Bonnie C. Miller

Amyloid-β peptide levels in brain are inversely correlated with insulysin activity levels in vivo

Factors that elevate amyloid-β (Aβ) peptide levels are associated with an increased risk for Alzheimers disease. Insulysin has been identified as one of several proteases potentially involved in Aβ degradation based on its hydrolysis of Aβ peptides in vitro. In this study, in vivo levels of brain Aβ40 and Aβ42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the γ-secretase-generated C-terminal fragment of the Aβ precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased ≈50%, brain Aβ peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Aβ peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimers disease.

Characterization of ionotrophic purinergic receptors in hepatocytes

Ionotrophic purinergic (P2X) receptors function as receptor‐gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na+ and Ca2+. Based on evidence that extracellular adenosine 5′‐triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP‐dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2′3′‐O‐(4‐benzoyl‐benzoyl)‐adenosine 5′‐triphosphate (BzATP; 10 μM) caused a rapid increase in membrane Na+ conductance. Similarly, with Fluo‐3 fluorescence, BzATP induced an increase in intracellular [Ca2+]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu2+, and the P2X4 modulators Zn2+ and ivermectin (0.3‐3 μM) each increased intracellular [Ca2+]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen‐rich liver samples. Conclusion: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na+ and Ca2+ transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism. (HEPATOLOGY 2007.)

Antiviral Cytokines Induce Hepatic Expression of the Granzyme B Inhibitors, Proteinase Inhibitor 9 and Serine Proteinase Inhibitor 6

Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-αβ or IFN-γ, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-α, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-γ and TNF-α also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-α, IFN-γ, or TNF-α. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.

Importin-α Protein Binding to a Nuclear Localization Signal of Carbohydrate Response Element-Binding Protein (ChREBP)

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. Circulating blood glucose levels affect ChREBP activity in hepatocytes largely by post-translational mechanisms that include phosphorylation-dependent subcellular localization. Previously, we showed that ChREBP is retained in the cytosol by phosphorylation-dependent binding to 14-3-3 protein dimers and identified the α2 helix (residues 125–135) phospho-Ser140 domain as the primary 14-3-3 binding site (Sakiyama, H., Wynn, R. M., Lee, W. R., Fukasawa, M., Mizuguchi, H., Gardner, K. H., Repa, J. J., and Uyeda, K. (2008) J. Biol. Chem. 283, 24899–24908). To enter the nucleus in response to high glucose, ChREBP must bind importin-α; this heterodimer then forms a complex with importin-β to interact with the nuclear pore complex. In this work, we recharacterized the importin-α binding nuclear localization signal (NLS) of rat ChREBP, identifying it as an extended classical bipartite NLS encompassing minimally residues 158–190. Replacing Lys159/Lys190 residues of ChREBP with alanine resulted in loss of importin-α binding, glucose-stimulated transcriptional activity and nuclear localization. A secondary 14-3-3 protein binding site also was identified, the α3 helix (residues 170–190) phospho-Ser196 domain. Importin-α and 14-3-3 were found to bind competitively to this secondary site. These results suggest an important mechanism by which importin-α and 14-3-3 control movement of ChREBP in and out of the nucleus in response to changes in glucose levels in liver and thus further suggest that the extended NLS of ChREBP is a critical glucose-sensing, glucose-responsive site.

The role of serpinb9/serine protease inhibitor 6 in preventing granzyme B–dependent hepatotoxicity†

Virally infected hepatocytes are resistant to cytotoxic lymphocyte killing by perforin‐dependent and granzyme‐dependent effector mechanisms. The present studies were designed to examine the role of serine protease inhibitor 6 (SPI‐6) in limiting granzyme B–dependent cytotoxic effector mechanisms in the liver. SPI‐6–specific small interfering RNA (siRNA) administration to C57Bl/6J (B6) mice elicited transient alanine aminotransferase (ALT) elevations that were not observed in either granzyme B–deficient B6 (B6.gzmb−/−) or natural killer (NK) cell–depleted B6 mice. When SPI‐6 expression was abolished by siRNA administration at the time of infection with a recombinant, replication‐deficient adenovirus [E1‐deleted adenovirus encoding β‐galactosidase (AdCMV‐LacZ)], earlier and dramatically increased, and earlier ALT elevations were observed in wild‐type B6 but not in B6.gzmb−/− or NK cell–depleted mice. When a 3‐fold higher dose of AdCMV‐LacZ was administered to B6 mice, the coadministration of SPI‐6 siRNA resulted in the early onset of lethal, acute liver failure. Of note, the accelerated clearance of AdCMV‐LacZ was observed in recipients of SPI‐6 siRNA. Conclusion: These results indicate that the regulated expression of SPI‐6 in hepatocytes during viral infection or following noninfectious causes of liver injury protects hepatocytes against excessively vigorous granzyme B–dependent killing but may also delay immune clearance of virally infected hepatocytes. (HEPATOLOGY 2007.)

Fas and TNFR1, but not cytolytic granule-dependent mechanisms, mediate clearance of murine liver adenoviral infection†

After intravenous injection of replication‐deficient adenovirus, hepatocytes are transduced and express high levels of adenovirus‐encoded genes. However, adenovirally encoded gene expression is ablated rapidly by CD8+ T‐cell–dependent mechanisms. Thus, this model is suitable for examining intrahepatic cytotoxic T lymphocyte (CTL) effector mechanisms. In the present studies, recombinant adenoviruses encoding secreted (human apolipoprotein A‐I) or intracellular (β‐galactosidase) gene products were infused into mice with genetic deficiencies affecting the granule exocytosis‐, Fas‐, or tumor necrosis factor receptor 1 (TNFR1)‐mediated pathways of CTL and natural killer cell effector function; the rates of clearance of adenovirus‐encoded gene products were assessed. Clearance of secreted or intracellular adenoviral gene products was not delayed in perforin‐deficient mice or dipeptidyl peptidase I‐deficient mice, which fail to process and activate granzyme A or granzyme B. TNFR1‐deficient mice also exhibited no delay in clearance of adenoviral gene products. However, adenoviral clearance from Fas‐deficient mice was delayed, and such delays were much greater in mice deficient in both TNFR1 and Fas. In contrast, chimeric mice lacking both hepatic Fas and lymphocyte perforin function exhibited no greater delay in adenoviral clearance than chimeras deficient only in hepatic Fas expression. In conclusion, Fas‐dependent mechanisms are required for efficient clearance of virally infected hepatocytes and, in Fas‐deficient animals, TNFR1‐dependent mechanisms provide an alternative mechanism for hepatic adenovirus clearance. In contrast, perforin‐ and granule protease–dependent cytotoxicity mechanisms play no apparent role in clearance of adenovirus from the liver. (HEPATOLOGY 2005;41:97–105.)

Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

Background: Glucose-responsive ChREBP binds 14-3-3 and localizes cytosolically during starvation. Results: ChREBP interacts with 14-3-3 through its α2 helix in a novel phosphorylation-independent conformation. Conclusion: 14-3-3 binds to ChREBP with high affinity to stabilize ChREBP in the cytosolic compartment under low glucose conditions. Significance: ChREBP·14-3-3 structure is the first step toward understanding the mechanism by which ChREBP responds to glucose levels. Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3β bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the α2 helix of ChREBP (residues 117–137) adopts a well defined α-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP α2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP α2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3β·ChREBP α2 complex.

CD28 Costimulation Induces δ Opioid Receptor Expression During Anti-CD3 Activation of T Cells

Previous studies have demonstrated that naive splenic mouse T cells express no or only very low levels of the δ-type opioid receptor (δOR), but stimulation of mouse splenocytes with Con A results in induction of δOR mRNA and protein. In this report we have shown that stimulation of highly purified populations of naive mouse T cells with anti-CD3 mAb alone results in T cell activation, as evidenced by sustained IL-2 secretion and cell proliferation, but fails to elicit δOR expression. However, δOR expression is induced by costimulation of these very pure T cells with anti-CD3 and anti-CD28 mAbs. The δOR induction by anti-CD3 and anti-CD28 costimulation was completely blocked by inhibition of phosphatidylinositol 3-kinase with wortmannin. Because phosphatidylinositol 3-kinase activation in T cells is linked to costimulation, these results suggest that induction of δOR expression during T cell activation is strictly dependent on costimulation. It also appears that costimulatory receptors other than CD28 can provide the signaling required for δOR expression because δOR mRNA was induced by Con A stimulation of splenocytes from CD28-deficient mice.

Susceptibility to acetaminophen (APAP) toxicity unexpectedly is decreased during acute viral hepatitis in mice

Acetaminophen (APAP) hepatotoxicity results from cytochrome P450 metabolism of APAP to the toxic metabolite, n-acetyl-benzoquinone imine (NAPQI), which reacts with cysteinyl residues to form APAP adducts and initiates cell injury. As APAP is commonly used during viral illnesses there has been concern that APAP injury may be additive to that of viral hepatitis, leading physicians to advise against its use in such patients; this has not been investigated experimentally. We infected C57BL/6 male mice with replication-deficient adenovirus to produce moderately severe acute viral hepatitis and observed that APAP doses that were hepatotoxic or lethal in control mice produced neither death nor additional increase in serum ALT when administered to infected mice at the peak of virus-induced liver injury. Moreover, the concentration of hepatic APAP-protein adducts formed in these mice was only 10% that in control mice. Protection from APAP hepatotoxicity also was observed earlier in the course of infection, prior to the peak virus-induced ALT rise. Hepatic glutathione limits APAP-protein adduct formation but glutathione levels were similar in control and infected mice. Cyp1a2 (E.C. 1.14.14.1) and Cyp2e1 (E.C. 1.14.13.n7) mRNA expression decreased by 3 days post-infection and hepatic Cyp2e1 protein levels were reduced almost 90% at 7 days, when adduct formation was maximally inhibited. In vitro, hepatocytes from virally infected mice also were resistant to APAP-induced injury but sensitive to NAPQI. Rather than potentiating APAP-induced liver injury, acute viral hepatitis in this model resulted in selective down-regulation of APAP metabolizing P450s in liver and decreased the risk of APAP hepatotoxicity.

Granzyme C Supports Efficient CTL-Mediated Killing Late in Primary Alloimmune Responses

It is well established that granzymes A and B play a role in CTL killing of target cells by the perforin-dependent granule exocytosis pathway. The functions of multiple additional granzymes expressed in CTL are less well defined. In the present studies, CTL generated from mice deficient in dipeptidyl peptidase 1 (DPP1) were used to investigate the contribution of granzyme C to CTL killing of allogeneic target cells. DPP1 is required for activation of granzymes A and B by proteolytic removal of their N-terminal dipeptide prodomains while a significant portion of granzyme C is processed normally in the absence of DPP1. Cytotoxicity of DPP1−/− CTL generated in early (5-day) MLC in vitro and in peritoneal exudate cells 5 days after initial allogeneic sensitization in vivo was significantly impaired compared with wild-type CTL. Following 3 days of restimulation with fresh allogeneic stimulators however, cytotoxicity of these DPP1−/− effector cells was comparable to that of wild-type CTL. Killing mediated by DPP1−/− CTL following restimulation was rapid, perforin dependent, Fas independent and associated with early mitochondrial injury, phosphatidyl serine externalization, and DNA degradation, implicating a granzyme-dependent apoptotic pathway. The increased cytotoxicity of DPP1−/− CTL following restimulation coincided with increased expression of granzyme C. Moreover, small interfering RNA inhibition of granzyme C expression during restimulation significantly decreased cytotoxicity of DPP1−/− but not wild-type CTL. These results indicate that during late primary alloimmune responses, granzyme C can support CTL-mediated killing by the granule exocytosis pathway in the absence of functional granzymes A or B.