Role of MAP4K4 Signaling in Adipocyte and Macrophage Derived Inflammation: A Dissertation

Human obesity is increasing globally at an impressive rate. The rise in obesity has led to an increase in diseases associated with obesity, such as type 2 diabetes. A major prerequisite for this disease is the development of insulin resistance in the muscle and adipose tissues. Interestingly, experiments in rodent models suggest that adipocytes and macrophages can profoundly influence the development of insulin resistance. Accordingly, the number of adipose tissue macrophages increases substantially during the development of obesity. Numerous research models have demonstrated that macrophages promote insulin resistance by secreting cytokines, like TNFα, which impair whole body insulin sensitivity and adipose tissue function. Additionally, enhancements of murine adipose function, particularly glucose disposal, prevent the development of insulin resistance in mice on a high fat diet. Thus, mechanisms which enhance adipose function or attenuate macrophage inflammation are of interest. Our lab previously identified mitogen activated protein kinase kinase kinase kinase 4 (MAP4K4) as a potent negative regulator of adipocyte function. In these studies, TNFα treatment increased the expression of adipocyte MAP4K4. Furthermore, the use of small interfering RNAs (siRNA) to block the increase in MAP4K4 expression protected adipocytes from some of the adverse effects of TNFα. Because MAP4K4 is a potent negative regulator of adipocyte function, an understanding of the mechanisms by which TNFα regulates MAP4K4 expression is of interest. Thus, for the first part of this thesis, I characterized the signaling pathways utilized by TNFα to regulate MAP4K4 expression in cultured adipocytes. Here I show that TNFα increases MAP4K4 expression through a pathway requiring the transcription factors activating transcription factor 2 (ATF2) and the JUN oncogene (cJUN). Through TNFα receptor 1 (TNFR1), but not TNFR2, TNFα increases MAP4K4 expression. This increase is highly specific to TNFα, as the inflammatory agents IL-1β, IL-6 and LPS did not affect MAP4K4 expression. In agreement, the activation of cJUN and ATF2 by TNFα is sustained over a longer period of time than by IL-1β in adipocytes. Finally, MAP4K4 is unique as the expression of other MAP kinases tested fails to change substantially with TNFα treatment. For the second part of this thesis, I assessed the role of MAP4K4 in macrophage inflammation in vitro and in vivo. To accomplish this task, pure β1,3-D-glucan shells were used to encapsulate siRNA. Glucan shells were utilized because they are effectively taken up by macrophages which express the dectin-1 receptor and they survive oral delivery. I demonstrate that these β1,3-D-glucan encapsulated RNAi particles (GeRPs) are efficiently phagocytosed and capable of mediating the silencing of multiple macrophage genes in vitro and in vivo. Importantly, oral treatment of mice with GeRPs fails to increase plasma IFNγ and TNFα or alter serum AST and ALT levels. Orally administered GeRPs are found in macrophages isolated from the spleen, liver, lung and peritoneal cavity and mediate macrophage gene silencing in these tissues. Utilizing this technology, I reveal that MAP4K4 augments the expression of TNFα in macrophages following LPS treatment. Oral delivery of MAP4K4 siRNA in GeRPs silences MAP4K4 expression by 70% and reduces basal TNFα and IL-1β expression significantly. The depletion of MAP4K4 in macrophages protects 40% of mice from death in the LPS/D- galactosamine (D-GalN) model of septicemia, compared to less than 10% in the control groups. This protection associates with significant decreases in serum TNFα concentrations following LPS/D-GalN challenge. Consistent with reduced macrophage inflammation, hepatocytes from mice treated orally with GeRPs targeting MAP4K4 present less apoptosis following LPS/D-GalN treatment. Thus, MAP4K4 is an important regulator of macrophage TNFα production in response to LPS. The results presented here add to the knowledge of MAP4K4 action in adipocyte and macrophage inflammation substantially. Prior to these studies, the mechanism by which TNFα controlled MAP4K4 expression in adipocytes remained unknown. Considering that MAP4K4 is a negative regulator of adipocyte function, identifying the mechanisms that control MAP4K4 expression was of interest. Furthermore, the role of macrophage MAP4K4 in LPS stimulated TNFα production was also unknown. To address this question in vivo, new technology specifically targeting macrophages was needed. Thus, we developed a technology for non toxic and highly specific macrophage gene silencing in vivo. Considering that macrophages mediate numerous diseases, the application of GeRPs to these disease models is an exciting new possibility

The Inflammatory Kinase MAP4K4 Promotes Reactivation of Kaposi's Sarcoma Herpesvirus and Enhances the Invasiveness of Infected Endothelial Cells

Kaposi's sarcoma (KS) is a mesenchymal tumour, which is caused by Kaposi's sarcoma herpesvirus (KSHV) and develops under inflammatory conditions. KSHV-infected endothelial spindle cells, the neoplastic cells in KS, show increased invasiveness, attributed to the elevated expression of metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). The majority of these spindle cells harbour latent KSHV genomes, while a minority undergoes lytic reactivation with subsequent production of new virions and viral or cellular chemo- and cytokines, which may promote tumour invasion and dissemination. In order to better understand KSHV pathogenesis, we investigated cellular mechanisms underlying the lytic reactivation of KSHV. Using a combination of small molecule library screening and siRNA silencing we found a STE20 kinase family member, MAP4K4, to be involved in KSHV reactivation from latency and to contribute to the invasive phenotype of KSHV-infected endothelial cells by regulating COX-2, MMP-7, and MMP-13 expression. This kinase is also highly expressed in KS spindle cells in vivo. These findings suggest that MAP4K4, a known mediator of inflammation, is involved in KS aetiology by regulating KSHV lytic reactivation, expression of MMPs and COX-2, and, thereby modulating invasiveness of KSHV-infected endothelial cells. © 2013 Haas et al

ICF, An Immunodeficiency Syndrome: DNA Methyltransferase 3B Involvement, Chromosome Anomalies, and Gene Dysregulation

The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) is the only disease known to result from a mutated DNA methyltransferase gene, namely, DNMT3B. Characteristic of this recessive disease are decreases in serum immunoglobulins despite the presence of B cells and, in the juxtacentromeric heterochromatin of chromosomes 1 and 16, chromatin decondensation, distinctive rearrangements, and satellite DNA hypomethylation. Although DNMT3B is involved in specific associations with histone deacetylases, HP1, other DNMTs, chromatin remodelling proteins, condensin, and other nuclear proteins, it is probably the partial loss of catalytic activity that is responsible for the disease. In microarray experiments and real-time RT-PCR assays, we observed significant differences in RNA levels from ICF vs. control lymphoblasts for pro- and anti-apoptotic genes (BCL2L10, CASP1, and PTPN13); nitrous oxide, carbon monoxide, NF-κB, and TNFa signalling pathway genes (PRKCH, GUCY1A3, GUCY1B3, MAPK13; HMOX1, and MAP4K4); and transcription control genes (NR2F2 and SMARCA2). This gene dysregulation could contribute to the immunodeficiency and other symptoms of ICF and might result from the limited losses of DNA methylation although ICF-related promoter hypomethylation was not observed for six of the above examined genes. We propose that hypomethylation of satellite 2at1qh and 16qh might provoke this dysregulation gene expression by trans effects from altered sequestration of transcription factors, changes in nuclear architecture, or expression of noncoding RNAs

Zielona Góra communication center – the concept of bus and train station revitalisation

Przystanki, stanice, a następnie dworce zielonogórskie historycznie były lokowane na obrzeżach terenu zabudowanego. Reguła ta była zachowana przy konstruowaniu linii kolejowej i dworca w 2. połowie XIX w. – linia ta została zbudowana ok. 1 km na północ od Ratusza i ok. 600 m od gra-nic zwartej zabudowy miejskiej. W pobliżu dworca kolejowego powstały duże zakłady przemysłowe, bazy przeładunkowe i zakłady usługowe. W toku rozwoju tego sektora, został on uzupełniony o zabudowę mieszkalną, usługową i tereny zieleni. Współcześnie zabudowa ma w dużej mierze charakter chaotyczny, co jest wynikiem mieszania się różnych wizji urbanistycznych. W wyniku znacznego rozwoju przestrzennego miasta, zarówno transport kolejowy, jak drogowy przebiegają przez jego środek. Przeszkodą wynikającą z tego faktu jest utrudniona przez torowisko komunikacja części miasta, które rozwinęły się po obu jego stronach. W pracy przedstawiono koncepcję przebudowy przestrzeni przylegającej do dworców PKP i PKS, zgodnie z zasadą zrównoważonego rozwoju i użytkowania terenu. Założeniem jest także ułatwienie komunikacji między sektorami miejskimi rozdzielonymi przez torowisko.Stops, stations, and then the railway station in Zielona Góra, historically have been located on the outskirts of built-up area. This rule was retained in the construction of the railway line and station in the 2nd half of the XIXth century – this line was built approx. 1 km north of City Hall and approx. 600 m from the boundaries of urban built-up area. Near the railway station a large industrial plants, depots and service plants were built. During the development of this sector, it has been supplemented by residential development, services and green areas. Today, building has large-lychaotic lay-out as a result of the mixing of different visions of urban planning. In a consequence of the significant growth of the city, both rail-road and roads for wheeled vehicles have been running through its center. The obstacle resulting from this fact is hindered communication between the both parts of the city that have developed on the both sides of the tracks. The paper presents the concept of remodeling the space adjacent to the railway and bus stations, in accordance to the principle of sustainable development and land use. The idea is also to facilitate communication between the urban sectors separated by track

A PP2A regulatory subunit regulates C. elegans insulin/IGF-1 signaling by modulating AKT-1 phosphorylation.

The C. elegans insulin/IGF-1 signaling (IIS) cascade plays a central role in regulating life span, dauer, metabolism, and stress. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine-specific protein kinases. An RNAi screen for serine/threonine protein phosphatases that counterbalance the effect of the kinases in the IIS pathway identified pptr-1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr-1 affects IIS pathway-associated phenotypes including life span, dauer, stress resistance, and fat storage. We show that PPTR-1 functions by regulating worm AKT-1 phosphorylation at Thr 350. With striking conservation, mammalian B56beta regulates Akt phosphorylation at Thr 308 in 3T3-L1 adipocytes. In C. elegans, this ultimately leads to changes in subcellular localization and transcriptional activity of the forkhead transcription factor DAF-16. This study reveals a conserved role for the B56 regulatory subunit in regulating insulin signaling through AKT dephosphorylation, thereby having widespread implications in cancer and diabetes research

Tumor necrosis factor alpha (TNFalpha) stimulates Map4k4 expression through TNFalpha receptor 1 signaling to c-Jun and activating transcription factor 2

Tumor necrosis factor alpha (TNFalpha) is a cytokine secreted by macrophages and adipocytes that contributes to the low grade inflammation and insulin resistance observed in obesity. TNFalpha signaling decreases peroxisome proliferator-activated receptor gamma and glucose transporter isoform 4 (GLUT4) expression in adipocytes, impairing insulin action, and this is mediated in part by the yeast Ste20 protein kinase ortholog Map4k4. Here we show that Map4k4 expression is selectively up-regulated by TNFalpha, whereas the expression of the protein kinases JNK1/2, ERK1/2, p38 stress-activated protein kinase, and mitogen-activated protein kinase kinases 4/7 shows little or no response. Furthermore, the cytokines interleukin 1beta (IL-1beta) and IL-6 as well as lipopolysaccharide fail to increase Map4k4 mRNA levels in cultured adipocytes under conditions where TNFalpha elicits a 3-fold effect. Using agonistic and antagonistic antibodies and small interfering RNA (siRNA) against TNFalpha receptor 1 (TNFR1) and TNFalpha receptor 2 (TNFR2), we show that TNFR1, but not TNFR2, mediates the increase in Map4k4 expression. TNFR1, but not TNFR2, also mediates a potent effect of TNFalpha on the phosphorylation of JNK1/2 and p38 stress-activated protein kinase and their downstream transcription factor substrates c-Jun and activating transcription factor 2 (ATF2). siRNA-based depletion of c-Jun and ATF2 attenuated TNFalpha action on Map4k4 mRNA expression. Consistent with this concept, the phosphorylation of ATF2 along with the expression and phosphorylation of c-Jun by TNFalpha signaling was more robust and prolonged compared with that of IL-1beta, which failed to modulate Map4k4. These data reveal that TNFalpha selectively stimulates the expression of a key component of its own signaling pathway, Map4k4, through a TNFR1-dependent mechanism that targets the transcription factors c-Jun and ATF2

The Small Intestine Converts Dietary Fructose into Glucose and Organic Acids

Excessive consumption of sweets is a risk factor for metabolic syndrome. A major chemical feature of sweets is fructose. Despite strong ties between fructose and disease, the metabolic fate of fructose in mammals remains incompletely understood. Here we use isotope tracing and mass spectrometry to track the fate of glucose and fructose carbons in vivo, finding that dietary fructose is cleared by the small intestine. Clearance requires the fructose-phosphorylating enzyme ketohexokinase. Low doses of fructose are ∼90% cleared by the intestine, with only trace fructose but extensive fructose-derived glucose, lactate, and glycerate found in the portal blood. High doses of fructose (≥1 g/kg) overwhelm intestinal fructose absorption and clearance, resulting in fructose reaching both the liver and colonic microbiota. Intestinal fructose clearance is augmented both by prior exposure to fructose and by feeding. We propose that the small intestine shields the liver from otherwise toxic fructose exposure

The Small Intestine Converts Dietary Fructose into Glucose and Organic Acids

Excessive consumption of sweets is a risk factor for metabolic syndrome. A major chemical feature of sweets is fructose. Despite strong ties between fructose and disease, the metabolic fate of fructose in mammals remains incompletely understood. Here we use isotope tracing and mass spectrometry to track the fate of glucose and fructose carbons in vivo, finding that dietary fructose is cleared by the small intestine. Clearance requires the fructose-phosphorylating enzyme ketohexokinase. Low doses of fructose are ∼90% cleared by the intestine, with only trace fructose but extensive fructose-derived glucose, lactate, and glycerate found in the portal blood. High doses of fructose (≥1 g/kg) overwhelm intestinal fructose absorption and clearance, resulting in fructose reaching both the liver and colonic microbiota. Intestinal fructose clearance is augmented both by prior exposure to fructose and by feeding. We propose that the small intestine shields the liver from otherwise toxic fructose exposure