Sepsis still remains a major cause for morbidity and mortality in patients. The molecular mechanisms underlying the disease are still enigmatic. A great number of therapeutic approaches have failed and treatment strategies are limited to date. Among those few admitted for clinical intervention, intensive insulin treatment has proven to be effective in the reduction of disease related complications in critically ill patients. Insulin effectively reduces glucose levels and thereby contributes to protection. On the other hand insulin is a potent signaling pathway activator. One of those is the PI3K signaling axis. Activation of PI3K is known to limit pro-inflammatory gene expression. Here we can show that in a mouse model of insulin hypersensitivity induced by the deletion of the PI3K antagonist PTEN, specifically in hepatic tissue, significant protection is conferred in murine models of lethal endotoxemia and sepsis. Acute inflammatory responses are diminished, glucose metabolism normalized and vascular activation is reduced. Furthermore we investigated the hepatic gene expression profile of relevant anti-inflammatory genes in PTEN deficient mice and found marked upregulation of PPARγ and HO-1. We conclude from our data that insulin hypersensitivity via sustained activation of the PI3K signaling pathway exerts protective effects in acute inflammatory processes.
Class 1 Phosphoinositide-3-Kinases (PI3Ks) have been widely studied and mediate essential roles in cellular proliferation, chemotaxis, insulin sensitivity and immunity. Here, we provide a comprehensive overview of how macrophage expressed PI3Ks and their downstream pathways orchestrate responses to metabolic stimuli and nutrients, polarizing macrophages, shaping their cellular identity and function. Particular emphasis will be given to adipose tissue macrophages, crucial players of insulin resistance and chronic metabolically triggered inflammation during obesity. An understanding of PI3K dependent wiring of macrophage responses is important as this is involved in various diseases ranging from obesity, type 2 diabetes to chronic inflammatory disease.
Summary The tumour suppressor phosphatase and tensin homologue (PTEN), mutated or lost in many human cancers, is a major regulator of angiogenesis. However, the cellular mechanism of PTEN regulation in endothelial cells so far remains elusive. Here, we characterise the urokinase receptor (uPAR, CD87) and its tumour-derived soluble form, suPAR, as a key molecule of regulating PTEN in endothelial cells. We observed uPAR-deficient endothelial cells to express enhanced PTEN mRNA- and protein levels. Consistently, uPAR expression in endogenous negative uPAR cells, down-regulated PTEN and activated the PI3K/Akt pathway. Additionally, we found that integrin adhesion receptors act as trans-membrane signaling partners for uPAR to repress PTEN transcription in a NF-κB-dependent manner. Functional in vitro assays with endothelial cells, derived from uPAR-deficient and PTEN heterozygous crossbred mice, demonstrated the impact of uPAR- dependent PTEN regulation on cell motility and survival. In an in vivo murine angiogenesis model uPAR-deficient PTEN heterozygous animals increased the impaired angiogenic phenotype of uPAR knockout mice and were able to reverse the high invasive potential of PTEN heterozygots. Our data provide first evidence that endogenous as well as exogenous soluble uPAR down-regulated PTEN in endothelial cells to support angiogenesis. The uPAR-induced PTEN regulation might represent a novel target for drug interference, and may lead to the development of new therapeutic strategies in anti-angiogenic treatment.
Abstract Tumor stroma crucially influences the growth of most epithelial tumors and is in focus of many therapeutic approaches. Several oncogenes and tumor suppressors coordinate complex signaling cascades in the tumor and its microenvironment. In this context, downregulation of the tumor suppressor PTEN was shown to be indispensable for (tumor-)angiogenesis and cancer outgrowth. However, the essential process leading to PTEN downregulation, favouring a pro-angiogenic phenotype, in tumor vasculature is unknown so far. Here we show that PTEN-dependent angiogenesis is centrally affected by tumor- secreted soluble uPAR. In detail, the presence of uPAR significantly influenced the levels of PTEN in vitro and in vivo. Endothelial cell stimulation with tumor- derived soluble uPAR remarkable decreased PTEN levels, which led to increased cell migration, enhanced wound healing as well as capillary-sprouting in vitro and enhanced endothelial cell invasion and functional vessel formation in a directed-in vivo angiogenesis assay. uPAR- derived PTEN regulation was not mediated via mRNA stability or decrease in PTEN degradation, but via regulation of PTEN transcription as revealed by actinomycin D experiments. uPAR thereby interacted with αv integrins, which enhanced FAK activation and - as a consequence - led to NFkB-dependent PTEN repression. Finally, the biological relevance of uPAR- dependent PTEN regulation was evaluated and confirmed via uPAR -/- crossbreds with either PTEN +/- or PTEN +/+ mice. Our results provide first evidence of the role of tumor-derived uPAR in regulation of the central phosphatase PTEN in angiogenic cell behaviour. Our findings give novel insights into the complex mechanism of the tumor- microenvironmental interaction and might lead to new therapeutic strategies in cancer. Citation Format: Matthias Unseld, Anastasia Chilla, Clemens Pausz, Johannes Breuss, Gernot Schabbauer, Gerald Prager. PTEN dependent angiogenesis is mainly regulated by (tumor secreted-) uPAR. [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 3602. doi:10.1158/1538-7445.AM2014-3602
Systemic bone loss in diseases such as osteoporosis or local bone destruction in rheumatoid arthritis, proriasis arthritis or ankylosing spondylitis often leads to disability and severely reduced quality of life. This damage to the bony structures is almost exclusively mediated by a special cell type, the osteoclasts. Therefore, it is important to understand factors and pathways regulating the generation of osteoclasts. In this study, the authors analysed the impact of the PI3-Kinase/PTEN axis on osteoclast generation and bone biology under basal and inflammatory conditions. PTEN is a lipid phosphatase and one of the main antagonists of the PI3-kinase. The authors show that mice with a monocyte/macrophage-specific deletion of PTEN (myeloid specific PTEN−/−) display increased osteoclastogenesis in vitro and in vivo compared to wild-type mice. However, under homeostatic conditions, enhanced osteoclastogenesis did not result in systemic bone loss. This was most likely due to a significantly increased bone formation evidenced by an enhanced mineral apposition rate in myeloid specific PTEN−/− mice, whereas osteoblast numbers were not different. In contrast, under inflammatory conditions in the hTNFtg mouse model of arthritis, myeloid specific PTEN−/− displayed enhanced local bone destruction as well as osteoclast formation in the inflamed joints. The extent of synovial inflammation, however, was not different between wt and myeloid specific PTEN−/− mice. These data demonstrate that enhanced PI3-Kinase activity in myeloid cells leads to increased osteoclastogenesis. Under homeostatic conditions, increased osteoclastogenesis is compensated by enhanced osteoblast activity. However, under inflammatory conditions, loss of control of PI3-Kinase activity by PTEN in myeloid cells leads to enhanced local bone destruction.
Abstract Infections induce complex host responses linked to antiviral defense, inflammation and tissue damage and repair. These processes are increasingly understood to involve systemic metabolic reprogramming. We hypothesized that the liver as a central metabolic hub may orchestrate many of these changes during infection. Thus, we investigated the systemic interplay between inflammation and metabolism in a mouse model of chronic viral infection and hepatitis. Here we show that virus-induced type I interferon (IFN-I) modulates wide-spread metabolic alterations of the liver in a hepatocyte-intrinsic Ifnar1-dependent way. Specifically, IFN-I repressed the transcription of numerous genes with metabolic function including Otc and Ass1 , which encode enzymes of the urea cycle. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8 T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation.
While the regulatory role of individual microRNAs (miRNAs) in rheumatoid arthritis (RA) is well established, the role of DICER1 in the pathogenesis of the disease has not yet been investigated. The purpose of this study was to analyze the expression of factors involved in miRNA biogenesis in fibroblast-like synoviocytes (FLS) from RA patients and to monitor the arthritis triggered by K/BxN serum transfer in mice deficient in the Dicer gene (Dicer(d/d) ).The expression of genes and precursor miRNAs was quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MicroRNA macroarray profiling was monitored by qRT-PCR. Cytokines were quantified by enzyme-linked immunosorbent assay. Experimental arthritis in mice was achieved by the transfer of serum from K/BxN donors. Apoptosis was quantified using an enzyme-linked immunosorbent assay.We found decreased DICER1 and mature miRNA expression in synovial fibroblasts from RA patients. These cells were hyperresponsive to lipopolysaccharide, as evidenced by their increased interleukin-6 secretion upon stimulation. Experimental serum-transfer arthritis in Dicer(d/d) mice confirmed that an unbalanced biogenesis of miRNAs correlated with an enhanced inflammatory response. Synoviocytes from both RA patients and Dicer(d/d) mice exhibited increased resistance to apoptotic stimuli.The findings of this study further substantiate the important role of DICER1 in the maintenance of homeostasis and the regulation of inflammatory responses.
Abstract Macrophages and antigen-presenting cells are important parts of the innate immune system as they fend off invading microbes, produce cytokines, activate other leukocytes and are implicated in the resolution of inflammatory processes and tissue remodeling. They have been found to contribute to tumor progression as they undergo phenotypic conversion from a ‘classical’ inflammatory state (M1) into an ‘alternatively’ activated, tumor tolerating state (M2). In our previous work we could show that the PI3K/PTEN signaling pathway plays a role in this fate decision process. Deletion of PTEN in myeloid cells leading to sustained PI3K activation results in an M2-like phenotype characterized by an increased release of anti-inflammatory factors. Alternative activation of myeloid cells is beneficial in acute models of infection and inflammation, but a diminished innate immune response could be detrimental in tumor development since tumor-associated macrophages show an immune-suppressive, M2-like phenotype. To address the question whether myeloid PTEN plays a role in tumor immune surveillance, we applied a model of colitis associated colon cancer (CAC) in myeloid cell specific PTEN-deficient mice. PTENfl/fl LysM cre conditional knock-out (KO) mice showed an increased tumor burden and increased mortality in this model of inflammation driven CAC. Isolated myeloid cells from these mice exhibited an up-regulation of M2-marker genes and a down-regulation of pro-inflammatory cytokines. Moreover we found an increase in immune-regulatory innate cells of the secondary lymphoid organs. T-cells isolated from PTEN KO-mice had decreased cytokine production as well as a reduced proliferative potential ex vivo. Therefore we suggest that myeloid PTEN deficiency leads to reduced T-cell stimulation thereby promoting intestinal tumor growth. Taken together this study highlights the importance of the PI3K/PTEN signaling axis in myeloid cells to control tumor immune surveillance and supports the idea that PI3K inhibitors currently used in clinical settings may have additional functions beyond tumor cell targeting. Citation Format: Mario Kuttke, Julia Pisoni, Emine Sahin, Sophie Percig, Andrea Vogel, Daniel Kraemmer, Leslie Hanzl, Johannes Schmid, Gernot Schabbauer. PTEN-deficiency in myeloid cells alters tumor immune surveillance in a murine model of inflammation driven colon cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B74.
Tissue factor (TF) is the primary cellular initiator of blood coagulation (1). It is constitutively expressed around blood vessels and plays an essential role in hemostasis. Under normal conditions, the surface of the endothelium is antithrombotic due to the binding of the anticoagulant protein tissue factor pathway inhibitor (TFPI) and the absence of TF. In addition, "resting" endothelium expresses the receptors thrombomodulin and endothelial protein C receptor, which permits the generation of the anticoagulant protein, activated protein C. However, under pathological conditions, the surface of the endothelium becomes prothrombotic due to the induction of TF and the downregulation of anticoagulants.
