AP-1 Family Research Articles

A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages.

The transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8-PU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRF-AP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites.

C-Fos Protects Neurons Through a Noncanonical Mechanism Involving HDAC3 Interaction: Identification of a 21-Amino Acid Fragment with Neuroprotective Activity.

Proteins belonging to the AP-1 family of transcription factors are known to be involved in the regulation of neuronal viability. While strides have been made to elucidate the mechanisms of how individual members regulate cell death, much remains unknown. We find that the expression of one AP-1 member, c-Fos, is reduced in cerebellar granule neurons (CGNs) induced to die by low potassium (LK) treatment. Restoration and increase of this expression protect CGNs against LK-induced death, whereas knockdown induces death of otherwise healthy neurons. Furthermore, forced expression can protect cortical neurons against homocysteic acid (HCA)-induced toxicity. Taken together, this suggests that c-Fos is necessary for neuronal survival and that elevating c-Fos expression has a neuroprotective effect. Consistent with this idea is the finding that c-Fos expression is reduced selectively in the striatum in two separate mouse models of Huntington's disease and forced expression protects against neuronal death resulting from mutant huntingtin (mut-Htt) expression. Interestingly, neuroprotection by c-Fos does not require its DNA-binding, transcriptional, or heteromerization domains. However, this protective activity can be inhibited by pharmacological inhibition of c-Abl, CK-I, and MEK-ERK signaling. Additionally, expression of point mutant forms of this protein has identified that mutation of a tyrosine residue, Tyr345, can convert c-Fos from neuroprotective to neurotoxic. We show that c-Fos interacts with histone deacetylase-3 (HDAC3), a protein that contributes to mut-Htt neurotoxicity and whose overexpression is sufficient to promote neuronal death. When co-expressed, c-Fos can protect against HDAC3 neurotoxicity. Finally, our study identifies a 21-amino acid region at the C-terminus of c-Fos that is sufficient to protect neurons against death induced by LK, HCA treatment, or mut-Htt expression when expressed via a plasmid transfection or as a cell-permeable peptide. This cell-permeable peptide, designated as Fos-CTF, could have potential as a therapeutic agent for neurodegenerative diseases.

The innate immune system in Parkinson′s disease: a novel target promoting endogenous neuroregeneration

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder clinically defined by key symptoms like bradykinesia, rigidity, resting tremor, and postural instability. Reduction of dopaminergic terminals in the caudate nucleus and putamen, i.e., the striatum, accompanied by decreased striatal levels of dopamine, and most likely consecutive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are tightly linked to the onset of the motor symptoms. With the etiology of PD largely not yet well defined, there is accumulating evidence for increased immune activation in PD. The majority of studies favor a detrimental effect of microglia, the resident innate immune cells of the brain, on PD pathogenesis. In the presence of alpha-synuclein, inflammatory responses elicited by microglia may be initiated by pattern recognition receptors, e.g., toll-like receptors (TLR) leading to the distinct activation of signal transduction pathways. For instance, TLRs couple to signal adaptor proteins like myeloid differentiation primary response gene 88 (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF), resulting ultimately in the activation of multiple, signal-dependent transcription factors including members of the nuclear factor κB (NF-κB) or AP-1 families. This leads to an enhanced production of pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) or IL-6 (Figure 1). Indeed, increased levels of pro-inflammatory mediators have been detected within the SNpc and striatum supporting the existence of an underlying neuroinflammatory signature in PD (see review by Hirsch and Hunot, 2009). As a consequence, these inflammatory mediators may directly or via astrocyte activation perturb neuronal homeostasis (Saijo et al., 2009). However, the precise temporal and spatial sequence of this immunogenic response during the course of PD is not defined yet.

Activation of transcription factors in a mouse lung following exposure to environmental chemical and biological agents.

Environmental biological and chemical agents can modulate innate and acquired immunity in the lung via the stimulation of Toll-like receptors (TLRs). To investigate the effect of environmental chemical agents on the activation of NF-κB and activator protein (AP)-1 subunits and the role of TLR4 signaling in the lung, C3H/HeN and C3H/HeJ (TLR4-defective) mice were exposed to 0 or 50 ppm of toluene for 6 hr/day, 5 days/week for 6 weeks. Some groups of mice were also stimulated with OVA or LPS as a biological agent. The DNA-binding activities of the NF-κB subunits (p50, p52, p65 and RelB) and AP-1 family members (FosB, c-Fos, +c-Jun, JunD) were compared using TransAM ELISA kits. Exposure to toluene alone produced no significant changes in both mice. Although stimulation with OVA or LPS alone significantly increased the DNA binding activities of p50 and p52 in C3H/HeN mice, there were no interactions between biological factors and toluene. In the C3H/HeJ mice, stimulation with OVA or LPS increased p65 and p52 binding activity and the combination of exposure to toluene and OVA significantly increased the DNA binding activities of the p65 and p52 in the lung. During AP-1 activation, co-exposure to toluene and OVA increased JunD binding activity in C3H/HeJ mice, while co-exposure to toluene and LPS influenced c-Fos binding activity in C3H/HeN mice. These results indicate that TLR4 may play an important role in activation of NF-κB or AP-1 family following exposure to environmental biological and chemical agents.

JUNB is a key transcriptional modulator of macrophage activation.

Activated macrophages are crucial for restriction of microbial infection but may also promote inflammatory pathology in a wide range of both infectious and sterile conditions. The pathways that regulate macrophage activation are therefore of great interest. Recent studies in silico have putatively identified key transcription factors that may control macrophage activation, but experimental validation is lacking. In this study, we generated a macrophage regulatory network from publicly available microarray data, employing steps to enrich for physiologically relevant interactions. Our analysis predicted a novel relationship between the AP-1 family transcription factor Junb and the gene Il1b, encoding the pyrogen IL-1β, which macrophages express upon activation by inflammatory stimuli. Previously, Junb has been characterized primarily as a negative regulator of the cell cycle, whereas AP-1 activity in myeloid inflammatory responses has largely been attributed to c-Jun. We confirmed experimentally that Junb is required for full expression of Il1b, and of additional genes involved in classical inflammation, in macrophages treated with LPS and other immunostimulatory molecules. Furthermore, Junb modulates expression of canonical markers of alternative activation in macrophages treated with IL-4. Our results demonstrate that JUNB is a significant modulator of both classical and alternative macrophage activation. Further, this finding provides experimental validation for our network modeling approach, which will facilitate the future use of gene expression data from open databases to reveal novel, physiologically relevant regulatory relationships.

MEK inhibitor PD-0325901 overcomes resistance to CK2 inhibitor CX-4945 and exhibits anti-tumor activity in head and neck cancer.

The serine-threonine kinase CK2 exhibits genomic alterations and aberrant overexpression in human head and neck squamous cell carcinomas (HNSCC). Here, we investigated the effects of CK2 inhibitor CX-4945 in human HNSCC cell lines and xenograft models. The IC50's of CX-4945 for 9 UM-SCC cell lines measured by MTT assay ranged from 3.4-11.9 μM. CX-4945 induced cell cycle arrest and cell death measured by DNA flow cytometry, and inhibited prosurvival mediators phospho-AKT and p-S6 in UM-SCC1 and UM-SCC46 cells. CX-4945 decreased NF-κB and Bcl-XL reporter gene activities in both cell lines, but upregulated proapoptotic TP53 and p21 reporter activities, and induced phospho-ERK, AP-1, and IL-8 activity in UM-SCC1 cells. CX-4945 exhibited modest anti-tumor activity in UM-SCC1 xenografts. Tumor immunostaining revealed significant inhibition of PI3K-Akt-mTOR pathway and increased apoptosis marker TUNEL, but also induced p-ERK, c-JUN, JUNB, FOSL1 and proliferation (Ki67) markers, as a possible resistance mechanism. To overcome the drug resistance, we tested MEK inhibitor PD-0325901 (PD-901), which inhibited ERK-AP-1 activation alone and in combination with CX-4945. PD-901 alone displayed significant anti-tumor effects in vivo, and the combination of PD-901 and CX-4945 slightly enhanced anti-tumor activity when compared with PD-901 alone. Immunostaining of tumor specimens after treatment revealed inhibition of p-AKT S129 and p-AKT T308 by CX-4945, and inhibition of p-ERK T202/204 and AP-1 family member FOSL-1 by PD-901. Our study reveals a drug resistance mechanism mediated by the MEK-ERK-AP-1 pathway in HNSCC. MEK inhibitor PD-0325901 is active in HNSCC resistant to CX-4945, meriting further clinical investigation.

Characterization of c-Jun from orange-spotted grouper, Epinephelus coioides involved in SGIV infection

The nuclear phosphoprotein c-Jun is a member of the AP1 family of transcription activating complex, can be induced by various extracellular stimuli such as virus infection. In this study, the c-Jun gene (Ec-c-Jun) was cloned from orange-spotted grouper, Epinephelus coioides. The full-length Ec-c-Jun cDNA is composed of 2046 bp and encodes a polypeptide of 328 amino acids with 81% identity of zebrafish. Amino acid alignment analysis indicated that Ec-c-Jun contained three conserved domains including a transactivation domain (TAD), a DNA-binding domain (DBD) and leucine zipper domain (LZD). RT-PCR results showed that Ec-c-Jun transcript was most abundant in spleen, kidney, heart and gill. The expression of Ec-c-Jun was up-regulated after challenged with Singapore grouper iridovirus (SGIV). To investigate the roles of Ec-c-Jun during SGIV infection, we constructed its dominant-negative mutant (DN-Ec-c-Jun) by deleting the major TAD that lacks amino acids 3–122. Fluorescence microscopy observation revealed that Ec-c-Jun and DN-Ec-c-Jun were expressed predominantly in the nucleus in transfected cells. Interestingly, the green fluorescence of Ec-c-Jun was congregated and co-localized with virus assembly sites at the late stage of SGIV infection. However, in DN-Ec-c-Jun transfected cells, no virus assembly sites were observed, and the distribution of fluorescence remained unchanged. Moreover, overexpression of DN-Ec-c-Jun in vitro delayed the occurrence of CPE induced by SGIV infection and inhibited the virus gene transcription. In addition, ectopic expression of DN-Ec-c-Jun was able to inhibit SGIV induced c-Jun/AP1 promoter activity in GS cells. Thus, we proposed that c-Jun transcription factor was essential for SGIV replication in vitro. Our results will contribute to understanding the crucial roles of JNK signaling pathway in fish virus infection.

Identification of drought-induced transcription factors in Sorghum bicolor using GO term semantic similarity.

Stress tolerance in plants is a coordinated action of multiple stress response genes that also cross talk with other components of the stress signal transduction pathways. The expression and regulation of stress-induced genes are largely regulated by specific transcription factors, families of which have been reported in several plant species, such as Arabidopsis, rice and Populus. In sorghum, the majority of such factors remain unexplored. We used 2DE refined with MALDI-TOF techniques to analyze drought stress-induced proteins in sorghum. A total of 176 transcription factors from the MYB, AUX_ARF, bZIP, AP2 and WRKY families of drought-induced proteins were identified. We developed a method based on semantic similarity of gene ontology terms (GO terms) to identify the transcription factors. A threshold value (≥ 90%) was applied to retrieve total 1,493 transcription factors with high semantic similarity from selected plant species. It could be concluded that the identified transcription factors regulate their target proteins with endogenous signals and environmental cues, such as light, temperature and drought stress. The regulatory network and cis-acting elements of the identified transcription factors in distinct families are involved in responsiveness to auxin, abscisic acid, defense, stress and light. These responses may be highly important in the modulation of plant growth and development.

Rice miR172 induces flowering by suppressing OsIDS1 and SNB, two AP2 genes that negatively regulate expression of Ehd1 and florigens.

BackgroundRice is a facultative short-day plant that flowers under long days (LD) after a lengthy vegetative phase. Although several inhibitors that delay flowering have been identified, the process by which rice eventually flowers under non-permissive LD conditions is not well understood.ResultsOverexpression of miR172 reduced flowering time significantly, suggesting its role as an inducer. Levels of miR172 increased as plants aged, further supporting our findings. Transcripts of SNB and OsIDS1, two members of the AP2 family that have the miR172 target site, were reduced in older plants as the level of miR172 rose. Overexpression of those AP2 genes delayed flowering; overexpression of miR172-resistant forms of SNB or OsIDS1 further delayed this process. This demonstrated that the AP2 genes function downstream of miR172. Two florigen genes -- Hd3a and RFT1 -- and their immediate upstream regulator Ehd1 were suppressed in the AP2 overexpression plants. This suggested that the AP2 genes are upstream repressors of Ehd1. In phytochrome mutants, miR172d levels were increased whereas those of SNB and OsIDS1 were decreased. Thus, it appears that phytochromes inhibit miR172d, an AP2 suppresser.ConclusionsWe revealed that miR172d developmentally induced flowering via repressing OsIDS1 and SNB, which suppressed Ehd1. We also showed that phytochromes negatively regulated miR172.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-014-0031-4) contains supplementary material, which is available to authorized users.

412 The Pediatric Preclinical Testing Program (PPTP): Analysis of the first 10 years in vivo testing

the AP-2 family (TFAP2) play important roles in embryonic development and differentiation. We aimed at investigating the role of TFAP2B in neuroblastoma pathogenesis and differentiation. Methods: The association of TFAP2B expression with prognostic markers and outcome was analyzed in 649 primary neuroblastomas using microarray data. Methylation of CpG sites related to TFAP2B was investigated by using Illumina 450K arrays in 105 primary neuroblastomas. To evaluate the functional relevance of TFAP2B in neuroblastoma, TFAP2B was re-expressed in IMR-32 and SH-EP neuroblastoma cells, and knocked down in SH-SY5Y and BE(2)-C neuroblastoma cells. Results: Low expression of TFAP2B was associated with adverse patient outcome (p 18 months, MYCN amplification, and unfavourable gene expression-based classification (p < 0.001 each). In addition, low TFAP2B expression was strongly correlated with methylation of promoter regions of the TFAP2B gene in high-risk neuroblastoma. In IMR-32 cells, demethylation with 5-Aza-2′-deoxyctidine induced TFAP2B expression. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP cells significantly impaired proliferation and led to G1-arrest (p < 0.01 each). Morphological signs of neuronal differentiation and senescence were observed after TFAP2B induction in IMR-32 and SH-EP cells, respectively. Knock-down of TFAP2B by lentiviral transduction of specific shRNAs effectively abrogated retinoic acid induced neuronal differentiation of SHSY5Y and BE(2)-C cells as indicated by microscopic examination and lack of up regulation of neuron related genes such as microtubule associated protein 2 (MAP2), neurofilament middle chain (NEFM) and synaptophysin (SYP). Conclusion: Our data suggest that TFAP2B expression is silenced in highrisk neuroblastoma by promoter methylation, and that TFAP2B expression might be critical for differentiation in neuroblastoma.

Angiotensin II limits NO production by upregulating arginase through a p38 MAPK–ATF-2 pathway

Enhanced vascular arginase activity can impair endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production and uncoupling NOS function. Elevated angiotensin II (Ang II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. In this study we explored the signaling pathway leading to increased arginase expression/activity in response to Ang II in bovine aortic endothelial cells (BAEC). Our previous studies indicate involvement of p38 mitogen activated protein kinase (MAPK) in Ang II-induced arginase upregulation and reduced NO production. In this study, we further investigated the Ang II-transcriptional regulation of arginase 1 in endothelial cells. Our results indicate the involvement of ATF-2 transcription factor of the AP1 family in arginase 1 upregulation and in limiting NO production. Using small interfering RNA (siRNA) targeting ATF-2, we showed that this transcription factor is required for Ang II-induced arginase 1 gene upregulation and increased arginase 1 expression and activity, leading to reduced NO production. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed the involvement of ATF-2. Moreover, our data indicate that p38 MAPK phosphorylates ATF-2 in response to Ang II. Collectively, our results indicate that Ang II increases endothelial arginase activity/expression through a p38 MAPK/ATF-2 pathway leading to reduced endothelial NO production. These signaling steps might be therapeutic targets for preventing vascular endothelial dysfunction associated with elevated arginase activity/expression.

Abstract 5315: Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation

Abstract Bryostatin 1 (bryo 1) is an anticancer agent currently in multiple clinical trials with a unique mechanism of action. Whereas bryo 1 binds to and activates protein kinase C (PKC) like the phorbol esters, it paradoxically antagonizes many but not all phorbol ester responses. In LNCaP prostate cancer cells the typical phorbol ester PMA induces TNFalpha secretion and apoptosis while bryo 1 fails to do so and antagonizes the PMA effect when used in combination. The mechanisms responsible for the unique effect of bryo 1 are still unknown. We have shown previously, using transcription factor analysis of microarray data from LNCaP cells, that multiple transcription factor families are involved in the dramatic transcriptional response to PMA and bryo 1 (3131 genes for PMA and 1569 genes for bryo 1). Further, analysis of 45 signaling pathways using reporter assays identified significant changes in 7 pathways induced both by PMA and bryo 1 at 6 hours. To further investigate the altered pathways, the novel Simple Western capillary immunoassay system (ProteinSimple) was used to analyze protein level changes following PKC activation. Simple Western system performs automated western analysis in capillaries using nanograms of lysate and provides digital quantitation with good sensitivity and excellent reproducibility. The system runs at a through-put of 96 sample / target combinations in a single overnight run. We analyzed 35 signals in total lysates (for detection of changes in protein levels and phosphorylation) and in nuclear extracts (for detection of translocation) from LNCaP cells treated for 60 min and 6 hrs. Changes downstream of PKC activation were measured, including PKC/PKD1 phosphorylation, activation of multiple members of the MEK/ERK pathway and levels of transcription factors of the NFKB, AP1, Stat and EGR families. Simple Western results correlated very well with conventional western analysis. The analysis validated the changes described previously using other methods on activation of MEK/ERK pathway and the changes in PKC phosphorylation, translocation and degradation. Bryo1 is different from PMA in failing to induce phosphorylation of PKCdelta at Tyr311 and the translocation of PKCalpha, delta, epsilon, and PKD1 to the nuclear fraction. Bryo1 is similar to PMA in inducing many of the early responses detected at 60 min (pPKCdeltaSer299, pMEK, pERK1/2, pPKD1 in total lysates, decrease in STAT3 levels etc.) but it fails to sustain the induced responses or to induce the late responses detected at 6 hours (pNFκBp65, increase in Fra1 and FosB levels, degradation of cMyc, translocation of cRel and RelB etc.). Using Simple Western system, we developed an approach for precise and accurate measurement of differential PMA and bryo 1 signaling in PKC activation. Further investigation needs to be performed to identify the mechanism/s responsible for the early termination of bryo 1 induced responses. Citation Format: Jin-Qiu Chen, Noemi Kedei, Michelle A. Herrmann, Peter M. Blumberg. Quantitative proteomic assessment of differential ligand responses downstream of protein kinase C activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5315. doi:10.1158/1538-7445.AM2014-5315

Abstract 1571: The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling

Abstract The basal sub-type, which shares features with triple-negative breast cancer (TNBC), is among the most lethal breast cancer subtype, characterized by a highly aggressive and metastatic phenotype. Although pathways that may represent targets for novel therapeutic intervention for basal like breast cancer (BLBC) have begun to be elucidated, the ability to define and selectively target the invasive and metastatic phenotype of basal-type/TNBC remains a major challenge facing the breast cancer field. Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is a known tumor suppressor in many cancers including breast. Low LKB1expression has been observed in breast cancer patients and we report a significant association between loss of LKB1 expression and poor prognosis specifically in the basal sub-type of breast cancer. Induction of LKB1 expression in BLBC cell lines inhibited invasiveness in vitro as well as lung and brain metastatic burden in an orthotopic xenograft tumor model. Further analysis of BLBC cell lines overexpressing LKB1 by next generation sequencing (RNA-seq) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extra cellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). We further demonstrated marked inhibition of matrix metalloproteinase 1 (MMP-1) expression and activity via regulation AP-1 family member cJun. Additionally, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating a reversal of the EMT phenotype in a triple-negative breast cancer cell line MDA-MB-231. We have demonstrated a role for LKB1 expression in the regulation of cell invasion and metastasis in addition to tumorigenesis. Taken together these data support future development of therapeutic agents to induce the LKB1 signaling pathway in BLCB/triple-negative breast cancer. Citation Format: Lyndsay V. Rhodes, Chandra R. Tate, Hope E. Burks, Van T. Hoang, Diari Gilliam, Elizabeth C. Martin, Steven Elliott, David FB Miller, Aaron Buechlein, Douglas Rusch, Haixu Tang, Kenneth P. Nephew, Matthew E. Burow, Bridgette M. Collins-Burow. The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2014-1571

Abstract 3395: Role of the activating protein 2 transcription factor in regulating cell invasion and migration in malignant glioma

Abstract Background: Malignant gliomas (MG) or high-grade astrocytomas are highly infiltrative brain tumors. Our previous work has shown that brain fatty acid binding protein (B-FABP) regulates the migratory properties of MG cells. B-FABP expression can be controlled by transcription factors such as NF1 and AP2. AP2 is a family of transcription factors involved in the regulation of genes responsible for cellular growth and differentiation during early development. Five AP2 genes have been identified in mice and humans: AP2α, AP2β, AP2γ, AP2δ and AP2ε. AP2 transcription factors are predominantly localized in the nucleus where they bind as homodimers or heterodimers to the consensus sequence GCCNNNGGC. Recent studies have shown that AP2α expression is inversely correlated with astrocytoma grade and that there are higher levels of AP2α in the cytoplasm of high-grade astrocytomas compared to low-grade astrocytomas. Objective: To study the regulation of B-FABP expression in response to AP2 in MG. Methodology: A panel of 10 MG cell lines was used to examine AP2α protein levels. Five of these cell lines were B-FABP +ve and five were B-FABP-ve. The effect of AP2 expression on endogenous B-FABP was examined by transfecting AP2 expression constructs into the B-FABP +ve U251 MG cell line. B-FABP levels were examined by western blot analysis and the subcellular localization of AP2 was studied by immunofluorescence analysis. Results: Western blot analysis indicates that AP2α is expressed in four out of five B-FABP-ve MG cell lines but in only one of the five B-FABP +ve MG cell lines tested. AP2 overexpression studies indicate that B-FABP levels are significantly reduced upon overexpression of AP2α, AP2β or AP2ε. Immunofluorescence data suggest preferential localization of AP2β in the cytoplasm of B-FABP +ve U251 MG cells. Our preliminary result shows difference in affinity towards heterodimerization between AP2 family members. Conclusion and future directions: AP2 is a negative regulator of B-FABP expression. Exclusion of AP2 from the nucleus allows transcription of B-FABP in MG cells, which in turn leads to increased cell migration. Experiments will be carried out to see the effect of AP2 heterodimerization on regulation of B-FABP transcription. Phosphorylation studies will be carried out to see whether AP2 phosphorylation affects its subcellular localization. This study may lead to new approaches to reduce B-FABP expression in MG, thereby reducing or preventing cell migration and tumor infiltration. Citation Format: Saket Jain, Ho Yin Poon, Roseline Godbout. Role of the activating protein 2 transcription factor in regulating cell invasion and migration in malignant glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3395. doi:10.1158/1538-7445.AM2014-3395

Lithium chloride suppresses LPS-mediated matrix metalloproteinase-9 expression in macrophages through phosphorylation of GSK-3β.

Abnormal degradation of matrix components due to dysregulated expression of matrix metalloproteinase (MMP)-9 in macrophages has been linked to progression of acute cerebral ischemia and atherosclerosis. We report that lithium chloride (LiCl) or CHIR99021, inhibitors of Wnt signaling pathway, enhance phosphorylation of glycogen synthase kinase-3beta and suppress lipopolysaccharide-mediated upregulation of MMP-9 expression in murine macrophage RAW264.7 cells in a dose-dependent manner. Suppression of MMP-9 expression by LiCl or CHIR99021 did not result after inhibition of kinases involved in NFκB or AP-1 family proteins, but from changes in the activity of histone deacetylases. Beneficial effects on atherosclerosis or cerebral ischemia in animal studies caused by LiCl may be in part explained by the suppression of MMP-9 gene expression.

Transcription factors and glyoxylate cycle genes prominent in the transition of soybean cotyledons to the first functional leaves of the seedling.

During early seedling growth, the cotyledons transition from a storage tissue to become effectively the first leaf-like structures of the plant. In this programmed developmental process, they likely undergo a massive change in gene expression to redirect their metabolism and physiological processes. To define the developmental shifts in gene expression and begin to understand the gene regulatory networks that set this transition in motion, we carried out high-throughput RNA sequencing of cotyledons from seven developmental stages of soybean seedlings. We identified 154 gene models with high expression exclusively in the early seedling stages. A significant number (about 25%) of those genes with known annotations were involved in carbohydrate metabolism. A detailed examination of glyoxylate cycle genes revealed the upregulation of their expression in the early stages of development. A total of approximately 50% of the highly expressed genes whose expression peaked in the mid-developmental stages encoded ribosomal family proteins. Our analysis also identified 219 gene models with high expression at late developmental stages. The majority of these genes are involved in photosynthesis, including photosystem I- and II-associated genes. Additionally, the advantage of RNA-Seq to detect genes expressed at low levels revealed approximately 460 transcription factors with notable expression in at least one stage of the developing soybean seedling. Relatively over-represented transcription factor genes encode AP2, zinc finger, NAC, WRKY, and MYB families. These transcription factor genes may lead to the transcriptional reprogramming during the transition of seedling cotyledons from storage tissue to metabolically active organs that serve as the first functional leaves of the plant.

Selection and characterization of a DNA aptamer that can discriminate between cJun/cJun and cJun/cFos.

The AP-1 family of transcriptional activators plays pivotal roles in regulating a wide range of biological processes from the immune response to tumorigenesis. Determining the roles of specific AP-1 dimers in cells, however, has remained challenging because common molecular biology techniques are unable to distinguish between the role of, for example, cJun/cJun homodimers versus cJun/cFos heterodimers. Here we used SELEX (systematic evolution of ligands by exponential enrichment) to identify and characterize DNA aptamers that are >100-fold more specific for binding cJun/cJun compared to cJun/cFos, setting the foundation to investigate the biological functions of different AP-1 dimer compositions.

Activation of the Aryl Hydrocarbon Receptor Dampens the Severity of Inflammatory Skin Conditions

SummaryEnvironmental stimuli are known to contribute to psoriasis pathogenesis and that of other autoimmune diseases, but the mechanisms are largely unknown. Here we show that the aryl hydrocarbon receptor (AhR), a transcription factor that senses environmental stimuli, modulates pathology in psoriasis. AhR-activating ligands reduced inflammation in the lesional skin of psoriasis patients, whereas AhR antagonists increased inflammation. Similarly, AhR signaling via the endogenous ligand FICZ reduced the inflammatory response in the imiquimod-induced model of skin inflammation and AhR-deficient mice exhibited a substantial exacerbation of the disease, compared to AhR-sufficient controls. Nonhematopoietic cells, in particular keratinocytes, were responsible for this hyperinflammatory response, which involved upregulation of AP-1 family members of transcription factors. Thus, our data suggest a critical role for AhR in the regulation of inflammatory responses and open the possibility for novel therapeutic strategies in chronic inflammatory disorders.

AP-2α is required after lens vesicle formation to maintain lens integrity.

Transcription factors are critical in regulating lens development. The AP-2 family of transcription factors functions in differentiation, cell growth and apoptosis, and in lens and eye development. AP-2α, in particular, is important in early lens development, and when conditionally deleted at the placode stage defective separation of the lens vesicle from the surface ectoderm results. AP-2α's role during later stages of lens development is unknown. To address this, the MLR10-Cre transgene was used to delete AP-2α from the lens epithelium beginning at embryonic day (E) 10.5. The loss of AP-2α after lens vesicle separation resulted in morphological defects beginning at E18.5. By P4, a small highly vacuolated lens with a multilayered epithelium was evident in the MLR10-AP-2α mutants. Epithelial cells appeared elongated and expressed fiber cell specific βB1 and γ-crystallins. Epithelial cell polarity and lens cell adhesion was disrupted and accompanied by the misexpression of ZO-1, N-Cadherin, and β-catenin. Cell death was observed in the mutant lens epithelium between postnatal day (P) 14 and P30, and correlated with altered arrangements of cells within the epithelium. Our findings demonstrate that AP-2α continues to be required after lens vesicle separation to maintain a normal lens epithelial cell phenotype and overall lens integrity and to ensure correct fiber cell differentiation.

Regulation of the HMOX1 gene by the transcription factor AP-2δ with unique DNA binding site.

AP-2 transcription factors are important sequence-specific DNA-binding regulators that are expressed in the neural crest and other tissues during mammalian development. The human AP-2 family of transcription factors consists of five members, AP-2α, -β, -γ, -δ and -ε, which have an important role in the regulation of gene expression during development and in the differentiation of multiple organs and tissues. The present study aimed to investigate the mechanism by which AP-2δ mediates heme oxygenase-1 (HMOX1) gene expression. It was identified that the human AP-2δ protein exhibited weak binding to a suboptimal AP-2 sequence, 5'-GCCN3GGC-3', to which all other AP-2 proteins bind invitro, providing the first example of DNA target specificity amongst the AP-2 family. AP-2δ protein bound to an optimized AP-2 consensus DNA sequence, 5'-GCCTGAGGC-3', invitro and transactivated gene expression in eukaryotic cells. The transactivation domain of Ap-2δ differs notably from those in the other AP-2 proteins as it lacks the PY motif (XPPXY) and several other conserved residues that are important for the transcriptional activity of AP-2 proteins, yet it functions as an equally strong activator.