The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.
J B Kim, P Sarraf, M Wright, K M Yao, E Mueller, G Solanes, B B Lowell, B M Spiegelman
Recent in vitro studies have shown that fibrinolytic activity may be attenuated by a thrombin-activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin, generated via the intrinsic pathway of coagulation in a factor XI-dependent way. Thus factor XI may play a role in the regulation of endogenous fibrinolysis. The aim of this study was to investigate the effect of in vivo inhibition of factor XI and TAFI in an experimental thrombosis model in rabbits. Incorporation of anti-factor XI antibodies in jugular vein thrombi resulted in an almost twofold increase in endogenous thrombolysis compared with a control antibody. A similar effect was observed when the anti-factor XI antibody was administered systemically. Inhibition of TAFI activity also resulted in a twofold increase in clot lysis whereas inhibition of both factor XI and TAFI activity had no additional effect. Thus, we provide the first in vivo evidence for enhanced thrombolysis through inhibition of clotting factor XI, demonstrating a novel role for the intrinsic pathway of coagulation. Furthermore we demonstrate that inhibition of TAFI had a similar effect on thrombolysis. We postulate that inhibition of factor XI activity enhances thrombolysis because of diminished indirect activation of TAFI.
M C Minnema, P W Friederich, M Levi, P A von dem Borne, L O Mosnier, J C Meijers, B J Biemond, C E Hack, B N Bouma, H ten Cate
Nitric oxide (NO) produced by NO synthase (NOS) modulates fetal pulmonary vascular tone and contributes to the fall in pulmonary vascular resistance (PVR) at birth. Although the inducible (type II) NOS isoform is present in human and rat fetal lungs, it is uncertain whether type II NOS activity contributes to vascular NO production in the fetal lung. To determine whether type II NOS is present in the ovine fetal lung and to study the potential contribution of type II NOS on the regulation of basal PVR in the fetus, we measured the hemodynamic effects of three selective type II NOS antagonists: aminoguanidine (AG), 2-amino-5,6-dihydro-6-methyl-4H-1,3 thiazine (AMT), and S-ethylisothiourea (EIT). Studies were performed after at least 72 h of recovery from surgery in 19 chronically prepared fetal lambs (133+/-3 d; 147 d, term). Brief intrapulmonary infusions of AG (140 mg), AMT (0.12 mg), and EIT (0.12 mg) increased basal PVR by 82, 69, and 77%, respectively (P < 0.05). The maximum increase in PVR occurred within 20 min, but often persisted up to 80 min. These agents also increased mean aortic pressure but did not alter the pressure gradient between the pulmonary artery and aorta, suggesting little effect on tone of the ductus arteriosus. Acetylcholine-induced pulmonary vasodilation remained intact after treatment with selective type II NOS antagonists, but not after treatment with the nonselective NOS blocker, nitro-L-arginine. Using Northern blot analysis with poly(A)+ RNA, we demonstrated the presence of two mRNA transcripts for type II NOS (4.1 and 2.6 kb) in the fetal lung. We conclude that the type II NOS isoform is present in the ovine fetal lung, and that selective type II NOS antagonists increase PVR and systemic arterial pressure in the late-gestation fetus. We speculate that type II NOS may play a physiological role in the modulation of vascular tone in the developing fetal lung.
R L Rairigh, T D Le Cras, D D Ivy, J P Kinsella, G Richter, M P Horan, I D Fan, S H Abman
To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression.
Z Wu, Y Xie, R F Morrison, N L Bucher, S R Farmer
The skin is constantly exposed to sunlight and frequently develops sun-induced skin cancers such as basal cell carcinoma (BCC). These epidermal-derived tumors escape local immune surveillance and infiltrate the dermis, requiring surgical removal. We report here that in contrast to keratinocytes in normal skin (n = 4), BCC tumor cells (n = 6) strongly and diffusely express Fas ligand (CD95L), but not Fas antigen (CD95). This CD95L expression in vivo by BCC tumor cells is associated with peritumoral T lymphocytes that are undergoing apoptosis. Moreover, CD95L can be induced on normal cultured keratinocytes after exposure to ultraviolet-B light (UV-B) irradiation. This induction of CD95L was confirmed at the mRNA and protein levels using multipassaged human keratinocytes and a keratinocyte cell line. Keratinocytes induced to express CD95L acquired the functional capacity to kill a CD95-positive lymphocyte cell line. Whereas hyperplastic keratinocytes in untreated psoriatic plaques do not express CD95L on their plasma membrane, after UV-B treatment there is strong and diffuse keratinocyte CD95L expression that coincided in a temporal fashion with depletion of intraepidermal T cells in all five patients studied. Our data suggest a novel molecular pathway by which UV light can contribute to the ability of a skin cancer to escape from immune attack by cytotoxic T lymphocytes, and a previously unrecognized therapeutic mechanism of action for UV-B light in psoriasis via keratinocyte CD95L expression. Such immunological events involving CD95L provide new insight and opportunity for novel treatment approaches not only for cutaneous neoplasms but also for various T cell-mediated dermatoses such as psoriasis.
C Gutierrez-Steil, T Wrone-Smith, X Sun, J G Krueger, T Coven, B J Nickoloff
We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.
M Arras, W D Ito, D Scholz, B Winkler, J Schaper, W Schaper
Loss of myofibril organization is a common feature of chronic dilated and progressive cardiomyopathy. To study how the heart compensates for myofibril degeneration, transgenic mice were created that undergo progressive loss of myofibrils after birth. Myofibril degeneration was induced by overexpression of tropomodulin, a component of the thin filament complex which determines and maintains sarcomeric actin filament length. The tropomodulin cDNA was placed under control of the alpha-myosin heavy chain gene promoter to overexpress tropomodulin specifically in the myocardium. Offspring with the most severe phenotype showed cardiomyopathic changes between 2 and 4 wk after birth. Hearts from these mice present characteristics consistent with dilated cardiomyopathy and a failed hypertrophic response. Histological analysis showed widespread loss of myofibril organization. Confocal microscopy of isolated cardiomyocytes revealed intense tropomodulin immunoreactivity in transgenic mice together with abnormal coincidence of tropomodulin and alpha-actinin reactivity at Z discs. Contractile function was compromised severely as determined by echocardiographic analyses and isolated Langendorff heart preparations. This novel experimentally induced cardiomyopathy will be useful for understanding dilated cardiomyopathy and the effect of thin filament-based myofibril degeneration upon cardiac structure and function.
M A Sussman, S Welch, N Cambon, R Klevitsky, T E Hewett, R Price, S A Witt, T R Kimball
Alopecia areata is a tissue-restricted autoimmune disease of the hair follicle, which results in hair loss and baldness. It is often psychologically devastating. The role of T lymphocytes in this disorder was investigated with cell transfer experiments. Scalp explants from patients were transplanted to severe combined immunodeficiency (SCID) mice and injected with autologous T lymphocytes isolated from involved scalp. T lymphocytes which had been cultured with hair follicle homogenate along with antigen-presenting cells were capable of inducing the changes of alopecia areata, including hair loss and perifollicular infiltrates of T cells, along with HLA-DR and ICAM-1 expression of the follicular epithelium. Similar changes were not noted in grafts injected with scalp-derived T cells that had not been cultured with follicular homogenate. These data indicate that alopecia areata is mediated by T cells which recognize a follicular autoantigen.
A Gilhar, Y Ullmann, T Berkutzki, B Assy, R S Kalish
Although glutamic acid decarboxylase (GAD) has been implicated in IDDM, there is no direct evidence showing GAD-reactive T cells are diabetogenic in vivo. To address this issue, 3-wk-old NOD mice received two injections of purified rat brain GAD; one mouse rapidly developed diabetes 3 wk later. Splenocytes from this mouse showed a proliferative response to purified GAD, and were used to generate a CD4+ T cell line, designated 5A, that expresses TCRs encoding Vbeta2 and Vbeta12. 5A T cells exhibit a MHC restricted proliferative response to purified GAD, as well as GAD65 peptide 524-543. After antigen-specific stimulation, 5A T cells secrete IFNgamma and TNFalpha/beta, but not IL-4. They are also cytotoxic against NOD-derived hybridoma cells (expressing I-Ag7) that were transfected with rat GAD65, but not nontransfected hybridoma cells. Adoptive transfer of 5A cells into NOD/SCID mice produced insulitis in all mice. Diabetes occurred in 83% of the mice. We conclude that GAD injection in young NOD mice may, in some cases, provoke diabetes due to the activation of diabetogenic T cells reactive to GAD65 peptides. Our data provide direct evidence that GAD65 autoimmunity may be a critical event in the pathogenesis of IDDM.
D Zekzer, F S Wong, O Ayalon, I Millet, M Altieri, S Shintani, M Solimena, R S Sherwin
In women, a relative hyperandrogenicity is statistically associated with insulin resistance and centralization of body fat, which are predictors for the development of non-insulin-dependent diabetes mellitus. The aim of this study was to evaluate the effect of androgenization of newborn female rats on insulin sensitivity at adult age. To mimic the neonatal androgen peak normally observed in male rats, female pups were administered one high dose of testosterone (T) subcutaneously within 3 h after birth. They were then given back to their mothers and followed to adult age. At the end of the week 9, tail samples were taken, showing no differences in fasting plasma concentrations of glucose, lactate, insulin, or free fatty acids between T-treated rats and controls. Plasma concentrations of T and progesterone were significantly lower in the T-treated rats, whereas no differences were found in the levels of corticosterone, estradiol, insulin-like growth factor I, or ACTH. After 10 wk, insulin sensitivity was studied with hyperglycemic and euglycemic hyperinsulinemic (5 mU insulin/kg/min) clamp techniques. The T-treated rats showed insulin resistance with both techniques, which was overcome with time and increasing insulin concentrations during the clamp measurements. The T-treated rats were also heavier and had increased relative weights of skeletal muscles and the spleen. Parametrial, retroperitoneal, and inguinal adipose tissues decreased in weight while mesenteric adipose tissue tended to increase, resulting in an approximately 30-50% larger mesenteric than other adipose tissues. It is concluded that neonatal T imprinting of female rats is followed by insulin resistance, changes in adipose tissue distribution, and an enlarged lean mass, without elevation of circulating T. Similar changes are seen in adult female rats or women receiving T.
C Nilsson, M Niklasson, E Eriksson, P Björntorp, A Holmäng
We have demonstrated previously that dichloroacetate can attenuate skeletal muscle fatigue by up to 35% in a canine model of peripheral ischemia (Timmons, J.A., S.M. Poucher, D. Constantin-Teodosiu, V. Worrall, I.A. Macdonald, and P.L. Greenhaff. 1996. J. Clin. Invest. 97:879-883). This was thought to be a consequence of dichloroacetate increasing acetyl group availability early during contraction. In this study we characterized the metabolic effects of dichloroacetate in a human model of peripheral muscle ischemia. On two separate occasions (control-saline or dichloroacetate infusion), nine subjects performed 8 min of single-leg knee extension exercise at an intensity aimed at achieving volitional exhaustion in approximately 8 min. During exercise each subject's lower limbs were exposed to 50 mmHg of positive pressure, which reduces blood flow by approximately 20%. Dichloroacetate increased resting muscle pyruvate dehydrogenase complex activation status by threefold and elevated acetylcarnitine concentration by fivefold. After 3 min of exercise, phosphocreatine degradation and lactate accumulation were both reduced by approximately 50% after dichloroacetate pretreatment, when compared with control conditions. However, after 8 min of exercise no differences existed between treatments. Therefore, it would appear that dichloroacetate can delay the accumulation of metabolites which lead to the development of skeletal muscle fatigue during ischemia but does not alter the metabolic profile when a maximal effort is approached.
J A Timmons, T Gustafsson, C J Sundberg, E Jansson, E Hultman, L Kaijser, J Chwalbinska-Moneta, D Constantin-Teodosiu, I A Macdonald, P L Greenhaff
No studies are available that have compared early defects in glucose metabolism in the offspring of insulin-deficient and insulin-resistant probands with non-insulin-dependent diabetes mellitus (NIDDM). To investigate this issue, we evaluated insulin secretion capacity with oral and intravenous glucose tolerance tests and with the hyperglycemic clamp, and insulin action with the euglycemic insulin clamp in 20 offspring of NIDDM patients with low fasting C-peptide (+/-450 pmol/liter), reflecting deficient insulin secretion (IS-group), 18 offspring of NIDDM patients with high fasting C-peptide (>/= 880 pmol/liter), reflecting insulin resistance (IR-group), and 14 healthy control subjects without a family history of NIDDM. The frequency of impaired glucose tolerance was 45.0% in the IS-group and 50% in the IR-group. The IS-group had lower insulin-glucose response at 30 min in the oral glucose tolerance test (85.2+/-10.0 pmol insulin per mmol glucose) than the control group (136.4+/-23.1 pmol insulin per mmol glucose; P < 0.05) and the IR-group (115.6+/-11.8 pmol insulin per mmol glucose; P = 0.05). Furthermore, the acute insulin response during the first 10 min of an intravenous glucose tolerance test was lower in the IS-group than in the IR-group. Maximal insulin secretion capacity evaluated by C-peptide levels during the hyperglycemic clamp did not differ between the groups. The IR-group had lower rates of whole body glucose uptake (60.1+/-4.6 micromol per lean body mass per minute) than did the control group (84.2+/-5.0 micromol per lean body mass per minute; P < 0.001) or the IS-group (82.6+/-5.9 micromol per lean body mass per minute; P < 0.01) and this was due to reduced glucose nonoxidation. To conclude, both impaired insulin secretion and insulin action seem to be inherited and could represent the primary defects in glucose metabolism in the offspring of NIDDM probands.
I Vauhkonen, L Niskanen, E Vanninen, S Kainulainen, M Uusitupa, M Laakso
The localization of circulating leukocytes within inflamed tissues occurs as the result of interactions with and migration across vascular endothelium, and is governed, in part, by the expression of adhesion molecules on both cell types. Recently, we have described a novel primary adhesion interaction between the structurally activated form of the adhesion molecule CD44 on lymphocytes and its major ligand hyaluronan on endothelial cells under physiologic laminar flow conditions, and have proposed that this interaction functions in an extravasation pathway for lymphocytes in vascular beds at sites of inflammation. While the regulation of activated CD44 on leukocytes has been characterized in depth, regulation of hyaluronate (HA) on endothelial cells has not been extensively studied. Here we demonstrate that the expression of HA on cultured endothelial cell lines and primary endothelial cultures is inducible by the proinflammatory cytokines TNFalpha and IL-1beta, as well as bacterial lipopolysaccharide. In addition, this inducibility appears strikingly restricted to endothelial cells derived from microvascular, but not large vessel, sources. The elevated HA levels thus induced result in increased CD44-dependent adhesive interactions in both nonstatic shear and laminar flow adhesion assays. Changes in mRNA levels for the described HA synthetic and degradative enzymes were not found, suggesting other more complex mechanisms of regulation. Together, these data add to the selectin and immunoglobulin gene families a new inducible endothelial adhesive molecule, hyaluronan, and help to further our understanding of the potential physiologic roles of the CD44/HA interaction; i.e., local cytokine production within inflamed vascular beds may enhance surface hyaluronan expression on endothelial cells, thereby creating local sites receptive to the CD44/HA interaction and thus extravasation of inflammatory cells.
M Mohamadzadeh, H DeGrendele, H Arizpe, P Estess, M Siegelman
Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase (4S). A feline MPS VI model used to demonstrate efficacy of enzyme replacement therapy is due to the homozygous presence of an L476P mutation in 4-sulfatase. An additional mutation, D520N, inherited independently from L476P and recently identified in the same family of cats, has resulted in three clinical phenotypes. L476P homozygotes exhibit dwarfism and facial dysmorphia due to epiphyseal dysplasia, abnormally low leukocyte 4S/betahexosaminidase ratios, dermatan sulfaturia, lysosomal inclusions in most tissues including chondrocytes, corneal clouding, degenerative joint disease, and abnormal leukocyte inclusions. Similarly, D520N/D520N and L476P/D520N cats have abnormally low leukocyte 4S/betahexosaminidase ratios, mild dermatan sulfaturia, lysosomal inclusions in some chondrocytes, and abnormal leukocyte inclusions. However, both have normal growth and appearance. In addition, L476P/D520N cats have a high incidence of degenerative joint disease. We conclude that L476P/D520N cats have a very mild MPS VI phenotype not previously described in MPS VI humans. The study of L476P/D520N and D520N/ D520N genotypes will improve understanding of genotype to phenotype correlations and the pathogenesis of skeletal dysplasia and joint disease in MPS VI, and will assist in development of therapies to prevent lysosomal storage in chondrocytes.
A C Crawley, G Yogalingam, V J Muller, J J Hopwood
The clinical potential of the p53 tumor suppressor gene is being evaluated currently for gene therapy of cancer. We have built a variant of wild-type p53, chimeric tumor suppressor 1 (CTS1), in which we have replaced the domains that mediate its inactivation. CTS1 presents some very interesting properties: (a) enhanced transcriptional activity; (b) resistance to the inactivation by oncogenic forms of p53; (c) resistance to the inactivation by MDM2; (d) lower sensitivity to E6-induced degradation; (e) ability to suppress cell growth; and (f ) faster induction of apoptosis. Thus, CTS1 is an improved tumor suppressor and an alternative for the treatment of wild-type p53-resistant human tumors by gene therapy.
E Conseiller, L Debussche, D Landais, C Venot, M Maratrat, V Sierra, B Tocque, L Bracco
Based on a consideration of the histopathology of nonbullous impetigo that shows localization of Streptococcus pyogenes to highly differentiated, subcorneal keratinocytes, we hypothesized that adherence of an impetigo strain of S. pyogenes would be promoted by terminal differentiation of keratinocytes. An assay was developed in which S. pyogenes adhered via pilus-like projections from the cell wall to the surface of cultured human keratinocytes in a time- and inoculum-dependent manner suggestive of a receptor-mediated process. Terminal differentiation of keratinocytes was induced by increasing the calcium concentration in the growth medium, and was confirmed by morphologic analysis using electron microscopy. Adherence of S. pyogenes was three and fourfold greater to keratinocytes differentiated in 1.0 and 1.5 mM calcium, respectively, compared with undifferentiated keratinocytes in 0.15 mM calcium. The presence of calcium during the adherence assay further enhanced adherence nearly twofold. Adherence occurred preferentially to sites of contact between adjacent keratinocytes, suggesting that the keratinocyte receptor may be a molecule involved in cell-to-cell adhesion. In contrast, nonpathogenic Streptococcus gordonii adhered poorly to keratinocytes regardless of their state of terminal differentiation, and adherence of a pharyngeal strain of S. pyogenes was twofold greater to undifferentiated than differentiated keratinocytes. This is the first report of in vitro adherence of S. pyogenes to keratinocytes in a manner that emulates human impetigo. Adherence of only the impetigo strain, and not the pharyngeal strain of S. pyogenes or the nonpathogenic S. gorgonii isolate, was promoted by keratinocyte differentiation. This result provides a model system for investigating the molecular pathogenesis of streptococcal skin infections.
G L Darmstadt, P Fleckman, M Jonas, E Chi, C E Rubens
The relationship between the number of circulating CD4+ T cells and the presence of particular CD8+ T cell subsets was analyzed by flow cytometry on PBL from asymptomatic HIV-1-infected patients whose specimens were collected every 2 mo for a total period of 32 mo. Only slight variations were detected in the absolute number of lymphocytes and percentage of CD3+ lymphocytes, whereas both CD4+ and CD8+ T cell subsets showed wide intrapatient variation. Variations in the number of CD8+CD28+ cells paralleled those of the CD4+ T cell subset in each patient tested, while the presence of CD8+CD28- T cells correlated inversely with CD4+ and CD8+CD28+ T cells. These data show that changes in the number of circulating CD4+-and CD8+CD28+ T cells are strongly related to the presence of CD8+CD28- T cells in these patients. Insight into the significance of CD8+CD28- T cell expansion will allow us to understand the mechanisms and significance of the HIV-1- driven change in CD4+CD8+ T cell homeostasis and the basic immunopathology of HIV disease.
A Caruso, S Licenziati, A D Canaris, A Cantalamessa, S Fiorentini, S Ausenda, D Ricotta, F Dima, F Malacarne, A Balsari, A Turano
Transgenic mice were generated with different levels of human apolipoprotein C1 (APOC1) expression in liver and skin. At 2 mo of age, serum levels of cholesterol, triglycerides (TG), and FFA were strongly elevated in APOC1 transgenic mice compared with wild-type mice. These elevated levels of serum cholesterol and TG were due mainly to an accumulation of VLDL particles in the circulation. In addition to hyperlipidemia, APOC1 transgenic mice developed dry and scaly skin with loss of hair, dependent on the amount of APOC1 expression in the skin. Since these skin abnormalities appeared in two independent founder lines, a mutation related to the specific insertion site of the human APOC1 gene as the cause for the phenotype can be excluded. Histopathological analysis of high expressor APOC1 transgenic mice revealed a disorder of the skin consisting of epidermal hyperplasia and hyperkeratosis, and atrophic sebaceous glands lacking sebum. In line with these results, epidermal lipid analysis showed that the relative amounts of the sebum components TG and wax diesters in the epidermis of high expressor APOC1 transgenic mice were reduced by 60 and 45%, respectively. In addition to atrophic sebaceous glands, the meibomian glands were also found to be severely atrophic in APOC1 transgenic mice. High expressor APOC1 transgenic mice also exhibited diminished abdominal adipose tissue stores (a 60% decrease compared with wild-type mice) and a complete deficiency of subcutaneous fat. These results indicate that, in addition to the previously reported inhibitory role of apoC1 on hepatic remnant uptake, overexpression of apoC1 affects lipid synthesis in the sebaceous gland and/or epidermis as well as adipose tissue formation. These APOC1 transgenic mice may serve as an interesting in vivo model for the investigation of lipid homeostasis in the skin.
M C Jong, M J Gijbels, V E Dahlmans, P J Gorp, S J Koopman, M Ponec, M H Hofker, L M Havekes
Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.
R K Ravi, E Weber, M McMahon, J R Williams, S Baylin, A Mal, M L Harter, L E Dillehay, P P Claudio, A Giordano, B D Nelkin, M Mabry
Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.
V Kolm-Litty, U Sauer, A Nerlich, R Lehmann, E D Schleicher
This study investigated the effect of chronic hypertonicity on the OKP cell Na/H antiporter, encoded by Na/H exchanger 3 (NHE3). Chronic (48 h) increases in extracellular glucose, mannitol, or raffinose concentration caused a significant increase in Na/H antiporter activity, while increases in urea concentration were without effect. This effect was seen with changes in osmolality of only 20 mOsm/liter, a magnitude that is observed clinically in poorly controlled diabetes mellitus. Increases in mannitol concentration acutely inhibited and chronically stimulated Na/H antiporter activity. The increase in Na/H antiporter activity induced by hypertonic incubation was resistant to 10(-7) and 5 x 10(-6) M but inhibited by 10(-4) M ethylisopropyl amiloride, consistent with regulation of NHE3. In addition, hypertonicity increased total cellular and plasma membrane NHE3 protein abundance twofold, with only a small increase in NHE3 mRNA abundance. We conclude that chronic pathophysiologically relevant increases in tonicity lead to increases in NHE3 protein abundance and activity. This may be responsible for increased proximal tubule apical membrane Na/H antiporter activity in poorly controlled diabetes mellitus, which could then contribute to hypertension, glomerular hyperfiltration and diabetic nephropathy.
P Ambühl, M Amemiya, P A Preisig, O W Moe, R J Alpern
Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.
M R Yeaman, A S Bayer, S P Koo, W Foss, P M Sullam
The renin-angiotensin system plays a major role in regulating blood pressure and maintaining electrolyte and volume homeostasis. Previously, the angiotensinogen gene, which encodes the key substrate for renin within this system, has been reported linked to and associated with essential hypertension in White Europeans, African-Caribbeans, and Japanese. Therefore, we investigated whether the angiotensinogen gene might be similarly implicated in the pathogenesis of essential hypertension in Chinese by carrying out linkage analysis in 310 hypertensive sibling pairs. Genotypes for two diallelic DNA polymorphisms observed at amino acid residues 174 (T174M) and 235 (M235T) within the coding sequence and for two highly informative dinucleotide (GT)-repeat sequences (one in the 3' flanking region, and one at a distance of 6.1 cM from the gene) were determined. Affected sibpair analysis conducted according to three different algorithms (S.A.G.E./SIBPAL, MAPMAKER/ SIBS, and APM methods) revealed no evidence for linkage of the angiotensinogen gene to hypertension. Our data indicate that molecular variants of this gene do not appear to contribute materially to the pathogenesis of primary hypertension among Chinese (a notion supported by concomitant, direct estimates of power), and that the disease relevance of this gene may vary therefore depending on ethnicity.
T Niu, X Xu, J Rogus, Y Zhou, C Chen, J Yang, Z Fang, C Schmitz, J Zhao, V S Rao, K Lindpaintner
Apoptosis of CD4+ lymphocytes is partially responsible for the depletion of these cells in HIV-infected individuals. CD4+ lymphocytes from HIV-1-infected patients express higher membrane levels of the Fas receptor, and are particularly susceptible to apoptosis after Fas triggering. IL-1beta- converting enzyme (ICE) is a key enzyme of the apoptotic machinery involved in Fas-mediated apoptosis of normal lymphocytes. The role of ICE in mediating the increased susceptibility of CD4+ lymphocytes from HIV-1-infected patients to apoptosis has not been examined. In this study, we found that ICE mRNA was present in T cells from both HIV-1-infected patients and controls. Active ICE proteins, p10 and p20, were demonstrated by immunoblot in lymphocytes from HIV-1-infected patients and in normal lymphocytes after treatment with Fas agonist, CH11 mAb. Cocultivation of lymphocytes from HIV-1-infected persons with Fas antagonist, antibody ZB4, resulted in decreased expression of ICE protein in lymphocytes from HIV-infected patients, and decreased apoptosis. Similar effects were obtained when cells were treated with synthetic ICE inhibitors, which blocked apoptosis in response to Fas triggering. When CD4+ and CD8+ cells were sorted by flow cytometry and analyzed by reverse transcriptase PCR, ICE mRNA was present in both CD8+ and CD4+ cells. However, flow cytometric analysis of lymphocytes with intracellular staining for ICE demonstrated ICE protein in the CD4+ but not the CD8+ cell fraction derived from blood of HIV-1-infected patients. These data suggest that activation of ICE occurs in vivo in CD4+ lymphocytes from HIV-1-infected individuals, and may account for the increased susceptibility of CD4+ cells to apoptosis.
E M Sloand, J P Maciejewski, T Sato, J Bruny, P Kumar, S Kim, F F Weichold, N S Young
This study was undertaken to characterize the insulin resistance and the mechanism thereof caused by chronic hyperinsulinemia produced in dogs by surgically diverting the veins of the pancreas from the portal vein to the vena cava. Pancreatic venous diversion (PVD, n = 8) caused a sustained increase in arterial insulin and decrease in portal insulin concentration compared with the control group (n = 6). Hyperinsulinemic euglycemic clamps were conducted 4 wk after surgery. The increase in the glucose disposal rate (GDR) was significantly less in the PVD group (39.0+/-5.0 vs. 27.9+/-3.2 micromol/kg/min, P < 0.01) compared with the control group, but the suppression of hepatic glucose production by insulin was similar for both groups. Muscle insulin receptor tyrosine kinase activity (IR-TKA) increased from 6.2+/-0.4 to 20.3+/-2.7 in the control group, but from 5.8+/-0.5 to only 12.7+/-1.7 fmol P/fmol IR in the PVD group (P < 0.01). With respect to the periphery, the time to half-maximum response (t1/2a) for arterial insulin was the same for both groups, whereas the t1/2a for lymph insulin (30+/-3 vs. 40+/-4 min, P < 0.05) and GDR (29+/-3 vs. 66+/-10 min, P < 0.01) were greater for the PVD group. Chronic hyperinsulinemia led to marked peripheral insulin resistance characterized by decreased insulin-stimulated GDR, and impaired activation of GDR kinetics due, in part, to reduced IR-TKA. Transendothelial insulin transport was impeded and was responsible for one third of the kinetic defect in insulin-resistant animals, while slower intracellular mechanisms of GDR were responsible for the remaining two thirds.
P D Miles, S Li, M Hart, O Romeo, J Cheng, A Cohen, K Raafat, A R Moossa, J M Olefsky
Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a member of the immunoglobulin superfamily that plays a role in a number of endothelial cell (EC) functions including migration, angiogenesis, and transmigration of leukocytes across endothelium. We postulated that one way PECAM-1 might exert its effects was by regulating intracellular EC levels of calcium. Using single-cell fluorometry, we found that engagement of PECAM-1 by mAbs induced a slow but sustained increase in intracellular calcium, both in EC and in an adherent PECAM-1-transfected cell line that models endothelium. Generation of this signal was specific for certain anti-PECAM-1 antibodies, required the presence of the cytoplasmic domain, depended on extracellular calcium and on tyrosine phosphorylation, but did not require cross-linking; in fact, calcium increases were stimulated by certain Fab fragments. Activation of EC by PECAM-1 also caused a time-dependent increase in prostacyclin release. Given the importance of intracellular calcium and prostacyclin release as signaling molecules, engagement of PECAM-1 during cell-cell interactions may alter a number of EC functions including secretion of vasoactive mediators.
I Gurubhagavatula, Y Amrani, D Pratico, F L Ruberg, S M Albelda, R A Panettieri Jr
Apolipoprotein (apo) E mediates lipoprotein binding to cellular lipoprotein receptors. Previously we reported that a synthetic peptide representing a linear dimeric repeat of amino acids 141-155 binds cellular LDL receptors. To prepare an apoE peptide that bound to both cholesterol-rich lipoproteins and lipoprotein receptors, an NH2-terminal acetylated apoE dimer peptide was synthesized. This acetylated peptide preferentially associated with lipoproteins in plasma, whereas nonacylated peptides were poor lipid binders. Acetylated peptide/LDL complexes (molar ratios of 4-5:1) enhanced the interaction of LDL with cultured human fibroblasts by 7-12-fold. Participation by both receptors and cell surface heparin sulfate proteoglycans was observed. When a preformed peptide/125I-LDL complex was injected intravenously into C57BL/6J apoE-deficient mice, its rate of removal was threefold higher than that of 125I-LDL alone. The liver and the spleen were major tissue distribution sites. Intravenous administration of free acetylated peptide resulted in a 30% reduction in total plasma cholesterol within 3-30 min, which reflected a 40-50% and 20-26% reduction in very low density lipoproteins and intermediate density lipoproteins, respectively. Therefore, this peptide selectively associated with cholesterol-rich lipoproteins and mediated their acute clearance in vivo.
I R Nikoulin, L K Curtiss
Normalization of the increased vascular nitric oxide (NO) generation with low doses of NG-nitro-L-arginine methyl ester (L-NAME) corrects the hemodynamic abnormalities of cirrhotic rats with ascites. We have undertaken this study to investigate the effect of the normalization of vascular NO production, as estimated by aortic cyclic guanosine monophosphate (cGMP) concentration and endothelial nitric oxide synthase (eNOS) protein expression in the aorta and mesenteric artery, on sodium and water excretion. Rats with carbon tetrachloride-induced cirrhosis and ascites were investigated using balance studies. The cirrhotic rats were separated into two groups, one receiving 0.5 mg/kg per day of L-NAME (CIR-NAME) during 7 d, whereas the other group (CIR) was administrated the same volume of vehicle. Two other groups of rats were used as controls, one group treated with L-NAME and another group receiving the same volume of vehicle. Sodium and water excretion was measured on days 0 and 7. On day 8, blood samples were collected for electrolyte and hormone measurements, and aorta and mesenteric arteries were harvested for cGMP determination and nitric oxide synthase (NOS) immunoblotting. Aortic cGMP and eNOS protein expression in the aorta and mesenteric artery were increased in CIR as compared with CIR-NAME. Both cirrhotic groups had a similar decrease in sodium excretion on day 0 (0.7 versus 0.6 mmol per day, NS) and a positive sodium balance (+0.9 versus +1.2 mmol per day, NS). On day 7, CIR-NAME rats had an increase in sodium excretion as compared with the CIR rats (sodium excretion: 2.4 versus 0.7 mmol per day, P < 0.001) and a negative sodium balance (-0.5 versus +0.8 mmol per day, P < 0.001). The excretion of a water load was also increased after L-NAME administration (from 28+/-5% to 65+/-7, P < 0.05). Plasma renin activity, aldosterone and arginine vasopressin were also significantly decreased in the CIR-NAME, as compared with the CIR rats. The results thus indicate that normalization of aortic cGMP and eNOS protein expression in vascular tissue is associated with increased sodium and water excretion in cirrhotic rats with ascites.
P Y Martin, M Ohara, P Gines, D L Xu, J St John, M Niederberger, R W Schrier
Anti-FcepsilonRIalpha autoantibodies (autoAbs) occur and may be of pathogenetic relevance in a subset of chronic urticaria (CU) patients. To analyze the prevalence and magnitude of the humoral anti-FcepsilonRIalpha response in cohorts of CU patients compared with individuals suffering from classic skin- related (auto)immune diseases, we developed an ELISA system for the measurement of anti-FcepsilonRIalpha autoAbs in nonfractionated serum samples. Results obtained using this assay correlated well with those generated by Western blotting. We found IgG anti-FcepsilonRIalpha autoreactivity in 38% of CU patients but not in atopic dermatitis patients, psoriatics, or healthy individuals. We frequently detected anti-FcepsilonRIalpha autoAbs in pemphigus vulgaris (PV, 39%), dermatomyositis (DM, 36%), systemic lupus erythematosus (SLE, 20%), and bullous pemphigoid (BP, 13%). While the autoAb titers in DM, SLE, BP, and PV were similar to those encountered in CU patients, only anti-FcepsilonRIalpha+ CU serum specimens displayed pronounced histamine-releasing activity. The anti-FcepsilonRIalpha autoAbs in CU patients belong predominantly to the complement-fixing subtypes IgG1 and IgG3, whereas in DM, PV, and BP, they were found to be mainly of the IgG2 or IgG4 subtype. Complement-activating properties of anti-FcepsilonRIalpha autoAbs can indeed be of pathogenetic relevance, because C5a receptor blockade on basophils as well as decomplementation reduced drastically the histamine-releasing capacity of most anti-FcepsilonRIalpha-reactive CU sera. As a consequence, therapeutic efforts in CU should aim at altering not only the quantity but also the complement-activating properties of IgG anti-FcepsilonRIalpha autoAbs.
E Fiebiger, F Hammerschmid, G Stingl, D Maurer
Interleukin 12 (IL-12), produced by myelomonocytic cells, plays a pivotal role in the development of T helper 1 (Th1) cells, which are involved in the pathogenesis of chronic inflammatory autoimmune disorders. 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] inhibits IL-12 production by activated macrophages and dendritic cells, thus providing a novel interpretation to its immunosuppressive properties. 1,25(OH)2D3 significantly inhibits mRNA expression for both IL-12 p35 and p40 subunits acting at the transcriptional level. The effect of 1,25(OH)2D3 on p40 promoter activation was analyzed by cotransfecting monocytic RAW264.7 cells with p40 promoter/reporter constructs and expression vectors for vitamin D3 receptor (VDR) and/or retinoid X receptor (RXRalpha). We observed transcriptional repression of the p40 gene by 1,25(OH)2D3, which required coexpression of VDR with RXR and an intact VDR DNA-binding domain. The repressive effect maps to a region in the p40 promoter containing a binding site for NF-kappaB (p40-kappaB). Deletion of the p40-kappaB site abrogates part of the inhibitory effect on the p40 promoter, confirming the functional relevance of this site. Activation of monocytic THP-1 cells in the presence of 1,25(OH)2D3 results in reduced binding to the p40-kappaB site. Thus, 1,25(OH)2D3 may negatively regulate IL-12 production by downregulation of NF-kappaB activation and binding to the p40-kappaB sequence.
D D'Ambrosio, M Cippitelli, M G Cocciolo, D Mazzeo, P Di Lucia, R Lang, F Sinigaglia, P Panina-Bordignon
HLA-B27 confers a very strong genetic predisposition to development of a reactive arthritis after infection by bacteria such as Salmonella typhimurium. This study examines the role of HLA-B27 in the initiation of the earliest host activities after exposure to Salmonella, namely activation of the immediate early genes in the epithelial cells. Our major finding is that in Hela cells, the expression of c-fos was induced by Salmonella invasion only when the cells expressed the transfected HLA-B27 gene, but not the HLA-A1 gene or a truncated HLA-B27 gene lacking the exons encoding the cytoplasmic domain. C-fos is potentially capable of complexing with members of the c-jun family to become the AP-1 transcription complex. Parallel to c-fos expression, we found that only with the HLA-B27 transfectant was there expression of AP-1. AP-1 potentially controls the expression of a large number of genes. On screening a panel of proinflammatory molecules, we found that Salmonella invasion induced expression of monocyte chemoattractant protein-1 in the HLA-B27 cells. Since each of these separate positive findings belong to the same cascade of events after cell activation, together they reinforce the hypothesis that HLA-B27 plays a modulatory role in the early signal transduction events induced by Salmonella invasion. This hypothesis adds another item to the list of allele-specific activities carried out by HLA class I molecules. If similar activation also occurs in the joints, it may play a major role in arthritis.
T Ikawa, M Ikeda, A Yamaguchi, W C Tsai, N Tamura, N Seta, M Trucksess, R B Raybourne, D T Yu
Only a few autoantibodies that are more or less specific for RA have been described so far. The rheumatoid factor most often tested for is not very specific for RA, while the more specific antiperinuclear factor for several reasons is not routinely used as a serological parameter. Here we show that autoantibodies reactive with synthetic peptides containing the unusual amino acid citrulline, a posttranslationally modified arginine residue, are specifically present in the sera of RA patients. Using several citrulline-containing peptide variants in ELISA, antibodies could be detected in 76% of RA sera with a specificity of 96%. Sera showed a remarkable variety in the reactivity pattern towards different citrulline-containing peptides. Affinity-purified antibodies were shown to be positive in the immunofluorescence-based antiperinuclear factor test, and in the so-called antikeratin antibody test, and were reactive towards filaggrin extracted from human epidermis. The specific nature of these antibodies and the presence of these antibodies early in disease, even before other disease manifestations occur, are indicative for a possible role of citrulline-containing epitopes in the pathogenesis of RA.
G A Schellekens, B A de Jong, F H van den Hoogen, L B van de Putte, W J van Venrooij