Am J Physiol Cell Physiol Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C1374-C1382, 2007. First published August 1, 2007; doi:10.1152/ajpcell.00220.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Videos
Right arrow All Versions of this Article:
293/4/C1374    most recent
00220.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, F.
Right arrow Articles by Sehgal, P. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, F.
Right arrow Articles by Sehgal, P. B.

RECEPTORS AND SIGNAL TRANSDUCTION

Live cell imaging of interleukin-6-induced targeting of "transcription factor" STAT3 to sequestering endosomes in the cytoplasm

Fang Xu,1 Somshuvra Mukhopadhyay,1 and Pravin B. Sehgal1,2

1Department of Cell Biology and Anatomy and 2Department of Medicine, New York Medical College, Valhalla, New York

Submitted 29 May 2007 ; accepted in final form 30 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Signal transducer and activator of transcription (STAT) family transcription factors are classically viewed as transducing cytokine- and growth factor-activated signals from the plasma membrane to the cell nucleus for the purpose of activating transcription. We report live cell imaging studies of fluorescently labeled STAT3 expressed in Hep3B hepatocytes that reveal interleukin (IL)-6-activated targeting of STAT3 and PY-STAT3 to relatively long-lived sequestering endosomes in the cytoplasm. This targeting was rapid but transient, required phosphorylation and integrity of Tyr 705 in STAT3, and was blocked by nocodazole, geldanamycin, and indirubin E804 and by overexpression of wild-type caveolin-1. Strikingly, overexpression of the dominant-negative (DN) mutant K44A of the GTPase dynamin II led to marked constitutive accumulation of STAT3 in the endocytic compartment with depletion of the STAT3 nuclear pool. Subsets of the native and K44A-generated STAT3- and PY-STAT3-sequestering endosomes colocalized with MyD88, an adapter protein that integrates pathways of Toll-like receptor and IL-1 transcriptional signaling and stabilization of mRNAs. These data provide direct evidence for the cytokine-induced "signal transduction" by STAT3 from the plasma membrane to a cytoplasmic membrane destination for yet to be elucidated function(s) in the cytoplasm including prolongation of signaling and/or cross talk.

cytokine-STAT signaling; transcytoplasmic transit; MyD88

SIGNAL TRANSDUCER AND ACTIVATOR of transcription (STAT) family proteins are classically viewed as transducing cytokine- and growth factor-activated signals from the plasma membrane to the cell nucleus for the purpose of activating transcription (4, 20, 22). In recent years several of the key tenets of the original cytokine-STAT signaling model (4) have had to be revised. First, the notion that nonphosphorylated "inactive" STATs are present in the cytoplasm as free monomers that then dimerize on Tyr phosphorylation has been replaced by the understanding that nonphosphorylated STATs in the cytoplasm exist as dimers and high-molecular-mass "statosome" complexes with other proteins (7, 10, 15, 17). Second, the notion that phosphorylation, 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 (34). Third, the notion that it is the activation by phosphorylation of STATs at the plasma membrane that then leads to their transcytoplasmic transit and import to the nucleus has been replaced by the recognition that even nonphosphorylated STATs traffic between the cytoplasm and nucleus at all times in a constitutive manner and that the increased accumulation of phosphorylated STATs in the nucleus on cytokine exposure reflects diminished export and not increased import per se (12, 18, 31). In this view, the rapid appearance of phosphorylated STATs in the nucleus within minutes of cytokine or growth factor exposure reflects the addition of Tyr-P, Ser-P, or both onto STAT molecules at the plasma membrane that were already in a constitutive shuttle to and from the nucleus. Fourth, the notion that the transcytoplasmic transit of STATs from the plasma membrane to the nuclear import machinery takes place as a free cytosolic process has been replaced by the understanding that this transcytoplasmic transit is initiated at plasma membrane raft microdomains and is mediated via membrane-associated caveolar and endocytic trafficking (the "signaling endosome" hypothesis) (2, 21–24). Nevertheless, in all of these refinements of the original STAT signaling model the focus has remained the transduction of a signal from the plasma membrane to the cell nucleus for transcriptional activation of target genes. Indeed, live cell imaging studies have been conducted extensively with fluorescently labeled STAT1 or STAT3 proteins demonstrating this transit to and from the nucleus (12, 18, 31, 32). Moreover, fluorescence relaxation spectroscopy in live Hep3B cells expressing STAT3-green fluorescent protein (GFP) has provided evidence for cytokine-induced generation of high-molecular-mass complexes of STAT3 >1 MDa (32). However, there have been no live cell observations reported of the targeting of cytokine-activated STAT "signal transducers" to cytoplasmic membrane destinations.

We report live cell imaging studies of fluorescently labeled STAT3 expressed in Hep3B hepatocytes that reveal interleukin (IL)-6-activated targeting of STAT3 and PY-STAT3 to relatively long-lived sequestering endosomes for function in the cytoplasm through cross talk with MyD88. These data require modification of yet another tenet of the original cytokine/STAT3 signaling paradigm (4)—that this signaling is exclusively to the nucleus for transcriptional effects—to now include considerations of STAT3-mediated "signal transduction" from the plasma membrane to cytoplasmic membrane destinations for function(s) in the cytoplasm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and plasmids. Growth of human Hep3B cells in six-well plates and in 100-mm petri dishes, IL-6 treatment, methods for cell fractionation, whole mount immunoelectron microscopy, fixation with cold paraformaldehyde, and immunofluorescence have been described previously (1, 21, 23–25, 28). Constitutive expression constructs for N1-enhanced GFP (EGFP) and STAT3-EGFP (32) were from Dr. Taddaki Miyazaki (Hokkaido University, Sapporo, Japan); for wild-type (wt) STAT3-yellow fluorescent protein (YFP), Y705F-STAT3-YFP, and STAT3D-YFP (E434A, E435A mutations) from Dr. Toshio Hirano (Osaka School of Medicine, Osaka, Japan); for dynamin II K44A and clathrin light chain from Dr. Lois Greene (National Institutes of Health, Bethesda, MD); for amphiphysin A1 and epsin2a fragment from Dr. Richard Jove (City of Hope Medical Center, Duarte, CA); for the S34N and Q79L dominant negative (DN) mutants of Rab5 from Dr. Marilyn Farquhar (University of California School of Medicine, San Diego, CA); and for wt caveolin-1 from Dr. Jeffrey Pessin (University of Iowa, Iowa City, IA). Expression constructs for wt dynamin II-FLAG and for the active dynamin MxA species were gifts from Dr. Richard D. Minshall (University of Illinois College of Medicine, Chicago, IL) and from Dr. Otto Haller (Universität Freiberg, Freiberg, Germany), respectively. Transient transfections were carried out with PolyFect (in all data shown here), Lipofectamine, or calcium phosphate methods (23, 24). Transfected cultures were usually used 18–24 h later; waiting for 48 h gave the same results.

Imaging. Images were collected with a MRC 1024 ES (Bio-Rad) confocal microscopy system with a black-and-white charge-coupled device camera and then rendered in pseudocolor. Data were collected with an Olympus Plan x10/NA 0.25 Phi objective. All data within each experiment were collected at identical imaging settings. Sequential frames in time-series data collection were rendered into movies with Windows MovieMaker software and converted into QuickTime with Quick Video Convert software. All data are presented without deconvolution.

Fluorescence protease protection assay. The topology of protein localization on the surface of or within cytoplasmic vesicles was evaluated with the fluorescence protease protection (FPP) assay essentially as described by Lorenz et al. (9). Briefly, Hep3B cultures transfected with the STAT3-GFP construct in six-well plates were washed extensively with warm phosphate-buffered saline and replenished with 1 ml of warm serum-free Dulbecco's modified Eagle's medium (DMEM) together with IL-6 and LysoTracker (100 nM). Thirty minutes later the cultures were switched to 1 ml of warm Hanks' balanced salt solution (HBSS), and cells displaying IL-6-induced STAT3-GFP cytoplasmic vesicles (in green) and lysosomes (in red) were imaged by confocal microscopy in a time-series mode (usually every 30 s). After commencement of the time-series recordings, 1 ml of a 2x strength digitonin solution in warm HBSS, then 1 ml of a 3x strength solution of trypsin in warm HBSS, and finally 1 ml of a 4x strength solution of Triton X-100 in warm HBSS were gently added sequentially at the times indicated in each experiment to give the following final concentrations: 50 µg/ml digitonin, 100 µg/ml trypsin, and 0.5% (vol/vol) Triton X-100.

Antibodies and reagents. IL-6 and additional cytokines investigated were purchased from R&D Systems (Minneapolis, MN). Staurosporine, genistein, sodium orthovanadate, nocodazole, filipin III, cytochalasin B, phenylarsine oxide (PAO), sodium meta-arsenite, and Escherichia coli lipopolysaccharide (LPS; serotype 0111:B4) were purchased from Sigma-Aldrich (St. Louis, MO). Cycloheximide, ionomycin, indirubin E804, piceatannol, and FTI-277 were from Calbiochem (San Diego, CA). Geldanamycin, MG132, poly(I)·poly(C), and leptomycin B were from Alexis Biochemicals (San Diego, CA), Myogenics/Millenium Pharmaceuticals (Cambridge, MA), Pharmacia Biotech/Amersham Biosciences (Amersham, UK) and Calbiochem (San Diego, CA), respectively. Reserveratrol was a gift from Dr. Joseph Wu (Dept. of Biochemistry and Molecular Biology, New York Medical College). LysoTracker, MitoTracker, and ER-Tracker were from Molecular Probes (Eugene, OR). Rabbit antibodies to STAT3, PY-STAT3, MyD88, IL-1 receptor-activated kinase-1 (IRAK-1), MyD88 adapter-like (MAL), Rab7, dynamin II, stathmin, catalase, HuR, {alpha}CP1/hnRNP-E1/E2, Sam68, and the FLAG peptide (Oct-A probe) and goat antibodies to Smad-anchor for receptor activation (SARA) and hepatocyte growth factor tyrosine kinase substrate (Hrs) were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal antibodies to early endosome antigen 1 (EEA1), Rab5, Rab11, lysosome membrane protein 1 (LAMP1), clathrin heavy chain, {alpha}-adaptin, and FAK were from BD Biosciences/Pharmingen (San Diego, CA), while that to vinculin was from Sigma-Aldrich. Respective Alexa Fluor-tagged secondary antibodies were from Molecular Probes. PolyFect transfection reagent and Lipofectamine were from Qiagen (Valencia, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In following up on our earlier immunofluorescence and cell fractionation studies of the IL-6-induced association of STAT3 and PY-STAT3 with cytoplasmic membrane fractions derived from Hep3B hepatocytes (20, 21, 23, 24), we carried out imaging of the targeting of GFP- or YFP-tagged STAT3 in live cells with a focus on cytoplasmic destinations. Figure 1A and Supplemental Video 1 show the sequestration of STAT3-GFP in relatively static cytoplasmic structures beginning within 5–10 min after IL-6 addition.1 Figure 1B shows that this sequestration was IL-6 induced and STAT3 specific in that it was not evident with the GFP tag alone or in the absence of IL-6. While there was phenotypic heterogeneity within each Hep3B culture, overall one-third to three-quarters of the transfected Hep3B cells in a culture exhibited this phenotype from experiment to experiment (Table 1). Supplemental Video 2 shows that these structures were somewhat motile within the cytoplasm along a linear disposition. Figure 1C demonstrates that IL-6-activated PY-STAT3 colocalized with STAT3-GFP in these sequestering cytoplasmic structures. The relatively low mobility of these STAT3-GFP-sequestering structures (Supplemental Videos 1 and 2) and their relatively long half-life (t1/2 of ~14 min in a staurosporine chase; Fig. 1D and Supplemental Video 3) suggested that these were distinct from the highly mobile signaling early endosomes or recycling endosomes (8, 19). These STAT3-sequestering structures were negative for LysoTracker, MitoTracker, and ER-tracker (Fig. 2, A and B) and, by immunofluorescence, for catalase (a peroxisome marker) (not shown). Moreover, a Z-series analysis confirmed that these IL-6-induced STAT3-GFP structures were in the same plane of the cytoplasm as the MitoTracker-labeled mitochondria (data not shown). As a positive control for the authenticity of the STAT3-GFP expressed in these cells, the data in Figs. 1 and 2 and Supplemental Videos 1–3 confirm the well-known IL-6-induced nuclear accumulation of STAT3-GFP in each of these experiments (18, 32).


Figure 1
View larger version (65K):
[in this window]
[in a new window]

  		Fig. 1. Interleukin (IL)-6-induced sequestration of transcription factor signal transducer and activator of transcription (STAT)3 in cytoplasmic endosomes. Human hepatocytes (Hep3B line) cultured in 6-well plates were transfected with the STAT3-green fluorescent protein (GFP) or N1-GFP constructs and imaged 20–48 h later with live cell confocal microscopy. A: IL-6 (25 ng/ml final concentration) was added immediately after the "0 min" frame, and the cells were imaged at 15-s intervals for the next 17 min. Supplemental Video 1 plays this time series at 4 frames/s. B: cultures transfected with the respective constructs were imaged before and 30 min after addition of IL-6. C: Hep3B cultures cotransfected with STAT3-GFP construct were treated with IL-6 for 30 min, fixed with paraformaldehyde, and immunostained with anti-PY-STAT3 polyclonal antibody (PAb). Data illustrate 2 independent experiments. D: Hep3B cultures transfected with STAT3-GFP construct were exposed to IL-6 for 20 min and then chased with staurosporine (10 µM), with images collected every minute for the next 45 min. The decay of the intensities of respective endocytic structures was quantitated with NIH ImageJ software and provided an average half-life of ~14 min after addition of staurosporine (see Supplemental Video 3). Scale bars = 25 µm.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Properties of IL-6-induced endosomes sequestering GFP- or YFP-tagged STAT3

 

Figure 2
View larger version (75K):
[in this window]
[in a new window]

  		Fig. 2. Characterization of STAT3-sequestering endosomes. A and B: Hep3B cultures transfected with the STAT3-GFP construct were exposed to IL-6 together with ER-Tracker, MitoTracker, and/or LysoTracker (per respective manufacturer's protocols) and imaged 30 min later. C: association of STAT3 with the cytoplasmic surface of membrane structures (fluorescence protease protection assay). Hep3B cultures transfected with the STAT3-GFP construct were first treated with IL-6 for 30 min in the presence of LysoTracker ("IL-6" frame). These were then sequentially imaged on treatment with digitonin (Dig, 50 µg/ml), trypsin (Tryp, 100 µg/ml), and then Triton X-100 (0.5% vol/vol) as indicated. D: sequestration of endogenous PY-STAT3 in cytoplasmic membrane structures. Replicate Hep3B cultures were exposed to IL-6 for 30 min and then sequentially to digitonin (50 µg/ml) in ice-cold 0.25 M sucrose-phosphate-buffered saline (sucrose buffer) and to Brij 58 (0.5% vol/vol in sucrose buffer), fixed with paraformaldehyde, and immunostained for PY-STAT3. Scale bars = 25 µm.

 
A combined detergent dissection-protease digestion approach (Ref. 9; FPP assay) was used to investigate whether the STAT3-GFP-sequestering cytoplasmic structures were membrane-delimited organelles and whether the STAT3 was present on its cytosolic surface or was within the organelle. Figure 2C summarizes data showing that STAT3-GFP associated with these cytoplasmic structures was largely digitonin resistant but could be digested away by subsequent addition of trypsin alone. As controls, LysoTracker within the lysosomal compartment and STAT3-GFP in the nucleus were relatively resistant until the cells were treated with Triton X-100.

The IL-6-induced targeting of STAT3-GFP to membrane-associated structures in the cytoplasm was not due to the GFP tag per se (12), in that experiment 2 (Table 1) shows that this targeting was also seen by using a YFP tag on STAT3 and Fig. 2D shows that endogenous PY-STAT3 in the cytoplasm of IL-6-treated Hep3B cells was also observed in punctuate structures arranged in linear arrays and that these puncta resisted digitonin but could be dissolved away by a stronger detergent such as Brij 58. Western blotting analyses of the solubilized fraction of endogenous PY-STAT3 and STAT3 in detergent-dissection experiments as in Fig. 2D showed that ~45–50% of cellular PY-STAT3 (i.e., in the cytoplasm plus nucleus), but only ~10–15% of cellular nonphosphorylated STAT3, was associated with digitonin-resistant but Brij 58-soluble structures in the cytoplasm (Fig. 3). Thus there was a preferential association of PY-STAT3 with digitonin-resistant punctuate structures in the cytoplasm. These cytoplasmic membrane-associated PY-STAT3 components could be enriched for by flotation through HistoDenz or sucrose gradients (1, 24, 28) in fractions that also contained clathrin heavy chain and the endosome marker Rab5 (Fig. 4, A and B). The vesicular nature of these gradient-enriched STAT3-bearing low-density structures was confirmed by whole mount anti-STAT3-immunogold assays (Fig. 4C).


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Detergent dissection studies of the IL-6-induced association of PY-STAT3 with cytoplasmic vesicles. Hep3B cultures in 100-mm dishes were exposed to IL-6 for 5 or 15 min, washed extensively with ice-cold phosphate-buffered saline and then sequentially with 4 washes (1 ml each time) in ice-cold 0.25 M sucrose buffer containing digitonin (50 µg/ml; D), then with 4 washes with sucrose buffer containing saponin (0.2%; S), then with 4 washes with sucrose buffer containing Brij 58 (0.5%; B), and then with 1 wash with sucrose buffer containing Triton X-100 (0.5%; T), and finally the nuclear residue was scraped into 1 ml of sucrose buffer containing Triton X-100 (0.5%) and SDS (0.2%) (Nuc). Representative aliquots of each wash were Western blotted for STAT3 and PY-STAT3, the images were quantitated with NIH ImageJ software, and the amounts of STAT3 and PY-STAT3 eluted with each of the detergents are summarized as % of that in the total of all cell fractions.

 

Figure 4
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 4. Cell fractionation studies of the IL-6-induced association of PY-STAT3 with cytoplasmic vesicles. A: Hep3B cultures in 100-mm plates (5 cultures/group) were treated with IL-6 for 30 min, harvested, and subjected to cell fractionation as described previously with the procedure of Storrie and Madden incorporating a sedimentation followed by a flotation gradient of the fractions at the Percoll/HistoDenz (17%) interface in the 1st gradient (24, 28). One-milliliter fractions were collected from the top of the 2nd gradient as indicated. Aliquots of the respective fractions were either immunoblotted (b; 30 µl/lane) or first immunopanned (IPN) with anti-PY-STAT3 magnetic beads (~500 µl/fraction) and then immunoblotted as shown (a) (23, 24). CHC, clathrin heavy chain; EEA1, early endosome antigen 1. B: Hep3B cultures in 100-mm plates (5 cultures/group) were treated with IL-6 for 30 min, harvested, and subjected to cell fractionation and a one-step flotation gradient procedure as described previously by Aniento and colleagues (1, 24) with the modification that additional sucrose layers were introduced into the gradient as indicated. The respective interfaces were collected, diluted to 11 ml, resedimented, and resuspended in 100 µl of homogenization buffer. Aliquots of the respective fractions were immunoblotted (30 µl/lane) as shown. LE, late endosome; E1–E3, endosome fractions; HM, heavy membranes; Sol, soluble. C: whole mount anti-STAT3 immunogold electron microscopy (EM). The washed resedimented fractions equivalent to the pooled E1, E2, and E3 from B, right, were spotted on Formvar-coated copper grids, fixed with paraformaldehyde, and processed for whole mount anti-STAT3 immunogold EM using a rabbit anti-STAT3 PAb and a donkey anti-rabbit PAb coupled to 10-nm gold beads (Electron Microscopy Sciences, Washington, PA). Several fields from the anti-STAT3-treated grids are shown; analyses using nonimmune IgG did not yield any association of the gold particles with the membrane structures (not shown).

 
To further characterize the STAT3-GFP-bearing endosomes we carried out an extensive double-label immunofluorescence survey using various endocytic and organellar markers. These data showed that the STAT3-GFP-sequestering membrane structures in the cytoplasm of IL-6-treated Hep3B cells were largely negative for Rab5 (marker for early endosomes), Rab7 (marker for late endosomes/lysosomes), Rab11 (marker for recycling endosome), EEA1, the IL-6-receptor chain gp130 chain, SARA, Smad proteins (using an anti-multi-Smad antibody), Hrs, LAMP1, clathrin heavy chain, {alpha}-adaptin, caveolin-1, and Toll-like receptors (TLR)1–10 (data not shown). Moreover, these were also largely devoid of vinculin (marker for focal adhesions) and stathmin (a STAT3-microtubule interacting protein), two previously reported destinations for activated STAT3 in the cytoplasm (16, 27) (data not shown). However, the IL-6-induced STAT3-sequestering membrane structures were dramatically affected by overexpression of several proteins known to have DN effects on endocytosis (dynamin II K44A, amphiphysin A1, epsin2a, clathrin light chain; Table 1, experiment 6; also see Figs. 5 and 6) (2, 3, 33). In contrast, two different DN mutants of Rab5, S34N and Q79L, had little effect on IL-6-induced STAT3-GFP cytoplasmic sequestration (data not shown). We conclude that the STAT3-sequestering membrane structures represent novel components of the cytoplasmic endocytic compartment.


Figure 5
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 5. K44A dominant-negative (DN) mutant of dynamin II leads to marked accumulation of STAT3 in cytoplasmic vesicles. A: Hep3B cultures cotransfected with the indicated GFP or yellow fluorescent protein (YFP) constructs (1 µg/well) and with either the K44A construct or control pCDNA3 (1 µg/well) were imaged 20 h later; wt, wild type. B: replicate cultures were cotransfected with 0.5 µg of the STAT3-GFP construct and varying amounts of the K44A construct with matching amounts of the control pCDNA3 to make a total of 4.5 µg of DNA/well. Approximately 150–200 cells were imaged ~20 h later and enumerated as indicated. P < 0.01 for the relationship between cytoplasmic accumulation of STAT3 and amount of cotransfected K44A DNA by ANOVA, and P < 0.02 for the inverse relationship between cytoplasmic accumulation of STAT3 and depletion of STAT3 from the nucleus. C: accumulation of IL-6-activated PY-STAT3 in both cytoplasmic vesicles and nucleus after K44A. Hep3B cells contransfected with STAT3-GFP and K44A constructs were treated with IL-6 for 30 min and imaged after fixation and immunostaining for PY-STAT3. D: accumulation of endogenous STAT3 and PY-STAT3 in cytoplasmic vesicles after K44A transfection. Hep3B cultures were transfected with the K44A construct alone and then treated with IL-6 for 30 min, followed by immunostaining for STAT3 and PY-STAT3. Scale bars = 25 µm.

 

Figure 6
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 6. Only cells overexpressing the K44A dynamin II mutant, but not wt dynamin II-FLAG or the MxA active dynamin species, show constitutive sequestration of STAT3-GFP in aberrant cytoplasmic vesicles and inhibition of IL-6-induced nuclear accumulation of STAT3-GFP. A: Hep3B cells cotransfected with the STAT3-GFP construct together with pCDNA, MxA, wt dynamin II-FLAG, or K44A dynamin II vectors were imaged without IL-6 and 30 min after treatment with IL-6. B: anti-dynamin II immunostaining of representative STAT3-GFP-expressing cells from the wt and K44A dynamin II cotransfected cultures in the experiment in A without and with IL-6 treatment. C: anti-FLAG immunostaining of representative cells from the experiment in A (STAT3-GFP cells overexpressing FLAG-tagged wt dynamin II and treated with IL-6 for 30 min). Scale bars = 25 µm.

 
IL-6-induced sequestration of STAT3 and PY-STAT3 by cytoplasmic endosomes was transient in that it decayed by 1–3 h (Table 1, experiment 1). It was observed irrespective of the particular transient transfection method used (PolyFect, Lipofectamine or calcium phosphate) (not shown) and required the integrity of Tyr 705 in STAT3 but not of its DNA-binding domain (Table 1, experiment 2). This cytoplasmic targeting was strongly inhibited by staurosporine (inhibitor of phosphorylation), genistein (inhibitor of Tyr phosphorylation), indirubin E804 (inhibitor of Src-mediated STAT3 activation; Ref. 14), PAO (inhibitor of endocytosis), nocodazole (inhibitor of microtubules), geldanamycin [inhibitor of heat shock protein (HSP)90], and sodium arsenite (stimulator of cytoplasmic stress granules or P bodies) but not by vanadate (phosphatase inhibitor), MG132 (proteosome inhibitor), filipin III (plasma membrane caveolar raft disrupter), cytochalasin B (inhibitor of microfilaments), FTI-277 (inhibitor of farnesyl transferase), ionomycin (calcium ionophore), leptomycin B (inhibitor of nuclear export), LPS (TLR activator), poly(I)·poly(C) (another TLR activator), or piceatannol or reserveratrol (supposed inhibitors of STAT3 activation) (Table 1 and data not shown). However, this cytoplasmic targeting of STAT3-GFP was dependent on preexisting proteins in that pretreatment of Hep3B cells with the protein synthesis inhibitor cycloheximide for 2–4 h or longer before IL-6 inhibited this targeting (Table 1, experiment 3).

In investigating the effect of membrane proteins that modulate trafficking along the caveolar and endocytic pathway on IL-6-induced STAT3-sequestering endosomes, we observed that overexpression of wt caveolin-1 inhibited this targeting (Table 1, experiment 6). Moreover, overexpression of proteins that inhibit endocytosis (amphiphysin A1 and clathrin light chain) inhibited STAT3-GFP-endosomal targeting (Table 1, experiment 6 and data not shown). Strikingly, overexpression of the K44A DN mutant of dynamin II and of epsin2a caused marked accumulation of STAT3 in cytoplasmic structures consistent with aberrant tubuloreticular elements of the endocytic pathway as has been previously characterized (3) (Fig. 5, A and B, and Table 1, experiment 6). This cytoplasmic accumulation was so marked that it led to a dramatic depletion of the nuclear STAT3 pool (Fig. 5, A–C). Nevertheless, the available cytoplasmic STAT3 was still sufficient for IL-6-induced activation to PY-STAT3 and accumulation of this PY-STAT3 in the nucleus and in cytoplasmic elements (Fig. 5C). However, this K44A-induced redistribution of bulk STAT3 to cytoplasmic membrane structures was constitutive and was also observed by using YFP-tagged wt STAT3 and the Y705F mutant of STAT3 (Fig. 5A) and, importantly, native endogenous STAT3 and PY-STAT3 as well (Fig. 5D). The enhanced localization to cytoplasmic vesicular structures following coexpression of dynamin II K44A was specific for STAT3 inasmuch as it was not seen when GFP alone was expressed (Fig. 5A).

Figure 6 illustrates a series of additional control experiments that verify that only cells overexpressing the K44A dynamin II mutant, but not cells overexpressing the wt dynamin II-FLAG species (26) or the active dynamin GTPase MxA species (citations in Ref. 24), show the aberrant and constitutive sequestration of STAT3-GFP in the cytoplasm as well as the marked inhibition of the nuclear STAT3-GFP pool on IL-6 treatment. This is consistent with our previous demonstration (24) that overexpression of the K44A dynamin species inhibited IL-6/STAT3-luciferase transcriptional signaling. In terms of STAT3 sequestration topology, FPP assay (as in Fig. 2C) provided evidence for the localization of STAT3-GFP on the surface of these aberrant endocytic elements in K44Adynamin-transfected cells and, additionally, confirmed that these were not lysosomal in that this compartment was LysoTracker negative (data not shown). That the membrane-active K44A DN mutant of dynamin II caused a profound change in the localization of STAT3-GFP to heterogeneous structures in the cytoplasmic endocytic compartment with marked depletion of STAT3 from the nucleus (Figs. 5 and 6) indicates that there must exist large-scale constitutive membrane-associated trafficking of STAT3 in the cytoplasm.

In exploring a possible function of activated STAT3 in the cytoplasm we observed that MyD88, an adapter protein that integrates TLR and IL-1 transcriptional signaling and mediates stabilization of cytokine-induced mRNAs (6, 29), colocalized with STAT3-sequestering endosomes in some of the Hep3B cells in cultures exposed to IL-6 (Fig. 7A). This colocalization was dramatically enhanced on overexpression with the dynamin K44A mutant with either GFP- or YFP-tagged STAT3 (wt or mutant Y705A), with almost every cell showing colocalization (Fig. 7, B and C). This was specific to MyD88 in that under the same experimental conditions we did not observe colocalization between STAT3-GFP and TLR1–10, the MyD88-like protein MAL, IRAK-1, p38 mitogen-activated kinase, phospho-p38, the mRNA stability-regulating proteins HuR, {alpha}CP1/hnRNP-E1/E2, and Sam68, or the Smad transcription factors with or without exposure to the cytokines IL-6, IL-1, transforming growth factor (TGF)-beta, TNF, LPS, or poly(I)·poly(C). Finally, Fig. 7D shows an example of colocalization of IL-6-induced endogenous PY-STAT3 with MyD88 in vesicles in the cytoplasm. As a control, the increased nuclear accumulation of endogenous PY-STAT3 in response to IL-6 is also evident in Fig. 7D. This K44A-induced cytoplasmic sequestration of STAT3-YFP was also observed in primary bovine pulmonary endothelial cells together with colocalization of MyD88 and PY-STAT3 (as in Figs. 5 and 7; data not shown).


Figure 7
View larger version (59K):
[in this window]
[in a new window]

  		Fig. 7. Colocalization of STAT3 with MyD88 in the cytoplasmic endocytic compartment. A: a Hep3B culture transfected with the STAT3-GFP construct was treated with IL-6 for 30 min, fixed, and immunostained for MyD88. One example of the colocalization of STAT3-GFP and MyD88 in a minority (1–5%) of cells is shown. B and C: marked colocalization of tagged STAT3 with MyD88 in Hep3B cells cotransfected with the K44A dynamin II construct. Almost all cells (>90%) showing tagged STAT3 in cytoplasmic vesicles showed colocalization of MyD88. D: colocalization of endogenous IL-6-activated PY-STAT3 with MyD88. A Hep3B culture transfected with the pCDNA3 control vector only was treated with IL-6 for 30 min and immunostained for endogenous PY-STAT3 and then for MyD88. Scale bars = 25 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It has been axiomatic in the STAT transcription factor literature that these "signal transducers" transmit cytokine and growth factor activation signals from the plasma membrane to the cell nucleus for purposes of regulating transcription. The present data add a new facet to the "signal transduction" capabilities of one of these STAT family members. The live cell imaging data convincingly demonstrate that substantial amounts of IL-6-activated STAT3 are targeted to cytoplasmic vesicles with a long-lived provenance. This cytoplasmic targeting to sequestering endosomes can be distinguished from the previously suggested association of PY-STAT3 with signaling endosomes in that the latter are presumed to be highly motile whereas the new live cell imaging data draw attention to a sequestering endosome. We suggest that this cytoplasmic targeting of PY-STAT3 likely subserves novel, yet to be elucidated functions.

The dramatic sequestration of bulk STAT3 in association with cytoplasmic vesicles following coexpression with the K44A DN mutant of dynamin II provides clear evidence for large-scale bulk trafficking of STAT3 to/through a membrane-associated station in the cytoplasm in a constitutive manner. These K44A-based data are consistent with our previous observation of the rapid constitutive association of bulk cytoplasmic STAT3 with cytoplasmic membranes within 15 min of treatment of Hep3B cells with the clathrin-mediated endocytosis inhibitor PAO (Fig. 4 in Ref. 24). Moreover, we previously showed (11, 25) an inverse relationship between caveolin-1 and the Tyr phosphorylation of STAT3. Thus we suggest that considerations of the epigenetic regulation of STAT3 function now need to include discussions of alterations in membrane trafficking in the cytoplasm.

The live cell imaging data showing the targeting of STAT3 to cytoplasmic endosomes stand in contrast to what is currently available in the STAT transcription factor literature. These new data demonstrate that this cytoplasmic targeting is rapid but transient. It is cytokine induced, requires active Tyr phosphorylation at Y705 in STAT3, is dependent on the integrity of microtubules, and requires short-lived protein(s) to direct the targeting as well as the integrity of HSP90 and c-Src function. However, in our hands thus far the STAT3-GFP-sequestering endosomes have proven negative for the IL-6 receptor chain gp130 despite clear evidence in the same cells for gp130-positive endosomes. Thus the structural basis for this endosomal targeting remains open.

It is remarkable that Mitsuyama et al. (13) have recently published (but not commented on) the dramatic sequestration of almost all of the cellular PY-STAT3 in cytoplasmic vesicular structures in CD4-positive T cells in the ileum of mice with genetically induced inflammatory bowel disease. In this instance, there was little apparent PY-STAT3 in the nucleus. In investigating PY-STAT3 subcellular localization in sections of human and rat lungs from individuals with pulmonary hypertension, we have observed the dramatic sequestration of PY-STAT3 in the cytoplasmic compartment in pulmonary arterial endothelial, smooth muscle, and lung parenchymal cells (Mukhopadhyay S, Shah M, Xu F, Patel K, and Sehgal PB, unpublished observations). Indeed, in these tissue studies the majority of cells showed cytoplasmic PY-STAT3, with some of the lung parenchymal cells showing clear PY-STAT3 sequestering cytoplasmic vesicles with almost no PY-STAT3 in the nucleus. These tissue-derived data, together with those in the present article, require a fresh look at STAT3 biology with a focus on cytoplasmic membrane-associated events involving activated STAT3.

In the absence of cotransfection with the K44A dynamin mutant, very few of the STAT3-sequestering vesicles corecruited MyD88. Nevertheless, after K44A coexpression the majority of cells showed colocalization of MyD88 to the STAT3- and PY-STAT3-sequestering vesicles. Nevertheless, these vesicles remained negative for TLR1–10 and for the adapters IRAK-1 and MAL and were unaffected by LPS, poly(I)·poly(C), or the cytokines TNF, IL-1, and TGF-beta. For the moment there remain two possible interpretations of these data: either the colocalization results from the effects of K44A on endocytic membrane trafficking that happen to affect the completely independent biology of STAT3 and MyD88 at the level of endosomes in a similar manner, or the activated STAT3 mediates some novel and as yet unknown function through the corecruited MyD88. Indeed, there is precedent for TLR-independent function of MyD88 in the cytoplasm (6, 29). MyD88 has been shown to mediate the stabilization of cytokine-induced mRNA species through their AU-rich elements in the 3' untranslated regions through the p38 MAPK pathways (29). Whether this takes place through MyD88 recruitment to endosomes, as does the integration with TLR signaling, is not known (6, 29). However, the possibility of enhanced mRNA stability is reminiscent of investigations carried out approximately two decades ago, which established that IL-6 and other acute-phase cytokines not only mediate transcriptional enhancement of target plasma protein genes in the liver but also enhance the stability of mRNAs for specific acute-phase proteins such as serum amyloid A, {alpha}2-macroglobulin, {alpha}1-acid glycoprotein, factor B, and complement C3 (5). However, thus far, in immunofluorescence colocalization in our hands the STAT3-GFP-sequestering vesicles have been negative for the mRNA stability regulation pathway proteins p38 MAPK, phospho-p38, HuR, {alpha}CP1/hnRNP-E1/E2, and Sam68.

The documentation of long-lived Trk-signaling endosomes that serve to greatly prolong nerve growth factor signaling from a cytoplasmic location (Ref. 30 and citations therein) suggests another possible function for the long-lived PY-STAT3-sequestering endosomal compartment. By analogy with long-lived Trk-signaling endosomes, the IL-6-induced cytoplasmic sequestration of activated STAT3 could contribute to temporal prolongation of nucleus-ward signaling.

To summarize, we provide the first live cell evidence showing IL-6-induced signal transduction by the "transcription factor" STAT3 to a sequestering endosome compartment in the cytoplasm. These data require modification of yet another tenet of the original cytokine-STAT3 signaling paradigm (4)—that this signaling is exclusively to the nucleus for transcriptional effects. Activated STAT3 also mediates signal transduction from the plasma membrane to cytoplasmic membrane destinations.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-077301.


   ACKNOWLEDGMENTS
 
We thank Drs. Lois Greene, Otto Haller, Toshio Hirano, Richard Jove, Richard D. Minshall, Taddaki Miyazaki, and Jeffrey Pessin for gifts of various plasmid expression vectors and Dr. Mehul Shah for compiling the STAT3 and PY-STAT3 enumeration in Fig. 3 and for carrying out the whole mount anti-STAT3-immunogold electron microscopy in Fig. 4C.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. B. Sehgal, Rm. 201 Basic Sciences Bldg., Dept. of Cell Biology & 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.

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Aniento F, Gu F, Parton RG, Gruenberg J. An endosomal beta-COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes. J Cell Biol 133: 29–41, 1996.[Abstract/Free Full Text]

2. 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]

3. 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]

4. 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]

5. Jiang SL, Samols D, Rzewnicki D, Macintyre SS, Greber I, Sipe J, Kushner I. Kinetic modeling and mathematical analysis indicate that acute phase gene expression in Hep 3B cells is regulated by both transcriptional and posttranscriptional mechanisms. J Clin Invest 95:1253–1261, 1995.[Web of Science][Medline]

6. Kawai T, Akira S. TLR signaling. Cell Death Differ 13: 816–825, 2006.[CrossRef][Web of Science][Medline]

7. 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]

8. Lakadamyali M, Rust MJ, Zhuang X. Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124: 997–1009, 2006.[CrossRef][Web of Science][Medline]

9. 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]

10. Mao X, Ren Z, Parker GN, Sondermann H, Pastorello MA, Wang W, McMurray JS, Demeler B, Darnell JE, Chen X. Structural bases of unphosphorylated STAT1 association and receptor binding. Mol Cell 17: 761–771, 2005.[CrossRef][Web of Science][Medline]

11. Mathew R, Huang J, Shah M, Patel K, Gewitz M, Sehgal PB. Disruption of endothelial cell caveolin-1{alpha}/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 110: 1499–1506, 2004.[Abstract/Free Full Text]

12. Meyer T, Begitt A, Vinkemeir U. Green fluorescent protein-tagging reduces the nucleocytoplasmic shuttling specifically of unphosphorylated STAT1. FEBS J 274: 815–826, 2007.[CrossRef][Medline]

13. Mitsuyama K, Matsumoto S, Rose-John S, Suzuki A, Hara T, Tomiyasu N, Handa K, Tsuruta O, Funabashi H, Scheller J, Toyonaga A, Sata M. STAT3 activation via interleukin 6 trans-signalling contributes to ileitis in SAMP1/Yit mice. Gut 55: 263–1269, 2006.

14. Nam S, Buettner R, Turkson J, Kim D, Cheng JQ, Muehlbeyer S, Hippe F, Vatter S, Merz KH, Eisenbrand G, Jove R. Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in cancer cells. Proc Natl Acad Sci USA 102: 5998–6003, 2005.[Abstract/Free Full Text]

15. 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]

16. Ng DC, Lin BH, Lim CP, Huang G, Zhang T, Poli V, Cao X. Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin. J Cell Biol 172: 245–257, 2006.[Abstract/Free Full Text]

17. Ota N, Brett TJ, Murphy TL, Fremont DH, Murphy KM. N-domain-dependent nonphosphorylated STAT4 dimers required for cytokine-driven activation. Nat Immunol 5: 208–215, 2004.[CrossRef][Web of Science][Medline]

18. 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]

19. Rink J, Ghigo E, Kalaidzidis Y, Zerial M. Rab conversion as a mechanism of progression from early to late endosomes. Cell 122: 735–749, 2005.[CrossRef][Web of Science][Medline]

20. Sehgal PB. Plasma membrane rafts and chaperones in cytokine/STAT signaling. Acta Biochim Pol 50: 583–594, 2003.[Web of Science][Medline]

21. 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]

22. Sehgal PB, Levy DE, Hirano T. (editors). Signal Transducers and Activators of Transcription: Activation and Biology, Dordrecht, The Netherlands: Kluwer Academic, 2003.

23. Shah M, Patel K, Fried VA, Sehgal PB. Interactions of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane rafts and cytosolic complexes: preservation of cytokine signaling during fever. J Biol Chem 277: 45662–45669, 2002.[Abstract/Free Full Text]

24. Shah M, Patel K, Mukhopadhyay S, Fang X, Guo G, Sehgal PB. Membrane-associated STAT3 and PY-STAT3 in the cytoplasm. J Biol Chem 281:7302–7308, 2006.[Abstract/Free Full Text]

25. 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]

26. Shajahan AN, Tiruppathi C, Smrcka AV, Malik AB, Minshall RD. Gbeta{gamma} activation of Src induces caveolae-mediated endocytosis in endothelial cells. J Biol Chem 279: 48055–48062, 2004.[Abstract/Free Full Text]

27. 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]

28. Storrie B, Madden EA. Isolation of subcellular organelles. Methods Enzymol 182: 203–225, 1990.[Web of Science][Medline]

29. Sun D, Ding A. MyD88-mediated stabilization of interferon-{gamma}-induced cytokine and chemokine mRNA. Nat Immunol 7: 375–381, 2006.[CrossRef][Web of Science][Medline]

30. Valdez G, Philippidou P, Rosenblum J, Akmentin W, Shao Y, Halegoua S. Trk-signaling endosomes are generated by Rac-dependent macroendocytosis. Proc Natl Acad Sci USA 104: 12270–12275, 2007.[Abstract/Free Full Text]

31. Vinkemeier U. Getting the message across, STAT! Design principles of a molecular signaling event. J Cell Biol 167: 197–201, 2004.[Abstract/Free Full Text]

32. 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]

33. 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]

34. 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:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
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]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Mukhopadhyay, M. Shah, F. Xu, K. Patel, R. M. Tuder, and P. B. Sehgal
Cytoplasmic provenance of STAT3 and PY-STAT3 in the endolysosomal compartments in pulmonary arterial endothelial and smooth muscle cells: implications in pulmonary arterial hypertension
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L449 - L468.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Videos
Right arrow All Versions of this Article:
293/4/C1374    most recent
00220.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xu, F.
Right arrow Articles by Sehgal, P. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xu, F.
Right arrow Articles by Sehgal, P. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.