Novel Mechanism Of Cross-talk Research Articles

Pyoluteorin regulates the biosynthesis of 2,4-DAPG through the TetR family transcription factor PhlH in Pseudomonas protegens Pf-5.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas.IMPORTANCEAntibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents.

1678P The interaction between cancer cells and cancer-associated fibroblasts promotes RAS signalling in squamous cell carcinoma, resulting in an immune-suppressive tumour microenvironment and poorer survival outcomes

Cancer-Associated Fibroblasts (CAFs) are a main component of the tumour microenvironment. Recent evidence suggests that high levels of CAFs correlate with poorer prognosis and a pro-tumorigenic microenvironment. The clinical relevance of CAF presence and the mechanisms by which they influence prognosis warrant further research. We used publicly available RNAseq data from The Cancer Genome Atlas (TCGA), gene expression data from cancer-cell (CC)-CAF co-cultures of three different cell lines. Further in vitro validation experiments were performed on human Head and Neck cancer cell lines, patient derived CAFs and macrophages from healthy donors. We generated a gene signature that predicts the abundance of CAF/CC interaction in clinical RNAseq data from a meta-analysis of transcriptomic data derived from Cancer Cell (CC) and CAF co-cultures. This gene signature is predictive of worse overall survival in datasets of Squamous Cell Carcinoma (SCC) patients. CAF abundance and the co-culture signature are strongly correlated with RAS signalling. Mechanistically, we showed that AP-1 transcription factor genes are strongly up-regulated when CCs and CAFs are in close contact. Direct contact between CCs and CAFs upregulates the RAS pathway in both cell types through membrane bound growth factors and growth factor receptors, activating AP-1 activity. We also demonstrated that the RAS / AP-1 axis is used by CCs to induce secretion of pro-tumorigenic and immune-inhibitory cytokines from CAFs, enhancing migration of potentially immune-suppressive macrophages. Of note, patients with high expression of CC-CAF coculture genes show an immune-suppressive microenvironment. Importantly, treatment with ERK inhibitors is able to disrupt the activation of this pathway, blocking cytokine production and impairing macrophage migration. In conclusion, we generated a gene signature predictive of overall survival in SCC patients, linked to cancer cell -CAF interaction and RAS signalling. This study reveals a novel mechanism of crosstalk between CCs and CAFs able to modulate the tumour immune microenvironment.

Inhibiting pyrimidine biosynthesis impairs Peste des Petits Ruminants Virus replication through depletion of nucleoside pools and activation of cellular immunity

Replication of peste des petits ruminants virus (PPRV) strongly depends on the cellular environment and resources of host cells including nucleoside pool. Thus, enzymes involved in nucleoside biosynthesis (such as pyrimidine biosynthesis pathway) are regarded as attractive targets for antiviral drug development. Here, we demonstrate that brequinar (BQR) and leflunomide (LFM) which are two specific inhibitors of DHODH enzyme and 6-azauracil (6-AU) which is an ODase enzyme inhibitor robustly inhibit PPRV replication in HEK293T cell line as well as in peripheral blood mononuclear cells isolated from goat. We further demonstrate that these agents exert anti-PPRV activity via the depletion of purimidine nucleotide. Interestingly, these inhibitors can trigger the transcription of antiviral interferon-stimulated genes (ISGs). However, the induction of ISGs is largely independent of the classical JAK-STAT pathway. Combination of BQR with interferons (IFNs) exerts enhanced ISG induction and anti-PPRV activity. Taken together, this study reveals an unconventional novel mechanism of crosstalk between nucleotide biosynthesis pathways and cellular antiviral immunity in inhibiting PPRV replication. In conclusion, targeting pyrimidine biosynthesis represents a potential strategy for developing antiviral strategies against PPRV.

Endothelial Reprogramming Stimulated by Oncostatin M Promotes Inflammation and Tumorigenesis in VHL-Deficient Kidney Tissue.

Clear-cell renal cell carcinoma (ccRCC) is the most prevalent subtype of renal cell carcinoma (RCC), and its progression has been linked to chronic inflammation. About 70% of the ccRCC cases are associated with inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene. However, it is still not clear how mutations in VHL, encoding the substrate-recognition subunit of an E3 ubiquitin ligase that targets the alpha subunit of hypoxia-inducible factor-α (HIFα), can coordinate tissue inflammation and tumorigenesis. We previously generated mice with conditional Vhlh knockout in kidney tubules, which resulted in severe inflammation and fibrosis in addition to hyperplasia and the appearance of transformed clear cells. Interestingly, the endothelial cells (EC), although not subject to genetic manipulation, nonetheless showed profound changes in gene expression that suggest a role in promoting inflammation and tumorigenesis. Oncostatin M (OSM) mediated the interaction between VHL-deficient renal tubule cells and the ECs, and the activated ECs in turn induced macrophage recruitment and polarization. The OSM-dependent microenvironment also promoted metastasis of exogenous tumors. Thus, OSM signaling initiates reconstitution of an inflammatory and tumorigenic microenvironment by VHL-deficient renal tubule cells, which plays a critical role in ccRCC initiation and progression. SIGNIFICANCE: A novel mechanism of cross-talk between ECs and VHL-deficient kidney tubules that stimulates inflammation and tumorigenesis is discovered, suggesting OSM could be a potential target for ccRCC intervention.

Protective Effects of Complement Component 8 Gamma Against Blood-Brain Barrier Breakdown.

The blood-brain barrier (BBB) regulates the traffic of micromolecules and macromolecules between the peripheral blood and the central nervous system, to maintain brain homeostasis. BBB disruption and dysfunction accompany a variety of neurological disorders and are closely related with the neuroinflammatory cascades that are triggered by leukocyte infiltration and glial activation. Here, we explored the role of complement component 8 gamma (C8G) in the maintenance of BBB integrity. Previously, C8G was shown to inhibit neuroinflammation by interfering with the sphingosine-1-phosphate (S1P)-S1PR2 interaction. The results of the present study revealed that C8G is localized in perivascular astrocytes, whereas S1PR2 is expressed in endothelial cells (ECs). In the lipopolysaccharide (LPS)-induced neuroinflammation model, the intracerebroventricular administration of the recombinant C8G protein protected the integrity of the BBB, whereas shRNA-mediated C8G knockdown enhanced BBB permeability and neutrophil infiltration. Using pharmacological agonists and antagonists of S1PR2, we demonstrated that C8G inhibited the inflammatory activation of ECs in culture by antagonizing S1PR2. In the in vitro BBB model, the addition of the recombinant C8G protein preserved endothelial integrity, whereas the knockdown of C8G exacerbated endothelial leakage under inflammatory conditions. Together, our findings indicate an important role for astrocytic C8G in protecting the BBB in the inflamed brain, suggesting a novel mechanism of cross talk between astrocytes and ECs in terms of BBB maintenance.

B cells sialylate bone marrow progenitors to inhibit granulocyte production

Abstract Effective hematopoiesis is predicated on the utilization of limited resources and space in the bone marrow during homeostasis and stress. During acute inflammation, secreted factors induce the egress of mature B cells from the medullary compartment, reclaiming space for the emergency proliferation of myeloid precursors. However, it remains unclear whether reciprocal mechanisms between B cells and myeloid cells exist during homeostasis. Recent work by our group has demonstrated the ability of extracellular glycosyltransferases to remodel plasma membrane glycans, altering cell sensitivity to growth factors. Here, we report that B cells secrete active ST6GAL1 sialyltransferase, capable of glycosylating cells by a non-cell autonomous, extrinsic mechanism. B cells secreted ST6GAL1 to glycosylate hematopoietic progenitors in co-culture, suppressing G-CSF-driven neutrophil production. In vivo, B cell-derived ST6GAL1 restored circulating enzyme levels and altered the sialylation of diverse hematopoietic populations after transplantation into St6gal1-deficient mice. In the bone marrow, colonizing IgD+ mature B cells colocalized with regions rich in sialylation. Finally, in the bone marrow of multiple myeloma patients, expression of ST6GAL1 correlated negatively with neutrophil abundance. Our findings underscore a novel mechanism of cross-talk between mature lymphocytes and myeloid progenitors involving paracrine extracellular sialylation, with potential implications for human disease.

CXCL11-CXCR3 Axis Mediates Tumor Lymphatic Cross Talk and Inflammation-Induced Tumor, Promoting Pathways in Head and Neck Cancers

Tumor metastasis to the draining lymph nodes is critical in patient prognosis and is tightly regulated by molecular interactions mediated by lymphatic endothelial cells (LECs). The underlying mechanisms remain undefined in the head and neck squamous cell carcinomas (HNSCCs). Using HNSCC cells and LECs we determined the mechanisms mediating tumor-lymphatic cross talk. The effects of a pentacyclic triterpenoid, methyl 2-trifluoromethyl-3,11-dioxoolean-1,12-dien-30-oate (CF3DODA-Me), a potent anticancer agent, were studied on cancer-lymphatic interactions. In response to inflammation, LECs induced the chemokine (C-X-C motif) ligand 9/10/11 chemokines with a concomitant increase in the chemokine (C-X-C motif) receptor 3 (CXCR3) in tumor cells. CF3DODA-Me showed antiproliferative effects on tumor cells, altered cellular bioenergetics, suppressed matrix metalloproteinases and chemokine receptors, and the induction of CXCL11-CXCR3 axis and phosphatidylinositol 3-kinase/AKT pathways. Tumor cell migration to LECs was inhibited by blocking CXCL11 whereas recombinant CXCL11 significantly induced tumor migration, epithelial-to-mesenchymal transition, and matrix remodeling. Immunohistochemical analysis of HNSCC tumor arrays showed enhanced expression of CXCR3 and increased lymphatic vessel infiltration. Furthermore, The Cancer Genome Atlas RNA-sequencing data from HNSCC patients also showed a positive correlation between CXCR3 expression and lymphovascular invasion. Collectively, our data suggest a novel mechanism for cross talk between the LECs and HNSCC tumors through the CXCR3-CXCL11 axis and elucidate the role of the triterpenoid CF3DODA-Me in abrogating several of these tumor-promoting pathways.

Activation of the mGlu1 metabotropic glutamate receptor has antipsychotic-like effects and is required for efficacy of M4 muscarinic receptor allosteric modulators

Recent clinical and preclinical studies suggest that selective activators of the M4 muscarinic acetylcholine receptor have potential as a novel treatment for schizophrenia. M4 activation inhibits striatal dopamine release by mobilizing endocannabinoids, providing a mechanism for local effects on dopamine signaling in the striatum but not in extrastriatal areas. G protein-coupled receptors (GPCRs) typically induce endocannabinoid release through activation of Gαq/11-type G proteins whereas M4 transduction occurs through Gαi/o-type G proteins. We now report that the ability of M4 to inhibit dopamine release and induce antipsychotic-like effects in animal models is dependent on co-activation of the Gαq/11-coupled mGlu1 subtype of metabotropic glutamate (mGlu) receptor. This is especially interesting in light of recent findings that multiple loss of function single nucleotide polymorphisms (SNPs) in the human gene encoding mGlu1 (GRM1) are associated with schizophrenia, and points to GRM1/mGlu1 as a gene within the “druggable genome” that could be targeted for the treatment of schizophrenia. Herein, we report that potentiation of mGlu1 signaling following thalamo-striatal stimulation is sufficient to inhibit striatal dopamine release, and that a novel mGlu1 positive allosteric modulator (PAM) exerts robust antipsychotic-like effects through an endocannabinoid-dependent mechanism. However, unlike M4, mGlu1 does not directly inhibit dopamine D1 receptor signaling and does not reduce motivational responding. Taken together, these findings highlight a novel mechanism of cross talk between mGlu1 and M4 and demonstrate that highly selective mGlu1 PAMs may provide a novel strategy for the treatment of positive symptoms associated with schizophrenia.

Par-4-dependent p53 up-regulation plays a critical role in thymoquinone-induced cellular senescence in human malignant glioma cells

Thymoquinone (TQ), the predominant bioactive constituent present in black cumin (Nigella sativa), exerts tumor suppressive activity against a wide variety of cancer cells. Cellular senescence, characterized by stable and long term loss of proliferative capacity, acts as a potent tumor suppressive mechanism. Here, we provide evidence for the first time that TQ suppresses growth of glioma cells by potentially inducing the expression of prostate apoptosis response-4 (Par-4) tumor suppressor protein. In turn, TQ-induced Par-4 expression triggers cellular senescence, as evidenced by increasing cellular size, β-galactosidase staining, G1 phase arrest, and increased expression of senescence markers such as p53, p21, Rb, and decreased expression of lamin B1, cyclin E and cyclin depended kinase-2 (CDK-2). Further, overexpression of Par-4 significantly increases the expression of p53 and its downstream target p21, and increases β-galactosidase positive cells, while siRNA/shRNA mediated-knockdown of Par-4 reverses the TQ-induced effects. Altogether, we describe a novel mechanism of cross talk between Par-4 and p53, that plays a critical role in TQ-induced senescence in human malignant glioma cells.

Abstract A03: FGF-WNT cooperativity and the role of lncRNAs in translational regulation in breast cancer

Abstract The purpose of this study is to understand translational mechanisms underlying the cooperativity between FGF and WNT signaling in cancer progression. FGF and WNT signaling, crucial pathways in mammary stem cell self-renewal, are frequently deregulated in human cancer. We previously showed that acute activation of FGF signaling in the mammary gland of pubertal mice with WNT hyperactivation dramatically accelerates cancer progression. FGF-WNT-hyperactive tumor cells exhibit enhanced activation of translational machinery. Given the potential involvement of translational regulation in FGF-WNT induced tumorigenesis, we employed ribosome profiling to identify selectively translated and ribosome associated RNAs induced by acute FGF activation in vivo. Many genes associated with RNA processing and transport were translationally upregulated. Interestingly, the most translationally upregulated transcript was Malat1, originally thought to be a nuclear-restricted long noncoding RNA (lncRNA) that regulates transcription of cancer-associated genes. Along with Malat1, many lncRNAs were associated with ribosomes in FGF-WNT tumors. Malat1's association with polysomes upon acute FGF activation was confirmed by direct polysome analysis. We also demonstrated that Malat1 transcripts are transported to the cytoplasm during G2/M transition, implicating that Malat1 may be translated into small novel peptides or involved in translation regulation during this particular phase of cell cycle. Additionally, there is evidence that Malat1 is a direct downstream target of WNT signaling. Our findings indicate that FGF activation potentially increases the translation efficiency of WNT-target oncogenes including lncRNAs like Malat1, unraveling a novel mechanism of crosstalk between lncRNAs and translational regulation in cancer progression. Citation Format: Tuan M. Nguyen, Elena B. Kabotyanski, Lucas C. Reineke, Qianxing Mo, Peng Zhang, Xiang Zhang, Yiwen Chen, Joel Neilson, Richard E. Lloyd, Jeffrey M. Rosen. FGF-WNT cooperativity and the role of lncRNAs in translational regulation in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A03.

Cowden syndrome-associated germline succinate dehydrogenase complex subunit D (SDHD) variants cause PTEN-mediated down-regulation of autophagy in thyroid cancer cells.

Thyroid cancer is a major component cancer of Cowden syndrome (CS), a disorder typically associated with germline mutations in PTEN. Germline variants in succinate dehydrogenase genes (SDHx) co-occurring with PTEN germline mutations confer a 2-fold increased prevalence (OR 2.7) of thyroid cancer compared to PTEN-associated CS but 50% decreased prevalence (OR 0.54) of thyroid cancer compared to SDHx-associated CS. We have previously shown that CS-associated SDHD variants G12S and H50R induce PTEN oxidation and nuclear accumulation in thyroid cancer. Our current study shows that SDHD-G12S and -H50R variants cause down-regulation of autophagy, demonstrating a role for SDHD in autophagy-associated pathogenesis of differentiated thyroid cancer. These findings could explain the increased prevalence of thyroid cancer in CS patients with SDHx germline mutations compared to those with PTEN mutations alone. Importantly, we demonstrate the dependence of this process on functional wild-type PTEN with reversal of decreased autophagy after PTEN knockdown. The latter could explain the clinically observed decrease in thyroid cancer prevalence in patients with co-existent PTEN mutations and SDHx variants. We also show that SDHD-G12S/H50R promotes mono-ubiquitination of PTEN, causing its translocation into the nucleus, upregulation of AKT and consequent phosphorylation of FOXO3a. Furthermore, SDHD-G12S/H50R-mediated increase in acetylation of FOXO3a further enhances AKT-associated phosphorylation of FOXO3a. This combination of phosphorylation and acetylation of FOXO3a results in its nuclear export for degradation and consequent down-regulation of FOXO3a-target autophagy-related gene (ATG) expression. Overall, our study reveals a novel mechanism of crosstalk amongst SDHD, PTEN and autophagy pathways and their potential roles in thyroid carcinogenesis.

HEY1 functions are regulated by its phosphorylation at Ser-68.

HEY1 (hairy/enhancer-of-split related with YRPW motif 1) is a member of the basic helix-loop-helix-orange (bHLH-O) family of transcription repressors that mediate Notch signalling. HEY1 acts as a positive regulator of the tumour suppressor p53 via still unknown mechanisms. A MALDI-TOF/TOFMS analysis has uncovered a novel HEY1 regulatory phosphorylation event at Ser-68. Strikingly, this single phosphorylation event controls HEY1 stability and function: simulation of HEY1 Ser-68 phosphorylation increases HEY1 protein stability but inhibits its ability to enhance p53 transcriptional activity. Unlike wild-type HEY1, expression of the phosphomimetic mutant HEY1-S68D failed to induce p53-dependent cell cycle arrest and it did not sensitize U2OS cells to p53-activating chemotherapeutic drugs. We have identified two related kinases, STK38 (serine/threonine kinase 38) and STK38L (serine/threonine kinase 38 like), which interact with and phosphorylate HEY1 at Ser-68. HEY1 is phosphorylated at Ser-68 during mitosis and it accumulates in the centrosomes of mitotic cells, suggesting a possible integration of HEY1-dependent signalling in centrosome function. Moreover, HEY1 interacts with a subset of p53-activating ribosomal proteins. Ribosomal stress causes HEY1 relocalization from the nucleoplasm to perinucleolar structures termed nucleolar caps. HEY1 interacts physically with at least one of the ribosomal proteins, RPL11, and both proteins cooperate in the inhibition of MDM2-mediated p53 degradation resulting in a synergistic positive effect on p53 transcriptional activity. HEY1 itself also interacts directly with MDM2 and it is subjected to MDM2-mediated degradation. Simulation of HEY1 Ser-68 phosphorylation prevents its interaction with p53, RPL11 and MDM2 and abolishes HEY1 migration to nucleolar caps upon ribosomal stress. Our findings uncover a novel mechanism for cross-talk between Notch signalling and nucleolar stress.

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication.

Cutting Edge: DNA in the Lung Microenvironment during Influenza Virus Infection Tempers Inflammation by Engaging the DNA Sensor AIM2.

Innate sensing of nucleic acids lies at the heart of antiviral immunity. During viral infection, dying cells may also release nucleic acids into the tissue microenvironment. It is unknown what effect such host signals have on the quality or duration of the immune response to viruses. In this study, we uncovered an immune-regulatory pathway that tempers the intensity of the host response to influenza A virus (IAV) infection. We found that host-derived DNA accumulates in the lung microenvironment during IAV infection. Ablation of DNA in the lung resulted in increased mortality, increased cellular recruitment, and increased inflammation following IAV challenge. The released DNA, in turn, was sensed by the DNA receptor absent in melanoma 2. Aim2(-/-) mice showed similarly exaggerated immune responses to IAV. Taken together, our results identify a novel mechanism of cross-talk between pathogen- and damage-associated molecular pattern-sensing pathways, wherein sensing of host-derived DNA limits immune-mediated damage to infected tissues.

Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis.

To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.

Subcellular gravin localization regulates PKA compartmentalization and PKA‐dependent signaling (539.7)

Scaffold proteins play a critical role in cellular homeostasis by positioning signaling molecules in proximity to downstream effectors. However, some scaffold proteins do not assemble static enzyme complexes, but form dynamic signalosomes that traffic to different subcellular compartments in response to stimuli. Gravin (AKAP12) ‐ a multivalent scaffold linked to cell cycle progression, cellular migration, β2‐adrenergic receptor regulation, and hippocampal learning and memory ‐ anchors PKA and other enzymes to the plasma membrane but is redistributed to the cytosol after [Ca2+]i elevation. We postulate that gravin redistribution represents a novel mechanism for cross‐talk between Ca2+‐dependent and cAMP‐dependent signaling pathways. To assess this, we measured the impact of gravin localization on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. Expression of gravin in cells lacking endogenous gravin caused an increase in forskolin‐dependent PKA activity in AKAR3 constructs targeted to the plasma membrane when compared to control cells lacking gravin or expressing a gravin construct lacking the PKA‐binding domain. Expression of a gravin mutant with reduced membrane localization showed no increase in membrane PKA activity compared to control cells lacking gravin. Interestingly, gravin expression caused a decrease in forskolin‐dependent PKA activity in cytosolic AKAR3 constructs compared to control cells lacking gravin. These results confirm that gravin localization regulates PKA signaling and support the hypothesis that gravin mediates cross‐talk between Ca2+ and cAMP‐dependent signaling in functions such as in cell cycle progression, cellular migration, β2‐adrenergic receptor regulation, and hippocampal learning and memory. Based on these results, dynamics in scaffold protein localization likely represents an important paradigm for the regulation of cellular signaling networks.Grant Funding Source: NIH P30GM103329

Cross-talk from β-Adrenergic Receptors Modulates α2A-Adrenergic Receptor Endocytosis in Sympathetic Neurons via Protein Kinase A and Spinophilin

Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α2ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α2AAR endocytosis and that α2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α2AARs. Importantly, we show that α2AR agonist-mediated α2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α2ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α2AARs and accelerating α2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α2AARs.

A novel mechanism of crosstalk between the p53 and NFκB pathways

The inflammation regulating transcription factor NFκB and the tumor-suppressing transcription factor p53 can act as functional antagonists. Chronic inflammation (NFκB activity) may contribute to the development of cancer through the inhibition of p53 function, while, conversely, p53 activity may dampen inflammation. Here we report that the E3 ubiquitin ligase MDM2, whose gene is transcriptionally activated by both NFκB and p53, can bind and inhibit the p65RelA subunit of NFκB. The interaction is mediated through the N-terminal and the acidic/zinc finger domains of MDM2 on the one hand and through the N-terminal Rel homology domain of p65RelA on the other hand. Co-expression of MDM2 and p65RelA caused ubiquitination of the latter in the nucleus, and this modification was dependent of a functional MDM2 RING domain. Conversely, inhibition of endogenous MDM2 by small-molecule inhibitors or siRNA significantly reduced the ubiquitination of ectopic and endogenous p65RelA. MDM2 was able to equip p65RelA with mutated ubiquitin moieties capable of multiple monoubiquitination but incapable of polyubiquitination; moreover, MDM2 failed to destabilize p65RelA detectably, suggesting that the ubiquitin modification of p65RelA by MDM2 was mostly regulatory rather than stability-determining. MDM2 inhibited the NFκB-mediated transactivation of a reporter gene and the binding of NFκB to its DNA binding motif in vitro. Finally, knockdown of endogenous MDM2 increased the activity of endogenous NFκB as a transactivator. Thus, MDM2 can act as a direct negative regulator of NFκB by binding and inhibiting p65RelA.

IL-4 Upregulates Igα and Igβ Protein, Resulting in Augmented IgM Maturation and B Cell Receptor–Triggered B Cell Activation

IL-4 is critical for optimal B cell activation and germinal center B cell expansion in T-dependent immune responses; however, the underlying mechanism remains elusive. In the current study, we found that primary B cells express little Igα and Igβ protein despite substantial levels of mRNA. IL-4 markedly upregulates Igα and Igβ protein expression that requires STAT6. Elevated Igα and Igβ protein form heterodimers that associate with IgM and significantly promote IgM maturation and surface IgM expression, resulting in amplified BCR-initiated signaling that is Lyn dependent. In vivo, we found that pregerminal center B cells express upregulated Igα, Igβ, and surface IgM expression, in conjunction with elevated BCR-triggered phosphorylated ERK ex vivo, that are dependent on IL-4 and reversed by in vivo administration of neutralizing anti-IL-4 Ab. Thus, this study elucidates a novel mechanism for cross-talk between the IL-4 and BCRs that programs enhancement of subsequent BCR signaling.

Insulin Receptor-Overexpressing β-Cells Ameliorate Hyperglycemia in Diabetic Rats through Wnt Signaling Activation

To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher therapeutic efficacy of IR-overexpressing β-cells.