Dependent Transcriptional Responses Research Articles

Abstract P014: Driving immune-dependent metabolic vulnerabilities in the breast tumor microenvironment

Abstract The tumor microenvironment (TME) is a complex arms race composed of host stroma and rapidly adapting cancer cells. This symbiosis is further complicated when dissecting the influence of inflammation, due to its multifaceted function within the TME as a driver of both pro- and anti-tumor responses. Comprising a major component of the TME, targeting or exploiting tumor-associated inflammation has long been sought for therapeutic purposes. However, the optimal amount and composition of inflammation for such purposes remains elusive. We have recently identified an interferon (IFN)-dependent transcriptional response in breast cancer cells that renders a unique sensitivity to oxidative stress produced by the biguanide-class complex I inhibitor, phenformin. Using syngeneic murine models representing luminal B (PyMT) and basal (4T1) breast cancer, we identified a modest sensitivity to phenformin administration in vivo. However, phenformin effectiveness is dramatically enhanced when used in combination with the toll-like receptor 3 (TLR3) agonist, polyinosinic:polycytidylic acid (poly (I:C)). Critically, the effectiveness of this combination treatment was lost when performed in immune-deficient mice (SCID-beige), indicating that an immune component was essential. These findings highlight a novel role for inflammation and the immune system to promote sensitivity to oxidative stress in the TME. By identifying cell types responsible for cultivating a microenvironment conducive to biguanide sensitivity in breast cancer, we can identify a novel immune-biomarker of complex I sensitivity. Therefore, using high-parameter flow cytometry, we are analyzing systemic and tumor-infiltrating leukocyte diversity and enumeration in both our genetically and phenotypically distinct breast cancer models. Combination-therapy-induced immune populations will be further characterized in their molecular and cellular response to TLR agonism and complex I inhibition. Functional responses such as inflammatory-mediator production, immunomodulatory activity, and direct and indirect tumor cytotoxicity will be assessed, ex vivo. Furthermore, monoclonal antibody-mediated depletion of candidate cell types will be used to validate their requirement and contribution to the observed synergy to the combination therapy. Examples of tumoricidal synergy between biguanides and the inflammatory TLR agonists have not yet been described, establishing these findings as novel additions to the field of tumor biology. Furthermore, identifying how sensitivity to oxidative stress can become situationally immune-dependent can greatly advance our understanding of how inflammation and metabolism intersect within the TME. Citation Format: John Heath, Stephanie Totten, Young Kyuen Im, Valerie Sabourin, Kathryn Hunt, Josie Ursini-Siegel. Driving immune-dependent metabolic vulnerabilities in the breast tumor microenvironment [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P014.

Research Highlights

Immune Regulation by Glucocorticoids Can Be Linked to Cell Type–dependent Transcriptional Responses Franco LM, Gadkari M, Howe KN, et al. J Exp Med. 2019;216(2):384–406. Glucocorticoids have been a mainstay for treatment of a range of inflammatory disorders for >60 years.1 While widely used, the biological basis underpinning their immunoregulatory effects is still poorly described. In general, glucocorticoids are known to act by binding to the glucocorticoid receptor (GR) in the cytosol, causing GR dimerization and subsequent translocation to the nucleus, where the ligand-bound GR binds to glucocorticoid response elements on the DNA increasing their transcriptional activity.2 Additionally, glucocorticoids are known to interfere with the activity of other transcription factors such as NF-êB.3 The genomic location of GR binding has been shown to differ greatly among different cell types. To further investigate how glucocorticoids differentially affect different cell types, Franco et al4 interrogated the genome-wide transcriptional response to glucocorticoids in 9 distinct human cell types in vitro and used this data to create a pathway-level map of the effects of glucocorticoids across these cell types. They found that the transcriptional response of different cell types to glucocorticoids is highly varied. The number of genes that responded to glucocorticoid treatment was greater in hematopoietic cells than in nonhematopoietic cells, with neutrophils having the greatest number of responsive genes. The specific glucocorticoid-responsive genes identified were also highly variable in each cell type, even when comparing cells of similar origin. Additionally, they found no correlation between the baseline expression of a gene and the average response of that gene to glucocorticoid treatment—many genes that were expressed at comparable baseline levels in hematopoietic and nonhematopoietic cells only responded to glucocorticoid treatment in one of these 2 groups. The authors also identified specific pathways affected by glucocorticoid stimuli. With a a specific focus on B cells, Franco et al found significant upregulation of IL-10, along with significant downregulation of essential B cell receptor signaling proteins, and a decrease in toll-like receptor expression. Functional studies confirmed that glucocorticoids diminished B cell receptor and toll-like receptor signaling. Glucocorticoids also increased expression of the gene encoding BLIMP-1, which is a central part of terminal differentiation in B cells. This could be a mechanism behind the reduced B cell proliferation seen following glucocorticoid administration. With detrimental effects of long-term glucocorticoid use well described, understanding the mechanistic pathways that underpin the immunoregulatory impact of these drugs may prove critical in finding novel agents that exert a similar response without adverse off-target effects. Mitochondrial Complex III Is Essential for Suppressive Function of Regulatory T Cells Weinberg SE, Singer BD, Steinert EM, et al. Nature. 2019;565(7740):495–499. In recent years, there has been a great interest in the potential of regulatory T cell (Treg) therapy for inducing tolerance after solid organ transplantation and for the treatment of graft-versus-host disease. With many promising trials,1 questions surrounding the perpetuation of the suppressive function of Tregs postinfusion remain. It has been shown that there are defined metabolic profiles associated with Treg differentiation and phenotype. Treg cells are largely dependent on mitochondrial metabolism and have been shown to oxidize pyruvate and lipids preferentially when compared with conventional T cells. This unique metabolic profile has been functionally linked to the suppressive phenotype of Treg cells; indeed, the plasticity of Treg suppressive function may well be linked to their metabolic plasticity. To investigate whether Treg suppression is reliant on the mitochondrial respiratory chain, Weinberg et al2 generated mice with a Treg-specific Rieske iron-sulfur protein (RISP) knockout. RISP is an essential part of the mitochondrial respiratory chain complex III, thus ablating mitochondrial complex III function. These mice displayed substantial inflammation by the age of 3 weeks, with enlarged lymph nodes and spleens, thymic atrophy, and increased CD4+ and CD8+ T cell activation and proliferation in the spleen and lymph nodes. This phenotype is highly reminiscent of the Treg deficient Scurfy mice3; however, the number of Tregs in RISP knockout mice remained unchanged. RISP knockout Tregs demonstrated significantly reduced suppressive capacity both in vitro and in vivo. Interestingly, Foxp3 expression levels did not change, demonstrating that Treg suppressive function is reliant on mitochondrial complex III. To further verify that this phenotype was due to a lack in mitochondrial complex III function, the authors also generated mice with a Treg-specific knockout of QPC, another essential subunit of mitochondrial complex III. A similar pattern of systemic inflammation and overt overproliferation of CD4+ and CD8+ T cells was observed, resulting in death of the mice by 3 weeks. These data show that mitochondrial complex III function is necessary for Treg suppressive function and global immune regulation. Understanding how mitochondrial function can affect Treg suppressive potency may prove important for their use as a cellular therapy posttransplantation, given that such therapies may be rendered nonfunctional in an ischemic microenvironment.

Immune regulation by glucocorticoids can be linked to cell type–dependent transcriptional responses

Glucocorticoids remain the most widely used immunosuppressive and anti-inflammatory drugs, yet substantial gaps exist in our understanding of glucocorticoid-mediated immunoregulation. To address this, we generated a pathway-level map of the transcriptional effects of glucocorticoids on nine primary human cell types. This analysis revealed that the response to glucocorticoids is highly cell type dependent, in terms of the individual genes and pathways affected, as well as the magnitude and direction of transcriptional regulation. Based on these data and given their importance in autoimmunity, we conducted functional studies with B cells. We found that glucocorticoids impair upstream B cell receptor and Toll-like receptor 7 signaling, reduce transcriptional output from the three immunoglobulin loci, and promote significant up-regulation of the genes encoding the immunomodulatory cytokine IL-10 and the terminal-differentiation factor BLIMP-1. These findings provide new mechanistic understanding of glucocorticoid action and emphasize the multifactorial, cell-specific effects of these drugs, with potential implications for designing more selective immunoregulatory therapies.

Comparative Transcriptomic and Epigenomic Analyses Reveal New Regulators of Murine Brown Adipogenesis

Increasing energy expenditure through brown adipocyte recruitment is a promising approach to combat obesity. We report here the comprehensive profiling of the epigenome and transcriptome throughout the lineage commitment and differentiation of C3H10T1/2 mesenchymal stem cell line into brown adipocytes. Through direct comparison to datasets from differentiating white adipocytes, we systematically identify stage- and lineage-specific coding genes, lncRNAs and microRNAs. Utilizing chromatin state maps, we also define stage- and lineage-specific enhancers, including super-enhancers, and their associated transcription factor binding motifs and genes. Through these analyses, we found that in brown adipocytes, brown lineage-specific genes are pre-marked by both H3K4me1 and H3K27me3, and the removal of H3K27me3 at the late stage is necessary but not sufficient to promote brown gene expression, while the pre-deposition of H3K4me1 plays an essential role in poising the brown genes for expression in mature brown cells. Moreover, we identify SOX13 as part of a p38 MAPK dependent transcriptional response mediating early brown cell lineage commitment. We also identify and subsequently validate PIM1, SIX1 and RREB1 as novel regulators promoting brown adipogenesis. Finally, we show that SIX1 binds to adipogenic and brown marker genes and interacts with C/EBPα, C/EBPβ and EBF2, suggesting their functional cooperation during adipogenesis.

Charon Mediates Immune Deficiency-Driven PARP-1-Dependent Immune Responses in Drosophila.

Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. In this article, we describe a novel molecular mechanism controlling NF-κB-dependent innate immune response. We show that a previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. Our results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, to our knowledge, our results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.

Abstract 420: G3BP1 Activation of NFATc Signaling is Inhibited by LRP6 in Aortic Vascular Smooth Muscle Cells

When fed a high fat diet, male LDL receptor -deficient (LDLR-/-) mice develop type 2 diabetes (T2D) with concomitant arteriosclerotic calcification. We recently identified that vascular smooth muscle (VSM) LRP6 expression inhibits pro-sclerotic noncanonical Wnt signaling, mediated in part by protein arginine (Arg) methylation relays that converge on NFAT- dependent transcriptional programs. To better understand the mechanisms whereby Arg methylation mediates LRP6 actions,immunoaffinity mass spectrometry analysis was performed on aortic VSM extracts from SM22-LRP6(fl/fl);LDLR-/- and LRP6(fl/fl);LDLR-/- control mice. Applying a 2.5- fold cutoff threshold, alterations were noted in 499 monomethylated (MMA), 109 symmetric dimethylated (SDMA), and 65 asymmetric dimethylated (ADMA) protein cognates. One protein, G3BP1 (GTPase activating-protein binding-protein 1), demonstrated increases in MMA on residues 433, 435 & 458 -- with concomitant reductions SDMA on residue 458 —with LRP6 deficiency. G3BP1 exhibited both nuclear and non-nuclear localization, consistent with NTF2 and RRM structural motifs. Transient transfection and MMA western blot analysis confirmed that protein Arg methyltransferase 1 recognized G3BP1 as a substrate. This modification was inhibited by co-expression of LRP6. G3BP1activated osteopontin (OPN)- and NFATc3- dependent transcriptional responses, with NFAT transcription enhanced by co-expression of either Fzd10 or constitutively active cdc42. LRP6 inhibited G3BP1+NFAT activation of artificial and native (OPN, TNF) NFATc3-targeted promoter - reporter constructs. Moreover, NFATc3 protein levels were increased in nuclear extracts of LRP6-deficient VSM and reduced by G3BP1-directed siRNA, with concomitant reduction in genomic targets. Structure-function analysis mapped transcriptional activation of NFAT signaling by G3BP1 to the 32 amino-acid C-terminal domain encompassing Arg methylation cognates. Thus, G3BP1 is a cellular target of Arg methylation that is negatively regulated by LRP6 and mediates inflammatory osteogenic signaling in VSM. Strategies targeting G3BP1 and/or its Arg-methylation may help limit arteriosclerotic calcification and vascular disease in the setting of type 2 diabetes.

Abstract A41: Shaping Myc-dependent transcriptional amplification

Abstract Myc deregulation, resulting in the elevated and constitutive expression of the protein, is a common oncogenic event that gives rise to numerous tumorigenic phenotypes in multiple tumor types. When, elevated, Myc acts as a general transcriptional regulator, non-linearly amplifying the expression of most transcriptionally active genes in the tumor cell. However the gene specific contexts that shape this non-linear response are poorly understood. Here we report that in B-cells, transcriptional response at active genes is largely a function of the change in Myc occupancy at promoter regions, consistent with Myc dependent transcriptional amplification. To explore determinants of Myc binding affinity, changes in Myc promoter occupancy were compared to sequence, transcription, and chromatin features in cells prior to Myc elevation. Changes in Myc occupancy correlate most strongly with promoter GC content rather than with the density of Myc E-box binding motifs or any other analyzed promoter feature. The ability of GC rich promoters to garner increased Myc occupancy levels suggest that general structural sequence and regulatory features of the promoter play a role in shaping Myc binding affinity. Importantly, Myc binding affinity, although generally correlating with transcriptional response, fails to articulate much of the observed gene expression variance suggesting that additional regulatory layers, including Myc interacting co-regulators such as MIZ1, likely contribute to shaping Myc dependent transcriptional responses. Citation Format: Charles Lin, James E. Bradner. Shaping Myc-dependent transcriptional amplification. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A41.

Repression of TIF1γ by SOX2 promotes TGF-β-induced epithelial-mesenchymal transition in non-small-cell lung cancer.

TIF1γ is a novel regulator of transforming growth factor (TGF)-β/Smad signaling. Our previous studies show that dysregulated expression of transcriptional intermediary factor 1 γ (TIF1γ) and abnormal TGF-β/Smad signaling are implicated in non-small-cell lung cancer (NSCLC) separately. However, how TIF1γ contributes to NSCLC by controlling TGF-β/Smad signaling is poorly understood. Here, we investigated the mechanistic role of TIF1γ in TGF-β-induced epithelial-mesenchymal transition (EMT), as well as a link between TIF1γ and SOX2 in NSCLC. We show that TIF1γ is a downstream target of SOX2 in NSCLC cells. SOX2 overexpression negatively regulated TIF1γ promoter activity and thereby attenuated TIF1γ mRNA and protein expression levels; SOX2 knockdown significantly enhanced TIF1γ promoter activity and augmented TIF1γ expression. Moreover, TIF1γ mRNA expression was downregulated in human NSCLC tissues and negatively correlated with SOX2 protein, which was upregulated in NSCLC tissues. Importantly, knockdown of TIF1γ or SOX2 overexpression augmented SMAD4 (human Mad (mothers against decapentaplegic)-related homologous protein 4)-dependent transcriptional responses, and enhanced TGF-β-induced EMT and human NSCLC cell invasion; knockdown of SOX2 impaired TGF-β-induced EMT and NSCLC cell invasion. In an in vivo model of metastasis, knockdown of TIF1γ promotes NSCLC cell metastasis. In addition, our data suggested that TIF1γ inhibited TGF-β-induced EMT through competing with SMAD4 in NSCLC cells. Taken together, our findings reveal a new mechanism by which SOX2-mediated transcription repression of TIF1γ promotes TGF-β-induced EMT in NSCLC.

Cholinergic transactivation of the EGFR in HaCaT keratinocytes stimulates a flotillin-1 dependent MAPK-mediated transcriptional response.

Acetylcholine and its receptors regulate numerous cellular processes in keratinocytes and other non-neuronal cells. Muscarinic acetylcholine receptors are capable of transactivating the epidermal growth factor receptor (EGFR) and, downstream thereof, the mitogen-activated protein kinase (MAPK) cascade, which in turn regulates transcription of genes involved in cell proliferation and migration. We here show that cholinergic stimulation of human HaCaT keratinocytes results in increased transcription of matrix metalloproteinase MMP-3 as well as several ligands of the epidermal growth factor family. Since both metalloproteinases and the said ligands are involved in the transactivation of the EGFR, this transcriptional upregulation may provide a positive feed-forward loop for EGFR/MAPK activation. We here also show that the cholinergic EGFR and MAPK activation and the upregulation of MMP-3 and EGF-like ligands are dependent on the expression of flotillin-1 which we have previously shown to be a regulator of MAPK signaling.

Mitogen-activated protein kinase Hog1 is activated in response to curcumin exposure in the budding yeast Saccharomyces cerevisiae.

BackgroundCurcumin (CUR), an active polyphenol derived from the spice turmeric, has been traditionally used for centuries in ancient Indian medicine to treat a number of diseases. The physiological effects of CUR have been shown to be diverse; however, the target molecules and pathways that CUR affects have yet to be fully described.ResultsHere, we demonstrate for the first time that the budding yeast mitogen-activated protein kinase (MAPK) Hog1 is essential for the response to CUR. Moreover, CUR-induced Hog1 phosphorylation was rescued by supplementation of iron to the growth medium. Hog1 was rapidly phosphorylated upon CUR treatment, but unlike the response to hyperosmotic shock (0.8 M NaCl), it remains activated for an extended period of time. A detailed analysis of HOG pathway mutants revealed that Pbs2p, Ptc2p, and Ssk2p are required for optimal CUR-induced Hog1 phosphorylation. We also observed a Hog1 dependent transcriptional response to CUR treatment that involved the up-regulation of glycerol-3-phosphate dehydrogenase 1 (GPD1), a factor that is essential for the hyperosmotic stress response.ConclusionsOur present finding revealed the role of Hog1 MAPK in regulation of CUR-induced transcriptional response. We anticipate that our finding will enhance the understanding on the molecular mode of action of CUR on S. cerevisiae.

PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS

Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.

Calcium responses to synaptically activated bursts of action potentials and their synapse-independent replay in cultured networks of hippocampal neurons

Both synaptic N-methyl-d-aspartate (NMDA) receptors and voltage-operated calcium channels (VOCCs) have been shown to be critical for nuclear calcium signals associated with transcriptional responses to bursts of synaptic input. However the direct contribution to nuclear calcium signals from calcium influx through NMDA receptors and VOCCs has been obscured by their concurrent roles in action potential generation and synaptic transmission. Here we compare calcium responses to synaptically induced bursts of action potentials with identical bursts devoid of any synaptic contribution generated using the pre-recorded burst as the voltage clamp command input to replay the burst in the presence of blockers of action potentials or ionotropic glutamate receptors. Synapse independent replays of bursts produced nuclear calcium responses with amplitudes around 70% of their original synaptically generated signals and were abolished by the L-type VOCC blocker, verapamil. These results identify a major direct source of nuclear calcium from local L-type VOCCs whose activation is boosted by NMDA receptor dependent depolarization. The residual component of synaptically induced nuclear calcium signals which was both VOCC independent and NMDA receptor dependent showed delayed kinetics consistent with a more distal source such as synaptic NMDA receptors or internal stores. The dual requirement of NMDA receptors and L-type VOCCs for synaptic activity-induced nuclear calcium dependent transcriptional responses most likely reflects a direct somatic calcium influx from VOCCs whose activation is amplified by synaptic NMDA receptor-mediated depolarization and whose calcium signal is boosted by a delayed input from distal calcium sources mostly likely entry through NMDA receptors and release from internal stores. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

Abstract 4828: A novel small molecule p53-pathway restoring compound suppresses colorectal cancer cells via p73

Abstract The tumor suppressor p53 regulates gene transcription impacting on cell cycle, cell death, angiogenesis, cell metabolism, or repair processes. Mutant p53 is found in approximately 50% of human cancers where it contributes to tumorigenesis, tumor progression and resistance to therapy. p53 pathway restoration is an outstanding target for cancer therapy because mutant p53 provides a basic difference between normal cells and tumor cells and because two decades ago wild-type p53 was shown to suppress colon cancer cell colony growth despite numerous other genetic alterations. More recently the power of p53 as a tumor suppressor was demonstrated in genetically engineered mouse models. To find novel drugs targeting p53 for cancer therapy, we set up a functional cell-based screening assay for tumor cell death-inducing small molecules that restore p53 pathway dependent transcriptional responses. Using the Chembridge library of 50,000 compounds we identified CB001 that restores p53 pathway signaling and induces cancer but not normal cell death. CB001 strongly induces p53-responsive transcriptional activity in a time- and dose-dependent manner in mutant-p53 containing human cancer cells. p53 target gene expression, including p21 and Puma, was upregulated in mutant-p53 containing cancer cells treated with CB001. Importantly, CB001 was found to induce cell death at low doses with minimal genotoxicity in human colorectal cancer cells with no evident toxicity towards normal cells. We further examined the role of p73, a member of p53 family, in the mechanism of action of CB001 towards p53 pathway restoration. Over-expression of p73 enhanced p53-responsive transcriptional activity induced by CB001, while knock-down of p73 reduced its induction of p53-responsive transcriptional activity in p53-deficient cancer cells. Protein lysate analysis showed that treatment with CB001 resulted in a significant increase in the p73 protein level and the phosphorylation of p73 in mutant-p53 containing cancer cells. Overexpression of p73 sensitized cancer cells to CB001 induced cell death. These results suggest that p53-responsive transcriptional activity and cell death induced by CB001 occur by targeting p73 in tumor suppressor p53-deficient cancer cells. Combination treatments demonstrate that CB001 can synergize with classical cytotoxic chemotherapy including 5-FU and CPT11 to induce cell death in mutant-p53 expressing colorectal cancer cells. Thus, CB001 may be used as a lead compound for the development of new anti-cancer drugs targeting the p53 pathway via a mechanism involving the p53 family member p73. This work was supported by the NCI Developmental Therapeutics Program through NCI Contract N01-CN-43302. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4828. doi:1538-7445.AM2012-4828

Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL

Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk−/− macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program.

Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass.

BackgroundThe progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansionResultsHere we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell.ConclusionsOur data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

RNAi Screening Implicates a SKN-1–Dependent Transcriptional Response in Stress Resistance and Longevity Deriving from Translation Inhibition

Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1–like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR–induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO), which is required for longevity that derives from reduced IIS. Our results indicate that the SKN-1 detoxification gene network monitors various metabolic and regulatory processes. Interference with one of these processes, translation initiation, leads to a transcriptional response whereby SKN-1 promotes stress resistance and functions together with DAF-16 to extend lifespan. This stress response may be beneficial for coping with situations that are associated with reduced protein synthesis.

Solid tumours arising from differently pre-oxygenated cells: Comparable growth rates despite dissimilar tissue oxygenation

Purpose: The hypoxia-inducible factor (HIF)-dependent transcriptional response is often very pronounced in hypoxic microregions of solid malignant tumours, leading to secretion of pro-angiogenic factors and activation of a hypoxia-tolerant, glycolytic metabolism. Here, the influence of the microenvironment of tumour-initiating cells as a factor determining intertumoural variations in the relative contributions of both processes has been examined.Material and methods: The oxygenation status was assessed in rat DS-sarcomas using polarographic needle electrodes. Tumours were generated by allografting cells from either normoxic cell culture or severely hypoxic/anoxic ascites. HIF-related marker expression and intercapillary distances were analysed using immunohistochemistry.Results: Cells preconditioned in hypoxic ascites form poorly vascularised, hypoxic tumours in rats, showing strong activation of HIF-1α and glucose transporter (GLUT)- 1. Conversely, tumour-initiating DS-cells derived from normoxic cell culture form highly angiogenic, normoxic tumours with a significantly lower expression of HIF-1α and GLUT-1. Growth rates and the fraction of Ki-67 positive cells for both tumour groups were comparable.Conclusions: The intensity of angiogenesis in this model is primarily determined by the state of metabolic adaptation of tumour-initiating cells, rather than being a function of HIF-activation during solid tumour growth, a finding which is highly relevant for the design of treatment regimens targeting the tumour vasculature.

Type I interferon receptor-independent and -dependent host transcriptional responses to mouse hepatitis coronavirus infection in vivo

BackgroundThe role of type I IFNs in protecting against coronavirus (CoV) infections is not fully understood. While CoVs are poor inducers of type I IFNs in tissue culture, several studies have demonstrated the importance of the type I IFN response in controlling MHV infection in animals. The protective effectors against MHV infection are, however, still unknown.ResultsIn order to get more insight into the antiviral gene expression induced in the brains of MHV-infected mice, we performed whole-genome expression profiling. Three different mouse strains, differing in their susceptibility to infection with MHV, were used. In BALB/c mice, which display high viral loads but are able to control the infection, 57 and 121 genes were significantly differentially expressed (≥ 1.5 fold change) upon infection at 2 and 5 days post infection, respectively. Functional association network analyses demonstrated a strong type I IFN response, with Irf1 and Irf7 as the central players. At 5 days post infection, a type II IFN response also becomes apparent. Both the type I and II IFN response, which were more pronounced in mice with a higher viral load, were not observed in 129SvEv mice, which are much less susceptible to infection with MHV. 129SvEv mice lacking the type I interferon receptor (IFNAR-/-), however, were not able to control the infection. Gene expression profiling of these mice identified type I IFN-independent responses to infection, with IFN-γ as the central player. As the BALB/c and the IFNAR-/- 129SvEv mice demonstrated very similar viral loads in their brains, we also compared their gene expression profiles upon infection with MHV in order to identify type I IFN-dependent transcriptional responses. Many known IFN-inducible genes were detected, several of which have previously been shown to play an important protective role against virus infections. We speculate that the additional type I IFN-dependent genes that we discovered may also be important for protection against MHV infection.ConclusionTranscriptional profiling of mice infected with MHV demonstrated the induction of a robust IFN response, which correlated with the viral load. Profiling of IFNAR-/- mice allowed us to identify type I IFN-independent and -dependent responses. Overall, this study broadens our present knowledge of the type I and II IFN-mediated effector responses during CoV infection in vivo.

Exposure to perfluorooctane sulfonate or fenofibrate causes PPAR-α dependent transcriptional responses in chicken embryo hepatocytes

Perfluorooctane sulfonate (PFOS) is a globally distributed environmental contaminant that is detected in the serum and liver of numerous mammalian and avian species. PFOS acts as a peroxisome proliferator in rodents, which occurs subsequent to activation of the nuclear receptor peroxisome proliferator activated receptor-alpha (PPAR-α). Activated PPAR-α up-regulates PPAR-α target genes, most of which are involved in lipid metabolism. Although several studies have investigated the effects of PFOS exposure on mammalian gene expression, there are few studies in avian species. To determine if PFOS is capable of activating avian PPAR-α, we exposed chicken embryo primary hepatocyte cultures ( N = 3 independent cell cultures) to PFOS or fenofibrate, a mammalian PPAR-α agonist, and examined the expression of PPAR-α and PPAR-α target genes using quantitative real-time PCR. The target genes examined were peroxisomal acyl-CoA oxidase (ACOX), liver fatty acid binding protein (L-FABP), enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase bifunctional enzyme (BIEN), peroxisomal 3-ketoacyl thiolase (PKT), and malic enzyme (ME). All five target genes were induced in response to PFOS exposure and all of the target genes, except L-FABP, were induced in response to fenofibrate. PPAR-α mRNA expression was not altered by PFOS or fenofibrate. This study provides the first evidence that PFOS can induce PPAR-α-dependent transcriptional responses in an avian species and provides the first characterization of fenofibrate induced transcriptional responses in chicken embryo hepatocyte cultures.

Gene expression analysis of BCR/ABL1‐dependent transcriptional response reveals enrichment for genes involved in negative feedback regulation

Philadelphia (Ph) chromosome-positive leukemia is characterized by the BCR/ABL1 fusion protein that affects a wide range of signal transduction pathways. The knowledge about its downstream target genes is, however, still quite limited. To identify novel BCR/ABL1-regulated genes we used global gene expression profiling of several Ph-positive and Ph-negative cell lines treated with imatinib. Following imatinib treatment, the Ph-positive cells showed decreased growth, viability, and reduced phosphorylation of BCR/ABL1 and STAT5. In total, 142 genes were identified as being dependent on BCR/ABL1-mediated signaling, mainly including genes involved in signal transduction, e.g. the JAK/STAT, MAPK, TGFB, and insulin signaling pathways, and in regulation of metabolism. Interestingly, BCR/ABL1 was found to activate several genes involved in negative feedback regulation (CISH, SOCS2, SOCS3, PIM1, DUSP6, and TNFAIP3), which may act to indirectly suppress the tumor promoting effects exerted by BCR/ABL1. In addition, several genes identified as deregulated upon BCR/ABL1 expression could be assigned to the TGFB and NFkB signaling pathways, as well as to reflect the metabolic adjustments needed for rapidly growing cells. Apart from providing important pathogenetic insights into BCR/ABL1-mediated leukemogenesis, the present study also provides a number of pathways/individual genes that may provide attractive targets for future development of targeted therapies. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.