Our laboratory previously found that protein-energy malnutrition (PEM) existing prior to brain ischemia impaired functional outcome measured in an open field test, and one-third of animals showed a marked increase in reactive gliosis. It was hypothesized that PEM worsened stroke outcome by increasing inflammation via increased activation of the transcription factor, nuclear factor kappa B (NFκB). Mongolian gerbils (11-12 wk old) were randomly assigned to a control diet (12.5% protein) or a protein-deficient diet (2%) for 28 days. The control group on average gained 4.9g and the PEM group lost 7.4g. PEM gerbils had significantly decreased food intake (P<0.001; unpaired t-test). Animals were then subjected to global ischemia or sham surgery, resulting in four experimental groups. Global ischemia was achieved by a 5 min bilateral common carotid artery occlusion with tympanic temperature regulated at 36.5 ± 0.2C. PEM independently increased hippocampal NFκB activation by three times higher than control diet animals at 6hr after surgery (p=0.014; 2-factor ANOVA) detected by electrophoretic mobility shift assay (EMSA). There was no significant effect of ischemia on NFκB activation and there was no interaction of diet and ischemia. Serum glucose and serum cortisol were also measured since both variables can be affected by PEM and can influence stroke outcome, but there was no significant effect of diet or ischemia. Because of the increased NFκB activation observed in PEM-Sham animals, a second experiment investigated if PEM also increased NFκB activation in the absence of surgery. Gerbils of the same age were randomly assigned to either control diet or PEM for 28 days but did not receive any surgery. PEM consistently increased NFκB activation. Since PEM exists in 16% of elderly stroke patients at admission, the data suggest that PEM may worsen stroke outcome through increased activation of NFκB. Because increased NFκB activation was also observed in PEM independent of ischemia, the data also have implications for the inflammatory response of protein-energy malnourished elderly in general.

Our laboratory previously found that protein-energy malnutrition (PEM) existing prior to brain ischemia impaired functional outcome measured in an open field test, and one-third of animals showed a marked increase in reactive gliosis. It was hypothesized that PEM worsened stroke outcome by increasing inflammation via increased activation of the transcription factor, nuclear factor kappa B (NFκB). Mongolian gerbils (11-12 wk old) were randomly assigned to a control diet (12.5% protein) or a protein-deficient diet (2%) for 28 days. The control group on average gained 4.9g and the PEM group lost 7.4g. PEM gerbils had significantly decreased food intake (P<0.001; unpaired t-test). Animals were then subjected to global ischemia or sham surgery, resulting in four experimental groups. Global ischemia was achieved by a 5 min bilateral common carotid artery occlusion with tympanic temperature regulated at 36.5 ± 0.2C. PEM independently increased hippocampal NFκB activation by three times higher than control diet animals at 6hr after surgery (p=0.014; 2-factor ANOVA) detected by electrophoretic mobility shift assay (EMSA). There was no significant effect of ischemia on NFκB activation and there was no interaction of diet and ischemia. Serum glucose and serum cortisol were also measured since both variables can be affected by PEM and can influence stroke outcome, but there was no significant effect of diet or ischemia. Because of the increased NFκB activation observed in PEM-Sham animals, a second experiment investigated if PEM also increased NFκB activation in the absence of surgery. Gerbils of the same age were randomly assigned to either control diet or PEM for 28 days but did not receive any surgery. PEM consistently increased NFκB activation. Since PEM exists in 16% of elderly stroke patients at admission, the data suggest that PEM may worsen stroke outcome through increased activation of NFκB. Because increased NFκB activation was also observed in PEM independent of ischemia, the data also have implications for the inflammatory response of protein-energy malnourished elderly in general.

Atherosclerosis is a chronic inflammatory disease of arterial blood vessels, characterized by deposition of lipoproteins in the arterial wall. Atherosclerotic plaques form preferentially in distinct regions of the vasculature such as branch points, curvatures and bifurcations, suggesting that local hemodynamic forces may contribute to disease susceptibility. Shear stress imparted on endothelial cells (ECs) by the flowing blood has been shown to modulate gene expression and remodelling of the artery. In this thesis, an in vitro model was established to recreate the contrasting environments found in atherosclerosis-prone and atherosclerosis-resistant regions of the vasculature to demonstrate a direct causal-relationship between shear stress and expression of endothelial nitric oxide synthase (eNOS) and p65 in ECs. In vitro assessment of cell shape and expression patterns of these anti- and atherogenic genes demonstrated that shear stress can induce cell morphology and gene expression patterns that are similar to ECs in atherosclerosis-prone and atherosclerosis-resistant regions of the mouse vasculature. Regulation of eNOS transcription by shear stress was demonstrated using a transgenic mouse model and in vitro heterogeneous nuclear RNA (hnRNA) quantification. Similar to ECs in atherosclerosis-prone regions, epithelial cells lining the small intestine lumen express high levels of p65. To investigate the effects of p65 expression levels on constitutive and tumour necrosis factor α (TNFα)-induced nuclear factor-κB (NF-κB) signalling, p65 expression was suppressed in HeLa cells by RNA interference. Lower p65 expression resulted in reduced TNFα-induced expression of NF-κB target genes, including many subunits of inhibitor of nuclear factor κB (IκB), demonstrating modulation of NF-κB priming by p65 expression levels. Suppression of p65 also affected constitutive expression levels of IκB, and resulted in re-setting of the NF-κB/IκB equilibrium. Experiments using inhibitors of canonical NF-κB signalling found that basal expression of NF-κB components is independent of nuclear factor κB kinase β (IKKβ) activity and proteasome-mediated degradation of IκBα. Together, these studies elucidate the mechanism of flow-mediated gene regulation and the effect of resulting changes in p65 expression on NF-κB signalling.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22695号 / 医博第4639号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 Shohab YOUSSEFIAN, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Loss of muscle due to aging is often associated with significant detrimental effects. Therefore, it is crucial to understand signaling molecules that may trigger the muscle loss or prevent the process. The transcription factor, Nuclear Factor Kappa-B (NF-κB), is associated with both catabolic and anabolic pathways of muscle metabolism and may be involved in age-related muscle loss. Leucine is an essential amino acid that is required for both protein synthesis and intracellular signaling pathways that regulate protein synthesis and degradation. The current study examined muscle NF-kB levels in male Sprague-Dawley rats, aged 6 (adult) and 21 months (old) fed a diet containing suboptimal leucine levels for 10-17 days. We found that old rats consumed less grams of food per body weight (BW) each day than adult rats (1.45% g diet/g BW vs. 2.4% g diet/g BW). Weight loss during the study was not significantly different between age groups. However the average mass of gastrocnemius and soleus muscles (g muscle/g BW) was significantly lower in old rats. Reduction in gastrocnemius (g muscle/g BW*10²) was associated with 1.8 fold higher muscle cell NF-κB in old vs. adult rats (p = 0.0443). There was also a higher level of ubiquitinated proteins in old gastrocnemius muscle cells relative to the adult gastrocnemius, however differences did not reach statistical significance. For tibialis anterior muscle, the average mass (g muscle/g BW*10²), NF-κB levels and ubiquitinated proteins were not significantly different between adult and old rats. Our findings suggest that aging affects muscle loss and NF-kB in a tissue-specific manner in rats fed a diet with suboptimal leucine levels.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPSinduced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.

No / 1 In this study we examined the signalling events that regulate lipopolysaccharide (LPS)-stimulated induction of interferon regulatory factor (IRF)-1 in human umbilical vein endothelial cells (HUVECS). 2 LPS stimulated a time- and concentration-dependent increase in IRF-1 protein expression, an effect that was mimicked by the cytokine, tumour necrosis factor (TNF)-¿. 3 LPS stimulated a rapid increase in nuclear factor kappa B (NFKB) DNA-binding activity. Preincubation with the NFKB pathway inhibitors, N-¿-tosyl-L-lysine chloromethyl ketone (TLCK) or pyrrolidine dithiocarbamate (PDTC), or infection with adenovirus encoding IKB¿, blocked both IRF-1 induction and NFKB DNA-binding activity. 4 LPS and TNF¿ also stimulated a rapid activation of gamma interferon activation site/gamma interferon activation factor (GAS/GAF) DNA-binding in HUVECs. Preincubation with the Janus kinase (JAK)-2 inhibitor, AG490 blocked LPS-stimulated IRF-I induction but did not affect GAS/ GAF DNA-binding. 5 Preincubation with TLCK, PDTC or infection with I¿Ba adenovirus abolished LPS-stimulated GAS/GAF DNA-binding. 6 Incubation of nuclear extracts with antibodies to RelA/p50 supershifted GAS/GAF DNA-binding demonstrating the involvement of NF¿B isoforms in the formation of the GAS/GAF complex. 7 These studies show that NF¿B plays an important role in the regulation of IRF-1 induction in HUVECs. This is in part due to the interaction of NF¿B isoforms with the GAS/GAF complex either directly or via an intermediate protein.

My first project was to investigate the role of Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI) on alpha-fetoprotein (AFP) promoter activity during liver development. AFP is highly expressed in the fetal liver, silenced at birth, and remains at very low levels in the adult liver. A GA substitution located at -119 of the human AFP promoter is associated with hereditary persistence of AFP (HPAFP) expression in the adult liver (Hum Molec Genet, 1993, 2:379). The -120 region harbors overlapping binding sites for HNF1 and NFI. While it has been shown that the GA substitution increases HNF1 binding, the role of NFI in AFP regulation has not been investigated. This overlapping HNF1/NFI site is conserved in other mammals, including mice. In this study, I used a combination of biochemical, tissue culture, and animal studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co-transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the mouse AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild-type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GGAA mutation (similar to the G-A human mutation) are more active in the adult liver. My data indicate that HNF1 and NFI compete for binding to the -120 region of the AFP promoter and this competition is involved in postnatal AFP repression. My second project was to study the control of Elongation of very long chain fatty acids like 3 (Elovl3) in the liver by Zinc fingers and homeoboxes 2 (Zhx2). The Zhx2 gene was originally characterized in our lab based on its ability to control the developmental repression of several hepatic genes, including AFP (PNAS, 102:401). Zhx2 is a member of a small family of proteins found only in vertebrates that also includes Zhx1 and Zhx3. These proteins all contain two zinc fingers and four homeodomains, suggesting that they function as regulators of gene expression. My study shows that Zhx2 regulates Elovl3 expression in female liver. Mouse strain-specific differences in adult liver Elovl3 mRNA levels and transgenic mouse data indicate that Zhx2 activates Elovl3 expression in the female adult liver. I also demonstrate that Elovl3 is repressed in the regenerating liver and that the level of Elovl3 repression is controlled by alpha-fetoprotein regulator 2 (Afr2). In addition, I show that Elovl3 expression is reduced in liver tumors, fibrotic livers and fatty livers, raising the possibility that Elovl3 can serve as a marker for HCC and liver damage.

The nuclear factor kappa B (NFKB) signalling pathway is essential in the establishment and propagation of inflammation in the intestine. An increased number of cells, predominantly of the macrophage and intestinal epithelial cell (IEC) type, are known to contain the active form of the NF1d3-p65 subunit in inflamed and noninflamed intestinal tissue from Crahn's disease (CD) patients, though this remains to be confirmed. However the stimuli that induce NFKB activation in IECs and the mechanism of NFKB activation in macrophages, are only poorly understood. As such, this thesis has investigated the NFKB signalling pathway and its role in intestinal inflammation. Increased levels of NFKB DNA-binding activity and inhibitor kappa B alpha (IKBa) protein levels were found in both inflamed and non-inflamed intestinal tissue from CD patients. However, Bcl-3 levels did not significantly change. In HeLa Ohio cells, a human mucosal epithelial cell line, interleukin-l ~ (IL-l ~), lipopolysaccharide (LPS) and Phorbol 12-myritate I3-acetate (PMA) were shown to induce NFKB activation. However, when these same stimuli were used in another human IEC line, Caco-2, little NFKB-mediated gene expression was observed unless a combination of stimuli, IL-l~, LPS and tumour necrosis factor alpha (TNFa), was used. In RAW 264.7 cells, a murine macrophage cell line, LPS-stimulated NFKBmediated NO production was shown to involve protein kinase C epsilon (PKCc). Subsequently, PKC€ protein levels were also shown to be up-regulated in inflamed intestinal tissue from TNBS-treated rats. This was associated with increased NFlcB activation and IKBa protein levels, increases that were absent in non~inflamed tissue from TNBS-treated rats. In addition, IKB~ and Bcl-3 protein levels did not differ between inflamed and non-inflamed tissues, although they did vary with intestinal region. In conclusion, this study shows that abnormal NFKB activation and IKBa expression occurs in CD, and also suggests increased NFKB activation IKBa expression can coexist within inflamed intestinal tissue. In addition, the IEC line Caco-2 is shown to be relatively unresponsive to NFKB activation. In the macrophage cell line, RAW 264.7, PKC£ is involved in NFKB-mediated gene expression, and PKC£ protein levels are increased in the inflamed, TNBS-treated, intestine.