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-1␣/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-1␣ levels in the lung was apparent within 48 hours, declining to Ϸ30% 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-1␣ 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-1␣ loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1␣ 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-1␣, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis. Conclusions-MCT-induced disruption of cav-1␣ chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

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 ϳ5% 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.Diverse cytokines and growth factors, including various interleukins and interferons, signal to the cell nucleus by activating the JAK 2 /STAT (Janus kinase/signal transducers and activators of transcription) pathway at the plasma membrane at the level of raft microdomains (reviewed in Refs. 1-3). This signal transduction from the plasma membrane to the nucleus by Tyr-and/or Ser-phosphorylated STAT proteins is widely represented exclusively as a soluble cytosolic process.Although until recently it was considered that latent STAT proteins in the cytoplasm were monomeric, work from this and other laboratories showed that latent STATs exist in the cytosol already in the form of at least dimers and included higher order complexes (200 -400 kDa statosome I and 1-to 2-MDa statosome II complexes) (4 -8, reviewed in Refs. 9 and 10). The absence of free STAT monomers in the cytoplasm has now been extensively confirmed (11)(12)(13)(14). Moreover, recent fluorescence transfer and fluorescence correlation spectroscopy data confirm the existence of STAT3 dimers and higher order statosome complexes (200 -400 kDa and 1-2 MDa) in the cytoplasm of live cells (15)(16)(17).That different growth factor and cytokine receptors are associated with the endocytic pathway and can even maintain their ongoing signaling function from this membrane-bound compartment has been clearly delineated (18 -20). Nevertheless, today, the widely represented model of IL-6/STAT3 signa...

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-␤-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°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.Fever ("calor") is a common response of the body to infection and injury (reviewed in Refs. 1 and 2). Interleukin-6 (IL-6) 1 is a major systemic mediator of this "acute-phase" response that includes stimulation of the liver to synthesize and secrete a large number of protective plasma proteins such as anti-proteinases, clotting factors, complement factors, and scavenger proteins that help to limit the site of injury or infection (1, 2). Furthermore, IL-6 is itself a centrally acting pyrogen (3-5).How is the major signaling pathway used by IL-6 to enhance acute-phase plasma protein gene expression in hepatocytes, the JAK-STAT3 pathway (Janus kinase-signal transducer and activator of transcription 3) (6 -11), maintained during an increase in body temperature? We report the identification of caveolin-1 (cav-1) and heat shock protein 90 (HSP90) as STAT3-associated proteins at the level of plasma membrane rafts and in high molecular mass cytoplasmic complexes, and we explore their role in cytokine signaling at normal and elevated temperatures.In previous cell fractionation studies of human hepatom...

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").

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.

Monocrotaline (MCT)-induced pulmonary hypertension (PH) in the rat is a widely used experimental model. We have previously shown that MCT pyrrole (MCTP) produces loss of caveolin-1 (cav-1) and endothelial nitric oxide synthase from plasma membrane raft microdomains in pulmonary arterial endothelial cells (PAEC) with the trapping of these proteins in the Golgi organelle (the Golgi blockade hypothesis). In the present study, we investigated the mechanisms underlying this intracellular trafficking block in experiments in cell culture and in the MCT-treated rat. In cell culture, PAEC showed trapping of cav-1 in Golgi membranes as early as 6 h after exposure to MCTP. Phenotypic megalocytosis and a reduction in anterograde trafficking (assayed in terms of the secretion of horseradish peroxidase derived from exogenously transfected expression constructs) were evident within 12 h after MCTP. Cell fractionation and immunofluorescence techniques revealed the marked accumulation of diverse Golgi tethers, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs), and soluble NSF attachment proteins (SNAPs), which mediate membrane fusion during vesicular trafficking (GM130, p115, giantin, golgin 84, clathrin heavy chain, syntaxin-4, -6, Vti1a, Vti1b, GS15, GS27, GS28, SNAP23, and ␣-SNAP) in the enlarged/ circumnuclear Golgi in MCTP-treated PAEC and A549 lung epithelial cells. Moreover, NSF, an ATPase required for the "disassembly" of SNARE complexes subsequent to membrane fusion, was increasingly sequestered in non-Golgi membranes. Immunofluorescence studies of lung tissue from MCT-treated rats confirmed enlargement of perinuclear Golgi elements in lung arterial endothelial and parenchymal cells as early as 4 days after MCT. Thus MCTinduced PH represents a disease state characterized by dysfunction of Golgi tethers, SNAREs, and SNAPs and of intracellular vesicular trafficking.soluble N-ethylmaleimide-sensitive factor attachment protein receptors; endothelium; intracellular membrane trafficking; Golgi blockade PULMONARY HYPERTENSION (PH) is a progressive disease with high morbidity and mortality (10). As the clinical manifestations of PH typically occur long after the initial injury, experimental models provide an opportunity to investigate the initiating mechanism at the cellular and biochemical levels (45). Among experimental models of PH, administration of the pyrrolizidine alkaloid monocrotaline (MCT) to the juvenile male rat has and continues to be used extensively (21,29). In this model, progressive PH develops 10 -14 days after a single injection of MCT. The injected MCT is converted to its active pyrrolic derivative [MCT pyrrole (MCTP)] by the cytochrome P-450 system in the liver (36). The bioactive MCTP, which has a short half-life (ϳ3 s in aqueous medium), affects the first vascular bed it encounters: the pulmonary arterial system. The major cellular targets of MCT in the rat lung include the pulmonary arterial endothelial cells (PAEC), pulmonary arterial smooth muscle cells (PASMC), and ...

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 ϳ10% 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-␥ receptor ␣ chain (IFN-␥R␣), 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-␤-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6-and IFN-␥-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").Signal transduction in mammalian cells is initiated by complex protein-protein interactions between ligands, receptors, and kinases at the level of the plasma membrane. It is now becoming clear that specialized microdomains at the cell surface, known as rafts and/or caveolae, are intimately involved in this process (1, 2). These lipid microdomains contain high concentrations of glycolipids, sphingomyelin, and cholesterol and represent platforms for conducting cellular functions such as vesicular trafficking and signal transduction (1, 2). Raft domains contain several integral raft proteins, which include caveolin and flotillin family members. Signaling processes shown to involve plasma membrane raft domains include immunoglobulin E signaling, T-cell antigen receptor signaling, B-cell receptor signaling, signaling involving epidermal growth factor, platelet-derived growth factor, insulin receptor, Ephrin B1 receptor, neurotrophin, Ha-Ras, nitric-oxide synthase and integrins (reviewed in Refs. 1-4). Caveolin-1 in rafts has been shown to modulate insulin-and Ha-Ras-mediated signaling (the "caveola signaling hypothesis") (3, 4). Despite these major advances in the understanding of rafts as...

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