969 resultados para Folate Receptor 1
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Human colon carcinoma Caco-2 cell monolayers undergo conversion into cells that share morphological and functional features of M cells when allowed to interact with B lymphocytes. A lymphotropic (X4) HIV-1 strain crosses M cell monolayers and infects underlying CD4(+) target cells. Transport requires both lactosyl cerebroside and CXCR4 receptors, which are expressed on the apical surface of Caco-2 and M cells. Antibodies specific for each receptor block transport. In contrast, a monotropic (R5) HIV-1 strain is unable to cross M cell monolayers and infect underlying monocytes, despite efficient transport of latex beads. Caco-2 and M cells do not express CCR5, but transfection of these cells with CCR5 cDNA restores transport of R5 virus, which demonstrates that HIV-1 transport across M cells is receptor-mediated. The follicle-associated epithelium covering human gut lymphoid follicles expresses CCR5, but not CXCR4, and lactosyl cerebroside, suggesting that HIV-1 infection may occur through M cells and enterocytes at these sites.
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The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.
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Glucagon-like peptide-1 (GLP-1) is the most potent stimulator of glucose-induced insulin secretion and its pancreatic beta-cell receptor is a member of a new subfamily of G-protein-coupled receptors which includes the receptors for vasoactive intestinal polypeptide, secretin and glucagon. Here we studied agonist-induced GLP-1 receptor internalization in receptor-transfected Chinese hamster lung fibroblasts using three different approaches. First, iodinated GLP-1 bound at 4 degrees C to transfected cells was internalized with a t 1/2 of 2-3 min following warming up of the cells to 37 degrees C. Secondly, exposure to GLP-1 induced a shift in the distribution of the receptors from plasma membrane-enriched to endosomes-enriched membrane fractions, as assessed by Western blot detection of the receptors using specific antibodies. Thirdly, continuous exposure of GLP-1 receptor-expressing cells to iodinated GLP-1 led to a linear accumulation of peptide degradation products in the medium following a lag time of 20-30 min, indicating a continuous cycling of the receptor between the plasma membrane and endosomal compartments. Potassium depletion and hypertonicity inhibited transferrin endocytosis, a process known to occur via coated pit formation, as well as GLP-1 receptor endocytosis. In contrast to GLP-1, the antagonist exendin-(9-39) did not lead to receptor endocytosis. Surface re-expression following one round of GLP-1 receptor endocytosis occurred with a half-time of about 15 min. The difference in internalization and surface re-expression rates led to a progressive redistribution of the receptor in intracellular compartments upon continuous exposure to GLP-1. Finally, endogenous GLP-1 receptors expressed by insulinoma cells were also found to be internalized upon agonist binding. Together our data demonstrate that the GLP-1 receptor is internalized upon agonist binding by a route similar to that taken by single transmembrane segment receptors. The characterization of the pathway and kinetics of GLP-1-induced receptor endocytosis will be helpful towards understanding the role of internalization and recycling in the control of signal transduction by this receptor.
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OBJECTIVE: Chronic activation of the nuclear factor-kappaB (NF-kappaB) in white adipose tissue leads to increased production of pro-inflammatory cytokines, which are involved in the development of insulin resistance. It is presently unknown whether peroxisome proliferator-activated receptor (PPAR) beta/delta activation prevents inflammation in adipocytes. RESEARCH DESIGN AND METHODS AND RESULTS: First, we examined whether the PPARbeta/delta agonist GW501516 prevents lipopolysaccharide (LPS)-induced cytokine production in differentiated 3T3-L1 adipocytes. Treatment with GW501516 blocked LPS-induced IL-6 expression and secretion by adipocytes and the subsequent activation of the signal transducer and activator of transcription 3 (STAT3)-Suppressor of cytokine signaling 3 (SOCS3) pathway. This effect was associated with the capacity of GW501516 to impede LPS-induced NF-kappaB activation. Second, in in vivo studies, white adipose tissue from Zucker diabetic fatty (ZDF) rats, compared with that of lean rats, showed reduced PPARbeta/delta expression and PPAR DNA-binding activity, which was accompanied by enhanced IL-6 expression and NF-kappaB DNA-binding activity. Furthermore, IL-6 expression and NF-kappaB DNA-binding activity was higher in white adipose tissue from PPARbeta/delta-null mice than in wild-type mice. Because mitogen-activated protein kinase-extracellular signal-related kinase (ERK)1/2 (MEK1/2) is involved in LPS-induced NF-kappaB activation in adipocytes, we explored whether PPARbeta/delta prevented NF-kappaB activation by inhibiting this pathway. Interestingly, GW501516 prevented ERK1/2 phosphorylation by LPS. Furthermore, white adipose tissue from animal showing constitutively increased NF-kappaB activity, such as ZDF rats and PPARbeta/delta-null mice, also showed enhanced phospho-ERK1/2 levels. CONCLUSIONS: These findings indicate that activation of PPARbeta/delta inhibits enhanced cytokine production in adipocytes by preventing NF-kappaB activation via ERK1/2, an effect that may help prevent insulin resistance.
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Camurati-Engelmann disease is characterized by hyperostosis of the long bones and the skull, muscle atrophy, severe limb pain, and progressive joint contractures in some patients. It is caused by heterozygous mutations in the transforming growth factor β1 (TGFβ1) believed to result in improper folding of the latency-associated peptide domain of TGFβ1 and thus in increased or deregulated bioactivity. Losartan, an angiotensin II type 1 receptor antagonist, has been found to downregulate the expression of TGFβ type 1 and 2 receptors. Clinical trials with losartan have shown a benefit in Marfan syndrome, while trials are underway for Duchenne muscular dystrophy and other myopathies associated with TGFβ1 signaling. We hypothesized that due to its anti-TGFβ1 activity, losartan might be beneficial in Camurati-Engelmann disease. This report concerns a boy who presented at age 13 years with severe limb pain and difficulty in walking. Clinical and radiographic evaluation results were compatible with Camurati-Engelmann disease and the diagnosis was confirmed by mutation analysis (c.652C > T [p.Arg218Cys]). The boy underwent an experimental treatment with losartan at a dosage of 50 mg/day, orally. During the treatment period of 18 months, the intensity and frequency of limb pain decreased significantly (as shown by a pain diary), and muscle strength improved, allowing the boy to resume walking and climbing stairs. No obvious side effects were observed. We cautiously conclude that TGFβ1 inhibition with losartan deserves further evaluation in the clinical management of Camurati-Engelmann disease.
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BACKGROUND: Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up. METHODS: BIG 1-98 is a randomised, phase 3, double-blind trial of postmenopausal women with hormone receptor-positive early breast cancer that compares 5 years of tamoxifen or letrozole monotherapy, or sequential treatment with 2 years of one of these drugs followed by 3 years of the other. Randomisation was done with permuted blocks, and stratified according to the two-arm or four-arm randomisation option, participating institution, and chemotherapy use. Patients, investigators, data managers, and medical reviewers were masked. The primary efficacy endpoint was disease-free survival (events were invasive breast cancer relapse, second primaries [contralateral breast and non-breast], or death without previous cancer event). Secondary endpoints were overall survival, distant recurrence-free interval (DRFI), and breast cancer-free interval (BCFI). The monotherapy comparison included patients randomly assigned to tamoxifen or letrozole for 5 years. In 2005, after a significant disease-free survival benefit was reported for letrozole as compared with tamoxifen, a protocol amendment facilitated the crossover to letrozole of patients who were still receiving tamoxifen alone; Cox models and Kaplan-Meier estimates with inverse probability of censoring weighting (IPCW) are used to account for selective crossover to letrozole of patients (n=619) in the tamoxifen arm. Comparison of sequential treatments to letrozole monotherapy included patients enrolled and randomly assigned to letrozole for 5 years, letrozole for 2 years followed by tamoxifen for 3 years, or tamoxifen for 2 years followed by letrozole for 3 years. Treatment has ended for all patients and detailed safety results for adverse events that occurred during the 5 years of treatment have been reported elsewhere. Follow-up is continuing for those enrolled in the four-arm option. BIG 1-98 is registered at clinicaltrials.govNCT00004205. FINDINGS: 8010 patients were included in the trial, with a median follow-up of 8·1 years (range 0-12·4). 2459 were randomly assigned to monotherapy with tamoxifen for 5 years and 2463 to monotherapy with letrozole for 5 years. In the four-arm option of the trial, 1546 were randomly assigned to letrozole for 5 years, 1548 to tamoxifen for 5 years, 1540 to letrozole for 2 years followed by tamoxifen for 3 years, and 1548 to tamoxifen for 2 years followed by letrozole for 3 years. At a median follow-up of 8·7 years from randomisation (range 0-12·4), letrozole monotherapy was significantly better than tamoxifen, whether by IPCW or intention-to-treat analysis (IPCW disease-free survival HR 0·82 [95% CI 0·74-0·92], overall survival HR 0·79 [0·69-0·90], DRFI HR 0·79 [0·68-0·92], BCFI HR 0·80 [0·70-0·92]; intention-to-treat disease-free survival HR 0·86 [0·78-0·96], overall survival HR 0·87 [0·77-0·999], DRFI HR 0·86 [0·74-0·998], BCFI HR 0·86 [0·76-0·98]). At a median follow-up of 8·0 years from randomisation (range 0-11·2) for the comparison of the sequential groups with letrozole monotherapy, there were no statistically significant differences in any of the four endpoints for either sequence. 8-year intention-to-treat estimates (each with SE ≤1·1%) for letrozole monotherapy, letrozole followed by tamoxifen, and tamoxifen followed by letrozole were 78·6%, 77·8%, 77·3% for disease-free survival; 87·5%, 87·7%, 85·9% for overall survival; 89·9%, 88·7%, 88·1% for DRFI; and 86·1%, 85·3%, 84·3% for BCFI. INTERPRETATION: For postmenopausal women with endocrine-responsive early breast cancer, a reduction in breast cancer recurrence and mortality is obtained by letrozole monotherapy when compared with tamoxifen montherapy. Sequential treatments involving tamoxifen and letrozole do not improve outcome compared with letrozole monotherapy, but might be useful strategies when considering an individual patient's risk of recurrence and treatment tolerability. FUNDING: Novartis, United States National Cancer Institute, International Breast Cancer Study Group.
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Clonally distributed inhibitory receptors negatively regulate natural killer (NK) cell function via specific interactions with allelic forms of major histocompatibility complex (MHC) class I molecules. In the mouse, the Ly-49 family of inhibitory receptors is found not only on NK cells but also on a minor (NK1.1+) T cell subset. Using Ly-49 transgenic mice, we show here that the development of NK1.1+ T cells, in contrast to NK or conventional T cells, is impaired when their Ly-49 receptors engage self-MHC class I molecules. Impaired NK1.1+ T cell development in transgenic mice is associated with a failure to select the appropriate CD1-reactive T cell receptor repertoire. In normal mice, NK1.1+ T cell maturation is accompanied by extinction of Ly-49 receptor expression. Collectively, our data imply that developmentally regulated extinction of inhibitory MHC-specific receptors is required for normal NK1.1+ T cell maturation and selection.
Interleukins (IL)-1 and IL-2 control IL-2 receptor alpha and beta expression in immature thymocytes.
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Functional high-affinity interleukin-2 receptors (IL-2R) contain three transmembrane proteins, IL-2R alpha, beta and gamma. We have investigated the expression of IL-2R alpha and beta genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL-2R alpha on their surface. Stimulation of IL-2R alpha+ and IL-2R alpha- immature thymocytes with phorbol 12-myristate 13-acetate and calcium ionophore induces synthesis of IL-2R alpha and IL-2R beta mRNA. Most of this response depends on autocrine stimulation by IL-2. IL-1 synergizes with IL-2 to induce a 120-fold increase in IL-2R alpha mRNA and a 14-fold increase in IL-2R beta mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL-2 plays a major role in the regulation of IL-2R expression in normal immature thymocyte. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.
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Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.
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Glucagon-like peptide-1(7-36)amide (tGLP-1), oxyntomodulin (OXM), and glucagon are posttranslational end products of the glucagon gene expressed in intestinal L-cells. In vivo, these peptides are potent inhibitors of gastric acid secretion via several pathways, including stimulation of somatostatin release. We have examined the receptors through which these peptides stimulate somatostatin secretion using the somatostatin-secreting cell line RIN T3. tGLP-1, OXM, and glucagon stimulated somatostatin release and cAMP accumulation in RIN T3 cells to similar maximum levels, with ED50 values close to 0.2, 2, and 50 nM and 0.02, 0.3, and 8 nM, respectively. Binding of [125I]tGLP-1, [125I]OXM, and [125I]glucagon to RIN T3 plasma membranes was inhibited by the three peptides, with relative potencies as follows: tGLP-1 > OXM > glucagon. Whatever the tracer used, the IC50 for tGLP-1 was close to 0.15 nM and was shifted rightward for OXM and glucagon by about 1 and 2-3 orders of magnitude, respectively. Scatchard analyses for the three peptides were compatible with a single class of receptor sites displaying a similar maximal binding close to 2 pmol/mg protein. In the hamster lung fibroblast cell line CCL39 transfected with the receptor for tGLP-1, binding of [125I]tGLP-1 was inhibited by tGLP-1, OXM, and glucagon, with relative potencies close to those obtained with RIN T3 membranes. Chemical cross-linking of [125I]tGLP-1, [125I]OXM, and [125I]glucagon revealed a single band at 63,000 mol wt, the intensity of which was dose-dependently reduced by all three peptides. These data suggest that in the somatostatin-secreting cell line RIN T3, OXM and glucagon stimulate somatostatin release through a tGLP-1-preferring receptor. This suggests that some biological effects, previously described for these peptides, might be due to their interaction with this receptor.
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BACKGROUND: Only 25% of IVF transfer cycles lead to a clinical pregnancy, calling for continued technical progress but also more in depth analysis of patients' individual characteristics. The interleukin-1 (IL-1) system and matrix metalloproteinases (MMPs) are strongly implicated in embryo implantation. The genes coding for IL-1Ra (gene symbol IL-1RN), IL-1beta, MMP2 and MMP9 bear functional polymorphisms. We analysed the maternal genetic profile at these polymorphic sites in IVF patients, to determine possible correlations with IVF outcome. METHODS: One hundred and sixty women undergoing an IVF cycle were enrolled and a buccal smear was obtained. The presence of IL-1RN variable number of tandem repeats and IL-1B + 3953, MMP2-1306 and MMP9-1562 single nucleotide substitutions were determined. Patients were divided into pregnancy failures (119), biochemical pregnancies (8) and clinical pregnancies (33). RESULTS: There was a 40% decrease in IL-1RN*2 allele frequency (P = 0.024) and a 45% decrease in IL-1RN*2 carrier status in the clinical pregnancy group as compared to the pregnancy failure group (P = 0.017). This decrease was still statistically significant after a multivariate logistic regression analysis. The likelihood of a clinical pregnancy was decreased accordingly in IL-1RN*2 carriers: odds ratio = 0.349, 95% confidence interval = 0.2-0.8, P = 0.017. The IL-1B, MMP2 and MMP9 polymorphisms showed no correlation with IVF outcome. CONCLUSIONS: IL-1RN*2 allele carriage is associated with a poor prognosis of achieving a pregnancy after IVF.
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TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.
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Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M(3) muscarinic receptor was implicated in the relaxing action of ACh and M(1) muscarinic receptor (M(1)AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M(1)AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M(1)AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M(1)AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M(1)AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."