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.

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200 -400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200 -400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosinephosphorylated STAT3 eluting at 80 -100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") coeluted with catalase at 230 kDa. In order to identify the protein components of the 200 -400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-coshifted p60 protein as the chaperone GRP58/ER-60/ ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g., stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membraneless metastable (shape-changing) condensates in the cytoplasm. In our studies, we used the same cell lines and methods as those used by previous investigators but concluded that wild-type MxA formed variably sized spherical or irregular bodies, filaments, and even a reticulum distinct from that of ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of green fluorescent protein (GFP)-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24 ± 0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase, respectively, in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature and provide evidence for membraneless MxA biomolecular condensates in the uninfected cell cytoplasm. IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human myxovirus resistance protein A (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation and further report that MxA forms membraneless metastable (shape-changing) condensates in the cytoplasm consisting of variably sized spherical or irregular bodies, filaments, and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1 to 3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

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...

In recent years several of the key tenets of the original cytokine-STAT signaling paradigm have had to be revised. First, that nonphosphorylated "inactive" STATs are present in the cytoplasm as free monomers which dimerized only subsequent to Tyr-phosphorylation has been replaced by the understanding that nonphosphorylated STATs in the cytoplasm exist largely as dimers and high molecular mass "statosome" complexes. Second, the notion that phosphorylation, either of Tyr or Ser residues or both, in STAT species is required for transcriptional activation has been replaced by the realization that nonphosphorylated STATs can be transcriptionally active albeit with respect to sets of target genes distinct from phosphorylated STATs. Third, the notion that it is the activation by phosphorylation of STATs at the plasma membrane that then leads to their import into the nucleus has been replaced by the recognition that even nonphosphorylated STATs shuttle between the cytoplasm and nucleus at all times in a constitutive manner. Fourth, the notion that the transcytoplasmic transit of STATs from the plasma membrane to the nuclear import machinery takes place exclusively as a free cytosolic process has been replaced by the understanding that at least a portion of this trans-cytoplasmic transit is mediated via membrane-associated caveolar and endocytic trafficking (the "signaling endosome" hypothesis). Fifth, the targeting and sequestration of activated STAT3 to long-lived endosomes in the cytoplasm requires consideration of STAT3-mediated "signal transduction" from the plasma membrane to cytoplasmic membrane destinations potentially for function(s) in the cytoplasm. Indeed, in tissue sections many discrete histologic cell types display PY-STAT3 almost exclusively in the cytoplasm with little, if any, in the nucleus. New challenges include determining the structural bases for the recruitment of nonphosphorylated dimeric STAT species to the cytosolic face of membranes including at the cytoplasmic tails of respective receptor complexes, the conformational changes subsequent to phosphorylation and the structural bases for the targeting and functions of STAT proteins within the cytoplasm per se.

Rationale: Pulmonary arterial hypertension is a progressive disease characterized by an elevation in the mean pulmonary artery pressure leading to right heart failure and a significant risk of death. Alterations in two transforming growth factor (TGF) signaling pathways, bone morphogenetic protein receptor II and the TGF-b receptor I, Alk1, have been implicated in the pathogenesis of pulmonary hypertension (PH). However, the role of TGF-b family signaling in PH and pulmonary vascular remodeling remains unclear. Objectives: To determine whether inhibition of TGF-b signaling will attenuate and reverse monocrotaline-induced PH (MCT-PH). Methods: We have used an orally active small-molecule TGF-b receptor I inhibitor, SD-208, to determine the functional role of this pathway in MCT-PH. Measurements and Main Results: The development of MCT-PH was associated with increased vascular cell apoptosis, which paralleled TGF-b signaling as documented by psmad2 expression. Inhibition of TGF-b signaling with SD-208 significantly attenuated the development of the PH and reduced pulmonary vascular remodeling. These effects were associated with decreased early vascular cell apoptosis, adventitial cell proliferation, and matrix metalloproteinase expression. Inhibition of TGF-b signaling with SD-208 in established MCT-PH resulted in a small but significant improvement in hemodynamic parameters and medial remodeling. Conclusions: These findings provide evidence that increased TGF-b signaling participates in the pathogenesis of experimental severe PH.Keywords: pulmonary hypertension; transforming growth factor-b; apoptosis; proliferation; matrix metalloproteinase Genetic studies of familial idiopathic pulmonary hypertension (PH) revealed that a germline mutation in one copy of bone morphogenetic protein (BMP) receptor II occurs in about 80% of patients with familial idiopathic PH (1, 2). The importance of transforming growth factor (TGF) signaling is underscored by the association of loss-of-function mutations of TGF-b receptor I, Alk1, with pulmonary arterial hypertension (PAH), as well as somatic microsatellite instability of the TGF-b receptor II gene in plexiform lesions present in pulmonary arteries of patients with idiopathic PAH (IPAH) (3, 4). On the other hand, there is also evidence of increased expression of TGF-b isoforms (5), TGF-b and BMP receptors, and enhanced TGF-b-dependent signaling in both familial PAH and IPAH lungs. These findings suggest that PH might develop due to unbalanced TGF-b signaling in pulmonary vascular cells rather than a simple loss of TGF-b signaling. The concept of imbalanced TGF-b signaling has been supported by the findings of enhanced TGF-b signaling in the setting of TGF-b receptor mutations in systemic vascular abnormalities (6-8).Activation of TGF-b, which is stored as an inactive dimer bound to the extracellular matrix, leads to its interaction with TGF-b receptor II, a constitutively active serine/threonine kinase that subsequently recruits and phosphorylates TGF-b receptor I. Two distinct ...

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...

We report unexpected nongenomic functions of signal transducer and activator of transcription (STAT) 5 species in the cytoplasm aimed at preserving the structure and function of the Golgi apparatus and rough endoplasmic reticulum (ER) in vascular cells. Immunoimaging and green fluorescent protein-tagged-STAT5a protein localization studies showed the constitutive association of nonphosphorylated STAT5a, and to a lesser extent STAT5b, with the Golgi apparatus and of STAT5a with centrosomes in human pulmonary arterial endothelial and smooth muscle cells. Acute knockdown of STAT5a/b species using small interfering RNAs (siRNAs), including in the presence of an mRNA synthesis inhibitor (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole), produced a dramatic phenotype within 1 day, consisting of dilatation and fragmentation of Golgi cisternae, a marked tubule-to-cyst change in the ER, increased accumulation of reticulon-4 (RTN4)/Nogo-B and atlastin-3 (ATL3) at cyst-zone boundaries, cystic separation of the outer and inner nuclear membranes, accompanied by scalloped/lunate distortion of the nucleus, with accumulation of RTN4 on convex sides of distorted nuclei. These cells showed inhibition of vesicular stomatitis virus G protein glycoprotein trafficking, mitochondrial fragmentation, and reduced mitochondrial function. STAT5a/b(-/-) mouse embryo fibroblasts also showed altered ER/Golgi dynamics. RTN4 knockdown using siRNA did not affect development of the cystic phenotype; ATL3 siRNA led to effacement of cyst-zone boundaries. In magnetic-bead cross-immunopanning assays, ATL3 bound both STAT5a and STAT5b. Remarkably, this novel cystic ER/lunate nucleus phenotype was characteristic of vascular cells in arterial lesions of idiopathic pulmonary hypertension, an unrelentingly fatal human disease. These data provide evidence of a STAT-family protein regulating the structure of a cytoplasmic organelle and implicate this mechanism in the pathogenesis of a human disease.