HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 294/3/L449    most recent 00377.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mukhopadhyay, S. Articles by Sehgal, P. B. Search for Related Content PubMed PubMed Citation Articles by Mukhopadhyay, S. Articles by Sehgal, P. B.

Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension

¹Department of Cell Biology and Anatomy and ²Department of Medicine, New York Medical College, Valhalla, New York; and ³Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 11 September 2007 ; accepted in final form 10 December 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Lung vascular lesions in pulmonary arterial hypertension (PAH) are characterized by enlarged, vacuolated ("megalocytotic") pulmonary arterial endothelial (PAEC) and smooth muscle cells (PASMC). We have recently proposed that dysfunction of vesicle tethers, soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), and SNAP receptors (SNAREs), leading to disruptions of intracellular trafficking in the Golgi to plasma membrane (centrifugal) and the plasma membrane to cell interior (centripetal) directions is a key causal mechanism in this disease. In PAH, there was a reciprocal relationship between loss of caveolin-1 (cav-1) in PAECs and increased expression of "activated" tyrosine-phosphorylated STAT3 (PY-STAT3) associated with a block in centrifugal trafficking to/through the Golgi organelle. In the present study, we investigated 1) whether centripetal trafficking of STAT3 and PY-STAT3 in PAECs and PASMCs was membrane-associated, and 2) whether this might be affected in PAH. Immunofluorescence and live cell imaging studies showed that, in both PAEC and PASMC, STAT3 was associated with cytoplasmic vesicles partially colocalizing with markers of the endolysosomal compartments (clathrin, EEA1, Rab5, Rab11, and LAMP1). Overexpression of cav-1 increased the targeting of STAT3 to lysosomes and inhibited STAT3 transcriptional activity. Exposure of PAECs to monocrotaline (MCT) pyrrole, which causes PAH in the rat, led to a loss of caveolar STAT3 with increased sequestration of STAT3 and PY-STAT3 in endosomes. In vivo, marked cytoplasmic sequestration of activated PY-STAT3 was a common feature in PAEC in the rat/MCT model and in cells in the proliferative arterial and plexiform lesions in PAH in humans. These data highlight the epigenetic regulation of centripetal cytokine and growth-factor signaling pathways and its modulation in PAH.

caveolin-1; signaling endosome; monocrotaline

IL-6 and its activation of STAT3 signaling is increasingly viewed as contributing to the pathobiology of PAH. IL-6 mRNA has been reported to be elevated in lungs of monocrotaline (MCT)-treated rats with PAH and inhibition of IL-6 production with dexamethasone-attenuated MCT-induced PAH (4). Serum IL-6 levels were increased in idiopathic PAH in human (18). Transgenic mice overexpressing IL-6 developed mild PAH, which could be exacerbated by hypoxia (13). Additionally, IL-6 levels were dramatically increased by more than 15-fold in transgenic mice expressing a mutant isoform of BMPR2 that spontaneously developed PAH (15). A negative feedback loop in which IL-6 inhibits bone morphogenetic protein receptor 2 (BMPR2) signaling has been proposed in this model (15).

In 2004, working with the rat/MCT model of PAH, we reported the reciprocal relationship between the loss of caveolin-1 (cav-1) in plasma membrane rafts/caveolae in PAECs and hyperactivation of the pro-proliferative, IL-6-activated transcription factor STAT3 [increase in tyrosine-phosphorylated STAT3 (PY-STAT3); Ref. 24]. The loss of cav-1 and hyperactivation of PY-STAT3 was observed within 2–4 days after administration of MCT to rats (i.e., before the onset of PAH at 10–14 days; Ref. 24). Close examination of individual cells in vivo showed that the specific endothelial cells that had a loss of cav-1 expression were the same cells with hyperactivation of PY-STAT3 and increased DNA synthesis (24). This reciprocal relationship between cav-1 downregulation and PY-STAT3 activation in PAH in the rat/MCT model was later confirmed by Jasmin and colleagues (19). That the increase in PY-STAT3 levels was seen before the onset of PAH in MCT-treated rats (19, 24) and that the introduction of a cav-1 scaffolding domain peptide blocked PY-STAT3 hyperactivation and ameliorated MCT-induced PAH (19) indicated that STAT3 played a role in the initiation of PAH, at least in this animal model. Moreover, Park and colleagues (33) reported the hyperactivation of PY-STAT3 in lungs of cav-1^–/– mice, which spontaneously developed PAH. Additionally, the increase in PY-STAT3 levels has now been confirmed in pulmonary arterial lesions of idiopathic PAH in humans (23), and a similar increase in STAT3 was also reported in lungs of mice with hypoxia-induced PAH (53). Thus the mechanisms of IL-6/STAT3 signal transduction from the plasma membrane to the cell interior in pulmonary arterial endothelial and vascular smooth muscle cells are of relevance to the pathobiology of PAH. However, considerations of centripetal signaling from the plasma membrane to the cell interior in the pulmonary hypertension literature have largely represented transcytoplasmic transit of activated signaling molecules as taking place exclusively as a soluble cytoplasmic process (reviewed in Refs. 42, 43).

To focus on IL-6/STAT3 signaling, STATs were viewed as monomeric cytosolic proteins that, upon cytokine stimulation, were phosphorylated on specific tyrosine residues and then dimerized, diffused through the cytosol to the region of nuclear pores, and then imported to the nucleus, which led to activation of gene transcription (reviewed in Refs. 11, 25, 40, 44, 51). Over the past few years, several key tenets of this classic JAK-STAT signaling paradigm have undergone revision. We and others have shown that "inactive" nonphosphorylated STATs were not free monomers in the cytoplasm but largely preexist in dimers and dimer-plus high molecular weight "statosome" complexes together with other proteins including clathrin heavy chain (CHC), glucose regulated protein 58 (GRP58), and heat shock protein 90 (HSP90) (14, 20, 29, 31). Moreover, we and others have established that the transcytoplasmic trafficking of PY-STAT3 was not a free cytosolic process but was initiated at plasma membrane raft microdomains (with or without cav-1) and proceeded along the cytoplasmic endocytic pathway at least in human hepatocytes (Hep3B cells) and in murine NIH/3T3 and BALB/c 3T3 fibroblast cells ("signaling endosome hypothesis"; Refs. 5, 39, 46). In these cell types, components of the endocytic pathway (such as dynamin, epsin, Eps15, and amphiphysin A1) were essential for optimal transcriptional activity of PY-STAT3 (5, 46). Additionally, other groups have established that that STAT3 constitutively shuttled between the nucleus and the cytoplasm (35) and that phosphorylation of STAT3 was not a prerequisite for transcriptional activity (57). However, nonphosphorylated STAT3 transcriptionally regulated different subsets of genes than PY-STAT3 (57).

With these insights, we asked whether the transcytoplasmic transit of STAT3 and PY-STAT3 in PAECs and PASMCs might also be membrane-associated and take place along the endocytic pathway, whether cav-1 might shunt this intracellular trafficking towards the lysosomal compartment as it does for TGF-β/Smad signaling (12), and whether this centripetal trafficking might be affected in PAH.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell culture. Growth of primary bovine PAECs, human PASMCs (HPASMC), and the human Hep3B cell line in T-75 flasks, six-well plates, or eight-well chamber slides was done essentially as described by us previously (24, 26–28, 41, 46). PAECs were used between passages 4–20. HPASMC cultures (from Cascade Biologics, Portland, OR) were initially grown in Medium 231 supplemented with smooth muscle growth supplement containing basic fibroblast growth factor, epidermal growth factor, and insulin (as supplied by Cascade Biologics) to stimulate proliferation. Before experimental use, HPASMC cultures were transferred to Medium 231 supplemented with smooth muscle differentiation supplement containing 30 µg/ml heparin (Cascade Biologics) for >7 days as per manufacturer's protocols.

Monocrotaline pyrrole (MCTP) was prepared from MCT (Trans World Chemicals, Rockville, MD) as described by us previously (41) and used at a final concentration of 200 µM for 4 days. By mass spectrometry, 30–50% of the input MCT was converted to active pyrrolic derivative (data not shown). Recombinant human IL-6 (R&D Systems, Minneapolis, MN) was used at 10 ng/ml for 30 min unless otherwise specified (24, 25, 29, 39). Phenylarsine oxide (PAO) was from Sigma-Aldrich (St. Louis, MO) and was used at 5 µM for 30 min (46).

Immunofluorescence analyses of cells in culture. Immunofluorescence analyses of PAECs and PASMCs were performed essentially as described by us earlier (24, 26–28, 41, 46). Cells were fixed and permeabilized using either the 4% cold paraformaldehyde-0.1% Triton X-100 protocol or 3.7% warm formalin (at 37^°C)-0.1% Triton X-100 protocol as previously described by us (24, 26–28, 41, 46). Secondary antibodies used were Alexa Fluor 488 and Alexa Fluor 594 (Molecular Probes, Eugene, OR). Nuclei were demarcated using 4,6-diamidino-2-phenylindole (DAPI). Images were captured using either a Leitz epifluorescence microscope system equipped with a black-and-white charge-coupled device (CCD) camera and then rendered in pseudocolor, or a MRC 1024 ES (Bio-Rad) confocal laser scanning microscopy system with a black-and-white CCD camera and then rendered in pseudocolor, or a Zeiss epifluorescent microscopy system equipped with an RGB camera (for immunomorphometry of lung sections). All images within each specific experiment were collected under identical imaging, laser, and microscopy settings. Images were deconvolved using the Iterative Deconvolve 3D plugin of the NIH ImageJ software.

Plasmids. The following constitutive expression constructs were used in our studies: 1) N1-EGFP and STAT3-EGFP from Dr. Taddaki Miyazaka, Hokkaido University, Sapporo, Japan (54); 2) dominant-negative (DN) dynamin II K44A (Dyn II K44A) from Dr. Lois Greene, National Institutes of Health (NIH), Bethesda, MD (55); 3) murine cav-1 wild-type (WT) from Dr. Ferruccio Galbiati, University of Pittsburgh, Pittsburgh, PA (52); 4) myc-tagged canine cav-1-WT and cav-1-S80A-myc and cav-1-S80E-myc, which are single point mutations in the scaffolding domain of cav-1 from Dr. Jeffery Pessin, SUNY Stony Brook, Stony Brook, NY (48); 5) a myc-tagged double point mutation in the scaffolding domain of cav-1 (canine species) cav-1-F92A/V94A and the corresponding myc-tagged cav-1-WT from Dr. Michael Quon, NIH (30); 6) human GRP58-WT from Drs. Mohammed Bourdi and Lace Pohl, NIH (8); and 7) p950M4-luciferase, a well-characterized STAT3 responsive luciferase construct, which contains four copies of STAT3 DNA-binding element from the human angiotensin promoter from Dr. Ashok Kumar, New York Medical College, Valhalla, NY (26, 46).

Live cell imaging and fluorescence protease protection assays. For live cell imaging experiments with STAT3-GFP, PAEC or Hep3B cells were cultured in six-well plates and transfected 24 h after plating either with STAT3-GFP or N1-GFP alone or in combination with Dyn II K44A or cav-1-WT using procedures described earlier (56). In cotransfection experiments, the ratio of the GFP-tagged plasmid to the non-GFP-tagged plasmid was 1:4 by mass (56). In each experiment, pcDNA3.1 was used as the "empty" control vector. Transfections were performed using PolyFect (Qiagen, Valencia, CA) as per manufacturer's protocol. Cultures were viewed 24–36 h after transfection using confocal laser microscopy. For dual-channel live cell imaging to visualize lysosomes, LysoTracker (Molecular Probes) was added to the medium at a final concentration of 100 nM as described by us earlier (28). Fluorescence protease protection (FPP) assays were performed as described by us recently (22, 56). Briefly, cultures were transferred to warm HBSS to a final volume of 1 ml. After identification of a region of interest, at discrete time intervals, 1 ml each of a 2x strength solution of digitonin in warm HBSS followed by a 3x strength solution of trypsin in warm HBSS and finally a 4x strength solution of Triton X-100 in warm HBSS were added. The final effective concentrations of digitonin, trypsin, and Triton X-100 were 50 µg/ml, 100 µg/ml, and 0.5% vol/vol, respectively (56). Images were captured using the MRC 1024 ES confocal microscope. There was no spillover between the red and green channels.

IL-6/STAT3-responsive reporter/luciferase assays. Luciferase reporter assays were carried out essentially as described by us earlier (26, 46). PAEC or Hep3B cells in six-well plates were transfected with the p950M4-luciferase reporter together with the constitutive β-galactosidase expression plasmid pCH110 and either murine cav-1-WT, myc-tagged canine cav-1-WT, cav-1-S80A-myc, cav-1-S80E-myc, cav-1-F92A/V94A, or GRP58-WT using PolyFect. All assays were carried out in triplicate for each variable. Cultures were incubated at 37°C for another 24 h and then treated with IL-6 (10 ng/ml) for 6 h followed by harvesting the cells in 300-µl passive lysis buffer (Promega, Madison, WI). Fifty-microliter aliquots of the clarified extracts were used to assay luciferase and β-galactosidase activity using respective assay kits/reagents from Promega and Roche (Indianapolis, IN), respectively, and the manufacturer's protocols. The luciferase activity was then normalized with reference to the β-galactosidase activity in the extract (to control for transfection efficiency) and expressed in terms of that for the IL-6-free, cav-1 and GRP58 cotransfection-free culture.

Immunofluorescence studies of MCT-treated rat lungs. Immunofluorescence analyses of rat lung sections were carried out essentially as described earlier (24, 41). All protocols were approved by the Institutional Animal Care and Use Committee. Four- to six-week-old juvenile male Sprague-Dawley rats were injected with MCT (60 mg/kg) subcutaneously. Control animals received equivalent amount of normal saline (vehicle). Lungs were harvested 4 wk later at a time when right ventricular hypertrophy was evident. Lungs were fixed in formaldehyde and embedded in paraffin, and 4-µm sections were processed for immunofluorescence. Antigen recovery was performed by boiling for 8–10 min in PBS.

Immunofluorescence studies of human PAH. Sections of formalin-fixed, paraffin-embedded archived lung tissue from patients with idiopathic PAH and from controls without PAH (n = 2 each) were obtained from the Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD. Antigen recovery and immunofluorescence analyses were carried out as for the MCT rat lung sections above.

Image analyses. All experimental and control images of the immunofluorescence data were collected at identical imaging settings. Image analyses were performed using NIH ImageJ (available as a free download from http://rsb.info.nih.gov/ij/). Per cell pixel intensities were measured by visually defining cell outlines and measuring integrated and mean pixel intensities for that area (28). For each image, the "background" was adjusted to 0 pixels to reduce variance in intensities between experimental and control sets. Colocalization analyses were performed using the NIH ImageJ Just Another Co-localization Plugin (JACoP) (6). JACoP directly provides the Pearson R correlation, Manders' M1 and M2 coefficients, and Costes' randomization coefficients for a pair of images in the red and green channels. Briefly, Pearson correlation coefficient (R) measures the pattern similarities between two images. Values of Pearson R range from +1, indicating complete colocalization, to –1, indicating negative colocalization. A Pearson R coefficient value of 0 indicates no correlation between the two images. Manders' M1 is defined as the ratio of the summed intensities of pixels from the green channel for which the intensity in the red channel is above 0 to the total intensity in the green channel; Manders' M2 is the reverse. These coefficients are good indicators of the level of colocalization between red and green channels (and vice versa) and are independent of the intensity of fluorescence. Costes' randomization is a useful tool to investigate whether the observed colocalization is purely due to chance or is statistically significant. This coefficient is based on a Monte Carlo protocol that creates random images by shuffling pixels in the green channel and then calculating a new Pearson R for each randomized image in the green channel (the image in the red channel is kept unchanged). The Pearson R of the original image in the green and red channel is then compared with each new Pearson R obtained from the randomized green images with the red channel. The final result is obtained as the likelihood that the Pearson R calculated by randomizing the green images is greater than the original Pearson R observed (7).

Antibody reagents. Rabbit polyclonal antibodies (pAbs) to STAT3, PY-STAT3, cav-1 and corresponding competing peptides were from Santa Cruz Biotechnology (Santa Cruz, CA). Murine MAbs to CHC, early endosome antigen 1 (EEA1), Golgi matrix 130 (GM130), lysosome-associated membrane protein 1 (LAMP1), Rab5, and Rab11 were from BD Biosciences (Eugene, OR).

Statistical analyses. Statistical analyses were performed using the two-tailed Student's t-test and Microsoft Excel software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Increased targeting of STAT3 and PY-STAT3 to cytoplasmic vesicles in MCTP-induced megalocytosis in PAECs. We (26) have previously shown that in MCTP-induced megalocytosis of PAEC in culture, there was a reciprocal relationship between loss of cellular cav-1 and hyperactivation of IL-6/STAT3 signaling as assayed in terms of an increase in PY-STAT3 in Western blotting assays. However, this increase in total and cytoplasmic PY-STAT3 did not convert into an enhancement of IL-6-activated STAT3 transcriptional luciferase-reporter activity compared with IL-6-treated PAEC not exposed to MCTP (26). Thus we asked whether PY-STAT3 was aberrantly sequestered in the cytoplasm of MCTP-treated PAEC.

Immunofluorescence assays for STAT3 localization in untreated PAECs in culture showed that STAT3 was constitutively localized in punctuate plasma membrane caveolae and in the nucleus (Fig. 1A, top, left). Exposure to IL-6 for 30 min led to a relative reduction in STAT3 in plasma membrane caveolae with an increase in the nuclear pool (Fig. 1A, top, right). Treatment of PAECs with MCTP for 4 days, which induced an increase in cell size (megalocytosis), led to a dramatic redistribution of STAT3 from plasma membrane caveolar puncta to large cytoplasmic vesicles (Fig. 1A, bottom, left). This cytoplasmic vesicular STAT3 persisted in megalocytotic PAECs exposed to IL-6 (Fig. 1A, bottom, right). Thus, in MCTP-induced megalocytosis in PAECs, there was a loss of caveolar STAT3 and sequestration of this transcription factor in cytoplasmic structures.

View larger version (98K): [in this window] [in a new window]   Fig. 1. Monocrotaline (MCT) pyrrole (MCTP) reduces targeting of STAT3 to plasma membrane caveolae but enhances that to the endolysosomal compartment. A: pulmonary arterial endothelial cells (PAECs) were plated in 6-well plates and 1 day later exposed to MCTP or vehicle dimethyl formamide (DMF). Four days later, the cultures were fixed using the cold paraformaldehyde-Triton X-100 protocol and processed for immunofluorescence using an anti-STAT3 rabbit polyclonal antibody (pAb). Nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI). Single arrowheads indicate caveolar STAT3, and double arrowheads indicate cytoplasmic, vesicular STAT3. Scale bar = 25 µm. B: PAECs in 6-well plates treated with MCTP as above were exposed to the thiol cross-linker phenylarsine oxide (PAO; 5 µM for 30 min) before fixation. Double-label immunofluorescence analyses were carried out using the rabbit pAb against STAT3 as in A and various murine MAbs against the endolysosomal markers Rab5, Rab11, lysosome-associated membrane protein 1 (LAMP1), and clathrin heavy chain (CHC). Arrowheads indicate colocalization of STAT3 with the endolysosomal markers. Costes' randomization analyses performed using NIH ImageJ showed that for each of the double-label immunofluorescence analyses, the probability that the observed colocalization was merely due to chance was <0.001 [Pearson R of the original image in the green and red channel (Rorig) > new Pearson R obtained from the randomized green images with the red channel (Rrand) in 100% of recreated images]. Scale bar = 25 µm. C: peptide competition assay confirming specificity of the anti-STAT3 pAb used. PAECs grown in 6-well plates were fixed and processed for immunofluorescence using either the STAT3 pAb alone or STAT3 pAb incubated with a 5-fold excess amount of relevant blocking peptide or an irrelevant blocking peptide [a caveolin-1 (cav-1) blocking peptide]. Incubation of the STAT3 pAb with the blocking peptides was done at room temperature for 1 h as described previously (47). Scale bar = 25 µm.

Figure 2, A and B, extends the above observations to activated PY-STAT3. Figure 2A, top, left, shows that, in control PAECs, there was constitutive presence of activated PY-STAT3 in plasma membrane caveolar puncta, in discrete cytoplasmic vesicles, as well as in the nucleus. Exposure to IL-6 led to an increase in nuclear PY-STAT3 (Fig. 2A, top, right). However, a pool of PY-STAT3 persisted in discrete puncta both at plasma membrane caveolae as well as in the cytoplasm. Exposure of PAECs to MCTP led 4 days later to a marked accumulation of PY-STAT3 in cytoplasmic vesicles and persistence in the nucleus. Additional exposure to IL-6 did not change this distribution in MCTP-treated PAEC (peptide competition controls that verify the specificity of the anti-PY-STAT3 pAb used are in Fig. 9, E and F; data confirming the detection of the correct PY-STAT3 protein by this antibody in Western blots has been shown in Refs. 24, 26, 46). This cytoplasmic sequestration of PY-STAT3 was not in the enlarged Golgi elements previously reported by us in such cells (26, 27, 28, 41) in that Fig. 2B shows that the bulk of this cytoplasmic PY-STAT3 did not colocalize with the Golgi tether GM130. The subcellular redistribution of STAT3 and PY-STAT3 after MCTP was accompanied by changes in the localization of the STAT3-associated protein and chaperone GRP58 (14) from the cytoplasm to the nucleus (Fig. 2C).

View larger version (138K): [in this window] [in a new window]   Fig. 2. Aberrant subcellular localization of tyrosine-phosphorylated STAT3 (PY-STAT3) and glucose regulated protein 58 (GRP58) in MCTP-induced megalocytosis in PAECs. PAECs in 6-well plates were treated with MCTP or DMF for 4 days and then fixed and processed for immunofluorescence using rabbit pAbs to PY-STAT3 (A and B) and GRP58 (C) and an MAb to Golgi matrix 130 (GM130) as indicated. Nuclei were visualized using DAPI in A. Single arrowheads in A and B indicate caveolar PY-STAT3, and double arrowheads indicate cytoplasmic, vesicular PY-STAT3. Scale bars = 25 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 9. Cytoplasmic provenance of PY-STAT3 in PAECs in PAECs in MCT-treated rats. Immunofluorescence analyses of formalin-fixed, paraffin-blocked sections of lung from rats with MCT-induced pulmonary arterial hypertension (PAH) (4 wk after MCT) and corresponding vehicle-treated control rats were performed as described in MATERIALS AND METHODS. A: triple-label immunofluorescence analyses of MCT and vehicle-treated rats was performed with anti-PY-STAT3 pAb, anti-smooth muscle actin (SMA) MAb, and DAPI (to visualize nuclei). Scale bar = 50 µm. B: double-label immunofluorescence analysis (different experiment from that in A) using anti-PY-STAT3 pAb and DAPI showing markedly increased in PY-STAT3 in luminal PAECs in rats administered MCT 4 wk earlier. Scale bar = 25 µm. C: summary of immunomorphometric imaging analyses of PY-STAT3 levels in luminal PAECs in MCT-treated rats. Per cell integrated pixel intensities were obtained using NIH ImageJ as described in MATERIALS AND METHODS. *P < 0.05 compared with the per cell pixel intensity of PAECs in vehicle-treated rats; n = 500 cells. D: high magnification panels of medium-sized pulmonary arteries in MCT-treated rats showing that PY-STAT3 was predominantly sequestered in the cytoplasm of luminal PAECs. Scale bar = 5 µm. E and F: peptide competition assay confirming the specificity of the PY-STAT3 pAb used throughout this study. PY-STAT3 pAb was preincubated with either no blocking peptide or a relevant PY-STAT3 blocking peptide (5-fold excess amount of blocking peptide to antibody) or an irrelevant blocking peptide (cav-1 peptide) followed by immunofluorescence analyses. Per cell integrated pixel intensities were obtained using NIH ImageJ and expressed in terms of the section stained with PY-STAT3 pAb without any blocking peptide. *P < 0.05 compared with the section stained with PY-STAT3 pAb without any peptide; n = 50–60 cells per variable. Scale bar for E = 25 µm. G: high magnification inset from E (arrow) showing the cytoplasmic vesicular subcellular localization of PY-STAT3 in PAECs in vivo in the rat/MCT-model of PAH. Scale bar = 5 µm.

View larger version (70K): [in this window] [in a new window]   Fig. 3. Live cell imaging of the subcellular localization of STAT3-GFP in PAECs. A: PAECs were plated in 6-well plates and transfected 24 h later with STAT3-GFP or N1-GFP expression constructs (1 µg of DNA per well) as described in MATERIALS AND METHODS. Twenty-four hours later, cultures were treated with IL-6 followed by addition of LysoTracker, and live cell imaging was performed 30 min later using confocal laser scanning microscopy. Arrowheads indicate vesicular accumulations of STAT3-GFP in the cytoplasm. Scale bar = 25 µm. B: PAECs in 6-well plates were either exposed to MCTP or solvent alone. Thirty-six hours later, the cultures were transfected with either STAT3-GFP and pcDNA3.1 or STAT3-GFP and dynamin II K44A (Dyn II K44A) expression constructs (1 µg of total DNA per well; ratio of 1:4 between the STAT3-GFP and the non-GFP plasmids). A further 24 h after transfection, half of the cultures were treated with IL-6 for 30 min, exposed to LysoTracker, and imaged using confocal laser scanning microscopy. Scale bar = 25 µm. C: the fluorescence protease protection (FPP) assay. PAECs in 6-well plates were transfected with STAT3-GFP, and 36 h later the medium was changed to warm HBSS (1 ml per well) containing LysoTracker and the cytoplasmic STAT3-GFP vesicles imaged by confocal microscopy. The FPP assay was carried out by imaging the culture every 15 s while sequentially exposing it to HBSS solutions containing digitonin, trypsin, and Triton X-100 as described in MATERIALS AND METHODS (effective final concentrations of each: 50 µg/ml, 100 µg/ml, and 0.5% vol/vol, respectively). Scale bar = 25 µm.

Cav-1 overexpression increases targeting of STAT3 to lysosomes and inhibits STAT3 transcriptional activity. WT cav-1 enhances the lysosomal targeting of the TGF-β/TGF-βRs endocytic complexes resulting in inhibition of transcriptional signaling (12). Thus we investigated whether cav-1 might have a similar effect on STAT3 endocytic trafficking and transcriptional activity. We first investigated an RNA interference (RNAi) approach to downregulate cav-1 expression. In preliminary experiments, although cav-1 RNAi enhanced IL-6/STAT3 transcriptional signaling, a significant reduction in cav-1 levels per se was difficult to verify by Western blotting, and a similar enhancement of IL-6/transcriptional signaling was observed using an irrelevant RNAi control oligonucleotide. Thus we turned to an alternative approach, overexpression of cav-1 and its mutants in the human Hep3B cell line that expresses only low levels of endogenous cav-1. Overexpression of WT cav-1 in these cells by transient transfection methods led to the targeting of STAT3-GFP to LysoTracker-positive lysosomes (Fig. 4A). Quantitative colocalization analyses performed using NIH ImageJ software (using multiple 983- x 983-µm images) showed that cotransfection of the STAT3-GFP construct together with one expressing WT cav-1 led to a statistically significant increase in the colocalization of STAT3-GFP with LysoTracker in both the Pearson R and Manders' M2 statistics (Fig. 4B). That the colocalization of LysoTracker (red) with STAT3-GFP (green) did not significantly change in the Manders' M1 statistic acts as an internal control in this experiment in that because 100% of cells pick up LysoTracker in the red channel (generating a large denominator approaching 100%) but only a minority of these are cotransfected by the STAT3-GFP construct (thus generating a small numerator to begin with), we do not expect significant changes in colocalization of the green STAT3-GFP pixels (small numerator and thus only small additional changes due to cav-1 overexpression) to be apparent against the background of a large denominator of red LysoTracker pixels. As a further internal control for this experiment, overexpression of cav-1 did not change the level of colocalization of N1-GFP with LysoTracker even when using the Manders' M2 statistic.

View larger version (50K): [in this window] [in a new window]   Fig. 4. Epigenetic regulation of STAT3 signaling: enhanced targeting of STAT3 to lysosomes by cav-1 and inhibition of transcriptional activity by cav-1 and GRP58. A: human Hep3B cells with low endogenous cav-1 expression were plated in 6-well plates and 1 day later transfected with STAT3-GFP and murine cav-1 wild-type (WT) expression vector (total DNA per well = 1 µg; 1:4 ratio of STAT3-GFP to cav-1). Thirty-six hours later, cultures were exposed to IL-6 (30 min) and LysoTracker and imaged. Scale bar = 25 µm. B: Hep3B cells in 6-well plates were transfected either with STAT3-GFP and pcDNA3.1 or STAT3-GFP and murine cav-1-WT expression vectors (1:4 ratio of STAT3-GFP to non-GFP plasmids; total DNA per well = 1 µg). Thirty-six hours later, the cultures were exposed to LysoTracker and extensively imaged (at least >5,983 µm x 983 µm fields per variable). Formal colocalization analyses (of the green STAT3-GFP with the rad LysoTracker pixels) were then carried out on all the imaged frames using the NIH ImageJ Just Another Co-localization Plugin (JACoP) and expressed in terms of the Pearson R and Manders' M1 and M2 coefficients as described in MATERIALS AND METHODS (means ± SE). *P < 0.05 in comparing cav-1-transfected culture with that transfected with the pcDNA3.1 control; n.s., not significant (P 0.05). C: Hep3B cells in 6-well plates were transfected (in triplicate per variable) with p950M4-luciferase, a constitutive β-galactosidase expression construct, and either of pcDNA3.1, murine cav-1-WT, canine cav-1-WT-myc, or cav-1 scaffolding domain mutants cav-1 S80A-myc or cav-1 S80E-myc expression constructs. Basal and IL-6-induced luciferase and β-galactosidase activities were assayed as described in MATERIALS AND METHODS. Luciferase activity, normalized to respective β-galactosidase activity, is expressed in terms of that in the IL-6-free, pcDNA3.1-transfected cultures (means ± SE). *P 0.05 in comparisons of basal (IL-6-free) luciferase activity in cav-1 cotransfected cultures to that of the control pcDNA3.1 transfected-free culture; **P 0.05 in comparisons of IL-6-induced luciferase activity in cav-1-cotransfected cultures to that in IL-6-treated pcDNA3.1 controls. A.U., arbitrary units. D: PAECs in 6-well plates were transfected with p950M4-luciferase, a constitutive β-galactosidase expression construct and either of pcDNA3.1, murine cav-1-WT, or the cav-1 scaffolding domain mutant cav-1 F92A/V94A. Basal and IL-6-induced luciferase and β-galactosidase activity was assayed as described in C. *P 0.05 in comparisons of basal (IL-6-free) and **P 0.05 in comparisons of IL-6-induced luciferase activity. E: Hep3B cells in 6-well plates were transfected with p950M4-luciferase, a constitutive β-galactosidase expression construct and either pcDNA3.1 or an expression construct for human GRP58-WT. Basal and IL-6-induced luciferase and β-galactosidase activity was assayed as described in MATERIALS AND METHODS and expressed as in C. **P 0.05 in comparisons of IL-6-induced luciferase activity.

Association of STAT3 and PY-STAT3 with caveolar and endolysosomal subcellular compartments in HPASMC. The pulmonary arterial vascular smooth muscle cell is an important contributor to the pathobiology of PAH in humans (reviewed in Refs. 42, 43). Data from double- and triple-label confocal immunofluorescence analyses of HPASMCs in culture summarized in Figs. 5–8 provide evidence for the association of STAT3 with the endolysosomal pathway in this cell type. Cytoplasmic vesicles bearing STAT3 in HPASMCs partially colocalized with both CHC and cav-1 as well as with -adaptin (Fig. 5), with the early endosome marker EEA1 (Fig. 6), and with the lysosome marker LAMP1 (Figs. 6 and 7A). The triple-label analyses in Fig. 7B show that, in HPASMCs, cav-1 colocalized with STAT3 in a subset of LAMP1-positive lysosomes (Fig. 7B, single arrowheads) as well as in vesicles free of LAMP1 (Fig. 7B, bottom, double arrowhead). Thus, overall, the colocalization of STAT3 with endolysosomal/caveolar markers was partial in each instance and pointed to further complexity among STAT3-positive endosomes and their vesicular trafficking in vascular smooth muscle cells.

View larger version (81K): [in this window] [in a new window]   Fig. 5. Targeting of STAT3 to the caveolar/endolysosomal compartment in human pulmonary arterial endothelial smooth muscle cells (HPASMCs). HPASMCs in 6-well plates were grown in the presence of smooth muscle differentiation medium as described in MATERIALS AND METHODS for 8 days. Cultures were fixed, and immunofluorescence analyses were carried out using anti-STAT3 pAb and MAbs against CHC, -adaptin, or with a cav-1 pAb covalently linked to FITC (cav-1-FITC). Arrowheads indicate colocalization of STAT3 to vesicles that also carried caveolar/endolysosomal markers. Costes' randomization analyses performed using NIH ImageJ showed that for each of the double-label immunofluorescence images, the probability that the colocalization was merely due to chance was <0.001 (Rorig > Rrand in 100% of recreated images). Scale bars = 25 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 8. Targeting of PY-STAT3 to cytoplasmic destinations in vascular smooth muscle cells. HPASMCs cultured in 6-well plates were treated with IL-6 for 30 min or left untreated and then fixed and stained with anti-STAT3 pAb (A), anti-PY-STAT3 pAb (B), or a combination of anti-PY-STAT3 pAb and anti-LAMP1 MAb. In A and B, the boxed areas are shown in high magnification (High mag). Scale bars in A and B: low magnification (top) = 25 µm; high magnification (bottom) = 5 µm. Scale bar in C = 10 µm. Arrowheads in B show targeting of PY-STAT3 to cytoplasmic vesicles, and those in C to focal adhesions.

View larger version (97K): [in this window] [in a new window]   Fig. 6. Colocalization of STAT3 with the early endosome and lysosome compartments in HPASMCs. HPASMCs grown as in Fig. 5 were fixed, and immunofluorescence analyses carried out using anti-STAT3 pAb and MAbs against early endosome antigen 1 (EEA1) and LAMP1. Arrowheads show colocalization of STAT3 vesicles with EEA1 and LAMP1. Costes' randomization analyses performed using NIH ImageJ showed that for each of the double-label immunofluorescence analyses, the probability that the colocalization was merely due to chance was <0.001 (Rorig > Rrand in 100% of recreated images). Scale bar = 25 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 7. Colocalization patterns of STAT3 and cav-1 with lysosomes in HPASMCs. HPASMCs grown in 6-well plates as in Fig. 5 were fixed and treated with MCTP for 4 days, and immunofluorescence analyses were carried out using anti-STAT3 pAb and LAMP1 MAb (A) and additionally with cav-1-FITC (B). Single arrowheads in A show colocalization between STAT3 and LAMP1. Single arrowheads in B show colocalization among all 3, STAT3, cav-1, and LAMP1, and double arrowheads in B show colocalization between STAT3 and cav-1 only in LAMP1-free vesicles. Costes' randomization analyses showed that, for each of the double-label immunofluorescence analyses and individually for a set of 2 labels in the triple-label analyses, the probability that the colocalization was merely due to chance was <0.001 (Rorig > Rrand in 100% of recreated images). Scale bars = 25 µm.

Almost exclusive cytoplasmic provenance of PY-STAT3 in PAECs in MCT-treated rats. The data in Figs. 1–8, taken together, revealed an unexpected level of complexity in the centripetal trafficking of STAT3 and PY-STAT3 in endothelial and vascular smooth muscle cells along the endocytic pathway. Moreover, the customary expectation that all activated PY-STAT3 automatically trafficked to the cell nucleus was clearly an oversimplification. With these insights, we investigated the subcellular localization of PY-STAT3 in endothelial cells in vivo in the rat/MCT model of PAH. The data in Fig. 9, A–C, show that there was a 5-fold increase in total PY-STAT3 on a per cell basis in PAECs in rats that had developed MCT-induced PAH (by 4 wk) compared with that in controls. This increase in PY-STAT3 was restricted to arterial luminal PAECs and did not extend to vascular smooth muscle (Fig. 9, B and C; Table 1). Unexpectedly, high magnification imaging of the luminal PAECs that had increased PY-STAT3 content showed that >75% of cells (n = 363) displayed an exclusively cytoplasmic sequestration of PY-STAT3 and that this PY-STAT3 was present in discrete, cytoplasmic vesicles (Fig. 9, D and G). The specificity of the PY-STAT3 immunostaining was confirmed in peptide competition assays using relevant and irrelevant peptides (Fig. 9, E and F). Thus, in the MCT-treated rat, the markedly increased content of PY-STAT3 in the luminal PAEC was predominantly sequestered in cytoplasmic vesicles.

View larger version (78K): [in this window] [in a new window]   Fig. 10. Cytoplasmic provenance of PY-STAT3 in PASMCs and PAECs in pulmonary arterial lesions in PAH in humans. Immunofluorescence analyses were performed on formalin-fixed paraffin-block sections of archived lung tissue from patients with idiopathic PAH and controls without PAH as described in MATERIALS AND METHODS. A: double-label immunofluorescence analyses were performed using anti-PY-STAT3 pAb and DAPI. Scale bar = 25 µm. Art, pulmonary artery; L, arterial lumen. B: summary of immunomorphometric imaging analyses showing greater than 3-fold increase in total PY-STAT3 levels on a per cell basis in the SMA-positive PASMCs in pulmonary arterial lesions as in A, B, and C. Integrated pixel intensities on a per cell basis were obtained using NIH ImageJ. *P < 0.05 compared with the per cell pixel intensity of PASMCs in normal human lung sections; n = 80 in controls and 516 cells in PAH. C: high magnification images showing the cytoplasmic nature of PY-STAT3 in nonluminal SMA-positive PASMCs and in luminal PAECs in pulmonary arterial lesions with medial and intimal proliferation. Scale bar = 5 µm. D: examples of nuclear PY-STAT3 in luminal PAECs and nonluminal SMA-positive PASMCs cells in pulmonary arterial lesions with medial hypertrophy and intimal proliferation. Scale bar = 25 µm. E: plexiform lesion in idiopathic PAH in humans showing cells with both cytoplasmic (single arrowheads) and nuclear (double arrowheads) PY-STAT3. Whereas some of the cells were positive for SMA (green) and factor VIII (data not shown), all cells were negative for the macrophage marker ED-1 (data not shown). Scale bar = 5 µm. N, nuclear PY-STAT3; C, cytoplasmic PY-STAT3. F: high magnification inset from E showing the cytoplasmic vesicular subcellular localization of PY-STAT3. Scale bar = 5 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 11. Relationship between dysfunctional intracellular trafficking and elevated cytoplasmic PY-STAT3 in rat/MCT and human PAH. Triple-label immunofluorescence analyses were performed using lung sections of MCT-treated rats (A) or a patient with idiopathic PAH (B; same as in Fig. 10). Sections were probed with the pAb against PY-STAT3 and MAbs against -soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP), Vti1a (a v-SNARE), and GS28 (a Golgi v-SNARE). Arrowheads point to cells with elevated PY-STAT3 and the indicated SNAP receptor (SNARE) or SNAP. Scale bars = 5 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

View larger version (35K): [in this window] [in a new window]   Fig. 12. Epigenetic regulation of centripetal transcytoplasmic transit of signaling by cytokines and growth-factors relevant in pulmonary hypertension. SARA, Smad anchor for receptor activation; ANG I, angiopoietin-1; TGF-βR, TGF-β receptor; MVB, multivesicular bodies; HRS, hepatocyte-stimulating factor receptor substrate; NF2, neurofibromatosis gene product 2.

Data derived from PAECs and PASMCs in culture revealed that STAT3 and PY-STAT3 were constitutively associated with heterogeneous components of the endolysosomal system including EEA1- and Rab5-positive early endosomes, cav-1-positive caveosomes, Rab11-positive recycling endosomes, and LAMP1-positive lysosomes. The targeting of STAT3 to lysosomes was novel in that STAT3 has not been known to traffic to this organelle. Due to the potential similarities between the TGF-βR/Smad signaling paradigm where cav-1 leads to lysosomal targeting and attenuates transcriptional signaling and IL-6/STAT3 signaling, we investigated the role of cav-1 on the lysosomal targeting and transcriptional activity of STAT3. That cav-1 increased the lysosomal targeting of STAT3 while simultaneously reducing IL-6/STAT3-responsive luciferase reporter activity provides a mechanistic basis for understanding the reciprocal relationship between activation of STAT3-signaling and cav-1 downregulation in PAH.

Achcar et al. (1) have demonstrated that both factor VIII- and SMA-positive cells in plexiform lesions in idiopathic PAH in humans have marked decreases in the expression levels of cav-1 and cav-2. However, PAECs and PASMCs in remodeled pulmonary arteries with medial hypertrophy in the same set of patients did not show a change in cav-1 (or cav-2) expression compared with controls. Interestingly, the loss of cav-1 observed in plexiform lesions of idiopathic PAH was specific for these cells in that these authors did not find a decrease in cav-1 levels by Western blot assays of whole lung extracts. A similar decrease in cav-1 and cav-2 was also observed in a model of severe PAH in the rat caused by administration of the VEGF receptor antagonist SU-5416 followed by exposure of the animals to 3 wk of chronic hypoxia (1, 37). Although there is evidence from several groups showing the pivotal role played by the downregulation of cav-1 in pulmonary arterial lesions of idiopathic PAH in humans and in diverse experimental models (MCT, hypoxia, and SU-5416 + hypoxia), a recent report by Patel et al. (34) appears to have reached a different conclusion. The latter investigators reported that although there was a decrease in total cav-1 levels in whole lung extracts assayed by Western blotting in idiopathic PAH patients compared with controls, there was a paradoxical increase in cav-1 expression and caveolae numbers in PASMCs in vivo in this disease and in cultured PASMCs derived from such patients. However, the immunomorphometric data showing increased cav-1 expression in PASMCs in idiopathic PAH are unclear (34). Furthermore, these authors (34) misquote Achcar et al. (1) when they state that Achcar et al. observed a decrease in cav-1 expression in whole lung extract Western blot assays in idiopathic PAH (whereas Achcar et al. explicitly mention the specific loss of cav-1 in plexiform lesions only). The present data highlighting the epigenetic regulation of STAT3 transcriptional activity underline the importance of a careful cell type-specific evaluation of the relevant parameters and components in discussions of the pathobiology of PAH.

A pertinent question to ask at this point is if PAH is a disease associated with downregulation of cav-1, what is the relevance of the cav-1-induced targeting of STAT3 to lysosomes in the pathobiology of PAH? The key to answering this question is to focus not on cav-1 content at the tissue or whole cell level but in specific subcompartments within the cytoplasm. We suggest that the role of cav-1 at the level of the plasma membrane raft with loss of cav-1 leading to hyperactivation of STAT3 signaling is different from the role of cav-1 in targeting signaling molecules to the lysosomal compartment. To clarify further, first, we have shown that STAT3 interacts with cav-1 in diverse subcellular compartments including plasma membrane lipid rafts, other intracellular membrane-associated compartments like endosomes, as well as in the membrane-free cytoplasmic compartment (see Fig. 5 and Ref. 45 for details). Second, whereas cav-1 is a negative regulator of STAT3 tyrosine phosphorylation at the plasma membrane lipid raft level (see Ref. 19 and citations therein for details of how cav-1 negatively regulates signaling at the plasma membrane), the effect of the interaction of STAT3 with cav-1 at other subcellular compartments (like endosomes) is likely different and not known. Third, from work in this laboratory in the MCT model of PAH, the downregulation of cav-1 seen at the whole cell level is largely representative of a loss of cav-1 from the plasma membrane rafts/caveolae (24). Thus this loss of cav-1 relates to hyperactivation of PY-STAT3 signaling at the level of the raft/caveolae. Mechanistically, this loss of cav-1 from caveolae/rafts results from the trapping of cav-1 in the Golgi organelle (reviewed in Refs. 42, 43) from where this trapped cav-1 can aberrantly traffic to other cytoplasmic compartments. Finally, that scaffolding domain mutants of cav-1 were equally effective in inhibiting transcriptional IL-6/STAT3 signaling compared with WT cav-1 (Fig. 4) suggests that overexpression of cav-1 can inhibit PY-STAT3 function independent of events at the level of the plasma membrane caveolae/rafts. Thus the role of cav-1 in PAH needs to be understood at the level of discrete intracytoplasmic subcompartments and not merely in cell surface caveolae.

Recently, Masri and colleagues (23) reported increased PY-STAT3 in cellular elements in vascular lesions in idiopathic PAH in humans. However, the immunoperoxidase assays used to detect PY-STAT3 histologically did not have sufficient resolution to reveal the subcellular localization of PY-STAT3. These investigators also reported on the activation of PY-STAT3 in cultures of endothelial cells derived from patients with PAH. However, these investigators carried out Western blot assays for PY-STAT3 and DNA-binding assays using whole cell extracts, and thus the data obtained are not informative with respect to the subcellular localization of the PY-STAT3. Indeed, we have extensively shown that PY-STAT3 in the cytoplasm as assayed by Western blotting is competent in DNA-binding gel-shift assays (29). The present data showing that PY-STAT3 was sequestered in the cytoplasm in the majority of PAECs and PASMCs in pulmonary arterial lesions in human and experimental PAH underlines the significance of evaluating the subcellular localization of activated transcription factors.

The observation that, at the single cell level in vivo, the increase in cytoplasmic PY-STAT3 occurred in the same cells that also showed increased accumulation -SNAP, Vti1a, and GS28 suggests that this cytoplasmic sequestration of PY-STAT3 may reflect a consequence of the dysfunction of vesicle tethers, SNAREs, and SNAPs in this disease. However, we do not exclude the possibility that cytoplasmically sequestered PY-STAT3 may mediate novel biological functions in the cytoplasm as such relevant to the pathobiology of PAH. One possibility is that the cytoplasmic sequestration of PY-STAT3 is a mechanism for the cell to temporally regulate the amount of STAT3 entering into the nucleus. Recently, we (56) reported that, in Hep3B cells, IL-6 induced the targeting of STAT3 and PY-STAT3 to long-lived, sequestering endosomes colocalizing with the signaling adaptor MyD88. MyD88 has primarily been thought of as an adaptor protein required for Toll-like receptor and IL-1 signaling (see our Ref. 56 and citations therein). Additional functions of MyD88 include enhanced mRNA stabilization via activation of the p38 MAPK pathway (see Ref. 56 and citations therein). We entertain the possibility that the cytoplasmic PY-STAT3 signals to MyD88-positive endosomes to regulate mRNA stability. IL-6 and other acute phase cytokines, in addition to increasing the transcription of certain target plasma protein genes (like ₂-macroglobulin, complement C3, etc.), also stabilize their mRNAs in the cytoplasm (see our Ref. 56 for details). Thus the same transcription factor (activated PY-STAT3) might enhance transcription of a gene in the nucleus and also mediate enhanced stabilization of the product mRNA in the cytoplasm. In the context of PAH (9), it has recently been shown that NO increases the decay of matrix metalloproteinase-9 (MMP-9) mRNA by modulating the p38 kinase/HuR pathway (2). Thus MMP-9 mRNA represents a potential target for investigations of the biological functions of cytoplasmic PY-STAT3 in endothelial cells in the specific context of PAH.

To summarize, we show that the transcription factor STAT3 and its tyrosine-activated form, PY-STAT3, traffic through the endothelial and vascular smooth muscle cell cytoplasm in association with components of the endolysosomal pathway and that MCTP affects this centripetal trafficking in PAECs. More dramatically, we demonstrate the marked sequestration of PY-STAT3 in cytoplasmic vesicles in lung vascular cells in vivo in the rat MCT model and in human PAH. These observations are consistent with disruptions of centripetal intracellular trafficking in this disease.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-077301 (P. B. Sehgal) and IPO50 HL-084946 (R. M. Tuder) and an American Heart Association Predoctoral Fellowship (S. Mukhopadhyay).

	ACKNOWLEDGMENTS

 
Present address of M. Shah: Dept. of Cellular and Molecular Medicine, Univ. of California at San Diego, La Jolla, CA 92093.

Present address of R. M. Tuder: Division of Pulmonary and Critical Care Medicine, Dept. of Medicine, Univ. of Colorado at Denver, Health Sciences Center, Denver, CO 80262.

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. B. Sehgal, Rm. 201 Basic Sciences Bldg., Dept. of Cell Biology and Anatomy, New York Medical College, Valhalla, NY 10595 (e-mail: pravin_sehgal{at}nymc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Achcar RO, Demura Y, Rai PR, Taraseviciene-Stewart L, Kasper M, Voelkel NF, Cool CD. Loss of caveolin and heme oxygenase expression in severe pulmonary hypertension. Chest 129: 696–705, 2006.[CrossRef][Web of Science][Medline]
2. Akool el-S, Kleinert H, Hamada FM, Abdelwahab MH, Fostermann U, Pfeilschifter J, Eberhardt W. Nitric oxide increases the decay of matrix mettaloproteinase 9 mRNA by inhibiting the expression of mRNA-stabilizing factor HuR. Mol Cell Biol 23: 4901–4916, 2003.[Abstract/Free Full Text]
3. Berishaj M, Gao SP, Ahmed S, Leslie K, Al-Ahmadie H, Gerald WL, Bornmann W, Bromberg JF. Stat3 is tyrosine-phosphorylated through the interleukin-6/glycoprotein 130/Janus kinase pathway in breast cancer. Breast Cancer Res 9: R32, 2007.[CrossRef][Medline]
4. Bhargava A, Kumar A, Yuan N, Gewitz MH, Mathew R. Monocrotaline induces interleukin-6 mRNA expression in rat lungs. Heart Dis 1: 126–132, 1999.[Medline]
5. Bild AH, Turkson J, Jove R. Cytoplasmic transport of Stat3 by receptor-mediated endocytosis. EMBO J 21: 3255–3263, 2002.[CrossRef][Web of Science][Medline]
6. Bogdanovic E, Nguyen VP, Dumont DJ. Activation of Tie2 by angiopoietin-1 and angiopoietin-2 results in their release and receptor internalization. J Cell Sci 119: 3551–3560, 2006.[Abstract/Free Full Text]
7. Bolte S, Cordelieres FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224: 213–232, 2006.[Web of Science][Medline]
8. Bourdi M, Demady D, Martin JL, Jabbour SK, Martin BM, George JW, Pohl LR. cDNA cloning and baculovirus expression of the human liver endoplasmic reticulum P58: characterization as a protein disulfide isomerase isoform, but not as a protease or a carnitine acryltransferase. Arch Biochem Biophys 323: 397–403, 1995.[CrossRef][Web of Science][Medline]
9. Cowan KN, Jones PL, Rabinovitch M. Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis. Circ Res 84: 1223–1233, 1999.[Abstract/Free Full Text]
10. Damke H, Baba T, Warnock DE, Schmid SL. Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 127: 915–934, 1994.[Abstract/Free Full Text]
11. Darnell JE, Kerr IM, Stark GR. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264: 1415–1421, 1994.[Abstract/Free Full Text]
12. Di Guglielmo GM, Le Roy C, Goodfellow AF, Wrana JL. Distinct endocytic pathways regulate TGF-β receptor signaling and turnover. Nat Cell Biol 5: 410–421, 2003.[CrossRef][Web of Science][Medline]
13. Golembeski SM, West J, Tada Y, Fagan KA. Interleukin-6 causes mild pulmonary hypertension and augments hypoxia-induced pulmonary hypertension in mice. Chest 128, Suppl 6: 572S–573S, 2005.
14. Guo G, Patel K, Kumar V, Shah M, Fried V, Etlinger JD, Sehgal PB. Association of the chaperone glucose-regulated protein 58 (GRP58/ ER-60/ERp57) with Stat3 in cytosol and plasma membrane complexes. J Interferon Cytokine Res 22: 555–563, 2002.[CrossRef][Web of Science][Medline]
15. Hagen M, Fagan K, Steudel W, Carr M, Kirk K, Rodman DM, West J. Interaction of interleukin-6 and the BMP pathway in pulmonary smooth muscle. Am J Physiol Lung Cell Mol Physiol 292: L1473–L1479, 2007.[Abstract/Free Full Text]
16. Hartung A, Bitton-Worms K, Rechtman MM, Wenzel V, Boergerman JH, Hassel S, Henis YI, Knaus P. Different routes of bone morphogenetic proten (BMP) receptor endocytosis influence BMP signaling. Mol Cell Biol 26: 7791–7805, 2006.[Abstract/Free Full Text]
17. Itoh F, Divecha N, Brocks L, Oomen L, Janssen H, Calafat J, Itoh S, Dijke Pt P. The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/Smad signalling. Genes Cells 7: 321–331, 2002.[Abstract]
18. Itoh T, Nagaya N, Ishibashi-Ueda H, Kyotani S, Oya H, Sakamaki F, Kimura H, Nakanishi N. Increased plasma monocyte chemoattractant protein-1 level in idiopathic pulmonary arterial hypertension. Respirology 11: 158–163, 2006.[CrossRef][Web of Science][Medline]
19. Jasmin JF, Mercier I, Dupuis J, Tanowitz HB, Lisanti MP. Short-term administration of a cell-permeable caveolin-1 peptide prevents the development of monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy. Circulation 114: 912–920, 2006.[Abstract/Free Full Text]
20. Lackmann M, Harpur AG, Oates AC, Mann RJ, Gabriel A, Meutermans W, Alewood PF, Kerr IM, Stark GR, Wilks AF. Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: prevalence of latent STAT1 in high-molecular weight complexes. Growth Factors 16: 39–51, 1998.[Web of Science][Medline]
21. Lampugnani MG, Orsenigo F, Gagliani MC, Tacchetti C, Dejana E. Vescular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol 174: 593–604, 2006.[Abstract/Free Full Text]
22. Lorenz H, Hailey DW, Wunder C, Lippincott-Schwartz J. The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology. Nat Protoc 1: 276–279, 2006.[CrossRef][Medline]
23. Masri FA, Xu W, Comhair SA, Asosingh K, Koo M, Vasanji A, Drazba J, Anand-Apte B, Erzurum SC. Hyperproliferative apoptosis-resistant endothelial cells in idiopathic pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 293: L548–L554, 2007.[Abstract/Free Full Text]
24. Mathew R, Huang J, Shah M, Patel K, Gewitz M, Sehgal PB. Disruption of endothelial-cell caveolin-1/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 110: 1499–1506, 2004.[Abstract/Free Full Text]
25. Mertens C, Darnell JE. Snapshot: JAK-STAT signaling. Cell 131: 612, 2007.[Medline]
26. Mukhopadhyay S, Sehgal PB. Discordant regulatory changes in monocrotaline-induced megalocytosis of lung arterial endothelial and alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 290: L1216–L1226, 2006.[Abstract/Free Full Text]
27. Mukhopadhyay S, Shah M, Patel K, Sehgal PB. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response. Toxicol Appl Pharmacol 211: 209–220, 2006.[CrossRef][Web of Science][Medline]
28. Mukhopadhyay S, Xu F, Sehgal PB. Aberrant cytoplasmic sequestration of eNOS in endothelial cells after monocrotaline, hypoxia, and senescence: live-cell caveolar and cytoplasmic NO imaging. Am J Physiol Heart Circ Physiol 292: H1373–H1389, 2007.[Abstract/Free Full Text]
29. Ndubuisi MI, Guo GG, Fried VA, Etlinger JD, Sehgal PB. Cellular physiology of STAT3: where's the cytoplasmic monomer? J Biol Chem 274: 25499–25509, 1999.[Abstract/Free Full Text]
30. Nystrom FH, Chen H, Cong LN, Li Y, Quon MJ. Caveolin-1 interacts with the insulin receptor and can differentially modulate insulin signaling in transfected Cos-7 cells and rat adipose cells. Mol Endocrinol 13: 2013–2024, 1999.[Abstract/Free Full Text]
31. Ota N, Brett TJ, Murphy TL, Fremont DH, Murphy KM. N-domain-dependent nonphosphorylated STAT4 dimers required for cytokine-driven activation. Nat Immun 5: 208–215, 2004.[CrossRef][Web of Science]
32. Panopoulou E, Gillooly DJ, Wrana JL, Zerial M, Stenmark H, Murphy C, Fotsis T. Early endosome regulation of Smad-dependent signaling in endothelial cells. J Biol Chem 277: 18046–18052, 2002.[Abstract/Free Full Text]
33. Park DS, Cohen AW, Frank PG, Razani B, Lee H, Williams TM, Chandra M, Shirani J, De Souza AP, Tang B, Jelicks LA, Factor SM, Weiss LM, Tanowitz HB, Lisanti MP. Caveolin-1 null (–/–) mice show dramatic reductions in life span. Biochemistry 42: 15124–15131, 2003.[CrossRef][Web of Science][Medline]
34. Patel HH, Zhang S, Murray F, Suda RY, Head BP, Yokoyama U, Swaney JS, Niesman IR, Schermuly RT, Pullamsetti SS, Thistlethwaite PA, Miyanohara A, Farquhar MG, Yuan JX, Insel PA. Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension. FASEB J 21: 2970–2979, 2007.[Abstract/Free Full Text]
35. Pranada AL, Metz S, Herrmann A, Heinrich PC, Muller-Newen G. Real time analysis of STAT3 nucleocytoplasmic shuttling. J Biol Chem 279: 15114–15123, 2004.[Abstract/Free Full Text]
36. Richter A, Yeager ME, Zaiman A, Cool CD, Voelkel NF, Tuder RM. Impaired transforming growth factor-beta signaling in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 170: 1340–1348, 2004.[Abstract/Free Full Text]
37. Sakao S, Taraseviciene-Stewart L, Lee JD, Wood K, Cool CD, Voelkel NF. Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells. FASEB J 19: 1178–1180, 2005.[Abstract/Free Full Text]
38. Santos SC, Miguel C, Domingues I, Calado A, Zhu Z, Wu Y, Dias S. VEGF and VEGFR-2 (KDR) internalization is required for endothelial recovery during wound healing. Exp Cell Res 313: 1561–1574, 2007.[CrossRef][Web of Science][Medline]
39. Sehgal PB, Guo GG, Shah M, Kumar V, Patel K. Cytokine signaling: STATs in plasma membrane rafts. J Biol Chem 277: 12067–12074, 2002.[Abstract/Free Full Text]
40. Sehgal PB, Levy DE, Hirano T. (editors). Signal Transducers and Activators of Transcription (STATs): Activation and Biology. Dordrecht, The Netherlands: Kluwer Academic Press, 2003, p. 1–746.
41. Sehgal PB, Mukhopadhyay S, Xu F, Patel K, Shah M. Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 292: L1526–L1542, 2007.[Abstract/Free Full Text]
42. Sehgal PB, Mukhopadhyay S. Dysfunctional intracellular trafficking in the pathobiology of pulmonary arterial hypertension. Am J Respir Cell Mol Biol 37: 31–37, 2007.[Abstract/Free Full Text]
43. Sehgal PB, Mukhopadhyay S. Pulmonary arterial hypertension: a disease of tethers, SNAREs and SNAPs? Am J Physiol Heart Circ Physiol 293: H77–H85, 2007.[Abstract/Free Full Text]
44. Sehgal PB. Plasma membrane rafts and chaperones in cytokine/STAT signaling. Acta Biochim Pol 50: 583–594, 2003.[Web of Science][Medline]
45. Shah M, Patel K, Fried VA, Sehgal PB. Interactions of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signaling during fever. J Biol Chem 277: 45662–45669, 2002.[Abstract/Free Full Text]
46. Shah M, Patel K, Mukhopadhyay S, Xu F, Guo G, Sehgal PB. Membrane associated STAT3 and PY-STAT3 in the cytoplasm. J Biol Chem 281: 7302–7308, 2006.[Abstract/Free Full Text]
47. Shah M, Patel K, Sehgal PB. Monocrotaline pyrrole-induced endothelial cell megalocytosis involves a Golgi blockade mechanism. Am J Physiol Cell Physiol 288: C850–C862, 2005.[Abstract/Free Full Text]
48. Shigematsu S, Watson RT, Khan AK, Pessin JE. The adipocyte plasma membrane caveolin functional/structural organization is necessary for the efficient endocytosis of GLUT4. J Biol Chem 278: 10683–10690, 2003.[Abstract/Free Full Text]
49. Silver DL, Naora H, Liu J, Cheng W, Montell DJ. Activated signal transducer and activator of transcription (STAT) 3: localization in focal adhesions and function in ovarian cancer cell motility. Cancer Res 64: 3550–3558, 2004.[Abstract/Free Full Text]
50. Tsukazaki T, Chaing TA, Davison AF, Attisano L, Wrana JL. SARA a FYVE domain protein that recruits Smad2 to the TGFβ receptor. Cell 95: 779–791, 1998.[CrossRef][Web of Science][Medline]
51. Turkson J. STAT proteins as novel targets for cancer drug discovery. Expert Opin Ther Targets 8: 409–422, 2004.[CrossRef][Web of Science][Medline]
52. Volonte D, Galbiati F, Lisanti MP. Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras. The subcellular distribution of caveolin-1 is modulated by cell-cell contact. FEBS Lett 445: 431–439, 1999.[CrossRef][Web of Science][Medline]
53. Wang GS, Qian GS, Bai L, Chen Y, Wu GM, Zhou DS, Qian P. Enhanced expression of signal transducers and activators of transcription in lung tissue of hypoxic pulmonary hypertension rat models. Zhonghua Nei Ke Za Zhi 42: 768–772, 2003.[Medline]
54. Watanabe K, Saito K, Matsuda T, Tamura M, Kon S, Miyazaki T, Uede T. Molecular dynamics of STAT3 on IL-6 signaling pathway in living cells. Biochem Biophys Res Commun 324: 1264–1273, 2004.[CrossRef][Web of Science][Medline]
55. Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE. Clathrin exchange during clathrin-mediated endocytosis. J Cell Biol 155: 291–300, 2001.[Abstract/Free Full Text]
56. Xu F, Mukhopadhyay S, Sehgal PB. Live cell imaging of interleukin-6-induced targeting of "transcription factor" STAT3 to sequestering endosomes in the cytoplasm. Am J Physiol Cell Physiol 293: C1374–C1382, 2007.[Abstract/Free Full Text]
57. Yang J, Chatterjee-Kishore M, Staugaitis SM, Nguyen H, Schlessinger K, Levy DE, Stark GR. Novel roles of unphosphorylated STAT3 in oncogenesis and transcriptional regulation. Cancer Res 65: 939–947, 2005.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. Mukhopadhyay, J. Lee, and P. B. Sehgal Depletion of the ATPase NSF from Golgi membranes with hypo-S-nitrosylation of vasorelevant proteins in endothelial cells exposed to monocrotaline pyrrole Am J Physiol Heart Circ Physiol, November 1, 2008; 295(5): H1943 - H1955. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 294/3/L449    most recent 00377.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mukhopadhyay, S. Articles by Sehgal, P. B. Search for Related Content PubMed PubMed Citation Articles by Mukhopadhyay, S. Articles by Sehgal, P. B.