Research Article
Abstract
Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). However, less is known about the chronic effects of Akt activation in the heart, and, paradoxically, Akt is activated in samples from patients with chronic heart failure. We generated Tg mice with cardiac-specific expression of either activated (myristoylated [myr]) or dominant-negative (dn) Akt and assessed their response to IRI in an ex vivo model. While dn-Akt hearts demonstrated a moderate reduction in functional recovery after IRI, no function was restored in any of the myr-Akt–Tg hearts. Moreover, infarcts were dramatically larger in myr-Akt–Tg hearts. Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of PI3K activity through both proteasome-dependent degradation of insulin receptor substrate–1 (IRS-1) and inhibition of transcription of IRS-1 as well as that of IRS-2. To test the functional significance of these signaling changes, we performed in vivo cardiac gene transfer with constitutively active PI3K in myr-Akt–Tg mice. Restoration of PI3K rescued function and reduced injury after IRI. These data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection and suggest a mechanism by which chronic Akt activation can become maladaptive.
Authors
Tomohisa Nagoshi, Takashi Matsui, Takuma Aoyama, Annarosa Leri, Piero Anversa, Ling Li, Wataru Ogawa, Federica del Monte, Judith K. Gwathmey, Luanda Grazette, Brian Hemmings, David A. Kass, Hunter C. Champion, Anthony Rosenzweig
Abstract
HIV infection leads to decreases in the number of CD4+ T lymphocytes and an increased risk for opportunistic infections and neoplasms. The administration of intermittent cycles of IL-2 to HIV-infected patients can lead to profound increases (often greater than 100%) in CD4 cell number and percentage. Using in vivo labeling with 2H-glucose and BrdU, we have been able to demonstrate that, although therapy with IL-2 leads to high levels of proliferation of CD4 as well as CD8 lymphocytes, it is a remarkable preferential increase in survival of CD4 cells (with half-lives that can exceed 3 years) that is critical to the sustained expansion of these cells. This increased survival was time-dependent: the median half-life, as determined by semiempirical modeling, of labeled CD4 cells in 6 patients increased from 1.7 weeks following an early IL-2 cycle to 28.7 weeks following a later cycle, while CD8 cells showed no change in the median half-life. Examination of lymphocyte subsets demonstrated that phenotypically naive (CD27+CD45RO–) as well as central memory (CD27+CD45RO+) CD4 cells were preferentially expanded, suggesting that IL-2 can help maintain cells important for host defense against new antigens as well as for long-term memory to opportunistic pathogens.
Authors
Joseph A. Kovacs, Richard A. Lempicki, Igor A. Sidorov, Joseph W. Adelsberger, Irini Sereti, William Sachau, Grace Kelly, Julia A. Metcalf, Richard T. Davey Jr., Judith Falloon, Michael A. Polis, Jorge Tavel, Randy Stevens, Laurie Lambert, Douglas A. Hosack, Marjorie Bosche, Haleem J. Issaq, Stephen D. Fox, Susan Leitman, Michael W. Baseler, Henry Masur, Michele Di Mascio, Dimiter S. Dimitrov, H. Clifford Lane
Abstract
The fusion peptide (FP) in the N terminus of the HIV envelope glycoprotein, gp41, functions together with other gp41 domains to fuse the virion with the host cell membrane. We now report that FP colocalizes with CD4 and TCR molecules, coprecipitates with the TCR, and inhibits antigen-specific T cell proliferation and proinflammatory cytokine secretion in vitro. These effects are specific: T cell activation by PMA/ionomycin or mitogenic antibodies is not affected by FPs, and FPs do not interfere with antigen-presenting cell function. In vivo, FPs inhibit the activation of arthritogenic T cells in the autoimmune disease model of adjuvant arthritis and reduce the disease-associated IFN-γ response. Hence, FPs might play 2 roles in HIV infection: mediating membrane fusion while downregulating T cell responses to itself that could block infection. Disassociated from HIV, however, the FP molecule provides a novel reagent for downregulating undesirable immune responses, exemplified here by adjuvant arthritis.
Authors
Francisco J. Quintana, Doron Gerber, Sally C. Kent, Irun R. Cohen, Yechiel Shai
The AML1-ETO fusion gene and the FLT3 length mutation collaborate in inducing acute leukemia in mice
Abstract
The molecular characterization of leukemia has demonstrated that genetic alterations in the leukemic clone frequently fall into 2 classes, those affecting transcription factors (e.g., AML1-ETO) and mutations affecting genes involved in signal transduction (e.g., activating mutations of FLT3 and KIT). This finding has favored a model of leukemogenesis in which the collaboration of these 2 classes of genetic alterations is necessary for the malignant transformation of hematopoietic progenitor cells. The model is supported by experimental data indicating that AML1-ETO and FLT3 length mutation (FLT3-LM), 2 of the most frequent genetic alterations in AML, are both insufficient on their own to cause leukemia in animal models. Here we report that AML1-ETO collaborates with FLT3-LM in inducing acute leukemia in a murine BM transplantation model. Moreover, in a series of 135 patients with AML1-ETO–positive AML, the most frequently identified class of additional mutations affected genes involved in signal transduction pathways including FLT3-LM or mutations of KIT and NRAS. These data support the concept of oncogenic cooperation between AML1-ETO and a class of activating mutations, recurrently found in patients with t(8;21), and provide a rationale for therapies targeting signal transduction pathways in AML1-ETO–positive leukemias.
Authors
Christina Schessl, Vijay P.S. Rawat, Monica Cusan, Aniruddha Deshpande, Tobias M. Kohl, Patricia M. Rosten, Karsten Spiekermann, R. Keith Humphries, Susanne Schnittger, Wolfgang Kern, Wolfgang Hiddemann, Leticia Quintanilla-Martinez, Stefan K. Bohlander, Michaela Feuring-Buske, Christian Buske
Abstract
Pollen exposure induces allergic airway inflammation in sensitized subjects. The role of antigenic pollen proteins in the induction of allergic airway inflammation is well characterized, but the contribution of other constituents in pollen grains to this process is unknown. Here we show that pollen grains and their extracts contain intrinsic NADPH oxidases. The pollen NADPH oxidases rapidly increased the levels of ROS in lung epithelium as well as the amount of oxidized glutathione (GSSG) and 4-hydroxynonenal (4-HNE) in airway-lining fluid. These oxidases, as well as products of oxidative stress (such as GSSG and 4-HNE) generated by these enzymes, induced neutrophil recruitment to the airways independent of the adaptive immune response. Removal of pollen NADPH oxidase activity from the challenge material reduced antigen-induced allergic airway inflammation, the number of mucin-containing cells in airway epithelium, and antigen-specific IgE levels in sensitized mice. Furthermore, challenge with Amb a 1, the major antigen in ragweed pollen extract that does not possess NADPH oxidase activity, induced low-grade allergic airway inflammation. Addition of GSSG or 4-HNE to Amb a 1 challenge material boosted allergic airway inflammation. We propose that oxidative stress generated by pollen NADPH oxidases (signal 1) augments allergic airway inflammation induced by pollen antigen (signal 2).
Authors
Istvan Boldogh, Attila Bacsi, Barun K. Choudhury, Nilesh Dharajiya, Rafeul Alam, Tapas K. Hazra, Sankar Mitra, Randall M. Goldblum, Sanjiv Sur
Abstract
Iron homeostasis plays a critical role in many physiological processes, notably synthesis of heme proteins. Dietary iron sensing and inflammation converge in the control of iron absorption and retention by regulating the expression of hepcidin, a regulator of the iron exporter ferroportin. Human mutations in the glycosylphosphatidylinositol-anchored protein hemojuvelin (HJV; also known as RGMc and HFE2) cause juvenile hemochromatosis, a severe iron overload disease, but the way in which HJV intersects with the iron regulatory network has been unclear. Here we show that, within the liver, mouse Hjv is selectively expressed by periportal hepatocytes and also that Hjv-mutant mice exhibit iron overload as well as a dramatic decrease in hepcidin expression. Our findings define a key role for Hjv in dietary iron sensing and also reveal that cytokine-induced inflammation regulates hepcidin expression through an Hjv-independent pathway.
Authors
Vera Niederkofler, Rishard Salie, Silvia Arber
Abstract
Hereditary hemochromatosis is an iron-overload disorder resulting from mutations in proteins presumed to be involved in the maintenance of iron homeostasis. Mutations in hemojuvelin (HJV) cause severe, early-onset juvenile hemochromatosis. The normal function of HJV is unknown. Juvenile hemochromatosis patients have decreased urinary levels of hepcidin, a peptide hormone that binds to the cellular iron exporter ferroportin, causing its internalization and degradation. We have disrupted the murine Hjv gene and shown that Hjv–/– mice have markedly increased iron deposition in liver, pancreas, and heart but decreased iron levels in tissue macrophages. Hepcidin mRNA expression was decreased in Hjv–/– mice. Accordingly, ferroportin expression detected by immunohistochemistry was markedly increased in both intestinal epithelial cells and macrophages. We propose that excess, unregulated ferroportin activity in these cell types leads to the increased intestinal iron absorption and plasma iron levels characteristic of the juvenile hemochromatosis phenotype.
Authors
Franklin W. Huang, Jack L. Pinkus, Geraldine S. Pinkus, Mark D. Fleming, Nancy C. Andrews
Abstract
Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor–mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe–/– mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36–/–Apoe–/– and Msr1–/–Apoe–/– mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed.
Authors
Kathryn J. Moore, Vidya V. Kunjathoor, Stephanie L. Koehn, Jennifer J. Manning, Anita A. Tseng, Jessica M. Silver, Mary McKee, Mason W. Freeman
Abstract
We have generated a line of mutant mouse that lacks βKlotho, a protein that structurally resembles Klotho. The synthesis and excretion of bile acids were found to be dramatically elevated in these mutants, and the expression of 2 key bile acid synthase genes, cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1), was strongly upregulated. Nuclear receptor pathways and the enterohepatic circulation, which regulates bile acid synthesis, seemed to be largely intact; however, bile acid–dependent induction of the small heterodimer partner (SHP) NR0B2, a common negative regulator of Cyp7a1 and Cyp8b1, was significantly attenuated. The expression of Cyp7a1 and Cyp8b1 is known to be repressed by dietary bile acids via both SHP-dependent and -independent regulations. Interestingly, the suppression of Cyp7a1 expression by dietary bile acids was impaired, whereas that of Cyp8b1 expression was not substantially altered in βklotho–/– mice. Therefore, βKlotho may stand as a novel contributor to Cyp7a1-selective regulation. Additionally, βKlotho-knockout mice exhibit resistance to gallstone formation, which suggests the potential future clinical relevance of the βKlotho system.
Authors
Shinji Ito, Toshihiko Fujimori, Akiko Furuya, Junko Satoh, Yoko Nabeshima, Yo-ichi Nabeshima
Abstract
Expression of voltage-gated K+ channel, shaker-related subfamily, member 5 (KCNA5) underlies the human atrial ultra-rapid delayed rectifier K+ current (IKur). The KCNA5 polymorphism resulting in P532L in the C terminus generates IKur that is indistinguishable from wild type at baseline but strikingly resistant to drug block. In the present study, truncating the C terminus of KCNA5 generated a channel with wild-type drug sensitivity, which indicated that P532 is not a drug-binding site. Secondary structure prediction algorithms identified a probable α-helix in P532L that is absent in wild-type channels. We therefore assessed drug sensitivity of IKur generated in vitro in CHO and HEK cells by channels predicted to exhibit or lack this C-terminal α-helix. All constructs displayed near-identical IKur in the absence of drug challenge. However, those predicted to lack the C-terminal α-helix generated quinidine-sensitive currents (43–51% block by 10 μM quinidine), while the currents generated by those constructs predicted to generate a C-terminal α-helix were inhibited less than 12%. Circular dichroism spectroscopy revealed an α-helical signature with peptides derived from drug-resistant channels and no organized structure in those associated with wild-type drug sensitivity. In conclusion, we found that this secondary structure in the KCNA5 C terminus, absent in wild-type channels but generated by a naturally occurring DNA polymorphism, does not alter baseline currents but renders the channel drug resistant. Our data support a model in which this structure impairs access of the drug to a pore-binding site.
Authors
Benoit Drolet, Chantale Simard, Laura Mizoue, Dan M. Roden
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