The goal of these studies was to define the rate-limiting steps in the inactivation of type 2 iodothyronine deiodinase (D2). We examined the effects of ATP depletion, a lysosomal protease inhibitor, and an inhibitor of actin polymerization on D2 activity in the presence or absence of cycloheximide or 3,3', 5'-triiodothyronine (reverse T3, rT3) in rat pituitary tumor cells (GH4C1). We also analyzed the effects of the proteasomal proteolysis inhibitor carbobenzoxy- L-leucyl-L-leucyl-L-leucinal (MG132). The half-life of D2 activity in hypothyroid cells was 47 min after cycloheximide and 60 min with rT3 (3 nM). rT3 and cycloheximide were additive, reducing D2 half-life to 20 min. D2 degradation was partially inhibited by ATP depletion, but not by cytochalasin B or chloroquine. Incubation with MG132 alone increased D2 activity by 30-40% for several hours, and completely blocked the cycloheximide- or rT3-induced decrease in D2 activity. These results suggest that D2 is inactivated by proteasomal uptake and that substrate reduces D2 activity by accelerating degradation through this pathway. This is the first demonstration of a critical role for proteasomes in the post-translational regulation of D2 activity.
J Steinsapir, J Harney, P R Larsen
Elastolytic matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of abdominal aortic aneurysms (AAA), a disorder characterized by chronic aortic wall inflammation and destruction of medial elastin. The purpose of this study was to determine if human macrophage elastase (HME; MMP-12) might participate in this disease. By reverse transcription-polymerase chain reaction, HME mRNA was consistently demonstrated in AAA and atherosclerotic occlusive disease (AOD) tissues (six of six), but in only one of six normal aortas. Immunoreactive proteins corresponding to proHME and two products of extracellular processing were present in seven of seven AAA tissue extracts. Total HME recovered from AAA tissue was sevenfold greater than normal aorta (P < 0.001), and the extracted enzyme exhibited activity in vitro. Production of HME was demonstrated in the media of AAA tissues by in situ hybridization and immunohistochemistry, but HME was not detected within the media of normal or AOD specimens. Importantly, immunoreactive HME was specifically localized to residual elastin fragments within the media of AAA tissue, particularly areas adjacent to nondilated normal aorta. In vitro, the fraction of MMP-12 sequestered by insoluble elastin was two- to fivefold greater than other elastases found in AAA tissue. Therefore, HME is prominently expressed by aneurysm-infiltrating macrophages within the degenerating aortic media of AAA, where it is also bound to residual elastic fiber fragments. Because elastin represents a critical component of aortic wall structure and a matrix substrate for metalloelastases, HME may have a direct and singular role in the pathogenesis of aortic aneurysms.
J A Curci, S Liao, M D Huffman, S D Shapiro, R W Thompson
The study of zinc finger proteins has revealed their potential to act as oncogenes or tumor suppressors. Here we report the molecular, biochemical, and functional characterization of KS1 (KRAB/zinc finger suppressor protein 1), a novel, ubiquitously expressed zinc finger gene initially isolated from a rat pancreas library. KS1 contains 10 C2H2 zinc fingers, a KRAB-A/B motif, and an ID sequence that has been shown previously to participate in growth factor-regulated gene expression. Northern blot analysis using pancreatic cell lines demonstrates that KS1 mRNA is inducible by serum and epidermal growth factor, suggesting a role for this gene in cell growth regulation. Biochemical analysis reveals that KS1 is a nuclear protein containing two transcriptional repressor domains, R1 and R2. R1 corresponds to the KRAB-A motif, whereas R2 represents a novel sequence. Transformation assays using NIH3T3 cells demonstrate that KS1 suppresses transformation by the potent oncogenes Ha-ras, Galpha12, and Galpha13. Deletion of the R1/ KRAB-A domain does not modify the transformation suppressive activity of KS1, whereas deletion of R2 abolishes this function. Thus, KS1 is a novel growth factor-inducible zinc finger transcriptional repressor protein with the potential to protect against neoplastic transformation induced by several oncogenes.
B Gebelein, M Fernandez-Zapico, M Imoto, R Urrutia
Donor-specific (DST) or nonspecific blood transfusions administered before transplantation can enhance survival of vascularized allografts both in humans and animals but the immunological mechanisms of this effect remain unclear. We have analyzed the expression and the role of endogenous TGF-beta1 in a model of heart allograft tolerance, induced by pregraft DST in adult rats. We reported previously that this tolerance occurs despite a strong infiltration of leukocytes into the graft that are unable to produce both Th1- and Th2-related cytokines in vivo. Allografts from DST-treated rats express high levels of TGF-beta1 mRNA and active protein. This phenomenon is correlated with the rapid infiltration of leukocytes producing high amounts of TGF-beta1. TGF-beta1-producing cells are virtually absent among early infiltrating cells in rejected grafts but are found at a later time point. The induction of allograft tolerance in vivo is abrogated by administration of neutralizing anti-TGF-beta mAb. Moreover, overexpression of active TGF- beta1 in heart allografts using a recombinant adenovirus leads to prolonged graft survival in unmodified recipients. Taken together, our results identify TGF-beta as a critical cytokine involved in the suppression of allograft rejection induced by DST and suggest that TGF-beta-producing regulatory cells are also involved in allograft tolerance.
R Josien, P Douillard, C Guillot, M Müschen, I Anegon, J Chetritt, S Menoret, C Vignes, J P Soulillou, M C Cuturi
Cellular expression of the beta2-adrenergic receptor (beta2AR) is controlled in part by a 19-amino acid peptide that regulates mRNA translation. This peptide is encoded by a short open reading frame, termed the 5' leader cistron (5'LC), which is 102 bp upstream of the beta2AR coding block. In 176 normal subjects we found a single nucleotide polymorphism resulting in either Arg (previously denoted wild-type) or Cys at position 19 of this peptide. Allele frequencies were 0.37 for Arg and 0.63 for Cys. To determine if these variants altered beta2AR expression, COS-7 cells were transfected with polymorphic constructs consisting of 1,989 bp encompassing the 5'LC and the beta2AR coding block exactly as found in the human gene. beta2AR density, as determined by [125I]CYP radioligand binding, was 72% higher in cells transfected with the 5'LC-Cys19 construct as compared with those transfected with the 5'LC-Arg19 construct and 110% higher when a cotransfection technique with a luciferase construct was used to control for transfection efficiency. Levels of the two mRNA transcripts were not different, confirming in vitro studies that the upstream peptide regulates receptor expression at the translational level. In human airway smooth muscle cells that natively express beta2AR, receptor expression was approximately twofold higher in those bearing the Cys versus the Arg polymorphism, confirming the phenotype in a relevant cell type. Linkage disequilibrium was observed between the 5'LC-Cys polymorphism and the beta2AR coding block polymorphisms Arg16 and Gln27 (P < 0.0001), although several different haplotypes were identified. Thus, beta2AR expression in the human population is controlled by a common polymorphism of this 5'LC, and may be responsible for interindividual variation in betaAR responsiveness.
D W McGraw, S L Forbes, L A Kramer, S B Liggett
Thymus and activation-regulated chemokine (TARC) is a recently identified lymphocyte-directed CC chemokine which specifically chemoattracts T helper type 2 CD4(+) T cells in human. To establish the pathophysiological roles of TARC in vivo, we investigated whether a monoclonal antibody (mAb) against TARC could inhibit the induction of hepatic lesions in murine model using Propionibacterium acnes and lipopolysaccharide (LPS). P. acnes-induced intrahepatic granuloma formation in the priming phase is essential to the subsequent liver injury elicited by a low dose of LPS. The priming phase appears to be dominated by Th1 type immune responses determined by the profile of chemokine and chemokine receptor expression. TARC was selectively produced by granuloma-forming cells, and CC chemokine receptor 4 (CCR4)-expressing CD4(+) T cells migrated into the liver after LPS administration. In vivo injection of anti-TARC mAb just before LPS administration protected the mice from acute lethal liver damage, which was accompanied by a significant reduction of both CCR4 mRNA expression and IL-4 production by liver-infiltrating CD4(+) T cells. Moreover, both TNF-alpha and Fas ligand expressions in the liver were decreased by anti-TARC treatment. These results suggest that recruitment of IL-4-producing CCR4(+) CD4(+) T cells by granuloma-derived TARC into the liver parenchyma may be a key cause of massive liver injury after systemic LPS administration.
H Yoneyama, A Harada, T Imai, M Baba, O Yoshie, Y Zhang, H Higashi, M Murai, H Asakura, K Matsushima
4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.
M Parola, G Robino, F Marra, M Pinzani, G Bellomo, G Leonarduzzi, P Chiarugi, S Camandola, G Poli, G Waeg, P Gentilini, M U Dianzani
Previously we described activating mutations of hbetac, the common signaling subunit of the receptors for the hematopoietic and inflammatory cytokines, GM-CSF, IL-3, and IL-5. The activated mutant, hbetacFIDelta, is able to confer growth factor-independent proliferation on the murine myeloid cell line FDC-P1, and on primary committed myeloid progenitors. We have used this activating mutation to study the effects of chronic cytokine receptor stimulation. Transgenic mice were produced carrying the hbetacFIDelta cDNA linked to the constitutive promoter derived from the phosphoglycerate kinase gene, PGK-1. Transgene expression was demonstrated in several tissues and functional activity of the mutant receptor was confirmed in hematopoietic tissues by the presence of granulocyte macrophage and macrophage colony-forming cells (CFU-GM and CFU-M) in the absence of added cytokines. All transgenic mice display a myeloproliferative disorder characterized by splenomegaly, erythrocytosis, and granulocytic and megakaryocytic hyperplasia. This disorder resembles the human disease polycythemia vera, suggesting that activating mutations in hbetac may play a role in the pathogenesis of this myeloproliferative disorder. In addition, these transgenic mice develop a sporadic, progressive neurological disease and display bilateral, symmetrical foci of necrosis in the white matter of brain stem associated with an accumulation of macrophages. Thus, chronic hbetac activation has the potential to contribute to pathological events in the central nervous system.
R J D'Andrea, D Harrison-Findik, C M Butcher, J Finnie, P Blumbergs, P Bartley, M McCormack, K Jones, R Rowland, T J Gonda, M A Vadas
We have used a human leukemia cell line that, after homologous recombination knockout of the gp91-phox subunit of the phagocyte respiratory-burst oxidase cytochrome b-558, mimics chronic granulomatous disease (X-CGD) to study the role of oxygen radicals in apoptosis. Camptothecin (CPT), a topoisomerase I inhibitor, induced significantly more apoptosis in PLB-985 cells than in X-CGD cells. Sensitivity to CPT was enhanced after neutrophilic differentiation, but was lost after monocytic differentiation. No difference between the two cell lines was observed after treatment with other apoptosis inducers, including etoposide, ultraviolet radiation, ionizing radiation, hydrogen peroxide, or 7-hydroxystaurosporine. After granulocytic differentiation of both cell lines, CPT still induced apoptosis, suggesting independence from replication in fully differentiated and growth-arrested cells. Pyrrolidine dithiocarbamate (an antioxidant inhibitor of NF-kappaB) and catalase partially inhibited CPT-induced DNA fragmentation in granulocytic-differentiated PLB-985 cells, but had no effect in X-CGD cells. Flow cytometry analysis revealed that reactive oxygen intermediates were generated in CPT-treated PLB-985 cells. These data indicate that oxygen radicals generated by NADPH oxidase may contribute directly or indirectly to CPT-induced apoptosis in human leukemia and in neutrophilic-differentiated cells.
W Hiraoka, N Vazquez, W Nieves-Neira, S J Chanock, Y Pommier
The study of cytotoxic T cell responses to measles antigens during infection and after vaccination may provide insight into the immunopathology of the infection. It will also provide a knowledge of the immunity conferred by wild or attenuated virus, which will help in the design of new vaccines. Direct cytotoxic T cell responses, which did not require in vitro restimulation, were measured from peripheral blood by a standard 51Cr-release assay in 35 patients with acute measles, using HLA class I matched allogeneic B cells as targets. 77% showed specific responses to measles fusion protein, 69% to the hemagglutinin, and 50% to the nucleoprotein. These responses, which were related to severity of disease and history of previous vaccination, had waned by 14-24 wk after measles when memory responses to the same antigens could be elicited by restimulation in 71% of the 13 patients tested. A similar pattern followed vaccination: direct cytotoxic responses to fusion and hemagglutinin proteins were shown in 70% of the 20 children tested while 50% responded to the nucleoprotein. These responses, which were mediated by both CD8(+) and CD4(+) cells, faded over 6 wk when memory responses could be restimulated. Thus, a vigorous cytotoxic T lymphocyte response to fusion, hemagglutinin, and nucleoproteins is important in both natural and vaccine-induced immunity to measles.
A Jaye, A F Magnusen, A D Sadiq, T Corrah, H C Whittle
Controlled ovarian hyperstimulation with gonadotropins is followed by Ovarian Hyperstimulation Syndrome (OHSS) in some women. An unidentified capillary permeability factor from the ovary has been implicated, and vascular endothelial cell growth/permeability factor (VEGF) is a candidate protein. Follicular fluids (FF) from 80 women who received hormonal induction for infertility were studied. FFs were grouped according to oocyte production, from group I (0-7 oocytes) through group IV (23-31 oocytes). Group IV was comprised of four women with the most severe symptoms of OHSS. Endothelial cell (EC) permeability induced by the individual FF was highly correlated to oocytes produced (r2 = 0.73, P < 0.001). Group IV FF stimulated a 63+/-4% greater permeability than FF from group I patients (P < 0. 01), reversed 98% by anti-VEGF antibody. Group IV fluids contained the VEGF165 isoform and significantly greater concentrations of VEGF as compared with group I (1,105+/-87 pg/ml vs. 353+/-28 pg/ml, P < 0. 05). Significant cytoskeletal rearrangement of F-actin into stress fibers and a destruction of ZO-1 tight junction protein alignment was caused by group IV FF, mediated in part by nitric oxide. These mechanisms, which lead to increased EC permeability, were reversed by the VEGF antibody. Our results indicate that VEGF is the FF factor responsible for increased vascular permeability, thereby contributing to the pathogenesis of OHSS.
E R Levin, G F Rosen, D L Cassidenti, B Yee, D Meldrum, A Wisot, A Pedram
The cortical thick ascending limb (CTAL) absorbs Cl- via a Na+-K+-Cl- cotransport at the apical membrane and several Cl- channels at the basolateral membrane, including a 9-pS channel having several properties of the cystic fibrosis transmembrane conductance regulator (CFTR). Having checked that CFTR mRNA is present in the mouse CTAL, we investigated whether this channel is a CFTR molecule by applying the patch-clamp technique to CTALs microdissected from CFTR knockout mice (cftrm1Unc). The 9-pS channel was active in cell-attached patches from tubules of mice homozygous for the disrupted cftr gene [CFTR (-/-)] at the same frequency and with the same activity (NPo) as in normal [CFTR (+/+)] or heterozygous [CFTR (+/-)] mice. The conductive properties of the channel, studied on inside-out patches, were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) tubules, as were the sensitivities to internal pH and internal ATP, two typical features of this channel. In addition, the Cl- absorption in isolated, microperfused CTALs and the Na+-K+-Cl- cotransport activity were identical in CFTR (-/-), CFTR (+/+), and CFTR (+/-) mice. These results show that the 9-pS Cl- channel is distinct from CFTR, and that the CFTR protein has no influence on the Cl- absorption in this part of the renal tubule.
P Marvão, M C De Jesus Ferreira, C Bailly, M Paulais, M Bens, R Guinamard, R Moreau, A Vandewalle, J Teulon
Thromboxane A2 (TXA2) is a labile metabolite of arachidonic acid that has potent biological effects. Its actions are mediated by G protein-coupled thromboxane-prostanoid (TP) receptors. TP receptors have been implicated in the pathogenesis of cardiovascular diseases. To investigate the physiological functions of TP receptors, we generated TP receptor-deficient mice by gene targeting. Tp-/- animals reproduce and survive in expected numbers, and their major organ systems are normal. Thromboxane agonist binding cannot be detected in tissues from Tp-/- mice. Bleeding times are prolonged in Tp-/- mice and their platelets do not aggregate after exposure to TXA2 agonists. Aggregation responses after collagen stimulation are also delayed, although ADP-stimulated aggregation is normal. Infusion of the TP receptor agonist U-46619 causes transient increases in blood pressure followed by cardiovascular collapse in wild-type mice, but U-46619 caused no hemodynamic effect in Tp-/- mice. Tp-/- mice are also resistant to arachidonic acid-induced shock, although arachidonic acid signifi-cantly reduced blood pressure in Tp-/- mice. In summary, Tp-/- mice have a mild bleeding disorder and altered vascular responses to TXA2 and arachidonic acid. Our studies suggest that most of the recognized functions of TXA2 are mediated by the single known Tp gene locus.
D W Thomas, R B Mannon, P J Mannon, A Latour, J A Oliver, M Hoffman, O Smithies, B H Koller, T M Coffman
Cellular pathways for induction of programmed cell death (PCD) have been identified, but little is known about specific extracellular matrix processes that may affect apoptosis along those pathways. In this study, a series of Burkitt's lymphoma (BL) cell lines were assayed for their expression of tissue inhibitor of metalloproteinases (TIMP)-1. Results indicate that TIMP-1-positive BL lines show resistance to cold-shock-induced apoptosis. Furthermore, recombinant TIMP-1, but not TIMP-2 or a synthetic metalloproteinase inhibitor (BB-94), confers resistance to apoptosis induced by both CD95-dependent and -independent (cold shock, serum deprivation, and gamma-radiation) pathways in TIMP-1-negative BL lines. TIMP-1 suppression of PCD is not due to metalloproteinase inhibition, as reduction and alkylation of the TIMP-1 did not abolish this activity. Retroviral induction of TIMP-1 not only resulted in cell survival but also in continued DNA synthesis for up to 5 d in the absence of serum, while controls underwent apoptosis. This resistance to apoptosis is reversed by anti-TIMP-1 antibodies, demonstrating that secreted TIMP-1 is active in blocking apoptosis. Furthermore, TIMP-1 upregulation induced expression of Bcl-XL but not Bcl-2 as well as decreased NF-kappaB activity as compared with controls. These results demonstrate that TIMP-1 suppresses apoptosis in B cells and suggests a novel activity for TIMP-1 in tissue homeostasis.
L Guedez, W G Stetler-Stevenson, L Wolff, J Wang, P Fukushima, A Mansoor, M Stetler-Stevenson
Intestinal epithelial cells express hPepT1, an apical transporter responsible for the uptake of a broad array of small peptides. As these could conceivably include n-formyl peptides, we examined whether hPepT1 could transport the model n-formylated peptide fMLP and, if so, whether such cellular uptake of fMLP influenced neutrophil-epithelial interactions. fMLP uptake into oocytes was enhanced by hPepT1 expression. In addition, fMLP competitively inhibited uptake of a known hPepT1 substrate (glycylsarcosine) in hPepT1 expressing oocytes. hPepT1 peptide uptake was further examined in a polarized human intestinal epithelial cell line (Caco2-BBE) known to express this transporter. Epithelial monolayers internalized apical fMLP in a fashion that was competitively inhibited by other hPepT1 recognized solutes, but not by related solutes that were not transported by hPepT1. Fluorescence analyses of intracellular pH revealed that fMLP uptake was accompanied by cytosolic acidification, consistent with the known function of hPepT1 as a peptide H+ cotransporter. Lumenal fMLP resulted in directed movement of neutrophils across epithelial monolayers. Solutes that inhibit hPepT1-mediated fMLP transport decreased neutrophil transmigration by approximately 50%. Conversely, conditions that enhanced the rate of hPepT1-mediated fMLP uptake (cytosolic acidification) enhanced neutrophil-transepithelial migration by approximately 70%. We conclude that hPepT1 transports fMLP and uptake of these peptide influences neutrophil-epithelial interactions. These data (a) emphasize the importance of hPepT1 in mediating intestinal inflammation, (b) raise the possibility that modulating hPepT1 activity could influence states of intestinal inflammation, and (c) provide the first evidence of a link between active transepithelial transport and neutrophil-epithelial interactions.
D Merlin, A Steel, A T Gewirtz, M Si-Tahar, M A Hediger, J L Madara
Septic shock induced by lipopolysaccharide (LPS) triggering of cytokine production from monocytes/macrophages is a major cause of morbidity and mortality. The major monocyte/macrophage LPS receptor is the glycosylphosphatidylinositol (GPI)-anchored glycoprotein CD14. Here we demonstrate that CD14 coimmunoprecipitates with Gi/Go heterotrimeric G proteins. Furthermore, we demonstrate that heterotrimeric G proteins specifically regulate CD14-mediated, LPS-induced mitogen-activated protein kinase (MAPK) activation and cytokine production in normal human monocytes and cultured cells. We report here that a G protein binding peptide protects rats from LPS-induced mortality, suggesting a functional linkage between a GPI-anchored receptor and the intracellular signaling molecules with which it is physically associated.
K R Solomon, E A Kurt-Jones, R A Saladino, A M Stack, I F Dunn, M Ferretti, D Golenbock, G R Fleisher, R W Finberg