AP-1 Transcriptional Activity Research Articles

IKAROS and AIOLOS directly regulate AP-1 transcriptional complexes and are essential for NK cell development.

Ikaros transcription factors are essential for adaptive lymphocyte function, yet their role in innate lymphopoiesis is unknown. Using conditional genetic inactivation, we show that Ikzf1/Ikaros is essential for normal natural killer (NK) cell lymphopoiesis and IKZF1 directly represses Cish, a negative regulator of interleukin-15 receptor resulting in impaired interleukin-15 receptor signaling. Both Bcl2l11 and BIM levels, and intrinsic apoptosis were increased in Ikzf1-null NK cells, which in part accounts for NK lymphopenia as both were restored to normal levels when Ikzf1 and Bcl2l11 were co-deleted. Ikzf1-null NK cells presented extensive transcriptional alterations with reduced AP-1 transcriptional complex expression and increased expression of Ikzf2/Helios and Ikzf3/Aiolos. IKZF1 and IKZF3 directly bound AP-1 family members and deletion of both Ikzf1 and Ikzf3 in NK cells resulted in further reductions in Jun/Fos expression and complete loss of peripheral NK cells. Collectively, we show that Ikaros family members are important regulators of apoptosis, cytokine responsiveness and AP-1 transcriptional activity.

The CRM1‐mediated nuclear export effectuates the role conversion of tumour suppressor gene BATF2 in colorectal cancer

The CRM1‐mediated nuclear export effectuates the role conversion of tumour suppressor gene BATF2 in colorectal cancer

Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3

CARD11 is a critical scaffold protein linking antigen receptor (AgR) engagement to transcriptional programs required for effector lymphocyte survival, proliferation, and differentiation. Prior work established that CARD11 mediates these effects by enabling AgR-induced signaling through NF-kB, mTORC1 and c-Jun N-terminal kinase 2 (JNK2). Although JNK influences helper CD4+ T cell differentiation, mechanisms governing JNK signal regulation and function in human lymphocytes remain incompletely understood. We therefore asked if/how dominant interfering (DI) CARD11 variants impact JNK signaling and JNK-specific transcriptomic changes in T cells. Wild-type (WT) and CARD11 knockout (KO) Jurkat T cells were transfected with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling. Additionally, RNA sequencing was performed on mitogen-activated WT and CARD11 KO Jurkat T cells +/– JNK inhibition. Our results revealed that CARD11 is required for AgR-dependent activation of JNK1 and JNK2. Furthermore, heterozygous DI CARD11 mutations found in patients with CARD11-associated atopy with dominant interference of NF-kB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, similar to the degree of impaired NF-kB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4+ T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. These results were recapitulated in effector Tcells generated from CADINS patient PBMC, with greater GATA3 induction observed after mitogen activation relative to healthy controls. Intriguingly, simultaneous reactivation of JNK signaling through a CARD11-independent mechanism blunted AgR-induced GATA3 induction, suggesting JNK activity can directly modulate GATA3 expression. In summation, our novel findings imply that defective CARD11-dependent JNK signaling in CD4+ T cells contributes to enhanced Th2 differentiation and the atopic diathesis observed in CADINS patients, unveiling a new potential therapeutic target for ameliorating severe allergic disease manifestations.

Dominant interfering CARD11 variants disrupt JNK signaling in T cells and impaired CARD11-JNK signaling increases expression of GATA3

Abstract Although prior research implicated c-Jun N-terminal kinase (JNK) in helper CD4 +T cell differentiation, mechanisms governing JNK signaling and function in human lymphocytes remain nebulous. CARD11 is a lymphocyte-specific scaffold protein connecting antigen receptor (AgR) engagement to transcriptional programs necessary for effector cell survival, proliferation, and differentiation. In addition to NF-κB, CARD11 is also required for AgR-induced mTORC1 and JNK2 signaling. We sought to define the impact of dominant interfering (DI) CARD11 variants on JNK signaling and elucidate transcriptomic changes governed by CARD11-dependent JNK activation in T cells. We transfected WT and CARD11 knockout (KO) Jurkat T cells with plasmids expressing WT or DI CARD11 variants to assess effects on CARD11-dependent JNK signaling and performed RNA sequencing on mitogen-activated WT and CARD11 KO Jurkat T cells ± JNK inhibition. Our results revealed that AgR-dependent JNK1 andJNK2 activation are CARD11-dependent. Additionally, heterozygous DI CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease disrupted AgR-dependent JNK1/2 phosphorylation, c-Jun accumulation, and AP-1 transcriptional activity with variable potency, mirroring the extent of impaired NF-κB activation. Intriguingly, JNK inhibition in Jurkat and primary CD4 +T cells enhanced AgR-induced expression of GATA3, the master transcriptional regulator of Th2 cell differentiation. Our novel findings suggest that defects in CARD11-dependent JNK signaling in CD4 +T cells may contribute to severe allergic disease noted in CADINS patients, unveiling a new potential therapeutic target. Supported by funding from the Jeffrey Modell Specific Defect Research Program (ALS) and the Henry M. Jackson Foundation for the Advancement of Military Medicine (BMB).

Abstract 4753: LSD1 &8211mediated FOXA2/AP1 transcription program drives lineage plasticity in prostate cancer

Abstract Background: While castration-resistant PCa (CRPC) can be further treated with second-generation androgen deprivation therapies (ADTs), the tumor can quickly generate resistance through multiple mechanisms. One critical mechanism is that tumor cells can progress to AR-indifferent stem cell-like (SCL-PCa or SCLPC) or neuroendocrine-like CRPC (NE-PCa or NEPC) through lineage plasticity. However, the underlying molecular basis remains to be determined, and clinical treatment options for these aggressive CRPC subtypes are currently limited. FOXA1 and FOXA2, which are members of the FOXA (Forkhead Box A) protein family, are pioneer transcription factors. While FOXA1 is well known for its function as a critical pioneer factor of AR and pivotal for maintaining AR signaling, the molecular function of FOXA2 in PCa cells is poorly understood despite that FOXA2 is known to be overexpressed in NEPC. Moreover, since FOXA2, like FOXA1, is currently undruggable in the clinic, there is an urgent need to decipher the molecular basis for the chromatin binding of FOXA2 and identify druggable targets that regulate FOXA2 binding and activity in the aggressive FOXA2-positive CRPC. Methods: In the current study, we first examined the role of FOXA2 in tumorigenesis and metastasis by using various in vitro and in vivo assays including mouse subcutaneous injection and zebrafish embryo injection approaches. We then conducted integrated whole genomic transcriptome and cistrome analyses in SCLPC and NEPC cell line models to characterize the activity of FOXA2 on chromatin and to identify its collaborating transcription factors that may play specific functions in promoting SCLPC or NEPC using a series of biochemical and bioinformatic approaches. Results: We found that FOXA2 silencing decreased the growth and metastasis of SCLPC and NEPC cells. Importantly, we discovered that FOXA2 chromatin binding is tightly associated with binding of JUN family proteins (primarily c-Jun) and FOXA2 silencing dramatically interrupted the global chromatin binding of c-Jun and FOSL1. The transcription targets of FOXA2/AP-1 are highly enriched for neuroendocrine and plasticity associated genes and associated with poor clinical outcomes. Furthermore, we also found that FOXA2 chromatin binding is globally enhanced by an epigenetic factor LSD1, and LSD1 inhibition can repress FOXA2/AP-1 activity in multiple CRPC models. Conclusion: Overall, our data indicate that FOXA2 functions to maintain tumor growth and metastasis in SCLPC and NEPC models. Mechanistically, FOXA2 can act as a pioneer factor of AP-1 (c-Jun/FOSL1) and reprogram AP-1 transcription activity. This FOXA2/AP-1 axis is regulated by LSD1 via demethylating FOXA2 protein and LSD1 inhibition represses FOXA2-dependent CRPC tumor progression. These findings provide novel mechanistic insights into the molecular mechanisms for PCa lineage plasticity and treatment resistance. Citation Format: Zifeng Wang, Mingyu Liu, Songqi Zhang, Muqing Li, Susan Patalano, Jill A. Macoska, Dong Han, Shuai Gao, Hansen He, Changmeng Cai. LSD1 &8211mediated FOXA2/AP1 transcription program drives lineage plasticity in prostate cancer. [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 4753.

Anti-Oxidative and Anti-Aging Effects of Ethanol Extract of the Officinal Breynia (Breynia vitis-idaea) In Vitro.

The skin is the largest organ of the human body, and it is also the one most exposed to external environmental contaminants. The skin is the body's first defense against harmful environmental stimuli, including ultraviolet B (UVB) rays and hazardous chemicals. Therefore, proper care of the skin is required to prevent skin-related diseases and age-related symptoms. In this study, we analyzed anti-aging and anti-oxidative effects of Breynia vitis-idaea ethanol extract (Bv-EE) in human keratinocytes and dermal fibroblasts. The Bv-EE had free radical scavenging activity and decreased the mRNA expression of MMPs and COX-2 in H2O2- or UVB-treated HaCaT cells. The Bv-EE also inhibited AP-1 transcriptional activity and phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and mitogen-activated protein kinase 14 (p38), which are major AP-1 activators upon H2O2 or UVB exposure. Furthermore, the promoter activity and mRNA expression of collagen type I (Col1A1) increased in HDF cells treated with Bv-EE, and Bv-EE recovered the collagen mRNA expression decreased by H2O2 or UVB exposure. These results suggest that Bv-EE has anti-oxidative effects by inhibiting the AP-1 signaling pathway, and shows anti-aging effects by upregulating collagen synthesis.

ATF6-Mediated Signaling Contributes to PARP Inhibitor Resistance in Ovarian Cancer.

This study highlights that a novel p38-ATF6-mediated AP-1 signaling axis contributes to PARPi resistance and provides a clinical rationale for combining PARPi and AP-1 signaling inhibitors.

CD147 mediates epidermal malignant transformation through the RSK2/AP-1 pathway

BackgroundMalignant transformation of the epidermis is an essential process in the pathogenesis of cutaneous squamous-cell carcinoma (cSCC). Although evidence has demonstrated that CD147 plays key roles in various tumors, the role of CD147 in epidermal malignant transformation in vivo remains unclear.MethodsEpidermal CD147-overexpression or knockout (EpiCD147-OE or EpiCD147-KO) transgenic mouse models were generated for in vivo study. RNA-sequencing and q-PCR were performed to identify the differentially expressed genes. Immunohistochemistry and flow cytometry were performed to investigate the role of CD147 in regulating myeloid-derived suppressor cells (MDSCs). Immunoprecipitation, EMSA and ChIP assays were performed to investigate the mechanism of CD147 in cell transformation.ResultsWe found that specific overexpression of CD147 in the epidermis (EpiCD147-OE) induces spontaneous tumor formation; moreover, a set of chemokines and cytokines including CXCL1, which play essential function in MDSC recruitment, were significantly upregulated in EpiCD147-OE transgenic mice. As expected, overexpression of CD147 in the epidermis remarkably facilitated tumorigenesis by increasing the rate of tumor initiation and the number and size of tumors in the DMBA/TPA mouse model. Interestingly, the expression of CXCL1 and the infiltration of MDSCs were dramatically increased in EpiCD147-OE transgenic mice. Our findings also showed that knockdown of CD147 attenuated EGF-induced malignant transformation as well as CXCL1 expression in HaCaT cells. Consistently, CD147 was found overexpressed in cutaneous squamous cell carcinoma (cSCC), and positively related with the expression of CD33, a myeloid-associated marker. We further identified RSK2, a serine/threonine kinase, as an interacting partner of CD147 at the binding site of CD147D207-230. The interaction of CD147 and RSK2 activated RSK2, thus enhancing AP-1 transcriptional activation. Furthermore, EMSAs and ChIP assays showed that AP-1 could associate with the CXCL1 promoter. Importantly, RSK2 inhibitor suppressed the tumor growth in DMBA/TPA mouse model by inhibiting the recruitment of MDSCs.ConclusionOur findings demonstrate that CD147 exerts a key function in epidermal malignant transformation in vivo by activating keratinocytes and recruiting MDSCs via the RSK2/AP-1 pathway.

Role of RNA Binding Protein with Multiple Splicing (RBPMS) Variants in Cell Proliferation, Invasion and Angiogenesis in Ovarian Cancer

Worldwide, the number of cancer‐related deaths continues to increase due to the ability of cancer cells to become chemotherapy‐resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front‐line therapy, cisplatin. RNA binding protein with multiple splicing (RBPMS) is a member of family proteins that bind to the nascent RNA transcripts regulate their processing, which includes splicing, transport, localization and stability. Evidence indicates that RBPMS interact with c‐Fos, a member of the AP‐1 family that dimerizes with c‐Jun to stimulate AP‐1 transcriptional activity. Furthermore, RBPMS interacted with transcription factor Smad3, which bind to c‐Jun increasing AP‐1 transcriptional activity. Three major RBPMS isoforms have been described, RBPMS‐A (21.8 kDa), RBPMS‐B (22.4 kDa) and RBPMS‐C (24.2 kDa). Reports have shown that each RBPMS isoform interacts with proteins and/or RNAs responsible for cell growth, proliferation, and tumor progression. Previous reports of our laboratory have found that RBPMS expression decrease in cisplatin resistance compared with cisplatin sensitive cells. However, the specific RBPMS isoform responsible of this observation has not been elucidated and less is known about its role on invasiveness ability and angiogenesis capacity. We overexpressed each RBPMS variants (A, B and C) in the cisplatin resistant ovarian cancer cells, A2780CP20, and performed cell viability, cell proliferation and invasion assays. Significant reduction in the number of colonies and their area were observed in both isoform A and isoform C clones, compared with isoform B and control empty vector clones. However, clones of isoform A were more sensitive to cisplatin treatment in comparation with isoform B and C clones. In vivo experiments showed a significant reduction in tumor growth between RBPMS A and C when compared with the controls. Tumor tissues were subjected to immunohistochemical analysis for proliferation (Ki67) and angiogenesis (CD31). Elevated signals of both antibodies were detected in tissues from control groups when compared with RBPMS A and C overexpressing tumor tissues. These studies along with the RNAseq suggest that RBPMS, regulate transcripts involved in cell proliferation and invasion capacity of ovarian cancer cells. Moreover, RBPMS A and C represses uncontrolled proliferation, invasiveness, and angiogenesis in ovarian cancer cells. However, only the isoform A contributed to the cisplatin sensitivity of ovarian cancer cells. Future efforts on this project will be focused on determining the RBPMS isoform that represses AP‐1 (c‐Fos and c‐Jun) dependent gene regulation in cisplatin sensitive ovarian cancer cells.

Anti-Inflammatory Activities of an Anti-Histamine Drug, Loratadine, by Suppressing TAK1 in AP-1 Pathway.

Loratadine is an anti-histamine routinely used for treating allergies. However, recent findings have shown that Loratadine may also have anti-inflammatory functions, while their exact mechanisms have not yet been fully uncovered. In this paper, we investigated whether Loratadine can be utilized as an anti-inflammatory drug through a series of in vitro and in vivo experiments using a murine macrophage cell line and an acute gastritis mouse model. Loratadine was found to dramatically reduce the expression of pro-inflammatory genes, including MMP1, MMP3, and MMP9, and inhibit AP-1 transcriptional activation, as demonstrated by the luciferase assay. Therefore, we decided to further explore its role in the AP-1 signaling pathway. The expression of c-Jun and c-Fos, AP-1 subunits, was repressed by Loratadine and, correspondingly, the expression of p-JNK, p-MKK7, and p-TAK1 was also inhibited. In addition, Loratadine was able to reduce gastric bleeding in acute gastritis-induced mice; Western blotting using the stomach samples showed reduced p-c-Fos protein levels. Loratadine was shown to effectively suppress inflammation by specifically targeting TAK1 and suppressing consequent AP-1 signaling pathway activation and inflammatory cytokine production.

A Double-Negative Feedback Interaction between miR-21 and PPAR-α in Clear Renal Cell Carcinoma.

Simple SummaryClear cell renal cell carcinoma (ccRCC) is the main histotype of kidney cancer, which is typically highly resistant to conventional systemic therapies and also known for abnormal lipid accumulation. Identifying the actors and deciphering the molecular mechanisms that lead to tumor progression is an important step in the development of new therapeutic strategies to cure ccRCC. In this context, we focused our attention on miR-21, an oncogenic miRNA upregulated in many solid tumors, and peroxysome proliferator-activated receptor-α (PPAR- α), the master regulator of lipid metabolism and one target of miR-21. In this study, our data show a double-negative feedback interaction between PPAR-α and miR-21. Thus, miR-21 silencing could be therapeutically exploited to restore PPAR-α expression and consequently inhibit the oncogenic events mediated by the aberrant lipid metabolism of ccRCC.Clear cell renal cell carcinoma (ccRCC) is the main histotype of kidney cancer, which is typically highly resistant to conventional therapies and known for abnormal lipid accumulation. In this context, we focused our attention on miR-21, an oncogenic miRNA overexpressed in ccRCC, and peroxysome proliferator-activated receptor-α (PPAR- α), one master regulator of lipid metabolism targeted by miR-21. First, in a cohort of 52 primary ccRCC samples, using RT-qPCR and immunohistochemistry, we showed that miR-21 overexpression was correlated with PPAR-α downregulation. Then, in ACHN and 786-O cells, using RT-qPCR, the luciferase reporter gene, chromatin immunoprecipitation, and Western blotting, we showed that PPAR-α overexpression (i) decreased miR-21 expression, AP-1 and NF-κB transcriptional activity, and the binding of AP-1 and NF-κB to the miR-21 promoter and (ii) increased PTEN and PDCD4 expressions. In contrast, using pre-miR-21 transfection, miR-21 overexpression decreased PPAR-α expression and transcriptional activity mediated by PPAR-α, whereas the anti-miR-21 (LNA-21) strategy increased PPAR-α expression, but also the expression of its targets involved in fatty acid oxidation. In this study, we showed a double-negative feedback interaction between miR-21 and PPAR-α. In ccRCC, miR-21 silencing could be therapeutically exploited to restore PPAR-α expression and consequently inhibit the oncogenic events mediated by the aberrant lipid metabolism of ccRCC.

Chondroprotective effects of CDK4/6 inhibition via enhanced ubiquitin-dependent degradation of JUN in synovial fibroblasts.

ObjectiveTargeting synovial fibroblasts (SF) using a cyclin-dependent kinase (CDK) 4/6 inhibitor (CDKI) could be a potent therapy for RA via inhibition of proliferation and MMP-3 production. This study was designed to elucidate the mechanism of chondroprotective effects on SFs by CDK 4/6 inhibition.MethodsCDK4/6 activity was inhibited using CDKI treatment or enhanced by adenoviral gene transduction. Chondroprotective effects were evaluated using a collagen-induced arthritis model (CIA). Gene and protein expression were evaluated with quantitative PCR, ELISA and Western blotting. The binding of nuclear extracts to DNA was assessed with an electrophoresis mobility shift assay. RNA-Seq was performed to identify gene sets affected by CDKI treatment.ResultsCDKI attenuated cartilage destruction and MMP-3 production in CIA. In RASFs, CDKI impaired the binding of AP-1 components to DNA and inhibited the production of MMP-1 and MMP-3, which contain the AP-1 binding sequence in their promoter. CDK4/6 protected JUN from proteasome-dependent degradation by inhibiting ubiquitination. The RNA-Seq analysis identified CDKI-sensitive inflammatory genes, which were associated with the pathway of RA-associated genes, cytokine-cytokine receptor interaction and IL-17 signalling. Notably, the AP-1 motif was enriched in these genes.ConclusionThe mechanism of chondroprotective effects by CDK4/6 inhibition was achieved by the attenuation of AP-1 transcriptional activity via the impaired stability of JUN. Because the pharmacologic inhibition of CDK4/6 has been established as tolerable in cancer treatment, it could also be beneficial in patients with RA due to its chondroprotective and anti-inflammatory effects.

FosL1 Is a Novel Target of Levetiracetam for Suppressing the Microglial Inflammatory Reaction

We previously showed that the antiepileptic drug levetiracetam (LEV) inhibits microglial activation, but the mechanism remains unclear. The purpose of this study was to identify the target of LEV in microglial activity suppression. The mouse microglial BV-2 cell line, cultured in a ramified form, was pretreated with LEV and then treated with lipopolysaccharide (LPS). A comprehensive analysis of LEV targets was performed by cap analysis gene expression sequencing using BV-2 cells, indicating the transcription factors BATF, Nrf-2, FosL1 (Fra1), MAFF, and Spic as candidates. LPS increased AP-1 and Spic transcriptional activity, and LEV only suppressed AP-1 activity. FosL1, MAFF, and Spic mRNA levels were increased by LPS, and LEV only attenuated FosL1 mRNA expression, suggesting FosL1 as an LEV target. FosL1 protein levels were increased by LPS treatment and decreased by LEV pretreatment, similar to FosL1 mRNA levels. The FosL1 siRNA clearly suppressed the expression of TNFα and IL-1β. Pilocarpine-induced status epilepticus increased hippocampus FosL1 expression, along with inflammation. LEV treatment significantly suppressed FosL1 expression. Together, LEV reduces FosL1 expression and AP-1 activity in activated microglia, thereby suppressing neuroinflammation. LEV might be a candidate for the treatment of several neurological diseases involving microglial activation.

Immediate-early transcriptional response to insulin receptor stimulation

Insulin binding to the insulin receptor triggers intracellular signaling cascades involving the activation of protein and lipid kinases. As a result, multiple biological functions of the cells are changed. Here, we analyzed the regulation and signaling cascades leading to insulin-induced activation of the stimulus-responsive transcription factors. For the analyses, we used chromatin-embedded reporter genes having a cellular nucleosomal organisation, and fibroblasts expressing human insulin receptors (HIRcB cells). The results show that stimulation of the insulin receptor induced the expression of the transcription factor Egr-1. Attenuation of Egr-1 promoter activation was observed following expression of a dominant-negative mutant of the ternary complex factor Elk-1. These data were corroborated by experiments showing that insulin receptor stimulation increased the transcriptional activation potential of Elk-1. In addition, the transcriptional activity of AP-1 was significantly elevated in insulin-stimulated HIRcB cells. Expression of the dominant-negative mutant of Elk-1 reduced insulin-induced activation of AP-1, indicating that Elk-1 controls both serum response element and AP-1-regulated transcription. Moreover, we show that stimulation of the insulin receptor activates cyclic AMP response element (CRE)-controlled transcription, involving the transcription factor CREB. Insulin-induced transcription of Elk-1 and CREB-controlled reporter genes was attenuated by overexpression of MAP kinase phosphatase-1 or a constitutively active mutant of calcineurin A, indicating that both phosphatases are part of a negative feedback loop for reducing insulin-mediated gene transcription. Finally, we show that expression of the adenoviral protein E1A selectively reduced CRE-mediated transcription following stimulation of the insulin receptor. These data indicate that insulin-regulated transcription of CRE-containing genes is under epigenetic control.

Histone deacetylase 7 mediates endothelin-1-induced connective tissue growth factor expression in human lung fibroblasts through p300 and activator protein-1 activation

BackgroundHistone deacetylase (HDAC) inhibition was reported to ameliorate lung fibrosis in animal models. However, little is known about the underlying mechanism of HDAC7 in the regulation of CTGF production in lung fibroblasts.MethodsThe role of HDAC7 in CTGF production caused by ET-1 stimulation in WI-38 cells (human lung fibroblast) was examined. We also evaluated the expression of HDAC7 in the lung of ovalbumin-induced airway fibrosis model. Statistical data were shown as mean ± standard error.ResultsET-1-stimulated CTGF and α-SMA expression was attenuated by small interfering (si)RNA interference of HDAC7. ET-1 promoted HDAC7 translocation from the cytosol to nucleus. ET-1-stimulated CTGF expression was reduced by the transfection of p300 siRNA. ET-1 induced an increase in p300 activity. Furthermore, the acetylation of c-Jun was time-dependently induced by ET-1 stimulation, which was reduced by transfection of either HDAC7 or p300 siRNA. Both transfection of HDAC7 and p300 siRNA suppressed the ET-1-increased activity of AP-1-luciferase. Moreover, the presence of HDAC7 was required for ET-1-stimulated formation of HDAC7, p300, and AP-1 complex and recruitment to the CTGF promoter region. In an ovalbumin-induced airway fibrosis model, the protein level of HDAC7 was increased in the lung tissue, and the distribution of HDAC7 was colocalized with α-SMA-positive cells in the subepithelial layer of the airway.ConclusionsET-1 activates HDAC7 to initiate AP-1 transcriptional activity by recruiting p300 and eventually promotes the production of CTGF. HDAC7 might play a vital role in airway fibrosis and have the potential to be developed as a therapeutic target.

99529 The Role of ATF6-Mediated Signaling in PARP Inhibitor Resistant Ovarian Cancer

ABSTRACT IMPACT: This work has the potential to identify targetable pathways conveying resistance to PARP inhibitors that may improve ovarian cancer patient outcomes. OBJECTIVES/GOALS: High grade serous ovarian cancer is the deadliest gynecologic malignancy. PARP inhibitors are an FDA approved targeted therapy that is being used more and more frequently in the clinic. It is vital to understand mechanisms driving resistance to this therapy in order to develop treatments to improve patient responses. METHODS/STUDY POPULATION: RNA-sequencing and transcription factor analysis was used to identify pathways of interest. An AP-1 transcriptional reporter assays was used to confirm results of the transcription factor analysis. An unbiased lentiviral shRNA screen was used to identify AP-1 subunits promoting PARP inhibitor resistance. Lentiviral transduction allowed for the knockdown ATF6. Comet assays and two-plasmid systems were used to determine levels of DNA damage and levels of DNA damage repair respectively. RESULTS/ANTICIPATED RESULTS: PARP inhibitor resistant cell lines have increased WNT signaling which promotes to increased DNA damage repair. PARP inhibitor resistant cell lines also have increased AP-1 transcriptional activity, ATF6 expression, and active p38. ATF6 knockdown and p38 inhibition is sufficient to resensitize cells to PARP inhibition. Upon treatment with PARP inhibitors, ATF6 knockdown as well as p38 inhibition lead to increased DNA damage in PARP inhibitor resistant cell lines. RNA-sequencing reveals a signifcant overlap in downregulated genes in cells treated with a β-catenin inhibitor and cells with an ATF6 knockdown. DISCUSSION/SIGNIFICANCE OF FINDINGS: Due to the increasing prevalence of PARP inhibitors in the clinic, it is vital to uncover mechanisms contributing to resistance. This work has the potential to identify targetable pathways conveying resistance to PARP inhibitors that may improve ovarian cancer patient outcomes.

Blocking Fra-1 sensitizes triple-negative breast cancer to PARP inhibitor.

The AP-1 member Fra-1 is overexpressed in TNBC and plays crucial roles in tumor progression and treatment resistance. In a previous large-scale screen, we identified PARP1 to be among 118 proteins that interact with endogenous chromatin-bound Fra-1 in TNBC cells. PARP1 inhibitor (olaparib) is currently in clinical use for treatment of BRCA-mutated TNBC breast cancer. Here, we demonstrate that the Fra-1-PARP1 interaction impacts the efficacy of olaparib treatment. We show that PARP1 interacts with and downregulates Fra-1, thereby reducing AP-1 transcriptional activity. Olaparib treatment, or silencing of PARP1, consequently, increases Fra-1 levels and enhances its transcriptional activity. Increased Fra-1 can have adverse effect, including treatment resistance. We also found that a large fraction of PARP1-regulated genes was dependent on Fra-1. We show that by inhibiting Fra-1/AP-1, non-BRCA-mutated TNBC cells can become sensitized to olaparib treatment. We identify that high PARP1 expression is indicative of a poor clinical outcome in breast cancer patients overall (P=0.01), but not for HER-2 positive patients. In conclusion, by exploring the functionality of the Fra-1 and PARP1 interaction, we propose that targeting Fra-1 could serve as a combinatory therapeutic approach to improve olaparib treatment outcome for TNBC patients.

Enhancement of skin barrier and hydration-related molecules by protopanaxatriol in human keratinocytes

BackgroundProtopanaxatriol (PPT) is a secondary intestinal metabolite of ginsenoside in ginseng. Although the effects of PPT have been reported in various diseases including cancer, diabetes and inflammatory diseases, the skin protective effects of PPT are poorly understood. MethodsHaCaT cells were treated with PPT in a dose-dependent manner. mRNA and protein levels which related to skin barrier and hydration were detected compared with retinol. Luciferase assay was performed to explore the relative signaling pathway. Western blot was conducted to confirm these pathways and excavated further signals. ResultsPPT enhanced the expression of filaggrin (FLG), transglutaminase (TGM)-1, claudin, occludin and hyaluronic acid synthase (HAS) −1, −2 and −3. The mRNA expression levels of FLG, TGM-1, HAS-1 and HAS-2 were suppressed under NF-κB inhibition. PPT significantly augmented NF-κB-luc activity and upregulated Src/AKT/NF-κB signaling. In addition, PPT also increased phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK, JNK and p38 and upstream MAPK activators (MEK and MKK). Furthermore, transcriptional activity of AP-1 and CREB, which are downstream signaling targets of MAPK, was enhanced by PPT. ConclusionPPT improves skin barrier function and hydration through Src/AKT/NF-κB and MAPK signaling. Therefore, PPT may be a valuable component for cosmetics or treating skin disorders.

Trypanosoma cruzi Induces the PARP1/AP-1 Pathway for Upregulation of Metalloproteinases and Transforming Growth Factor β in Macrophages: Role in Cardiac Fibroblast Differentiation and Fibrosis in Chagas Disease.

Chagas disease (CD), caused by Trypanosoma cruzi, is a degenerative heart condition. In the present study, we investigated the role of poly [ADP-ribose] polymerase 1/activator protein 1 (PARP1/AP-1) in upregulation of profibrotic macrophages (Mϕ) and subsequent development of cardiac fibrosis in CD. We used in vitro and in vivo models of T. cruzi infection and chemical and genetic inhibition of Parp1 to examine the molecular mechanisms by which Mϕ might augment profibrotic events in CD. Cultured (RAW 264.7 and THP-1) Mϕ infected with T. cruzi and primary cardiac and splenic Mϕ of chronically infected mice exhibited a significant increase in the expression, activity, and release of metalloproteinases (MMP2, MMP9, and MMP12) and the cytokine transforming growth factor β (TGF-β). Mϕ release of MMPs and TGF-β signaled the cardiac fibroblast to myofibroblast differentiation, as evidenced by a shift from S100A4 to alpha smooth muscle actin (α-SMA) expression. Incubation of infected Mϕ with MMP2 and MMP9 inhibitors resulted in 60 to 74% decline in TGF-β release, and MMP9 and PARP1 inhibitors resulted in 57 to 70% decline in Mϕ TGF-β-driven cardiac fibroblast differentiation. Likewise, histological studies showed a 12- to 16-fold increase in myocardial expression of CD68 (Mϕ marker) and its colocalization with MMP9/TGF-β, galectin-3, and vimentin in wild-type mice with CD. In comparison, chronically infected Parp1-/- mice exhibited a >50% decline in myocardial levels of Mϕ and associated fibrosis markers. Further study showed that PARP1 synergized with c-Fos and JunB AP-1 family members for transcriptional activation of profibrotic response after T. cruzi infection. We conclude that PARP1 inhibition offers a potential therapy for controlling the T. cruzi-driven fibroblast differentiation in CD through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-β pathway.IMPORTANCE Cardiomyopathy is the most important clinical manifestation of T. cruzi-driven CD. Recent studies have suggested the detrimental role of the matrix metalloproteinases MMP2 and MMP9 in extracellular matrix (ECM) degradation during cardiac remodeling in T. cruzi infection. Peripheral TGF-β levels are increased in clinically symptomatic CD patients over those in clinically asymptomatic seropositive individuals. We provide the first evidence that during T. cruzi infection, Mϕ release of MMP2 and MMP9 plays an active role in activation of TGF-β signaling of ECM remodeling and cardiac fibroblast-to-myofibroblast differentiation. We also determined that PARP1 signals c-Fos- and JunB-mediated AP-1 transcriptional activation of profibrotic gene expression and demonstrated the significance of PARP1 inhibition in controlling chronic fibrosis in Chagas disease. Our study provides a promising therapeutic approach for controlling T. cruzi-driven fibroblast differentiation in CD by PARP1 inhibitors through modulation of the Mϕ signaling of the AP-1-MMP9-TGF-β pathway.

Antiphotoaging Effect of 3,5-Dicaffeoyl-epi-quinic Acid against UVA-Induced Skin Damage by Protecting Human Dermal Fibroblasts In Vitro.

Cutaneous aging is divided into intrinsic and exogenous aging correspondingly contributing to the complex biological phenomenon in skin. Intrinsic aging is also termed chronological aging, which is the accumulation of inevitable changes over time and is largely genetically determined. Superimposed on this intrinsic process, exogenous aging is associated with environmental exposure, mainly to ultraviolet (UV) radiation and more commonly termed as photoaging. UV-induced skin aging induces increased expression of matrix metalloproteinases (MMPs) which in turn causes the collagen degradation. Therefore, MMP inhibitors of natural origin are regarded as a primary approach to prevent or treat photoaging. This study investigated the effects of 3,5-dicaffeoyl-epi-quinic acid (DEQA) on photoaging and elucidated its molecular mechanisms in UVA-irradiated human dermal fibroblasts (HDFs). The results show that treatment with DEQA decreases MMP-1 production and increases type I collagen production in UVA-damaged HDFs. In addition, treatment of UVA-irradiated HDFs with DEQA downregulates MMP-1, MMP-3 and MMP-9 expression via blocking MAPK-cascade-regulated AP-1 transcriptional activity in UVA-irradiated HDFs. Furthermore, DEQA relieves the UVA-mediated suppression of type I procollagen and collagen expression through stimulating TGF-β/Smad signaling, leading to activation of the Smad 2/3 and Smad 4 nuclear translocation. These results suggest that DEQA could be a potential cosmetic agent for prevention and treatment of skin photoaging.