Biomedical subjects

Mehul Shah

Publications and source records attributed to Mehul Shah.

10 recordsLinked to original sources

PubMed2006

Upregulation of human angiotensinogen (AGT) gene transcription by interferon-gamma: involvement of the STAT1-binding motif in the AGT promoter.

Mechanisms to maintain blood pressure in the face of infection are critical to survival. The angiotensinogen (AGT) gene locus is an important component of this response. Thus the AGT gene, expressed predominantly by liver cells, is known to be a positive acute phase reactant. We have previously demonstrated activation of the AGT promoter in hepatocytes through the IL6/STAT3 signaling mechanism. We have now investigated whether IFN-gamma, a cytokine also induced in response to diverse infections, can regulate AGT gene expression, and have elucidated the molecular mechanism involved. IFN gamma treatment up-regulated AGT mRNA level and promoter activity in Hep3B hepatocytes. Sequential deletion of the promoter from the 5' side suggested the major IFN gamma responsive DNA element to be between -303 and -103. This region contained a candidate STAT1-binding site between -271 and -279. EMSA and chromatin immuno-precipitation (ChIP) assays confirmed that IFN-gamma treatment induced the binding of STAT1 to this element. Reporter constructs containing this AGT promoter derived element in a multimerized context but not a mutant version were responsive to IFN gamma. Moreover mutating this STAT1 element in the context of the wild-type AGT holo promoter reduced responsiveness to IFN gamma. In contrast to the clear synergism between dexamethasone and IL 6 in the upregulation of the AGT promoter (through interaction between GR and STAT3), the combination of IFN gamma with IL 6 or with dexamethasone did not further increase AGT promoter activity suggesting that the IFN gamma/STAT1 pathway represents a separate signaling mechanism. These data highlight the redundancy in cytokine-mediated host response pathways aimed at the maintenance of blood pressure during infection.

Angiotensinogen↗

PubMed2005

Membrane-associated STAT3 and PY-STAT3 in the cytoplasm.

Signal transduction from the plasma membrane to the nucleus by STAT proteins is widely represented as exclusively a soluble cytosolic process. Using cell-fractionation methods, we observed that approximately 5% of cytoplasmic STAT3 was constitutively associated with the purified early endosome (EE) fraction in human Hep3B liver cells. By 15-30 min after interleukin-6 (IL-6) treatment, up to two-thirds of cytoplasmic Tyr-phosphorylated STAT3 can be associated with the purified early endosome fraction (Rab-5-, EEA1-, transferrin receptor-, and clathrin-positive fraction). Electron microscopy, immunofluorescence, and detergent dissection approaches confirmed the association of STAT3 and PY-STAT3 with early endosomes. STAT3 was constitutively associated with clathrin heavy chain in membrane and in the 1- to 2-MDa cytosolic complexes. The membrane association was dynamic in that, within 15 min of treatment with the vicinal-thiol cross-linker phenylarsine oxide, there was a dramatic increase in bulk STAT3 association with sedimentable membranes. The functional contribution of PY-STAT3 association with the endocytic pathway was evaluated in transient transfection assays using IL-6-inducible STAT3-reporter-luciferase constructs and selective regulators of this pathway. STAT3-transcriptional activation was inhibited by expression constructs for dominant negative dynamin K44A, epsin 2a, amphiphysin A1, and clathrin light chain but enhanced by that for the active dynamin species MxA. Taken together, these studies emphasize the contribution of the endocytic pathway to productive IL-6/STAT3 signaling.

Adaptor Proteins, Vesicular Transport↗

PubMed2005

Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response.

The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a "megalocytosis" phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the "Golgi blockade" hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and alpha-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1alpha and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis.

Autoantigens↗

PubMed2004

Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism.

Pyrrolizidine alkaloids initiate disease in the lung (pulmonary hypertension), liver (veno-occlusive disease and cirrhosis), and kidneys (afferent arteriolar block and mesangiolysis) by inducing a megalocytotic phenotype in target endothelial and parenchymal cells. A "hit-and-run" type of exposure to the bioactive pyrrolizidine results, within 2-3 days, in enlarged cells with large nuclei and enlarged Golgi and endoplasmic reticulum, while the cells remain in G2/M block. In the present study, we recapitulated monocrotaline pyrrole (MCTP)-induced megalocytosis in cultures of bovine pulmonary arterial endothelial cells (PAEC), human Hep3B hepatocytes, human type II-like alveolar epithelial cells (A549), and human pulmonary arterial smooth muscle cells (PASMC) and investigated the subcellular mechanism involved. There was an inverse relationship between reduction in caveolin (Cav)-1 levels and stimulation of promitogenic STAT3 and ERK1/2 cell signaling. In megalocytotic PAEC, the Golgi scaffolding protein GM130 was shifted from membranes with heavy density to those with a lighter density. This lighter Golgi fraction was enriched for hypo-oligomeric Cav-1, indicating dysfunctional trafficking of cargo. Immunofluorescence imaging studies confirmed the trapping of Cav-1 in a GM130-positive Golgi compartment. There was an increase in Ser25 phosphorylation of GM130 (typically a prelude to Golgi fragmentation and mitosis) and increased association between pGM130, cdc2 kinase, and Cav-1. Nevertheless, megalocytotic MCTP-treated cells showed reduced entry into mitosis upon stimulation with 2-methoxyestradiol (2-ME), reduced 2-ME-induced Golgi fragmentation, and a slowing of Golgi reassembly after nocodazole-induced fragmentation. These data suggest that a disruption of the trafficking and mitosis sensor functions of the Golgi may represent the subcellular mechanism leading to MCTP-induced megalocytosis ("the Golgi blockade hypothesis").

Animals↗

PubMed2004

Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension.

BACKGROUND: In the monocrotaline (MCT)-treated rat, there is marked stimulation of DNA synthesis and megalocytosis of pulmonary arterial endothelial cells (PAECs) within 3 to 4 days, followed by pulmonary hypertension (PH) 10 to 14 days later. Growing evidence implicates caveolin-1 (cav-1) in plasma membrane rafts as a negative regulator of promitogenic signaling. We have investigated the integrity and function of endothelial cell-selective cav-1alpha/raft signaling in MCT-induced PH. METHODS AND RESULTS: Although PH and right ventricular hypertrophy developed by 2 weeks after MCT, a reduction in cav-1alpha levels in the lung was apparent within 48 hours, declining to approximately 30% by 2 weeks, accompanied by an increase in activation of the promitogenic transcription factor STAT3 (PY-STAT3). Immunofluorescence studies showed a selective loss of cav-1alpha and platelet endothelial cell adhesion molecule-1 in the PAEC layer within 48 hours after MCT but an increase in PY-STAT3. PAECs with cav-1alpha loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1alpha from the detergent-resistant lipid raft fraction concomitant with hyperactivation of STAT3. Moreover, cultured PAECs treated with MCT-pyrrole for 48 hours developed megalocytosis associated with hypo-oligomerization and reduction of cav-1alpha, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis. CONCLUSIONS: MCT-induced disruption of cav-1alpha chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

Animals↗

PubMed2002

Interactions of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signaling during fever.

Interleukin-6 (IL-6) initiates STAT3 signaling in plasma membrane rafts with the subsequent transit of Tyr-phosphorylated STAT3 (PY-STAT3) through the cytoplasmic compartment to the nucleus in association with accessory proteins. We initially identified caveolin-1 (cav-1) as a candidate STAT3-associated accessory protein due to its co-localization with STAT3 and PY-STAT3 in flotation raft fractions, and heat shock protein 90 (HSP90) due to its inclusion in cytosolic STAT3-containing 200-400-kDa complexes. Subsequent immunomagnetic bead pullout assays showed that STAT3, PY-STAT3, cav-1, and HSP90 interacted in plasma membrane and cytoplasmic complexes derived from uninduced and stimulated Hep3B cells. This was a general property of STAT3 in that these interactions were also observed in alveolar epithelial type II-like cells, lung fibroblasts, and pulmonary arterial endothelial cells. Exposure of Hep3B cells to the raft disrupter methyl-beta-cyclodextrin for 1-10 min followed by IL-6 stimulation for 15 min preferentially inhibited the appearance of PY-STAT3 in the cav-1-enriched sedimentable cytoplasmic fraction, suggesting that these complexes may represent a trafficking intermediate immediately downstream from the raft. Because IL-6 is known to function in the body in the context of fever, the possibility that HSP90 may help preserve IL-6-induced STAT3 signaling at elevated temperature was investigated. Geldanamycin, an HSP90 inhibitor, markedly inhibited IL-6-stimulated STAT3 signaling in Hep3B hepatocytes cultured overnight at 39.5 degrees C as evaluated by DNA-shift assays, trafficking of PY-STAT3 to the nucleus, cross-precipitation of HSP90 by anti-STAT3 polyclonal antibody, and reporter/luciferase construct experiments. Taken together, the data show that IL-6/raft/STAT3 signaling is a chaperoned pathway that involves cav-1 and HSP90 as accessory proteins and suggest a mechanism for the preservation of this signaling during fever.

Animals↗

PubMed2002

Cytokine signaling: STATS in plasma membrane rafts.

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to approximately 10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-gamma receptor alpha chain (IFN-gammaRalpha), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-beta-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6- and IFN-gamma-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis").

Blotting, Western↗

PubMed2002

Does racial bias exist in the medical management of heart failure?

BACKGROUND: It is suspected that effective therapies are often underutilized in black compared with white patients with coronary artery disease (CAD). HYPOTHESIS: We hypothesized that an unfavorable bias may exist against black patients in the medical management of heart failure. METHODS: In 566 consecutive adult subjects who were discharged alive from the hospital with a principal discharge diagnosis of heart failure, we assessed the effect of patient race on utilization of classes of medications (angiotensin-converting enzyme inhibitors [ACEI], digitalis, diuretic agents) and combinations of medications (effective vasodilators, i.e., ACEI or combined hydralazine and nitrate; effective combination therapy, i.e., effective vasodilator with digitalis and diuretic) known to be beneficial in symptomatic heart failure. RESULTS: Compared with black patients (n = 182), white patients were older, had a higher incidence of coronary artery disease, lower incidence of hypertension, and lower serum creatinine and left ventricular end-diastolic diameter. In crude analyses, the utilization of all medications was similar between white and black patients. After adjustment for clinical differences, black patients were more likely to receive ACEI (adjusted odds ratio [OR] = 1.84; 95% confidence interval [CI] 1.13-3.01), effective vasodilators (OR = 1.97; CI 1.20-3.23), and effective combination therapy (OR = 1.66; CI 1.02-2.69) than white patients at the time of discharge from the hospital. No multivariate association was seen between patient race and use of digoxin or diuretics. In an analysis of subsets of patients with ejection fraction < 45% (n = 260), no association was seen between patient race and utilization of effective medical therapy. CONCLUSION: Our results show no unfavorable bias against black patients with decompensated heart failure.

Adult↗

PubMed2002

The relationship between left ventricular shape and QT interval dispersion.

Left ventricular geometry is suspected to affect heterogeneity of myocardial repolarization; therefore, it is plausible but unproven that increased sphericity of the left ventricle is associated with greater QT interval dispersion. In 60 patients with dilated cardiomyopathy with left ventricular ejection fraction < or = 30%, we found that spherical distortion of the left ventricle was associated with increased QT dispersion, implying increased heterogeneity of myocardial repolarization.

Aged↗

PubMed2002

Association of the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57) with Stat3 in cytosol and plasma membrane complexes.

Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticulum lumen, was previously identified by us as one of several accessory proteins in the S100 cytosol fraction of human hepatoma Hep3B cells that was differentially coshifted by anti-Stat3 antibody in an antibody-subtracted differential protein display assay. In the present study, the association between GRP58 and Stat3 in different cytoplasmic compartments was evaluated using cross-immunoprecipitation and cell-fractionation techniques. In the S100 cytosol fraction, three different anti-GRP58 polyclonal antibodies (pAb) cross-immunoprecipitated Stat3 (but not Stat1), and, conversely, anti-Stat3 pAb cross-immunoprecipitated GRP58. Both cytosolic Stat3 and GRP58 eluted during Superose-6 gel-filtration chromatography in complexes of size 200-400 kDa (statosome I), and anti-Stat3 pAb cross-immunoprecipitated GRp58 from these FPLC elution fractions. Using differential sedimentation and density equilibrium flotation methods, Stat3 and GRP58 were observed to be coassociated with cytoplasmic membranes enriched for the plasma membrane marker 5' nucleotidase but not with those containing the endoplasmic reticulum marker BiP/GRP78. The Stat3 and GRP58-containing plasma membrane fraction also contained Stat1, Stat5b, and gp130. Stat activation by orthovanadate caused the accumulation of PY-Stat3 in the GRP58-containing plasma membrane fraction. However, this PY-Stat3 was DNA-binding deficient. Likewise, excess exogenous recombinant human GRP58 prepared using a baculovirus expression system preferentially inhibited Stat3 DNA-binding activity in the S100 cytosol, suggesting that GRP58 may sequester activated Stat3. The new data confirm the association between GRP58 and Stat3 in cytosolic 200-400-kDa statosome I complexes and show that both GRP58 and Stat family members coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3.

Antigens, CD↗