the disease course, there was a notable upsurge in CXCL10 and ROS levels. CXCL10 was found to facilitate NK cell migration through CXCR3 receptor activation. Furthermore, evidence of excessive autophagy in patient NK cells was linked to ROS accumulation, as indicated by immunofluorescence and Western blot analyses. The association between the NKG2D receptor and its adaptor proteins (AP2 subunits AP2A1 and AP2M1), LC3, and lysosomes was intensified after radiation exposure, as demonstrated by immunoprecipitation. This interaction led to NKG2D receptor endocytosis and subsequent lysosomal degradation. Our findings delineate a mechanism by which radiation-induced lung injury may suppress NK cell function through an autophagy-dependent pathway. The dysregulation observed suggests potential therapeutic targets; hence, modulating autophagy and enhancing NK cell activity could represent novel strategies for mitigating radiation pneumonitis.

Protective effect of hydrangenol on lipopolysaccharide-induced endotoxemia by suppressing intestinal inflammation

Hydrangenol, a dihydroisocoumarin, isolated from the leaves of Hydrangea serrata, possesses anti-inflammatory, anti-obesity, and anti-photoaging activities. In this study, we investigated the protective effects of hydrangenol (HG) against lipopolysaccharide (LPS)-induced endotoxemia and elucidated the underlying molecular mechanisms of action in C57BL/6 mice. Oral administration of HG (20 or 40 mg/kg) significantly restored the survival rate and population of macrophages, T helper cells (CD3+/CD4+), and Th17 cells (CD3+/CD4+/CCR6+) in the spleens of mice with LPS-induced endotoxemia. HG suppressed the expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and Interferon (IFN)-γ and the mRNA and protein expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the intestine and lung of LPS-treated mice. Molecular data showed that HG ameliorated the activation of nuclear factor kappa B (NF-κB) p65, signal transducers and activators of transcription 3 (STAT3), and c-Fos and c-Jun (AP-1 subunits) via the myeloid differentiation primary response 88 (MyD88) dependent toll-like receptor 4 (TLR4) signaling pathway in the LPS-treated mouse intestines. HG treatment caused the recovery of LPS-induced impaired tight junction (occludin and claudin-2) protein and mRNA expressions. Furthermore, HG improved LPS-induced gut dysbiosis in mice. Taken together, our results suggest that HG protects against LPS-induced endotoxemia by restoring immune cells and the capacity of the intestinal barrier, reducing intestinal inflammation, and improving the composition of the gut microbiota.

The novel small molecule TPN10518 alleviates EAE pathogenesis by inhibiting AP1 to depress Th1/Th17 cell differentiation

Multiple sclerosis (MS) is one of the most common autoimmune diseases of central nervous system (CNS) demyelination. Experimental autoimmune encephalomyelitis (EAE) is the most classic animal model for simulating the onset of clinical symptoms in MS. Previous research has reported the anti-inflammatory effects of artemisinin on autoimmune diseases. In our study, we identified a novel small molecule, TPN10518, an artemisinin derivative, which plays a protective role on the EAE model. We found that TPN10518 reduced CNS inflammatory cell infiltration and alleviated clinical symptoms of EAE. In addition, TPN10518 downregulated the production of Th1 and Th17 cells in vivo and in vitro, and decrease the levels of related chemokines. RNA-seq assay combined with the experimental results demonstrated that TPN10518 lowered the mRNA and protein levels of the AP1 subunits c-Fos and c-Jun in EAE mice. It was further confirmed that TPN10518 was dependent on AP1 to inhibit the differentiation of Th1 and Th17 cells. The results suggest that TPN10518 reduces the production of Th1 and Th17 cells through inhibition of AP1 to alleviate the severity of EAE disease. It is expected to be a potential drug for the treatment of MS.

Abstract 5167: Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling

Abstract Metastatic breast cancer remains one of the leading causes of global cancer incidence in women, despite the benefit of immune checkpoint inhibitors (ICIs) in managing various cancers and improving patient quality of life. Metastatic breast cancer is characterized by extensive infiltration of the tumor microenvironment (TME) with immunosuppressive cells, such as myeloid derived suppressor cells (MDSCs), that inhibit anti-tumoral immune cells and prevent effective activation of the adaptive immune system by ICIs. Previously, our group has shown in the breast TME that epigenetic reprogramming of MDSCs by entinostat, a histone deacetylase inhibitor, decreases MDSC immunosuppressive function and enhances response to ICIs, in part mediated by altered signaling within the Signal transducer and activator of transcription 3 (STAT3) and Nuclear factor kappa B (NFκB) axis. Next, we sought to examine the effects of entinostat on MDSC reprogramming in a distant metastatic TME. Using the syngeneic NT2.5LM NeuN mouse model of metastatic breast cancer, we established spontaneous lung metastases and treated with either vehicle or entinostat (5 mg/kg by oral gavage, 5x/week) for 3 weeks. Single cell RNA sequencing (scRNAseq) of macro-dissected lung metastases revealed a large MDSC population, and unsupervised pathways analysis of the MDSC cluster showed differential regulation of the IL6-JAK-STAT3 and TNFα-signaling-via-NFκB pathways upon entinostat treatment. In line with reports of crosstalk among STAT3, NFκB, and AP-1 pathways regulating inflammation in breast cancer, we also found through scRNAseq differential expression of AP-1 subunits JunB and FOSL1 upon entinostat treatment. At the protein level, western blot analysis of isolated intratumoral MDSCs from lung metastases revealed decreased STAT3 phosphorylation upon entinostat treatment. Using J774M cells, an MDSC-like cell line, we found decreased JunB phosphorylation and decreased FOSL1 protein upon entinostat treatment. Furthermore, preliminary evaluation of imaging mass cytometry (IMC) from tumor biopsies in selected patients with metastatic breast cancer treated with entinostat during the clinical trial NCI-9844 confirmed decreased STAT3 phosphorylation in MDSCs. Taken together, we provide new evidence in the metastatic lung TME that implicates a STAT3-NFκB-AP-1 mediated mechanism leading to decreased MDSC suppression. Evaluating this mechanism in MDSCs taken directly from treated lung metastases represents the most biologically relevant mechanistic study to date, and preliminary translation of findings in patients suggests that planned studies will lead to identification of the mechanism driving response. Citation Format: Aaron G. Baugh, Edgar Gonzalez, Valerie H. Narumi, Sofi Castanon, Jesse Kreger, James Leatherman, Won Jin Ho, Ashley Cimino-Mathews, Vered Stearns, Roisin M. Connolly, Elizabeth M. Jaffee, Adam L. MacLean, Evanthia T. Roussos Torres. Entinostat's modulation of myeloid derived suppressor cells through the STAT3-NFκB-AP-1 axis decreases suppressive signaling. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5167.

TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFβ1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFβ1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFβ1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFβ1 in diverse cellular backgrounds, showed positively correlated expression with TGFβ signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFβ1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.

Gene regulatory network reconfiguration in direct lineage reprogramming.

In direct lineage conversion, transcription factor (TF) overexpression reconfigures gene regulatory networks (GRNs) to reprogram cell identity. We previously developed CellOracle, a computational method to infer GRNs from single-cell transcriptome and epigenome data. Using inferred GRNs, CellOracle simulates gene expression changes in response to TF perturbation, enabling in silico interrogation of network reconfiguration. Here, we combine CellOracle analysis with lineage tracing of fibroblast to induced endoderm progenitor (iEP) conversion, a prototypical direct reprogramming paradigm. By linking early network state to reprogramming outcome, we reveal distinct network configurations underlying successful and failed fate conversion. Via in silico simulation of TF perturbation, we identify new factors to coax cells into successfully converting their identity, uncovering a central role for the AP-1 subunit Fos with the Hippo signaling effector, Yap1. Together, these results demonstrate the efficacy of CellOracle to infer and interpret cell-type-specific GRN configurations, providing new mechanistic insights into lineage reprogramming.

(-)-Epigallocatechin-3-Gallate Prevents IL-1β-Induced uPAR Expression and Invasiveness via the Suppression of NF-κB and AP-1 in Human Bladder Cancer Cells.

(-)-Epigallocatechin-3-O-gallate (EGCG), a primary green tea polyphenol, has powerful iron scavengers, belongs to the family of flavonoids with antioxidant properties, and can be used to prevent cancer. Urokinase-type plasminogen activator receptors (uPARs) are glycosylphosphatidylinositol (GPI)-anchored cell membrane receptors that have crucial roles in cell invasion and metastasis of several cancers including bladder cancer. The mechanism of action of EGCG on uPAR expression has not been reported clearly yet. In this study, we investigated the effect of EGCG on interleukin (IL)-1β-induced cell invasion and uPAR activity in T24 human bladder cancer cells. Interestingly, nuclear factor (NF)-κB and activator protein (AP)-1 transcription factors were critically required for IL-1β-induced high uPAR expression, and EGCG suppressed the transcriptional activity of both the ERK1/2 and JNK signaling pathways with the AP-1 subunit c-Jun. EGCG blocked the IL-1β-stimulated reactive oxygen species (ROS) production, in turn suppressing NF-κB signaling and anti-invasion effects by inhibiting uPAR expression. These results suggest that EGCG may exert at least part of its anticancer effect by controlling uPAR expression through the suppression of ERK1/2, JNK, AP-1, and NF-κB.

Nuciferine attenuates lipopolysaccharide-stimulated inflammatory responses by inhibiting p38 MAPK/ATF2 signaling pathways.

Nuciferine, isolated from Nelumbo nucifera (commonly known as lotus) leaves, has been shown to have beneficial effects, including antioxidant, anti-obesity, anti-diabetic, and anti-inflammatory properties. However, little is known about the mechanism of nuciferine action on the inflammatory response. This study aimed to investigate the anti-inflammatory effects of nuciferine and its underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated murine macrophages. In this study, nuciferine reduced LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production and mRNA expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Nuciferine also decreased the production of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. Furthermore, nuciferine inhibited the LPS-mediated transcriptional activity of nuclear factor (NF)-κB and activator protein (AP)-1, and the nuclear translocation of NF-κB p65 and activating transcription factor 2 (ATF2), an AP-1 subunit. Nuciferine also decreased the phosphorylation of IκB kinase (IKK), inhibitor of NF-κB (IκB), NF-κB, mitogen-activated protein kinase 3 (MKK3), MKK6, p38 mitogen-activated protein kinase (MAPK), and ATF2. Overall, our findings suggest that nuciferine may exert anti-inflammatory effects in LPS-induced macrophages by inhibiting the NF-κB and p38 MAPK/ATF2 signaling pathways.

MLK3 Regulates Inflammatory Response via Activation of AP-1 Pathway in HEK293 and RAW264.7 Cells.

Inflammation is a critically important barrier found in innate immunity. However, severe and sustained inflammatory conditions are regarded as causes of many different serious diseases, such as cancer, atherosclerosis, and diabetes. Although numerous studies have addressed how inflammatory responses proceed and what kinds of proteins and cells are involved, the exact mechanism and protein components regulating inflammatory reactions are not fully understood. In this paper, to determine the regulatory role of mixed lineage kinase 3 (MLK3), which functions as mitogen-activated protein kinase kinase kinase (MAP3K) in cancer cells in inflammatory response to macrophages, we employed an overexpression strategy with MLK3 in HEK293 cells and used its inhibitor URMC-099 in lipopolysaccharide (LPS)-treated RAW264.7 cells. It was found that overexpressed MLK3 increased the mRNA expression of inflammatory genes (COX-2, IL-6, and TNF-α) via the activation of AP-1, according to a luciferase assay carried out with AP-1-Luc. Overexpression of MLK3 also induced phosphorylation of MAPKK (MEK1/2, MKK3/6, and MKK4/7), MAPK (ERK, p38, and JNK), and AP-1 subunits (c-Jun, c-Fos, and FRA-1). Phosphorylation of MLK3 was also observed in RAW264.7 cells stimulated by LPS, Pam3CSK, and poly(I:C). Finally, inhibition of MLK3 by URMC-099 reduced the expression of COX-2 and CCL-12, phosphorylation of c-Jun, luciferase activity mediated by AP-1, and phosphorylation of MAPK in LPS-treated RAW264.7 cells. Taken together, our findings strongly suggest that MLK3 plays a central role in controlling AP-1-mediated inflammatory responses in macrophages and that this enzyme can serve as a target molecule for treating AP-1-mediated inflammatory diseases.

FRA1:c-JUN:HDAC1 complex down-regulates filaggrin expression upon TNFα and IFNγ stimulation in keratinocytes

Filaggrin (FLG), an essential structural protein for skin barrier function, is down-regulated under chronic inflammatory conditions, leading to disruption of the skin barrier. However, the detailed molecular mechanisms of how FLG changes in the context of chronic inflammation are poorly understood. Here, we identified the molecular mechanisms by which inflammatory cytokines inhibit FLG expression in the skin. We found that the AP1 response element within the -343/+25 of the FLG promoter was necessary for TNFα + IFNγ-induced down-regulation of FLG promoter activity. Using DNA affinity precipitation assay, we observed that AP1 subunit composition binding to the FLG promoter was altered from c-FOS:c-JUN (at the early time) to FRA1:c-JUN (at the late time) in response to TNFα + IFNγ stimulation. Knockdown of FRA1 or c-JUN abrogated TNFα + IFNγ-induced FLG suppression. Histone deacetylase (HDAC) 1 interacted with FRA1:c-JUN under TNFα + IFNγ stimulation. Knockdown of HDAC1 abrogated the inhibitory effect of TNFα + IFNγ on FLG expression. The altered expression of FLG, FRA1, c-JUN, and HDAC1 was confirmed in mouse models of 2,4-dinitrochlorobenzene-induced atopic dermatitis and imiquimod-induced psoriasis. Thus, the current study demonstrates that TNFα + IFNγ stimulation suppresses FLG expression by promoting the FRA1:c-JUN:HDAC1 complex. This study provides insight into future therapeutic strategies targeting the FRA1:c-JUN:HDAC1 complex to restore impaired FLG expression in chronic skin inflammation.

JUN Regulation of Injury-Induced Enhancers in Schwann Cells.

Schwann cells play a critical role after peripheral nerve injury by clearing myelin debris, forming axon-guiding bands of Büngner, and remyelinating regenerating axons. Schwann cells undergo epigenomic remodeling to differentiate into a repair state that expresses unique genes, some of which are not expressed at other stages of Schwann cell development. We previously identified a set of enhancers that are activated in Schwann cells after nerve injury, and we determined whether these enhancers are preprogrammed into the Schwann cell epigenome as poised enhancers before injury. Poised enhancers share many attributes of active enhancers, such as open chromatin, but are marked by repressive histone H3 lysine 27 (H3K27) trimethylation rather than H3K27 acetylation. We find that most injury-induced enhancers are not marked as poised enhancers before injury indicating that injury-induced enhancers are not preprogrammed in the Schwann cell epigenome. Injury-induced enhancers are enriched with AP-1 binding motifs, and the c-JUN subunit of AP-1 had been shown to be critical to drive the transcriptional response of Schwann cells after injury. Using in vivo chromatin immunoprecipitation sequencing analysis in rat, we find that c-JUN binds to a subset of injury-induced enhancers. To test the role of specific injury-induced enhancers, we focused on c-JUN-binding enhancers upstream of the Sonic hedgehog (Shh) gene, which is only upregulated in repair Schwann cells compared with other stages of Schwann cell development. We used targeted deletions in male/female mice to show that the enhancers are required for robust induction of the Shh gene after injury.SIGNIFICANCE STATEMENT The proregenerative actions of Schwann cells after nerve injury depends on profound reprogramming of the epigenome. The repair state is directed by injury-induced transcription factors, like JUN, which is uniquely required after nerve injury. In this study, we test whether the injury program is preprogrammed into the epigenome as poised enhancers and define which enhancers bind JUN. Finally, we test the roles of these enhancers by performing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated deletion of JUN-bound injury enhancers in the Sonic hedgehog gene. Although many long-range enhancers drive expression of Sonic hedgehog at different developmental stages of specific tissues, these studies identify an entirely new set of enhancers that are required for Sonic hedgehog induction in Schwann cells after injury.

AP1/2β-mediated exocytosis of tapetum-specific transporters is required for pollen development in Arabidopsis thaliana.

AP-1 and AP-2 adaptor protein (AP) complexes mediate clathrin-dependent trafficking at the trans-Golgi network (TGN) and the plasma membrane, respectively. Whereas AP-1 is required for trafficking to plasma membrane and vacuoles, AP-2 mediates endocytosis. These AP complexes consist of four subunits (adaptins): two large subunits (β1 and γ for AP-1 and β2 and α for AP-2), a medium subunit μ, and a small subunit σ. In general, adaptins are unique to each AP complex, with the exception of β subunits that are shared by AP-1 and AP-2 in some invertebrates. Here, we show that the two putative Arabidopsis thaliana AP1/2β adaptins co-assemble with both AP-1 and AP-2 subunits and regulate exocytosis and endocytosis in root cells, consistent with their dual localization at the TGN and plasma membrane. Deletion of both β adaptins is lethal in plants. We identified a critical role of β adaptins in pollen wall formation and reproduction, involving the regulation of membrane trafficking in the tapetum and pollen germination. In tapetal cells, β adaptins localize almost exclusively to the TGN and mediate exocytosis of the plasma membrane transporters such as ATP-binding cassette (ABC)G9 and ABCG16. This study highlights the essential role of AP1/2β adaptins in plants and their specialized roles in specific cell types.

ADAPTOR PROTEIN-1 complex-mediated post-Golgi trafficking is critical for pollen wall development in Arabidopsis.

Summary Primexine deposition is essential for the formation of pollen wall patterns and is precisely regulated by the tapetum and microspores. While tapetum‐ and/or microspore‐localized proteins are required for primexine biosynthesis, how their trafficking is established and controlled is poorly understood.In Arabidopsis thaliana, AP1σ1 and AP1σ2, two genes encoding the σ subunit of the trans‐Golgi network/early endosome (TGN/EE)‐localized ADAPTOR PROTEIN‐1 complex (AP‐1), are partially redundant for plant viability, and the loss of AP1σ1 function reduces male fertility due to defective primexine formation. Here, we investigated the role of AP‐1 in pollen wall formation.The deposition of Acyl‐CoA SYNTHETASE5 (ACOS5) and type III LIPID TRANSFER PROTEINs (LTPs) secreted from the anther tapetum, which are involved in exine formation, were impaired in ap1σ1 mutants. In addition, the microspore plasma membrane (PM) protein RUPTURED POLLEN GRAIN1 (RPG1), which regulates primexine deposition, accumulated abnormally at the TGN/EE in ap1σ1 mutants. We show that AP‐1μ recognizes the YXXΦ motif of RPG1, thereby regulating its PM abundance through endocytic trafficking, and that loss of AP1σ1 decreases the levels of other AP‐1 subunits at the TGN/EE.Our observations show that AP‐1‐mediated post‐Golgi trafficking plays a vital role in pollen wall development by regulating protein transport in tapetal cells and microspores.