Abstract
TNF-α is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.
Authors
Bruno Ségui, Olivier Cuvillier, Sabine Adam-Klages, Virginie Garcia, Sophie Malagarie-Cazenave, Sophie Lévêque, Sylvie Caspar-Bauguil, Jérôme Coudert, Robert Salvayre, Martin Krönke, Thierry Levade
Abstract
Using cre/loxP gene targeting, transgenic mice with muscle-specific inactivation of the GLUT4 gene (muscle GLUT4 KO) were generated and shown to develop a diabetes phenotype. To determine the mechanism, we examined insulin-stimulated glucose uptake and metabolism during hyperinsulinemic-euglycemic clamp in control and muscle GLUT4 KO mice before and after development of diabetes. Insulin-stimulated whole body glucose uptake was decreased by 55% in muscle GLUT4 KO mice, an effect that could be attributed to a 92% decrease in insulin-stimulated muscle glucose uptake. Surprisingly, insulin’s ability to stimulate adipose tissue glucose uptake and suppress hepatic glucose production was significantly impaired in muscle GLUT4 KO mice. To address whether these latter changes were caused by glucose toxicity, we treated muscle GLUT4 KO mice with phloridzin to prevent hyperglycemia and found that insulin-stimulated whole body and skeletal muscle glucose uptake were decreased substantially, whereas insulin-stimulated glucose uptake in adipose tissue and suppression of hepatic glucose production were normal after phloridzin treatment. In conclusion, these findings demonstrate that a primary defect in muscle glucose transport can lead to secondary defects in insulin action in adipose tissue and liver due to glucose toxicity. These secondary defects contribute to insulin resistance and to the development of diabetes.
Authors
Jason K. Kim, Ariel Zisman, Jonathan J. Fillmore, Odile D. Peroni, Ko Kotani, Pascale Perret, Haihong Zong, Jianying Dong, C. Ronald Kahn, Barbara B. Kahn, Gerald I. Shulman
Abstract
Expression of angiogenic factors such as VEGF under conditions of hypoxia or other kinds of cell stress contributes to neovascularization during wound healing. The inducible endoplasmic reticulum chaperone oxygen-regulated protein 150 (ORP150) is expressed in human wounds along with VEGF. Colocalization of these two molecules was observed in macrophages in the neovasculature, suggesting a role of ORP150 in the promotion of angiogenesis. Local administration of ORP150 sense adenovirus to wounds of diabetic mice, a treatment that efficiently targeted this gene product to the macrophages of wound beds, increased VEGF antigen in wounds and accelerated repair and neovascularization. In cultured human macrophages, inhibition of ORP150 expression caused retention of VEGF antigen within the endoplasmic reticulum (ER), while overexpression of ORP150 promoted the secretion of VEGF into hypoxic culture supernatants. Taken together, these data suggest an important role for ORP150 in the setting of impaired wound repair and identify a key, inducible chaperone-like molecule in the ER. This novel facet of the angiogenic response may be amenable to therapeutic manipulation.
Authors
Kentaro Ozawa, Toshikazu Kondo, Osamu Hori, Yasuko Kitao, David M. Stern, Wolfgang Eisenmenger, Satoshi Ogawa, Tohru Ohshima
Abstract
Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8+ T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8+ T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-γ–dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-γ production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-γ and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8+ T cell–mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.
Authors
Jon M. Wigginton, Eilene Gruys, Lisa Geiselhart, Jeffrey Subleski, Kristin L. Komschlies, Jong-Wook Park, Theresa A. Wiltrout, Kunio Nagashima, Timothy C. Back, Robert H. Wiltrout
Abstract
In NOD (nonobese diabetic) mice, a model of autoimmune diabetes, various immunomodulatory interventions prevent progression to diabetes. However, after hyperglycemia is established, such interventions rarely alter the course of disease or allow sustained engraftment of islet transplants. A proteasome defect in lymphoid cells of NOD mice impairs the presentation of self antigens and increases the susceptibility of these cells to TNF-α–induced apoptosis. Here, we examine the hypothesis that induction of TNF-α expression combined with reeducation of newly emerging T cells with self antigens can interrupt autoimmunity. Hyperglycemic NOD mice were treated with CFA to induce TNF-α expression and were exposed to functional complexes of MHC class I molecules and antigenic peptides either by repeated injection of MHC class I matched splenocytes or by transplantation of islets from nonautoimmune donors. Hyperglycemia was controlled in animals injected with splenocytes by administration of insulin or, more effectively, by implantation of encapsulated islets. These interventions reversed the established β cell–directed autoimmunity and restored endogenous pancreatic islet function to such an extent that normoglycemia was maintained in up to 75% of animals after discontinuation of treatment and removal of islet transplants. A therapy aimed at the selective elimination of autoreactive cells and the reeducation of T cells, when combined with control of glycemia, is thus able to effect an apparent cure of established type 1 diabetes in the NOD mouse.J. Clin. Invest.108:63–72 (2001). DOI:10.1172/JCI200112335.
Authors
Shinichiro Ryu, Shohta Kodama, Kazuko Ryu, David A. Schoenfeld, Denise L. Faustman
Abstract
Mitogen-activated protein kinase (MAPK) cascades are involved in inflammation and tissue destruction in rheumatoid arthritis (RA). In particular, c-Jun N-terminal kinase (JNK) is highly activated in RA fibroblast-like synoviocytes and synovium. However, defining the precise function of this kinase has been difficult because a selective JNK inhibitor has not been available. We now report the use of a novel selective JNK inhibitor and JNK knockout mice to determine the function of JNK in synoviocyte biology and inflammatory arthritis. The novel JNK inhibitor SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one) completely blocked IL-1–induced accumulation of phospho-Jun and induction of c-Jun transcription in synoviocytes. Furthermore, AP-1 binding and collagenase mRNA accumulation were completely suppressed by SP600125. In contrast, complete inhibition of p38 had no effect, and ERK inhibition had only a modest effect. The essential role of JNK was confirmed in cultured synoviocytes from JNK1 knockout mice and JNK2 knockout mice, each of which had a partial defect in IL-1–induced AP-1 activation and collagenase-3 expression. Administration of SP600125 modestly decreased the rat paw swelling in rat adjuvant-induced arthritis. More striking was the near-complete inhibition of radiographic damage that was associated with decreased AP-1 activity and collagenase-3 gene expression. Therefore, JNK is a critical MAPK pathway for IL-1–induced collagenase gene expression in synoviocytes and in joint arthritis, indicating that JNK is an important therapeutic target for RA.
Authors
Zuoning Han, David L. Boyle, Lufen Chang, Brydon Bennett, Michael Karin, Li Yang, Anthony M. Manning, Gary S. Firestein
Abstract
Most patients succumbing to colorectal cancer fail with liver-predominant metastases. To make a clinical impact in this disease, a systemic or whole-liver therapy may be required, whereas most cancer gene therapy approaches are limited in their ability to treat beyond local disease. As a preclinical model for cancer gene therapy, recombinant adenovirus containing the human IFN-β (hIFN-β) cDNA was delivered systemically in nude mouse xenograft models of human colorectal cancer liver metastases. The vector targeted hepatocytes that produced high levels of hIFN-β in the liver, resulting in a profound apoptotic response in the tumors and significant tumor regression. hIFN-β gene therapy not only resulted in improved survival and long-term cure in a micrometastatic model, but provided similar benefits in a clinically relevant gross disease model. A similar recombinant adenovirus containing the murine IFN-β (mIFN-β) cDNA also resulted in a therapeutic response and improved survival in syngeneic mouse models of colorectal cancer liver metastases. Depletion studies demonstrate a contribution of natural killer cells to this therapeutic response. The toxicity of an adenoviral vector expressing murine IFN-β in a syngeneic model is also presented. These encouraging results warrant further investigation of the use of cancer gene therapy for targeting metastatic disease.
Authors
Hiroomi Tada, David J. Maron, Eugene A. Choi, James Barsoum, Hanqin Lei, Qing Xie, Wenbiao Liu, Lee Ellis, A. David Moscioni, John Tazelaar, Stephen Fawell, Xiao Qin, Kathleen J. Propert, Alan Davis, Douglas L. Fraker, James M. Wilson, Francis R. Spitz
Abstract
In newborns and small mammals, cold-induced adaptive (or nonshivering) thermogenesis is produced primarily in brown adipose tissue (BAT). Heat production is stimulated by the sympathetic nervous system, but it has an absolute requirement for thyroid hormone. We used the thyroid hormone receptor-β–selective (TR-β–selective) ligand, GC-1, to determine by a pharmacological approach whether adaptive thermogenesis was TR isoform–specific. Hypothyroid mice were treated for 10 days with varying doses of T3 or GC-1. The level of uncoupling protein 1 (UCP1), the key thermogenic protein in BAT, was restored by either T3 or GC-1 treatment. However, whereas interscapular BAT in T3-treated mice showed a 3.0°C elevation upon infusion of norepinephrine, indicating normal thermogenesis, the temperature did not increase (<0.5°C) in GC-1–treated mice. When exposed to cold (4°C), GC-1–treated mice also failed to maintain core body temperature and had reduced stimulation of BAT UCP1 mRNA, indicating impaired adrenergic responsiveness. Brown adipocytes isolated from hypothyroid mice replaced with T3, but not from those replaced with GC-1, had normal cAMP production in response to adrenergic stimulation in vitro. We conclude that two distinct thyroid-dependent pathways, stimulation of UCP1 and augmentation of adrenergic responsiveness, are mediated by different TR isoforms in the same tissue.
Authors
Miriam O. Ribeiro, Suzy D. Carvalho, James J. Schultz, Grazia Chiellini, Thomas S. Scanlan, Antonio C. Bianco, Gregory A. Brent
Abstract
Cardiac neural crest ablation results in depressed myocardial calcium transients and elevated proliferation in myocardium at a stage when cardiac neural crest cells are not in contact with the myocardium. To test the hypothesis that cardiac neural crest–derived cells, which migrate into the caudal, ventral pharynx at stage 14, block a signal from the ventral pharynx, we cultured stage 12 chick heart tube or myocardial strips in the presence or absence of ventral pharynx. We found that myocardium cultured with ventral pharynx that had not yet contacted neural crest cells had significantly reduced calcium transients and an increased rate of proliferation. Ventral pharynx from intact embryos at a stage when neural crest–derived cells had reached the pharynx had no effect on myocardial calcium transients. Ventral pharynx from neural crest–ablated embryos continued to suppress myocardial calcium transients at this later stage. Myocardium cultured with FGF-2 also showed a significant reduction in calcium transients. An FGF-2–neutralizing Ab reversed the deleterious effect of the ventral pharynx on myocardial calcium transients and proliferation. We therefore examined the expression of FGF-2 and similar FGFs in the ventral pharynx. Only FGF-8 was expressed in a temporospatial pattern that made it a viable candidate for altering the myocardial calcium transient during stages 14–18. In explant cultures, neutralizing Ab for FGF-8 rescued development of the myocardial calcium transient in neural crest–ablated chick embryos.
Authors
Michael J. Farrell, Jarrett L. Burch, Kathleen Wallis, Linda Rowley, Donna Kumiski, Harriet Stadt, Robert E. Godt, Tony L. Creazzo, Margaret L. Kirby
Abstract
It has been postulated that TNF has a pivotal role in a cytokine cascade that results in joint inflammation and destruction in rheumatoid arthritis (RA). To evaluate this, we examined the response of TNF-deficient (Tnf–/–) mice in two models of RA. Collagen-induced arthritis (CIA) was induced by injection of chick type II collagen (CII) in CFA. Tnf–/– mice had some reduction in the clinical parameters of CIA and, on histology, significantly more normal joints. However, severe disease was evident in 54% of arthritic Tnf–/– joints. Tnf–/– mice had impaired Ig class switching, but preserved T cell proliferative responses to CII and enhanced IFN-γ production. Interestingly, CII-immunized Tnf–/– mice developed lymphadenopathy and splenomegaly associated with increased memory CD4+ T cells and activated lymph node B cells. Acute inflammatory arthritis was also reduced in Tnf–/– mice, although again some mice exhibited severe disease. We conclude that TNF is important but not essential for inflammatory arthritis; in each model, severe arthritis could proceed even in the complete absence of TNF. These results call into doubt the concept that TNF is obligatory for chronic autoimmune and acute inflammatory arthritis and provide a rationale for further studies into TNF-independent cytokine pathways in arthritis.
Authors
Ian K. Campbell, Kristy O’Donnell, Kate E. Lawlor, Ian P. Wicks
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