EGF induces nuclear translocation of STAT2 without tyrosine phosphorylation in intestinal epithelial cells

Leonard R. Johnson1, Shirley A. McCormack1, Chuan-He Yang2, Susan R. Pfeffer2, and Lawrence M. Pfeffer2

1 Department of Physiology and Biophysics and 2 Department of Pathology, College of Medicine, University of Tennessee, Memphis, Tennessee 38163


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Signal transducers and activators of transcription (STATs) are cytoplasmic proteins that bind to activated membrane receptors, undergo ligand-dependent phosphorylation on tyrosine residues, and translocate to the nucleus, where they induce transcription of specific genes in response to a variety of ligands, including cytokines and some growth factors. Using immunocytochemical and biochemical techniques, we investigated the localization and responses of STAT1 and STAT2 to epidermal growth factor (EGF) stimulation in IEC-6 intestinal epithelial cells and HeLa cells. These studies provide the first description of STAT activation and localization in response to EGF in intestinal epithelial cells and some novel findings regarding the activation and localization of STATs in general. These include the following. First, EGF promoted the tyrosine phosphorylation of STAT1 in IEC-6 cells and caused its translocation to the nucleus. Second, in the absence of EGF stimulation both STAT1 and STAT2 were localized to the Golgi apparatus in IEC-6 cells. Third, EGF caused the translocation of STAT2 to the nucleus in both IEC-6 and HeLa cells without inducing the tyrosine phosphorylation of STAT2.

signal transducers and activators of transcription; immunocytochemistry; Golgi apparatus; interferon; epidermal growth factor receptor


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

SIGNAL TRANSDUCERS AND ACTIVATORS of transcription (STATs) are known primarily for their mediation of the responses to cytokines, which usually involve some aspect of immunity (12). The binding of a cytokine to its membrane receptor results in the activation of tyrosine kinases and the subsequent phosphorylation of the cytoplasmic domain of the receptor (6). The phosphorylation of the receptor creates a docking site for SH2 domain-containing proteins. Each STAT contains a DNA binding domain and an SH2 domain (7, 8), which binds to the phosphorylated tyrosine residue of the receptor. The STATs are then tyrosine phosphorylated and released from the docking sites on the receptors. Activated STATs are recognized by other STAT molecules, resulting in dimerization and translocation to the nucleus (8). Within the nucleus STATs bind to specific DNA elements and enhance gene transcription.

To date at least six distinct forms of STATs have been described (12). The first two members of the STAT family, STAT1 and STAT2, were described originally because of their activation in response to type 1 interferons (IFN-alpha and -beta ) (18). Whereas numerous cytokines and growth factors activate STAT1, only type 1 IFNs have been found to activate STAT2 (19). Besides members of the Janus kinase family (JAKs), which were originally described as the tyrosine kinases activating STATs, numerous other protein tyrosine kinases, such as the epidermal growth factor receptor (EGFR) kinase, are able to activate STATs independently of JAKs (4, 5, 14).

Growth factors such as EGF and platelet-derived growth factor stimulate cell proliferation in many cell lines, whereas in others the same peptides prevent cell growth and induce apoptosis (1, 2). Whether a particular cell line responds to EGF by increasing growth or by undergoing programmed cell death probably depends on whether the dominant signal transduction pathway activated is the mitogen-activating protein (MAP) kinase cascade or STATs. Chin et al. (3) have recently shown that IFN-gamma activated STAT1 and induced apoptosis in both A-431 cells and HeLa cells. EGF, on the other hand, activated STAT1 and induced apoptosis in A-431 cells but not in HeLa cells, where it is a strong stimulator of proliferation and activates MAP kinase.

We were concerned primarily with the effect of EGF on STAT localization in intestinal epithelial cells, for which little is known about STAT signaling. Studies using intestinal epithelial cell line IEC-6, originally derived from fetal rat crypt cells (13), have shown that EGF and EGFR activation are required for both migration and proliferation (10, 15, 17). In the present experiments, we examined the effect of EGF on the activation and immunocytochemical localization of STAT1 and STAT2 in IEC-6 cells and HeLa cells. HeLa cells were used in part as a control because the effects of EGF on STATs in this cell line have been described (3). In IEC-6 cells, EGF induced the phosphorylation of STAT1 and increased its localization in both the nucleus and the Golgi apparatus. STAT2 localization increased in both the nucleus and Golgi as well, but without STAT2 being phosphorylated. EGF did not cause the phosphorylation of either STAT1 or STAT2 in HeLa cells but caused a pronounced translocation of STAT2 to the nucleus.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials

DMEM, Hanks' balanced salt solution (HBSS), and PBS were obtained from GIBCO BRL (Grand Island, NY). Fetal bovine serum (FBS), Triton X-100, insulin, gentamicin, BSA, and other chemicals and biochemicals were from Sigma (St. Louis, MO). Matrigel and EGF were purchased from Collaborative Research (Bedford, MA). The IEC-6 and HeLa cell lines were from the American Type Culture Collection (Manassas, VA). STAT1 and STAT2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The beta -coat protein (beta -COP) antibody was from Sigma. FITC- and rhodamine (LRSC)-conjugated anti-rabbit IgGs and LRSC-conjugated anti-mouse IgG came from Jackson ImmunoResearch (West Grove, PA), and VectaShield (a fluorescence-protecting mounting medium) came from Vector Laboratories (Burlingame, CA). Rat IFN-beta was a generous gift from Dr. H. Schellekens, University of Utrecht. Human consensus IFN (IFN con1) was a generous gift from Amgen (Thousand Oaks, CA).

Methods

Cell culture. IEC-6 cells were maintained as stock in DMEM containing 5% heat-inactivated FBS and 50 µg gentamicin plus 10 µg insulin per milliliter (DMEM-FBS). HeLa cells received 10% serum but did not receive insulin. Stock was passaged weekly and used for four passages only. Cells were incubated at 37.5°C in 90% air-10% CO2, and the medium was changed twice per week. For experiments, the cells were plated (day 0) in DMEM-dialyzed FBS on Matrigel-coated coverslips or in T-75 flasks and fed on day 2. Serum was removed on day 3, and the cells were analyzed 24 h later. For treatment with EGF, the cells were rinsed with HBSS, medium containing 20 ng/ml EGF was added, and the cells were reincubated for 2 or 30 min. The same procedure was followed when cells were treated with IFN. IEC-6 cells were incubated for 15 min with rat IFN (1,000 U/ml), and HeLa cells were incubated for 15 min with human IFN (1,000 U/ml).

Immunoprecipitations and immunoblot analysis. For immunoprecipitation studies, 1 × 108 cells were treated with EGF or IFN at 37°C for the periods of time indicated above and then washed with ice-cold PBS and lysed for 20 min in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 15% glycerol) containing 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin, and 1.75 µg/ml benzamidine. Samples were centrifuged (12,000 g, 15 min) at 4°C, and supernatants were immunoprecipitated overnight at 4°C with anti-phosphotyrosine (anti-pTyr; 4 µl of IG2 monoclonal antibody, a generous gift from A. R. Frackleton, Brown University). Immune complexes were collected with protein A-Sepharose beads (Pharmacia) and eluted in sample buffer. Samples were run on SDS-7.5% PAGE gel, transferred to polyvinylidene difluoride membranes (Millipore), and probed with anti-pTyr, anti-STAT1, or anti-STAT2 (Santa Cruz Laboratories), followed by anti-rabbit IgG coupled with horseradish peroxidase (Amersham). Blots were developed by enhanced chemiluminescence (Amersham).

Immunocytochemistry. The cells were fixed for 15 min, without being rinsed, in 3.7% formaldehyde in PBS, permeabilized for 10 min with 0.1% Triton X-100 in PBS, and blocked with PBS-2.0% BSA for 20 min. For double staining STAT1 and STAT2, cells were incubated successively with rabbit polyclonal anti-STAT1, FITC-conjugated anti-rabbit IgG, rabbit polyclonal anti-STAT2, and LRSC-conjugated anti-rabbit IgG, each for 1 h. For double staining of STAT2 and beta -COP, cells were incubated successively with rabbit polyclonal anti-STAT2, FITC-conjugated anti-rabbit IgG, mouse monoclonal anti-beta -COP, and LRSC-conjugated anti-mouse IgG, each for 1 h. The cells were washed five times with PBS-1% BSA at the end of each incubation with the antibody and finally three times with PBS before being mounted with VectaShield.

Microscopy. Images were captured by confocal laser scanning microscopy with the Bio-Rad MRC-1024 LaserSharp scanning system. The numerical aperture of the oil-immersion PlanApo 100× objective was 1.4. The images were obtained by sequential z series. Each image is a merged section of 0.5-µm slices, each chosen to include the same relative position in the cells. All images in the same experiment were obtained with the same settings of laser power, iris, and gain. The images were imported into Adobe Photoshop software for processing, which was also done with uniform settings for each experiment.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Effect of EGF on Distribution of STAT1 and STAT2 in IEC-6 Cells

In control IEC-6 cells both STAT1 and STAT2 were distributed primarily in the cytoplasm with little (STAT2) or no nuclear staining (Fig. 1). Most notable was a significant colocalization of both STATs to a perinuclear ring, with STAT2 appearing specifically in the Golgi apparatus. After a 2-min exposure to EGF there was an increase in STAT1 in the cytoplasm and Golgi. In both areas this consisted of a heavily punctate distribution. There also appeared to be some redistribution of STAT1 to the nucleus. The staining of STAT2 decreased in the cytoplasm and Golgi 2 min after EGF addition, whereas it increased slightly in the nucleus. Thirty minutes after the addition of EGF, STAT1 and STAT2 were clearly colocalized in the Golgi apparatus. For STAT2 the intensity of staining increased considerably compared with that at the 2-min time point. By 30 min cytoplasmic staining of both STATs and nuclear staining of STAT1 had nearly disappeared. STAT2, however, showed a heavy redistribution to the nucleus by 30 min.


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Fig. 1.   Distribution of STAT1 and STAT2 in IEC-6 cells after an exposure to epidermal growth factor (EGF) of 0 (A), 2 (B), or 30 (C) min. EGF was present at a concentration of 20 ng/ml and was added on day 4 after plating and 24 h after serum removal. Colored images (column 1), staining for STAT1 (green) and STAT2 (red) superimposed; black-and-white images, staining for STAT1 (column 2) and STAT2 (column 3) separately. Bar = 15 µm.

Effect of EGF on Distribution of STAT1 and STAT2 in HeLa Cells

The distributions of STAT1 and STAT2 in HeLa epidermal cancer cells were significantly different from the corresponding distributions in IEC-6 cells. Both STATs were diffusely distributed, as indicated by punctate staining throughout the cytoplasm of HeLa cells before the addition of EGF (Fig. 2). The proteins showed some colocalization, and there was slightly more staining for STAT2 than STAT1. In addition, a low level of nuclear staining of STAT2 was present. EGF had little or no effect on STAT1 at either time point, for STAT1 remained diffusely localized in the cytoplasm and the staining became more punctate with time. On the other hand, after a 2-min exposure to EGF there was a dramatic increase in the nuclear staining for STAT2. Interestingly, the general staining for STAT2 also increased, and there was a definite increase in the cytoplasm as well. After a 30-min incubation with EGF, a significant amount of STAT2 remained in the nuclei of the HeLa cells, and some remained in the cytoplasm, as indicated by punctate staining (Fig. 2). Unlike what was found for the IEC-6 cells, little or no staining of STAT2 appeared in the Golgi either before or after EGF addition. Therefore, in HeLa cells, EGF stimulated a redistribution of STAT2 to the nucleus but had little or no effect on the localization of STAT1.


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Fig. 2.   Distribution of STAT1 and STAT2 in HeLa cells after 0 (A), 2 (B), or 30 (C) min of exposure to 20 ng/ml EGF. Procedure was identical to that described in legend for Fig. 1. Column 1, staining for STAT1 (green) and STAT2 (red) superimposed; column 2, STAT1 staining; column 3, STAT2 staining. Bar = 15 µm.

To confirm the localization of STAT2 to the Golgi apparatus in IEC-6 cells, we stained for Golgi protein beta -COP, as well as for STAT2, in both IEC-6 and HeLa cells. As shown in Fig. 3, columns 1 and 3, beta -COP was evident in both cell types, although the IEC-6 cells had a more prominent Golgi. STAT2 (Fig. 3, columns 1 and 2) colocalized with beta -COP only in the IEC-6 cells.


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Fig. 3.   Distribution of STAT2 and beta -COP in control IEC-6 (A) and HeLa (B) cells. Colored images (column 1), staining for STAT2 (green) and beta -COP (red) superimposed; black-and-white images, staining for STAT2 (column 2) and beta -COP (column 3) separately in the same cell. Bar = 15 µm.

Effect of IFN on STAT1 and STAT2 Distribution

Because IFN activates and translocates both STAT1 and STAT2 to the nuclei of cells of other cell types, we examined the effects of IFN in IEC-6 cells. As shown in Fig. 4, 30 min of incubation with IFN resulted in a dramatic translocation of both STAT1 and STAT2 to the nuclei of IEC-6 cells. The staining of STAT1 and STAT2 also intensified in the cytoplasm, and STAT2 staining intensified in the Golgi apparatus after incubation with IFN.


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Fig. 4.   Effect of interferon (IFN) on STAT1 (A and B) and STAT2 (C and D) distribution in IEC-6 cells. Cells were grown in control medium, and serum was removed on day 3. Fresh serumless medium with 1,000 U/ml of rat IFN-beta (B and D) or without rat IFN-beta (A and C) was added 24 h later, and incubation continued for 30 min. After fixation and immunocytochemistry, cells were imaged by confocal microscopy. Bar = 5 µm.

Effect of EGF on Tyrosine Phosphorylation of STAT1 and STAT2

Because we observed that EGF induced nuclear translocation of STAT2 in IEC-6 and HeLa cells, we examined whether EGF caused tyrosine phosphorylation of either STAT1 or STAT2. As shown in Fig. 5, EGF induced a marked increase in the tyrosine phosphorylation of several proteins in IEC-6 cells. Most notable among these was the EGFR (180 kDa). Phosphorylation of the EGFR increased dramatically at 2 min and remained elevated at 30 min. EGF also induced the tyrosine phosphorylation of STAT1 (91 kDa) within 2 min, and low levels of tyrosine-phosphorylated STAT1 were still detectable after a 30-min incubation with EGF. In contrast, EGF had no effect on the tyrosine phosphorylation of STAT2 (113 kDa) in IEC-6 cells after either 2 or 30 min of exposure (Fig. 5).


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Fig. 5.   Representative Western blots of EGF receptor (EGFR), STAT1, and STAT2 phosphorylation in IEC-6 cells incubated for 0, 2, or 30 min with 20 ng/ml EGF or for 15 min with 1,000 U/ml rat IFN-beta . Samples were immunoprecipitated with anti-phosphotyrosine (anti-pTyr) antibody, collected, run on SDS-7.5% PAGE gel, transferred to polyvinylidene difluoride membranes, and probed with pTyr, STAT1, or STAT2 antibody followed by anti-rabbit IgG coupled to horseradish peroxidase. Blots were developed by enhanced chemiluminescence. Nos. at left are molecular masses in kDa.

In HeLa cells, although EGF induced the tyrosine phosphorylation of the EGFR, it did not lead to the tyrosine phosphorylation of either STAT1 or STAT2 (Fig. 6). The failure to detect STAT2 phosphorylation in response to EGF in either cell type and STAT1 phosphorylation in HeLa cells was not due to a general inability to detect cytokine-induced STAT2 tyrosine phosphorylation, inasmuch as IFN treatment of either IEC-6 or HeLa cells induced tyrosine phosphorylation of STAT2 (Figs. 5 and 6). IFN also induced tyrosine phosphorylation of STAT1 in both cell types.


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Fig. 6.   Representative Western blots of EGFR, STAT1, and STAT2 phosphorylation in HeLa cells. Procedure was identical to that described in legend for Fig. 5, except that human consensus IFN (IFN con1) was used instead of rat IFN-beta . Nos. at left are molecular masses in kDa.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

We were interested in learning about the effect of EGF on STAT1 and STAT2 localization and activation in intestinal epithelial cells. In fact, to our knowledge there are no reports of STAT activation and localization in gut epithelial cells and very few published studies on the immunocytochemical localization of STATs in other cell types. Using immunofluorescence, Schindler et al. (18) demonstrated the translocation of STAT1 and STAT2 proteins from the cytoplasm to the nucleus 30 min after treatment with IFN-alpha in both HeLa and human FS2 cells, a finding we reproduced with IEC-6 cells (Fig. 4). Most investigators, however, have measured the translocation of activated STATs to the nucleus by immunoprecipitation of 32P-labeled proteins from nuclear extracts or by induction of tyrosine phosphorylation by immunoblotting (8, 20). Thus if a protein is not phosphorylated, it will not be detected.

A summary of our immunocytochemical results is shown in Table 1. In IEC-6 cells, STAT1 was cytoplasmic and, after EGF treatment, moved to the nucleus and the Golgi, as well as intensifying in the cytoplasm. Because EGF caused the tyrosine phosphorylation of STAT1 in IEC-6 cells (Fig. 5), the translocation of STAT1 to the nucleus fits the standard paradigm. Before treatment with EGF, STAT2 was found to be heavily stained in the cytoplasm and Golgi apparatus of IEC-6 cells. EGF produced a marked translocation of STAT2 to the nucleus, its disappearance from the Golgi at 2 min, and its reappearance after 30 min. There was no evidence that EGF caused the tyrosine phosphorylation of STAT2. One of the major unexpected findings among these data is that STAT2 translocated to the nuclei of IEC-6 cells without being tyrosine phosphorylated after exposure to EGF.

                              
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Table 1.   Distribution of STAT1 and STAT2 in IEC-6 cells and HeLa cells before and after stimulation with EGF

The effects of EGF on IEC-6 cells are essentially the same as its effects on HeLa cells. In IEC-6 cells EGF activates MAP kinase and increases the incorporation of [3H]thymidine into DNA (11). Long-term treatment of IEC-6 cultures with EGF results in a significant decrease in doubling time and a significant increase in cell number (10). In HeLa cells EGF also has a strong mitogenic effect (3). Thus we compared the effect of EGF on the responses of STAT1 and STAT2 in IEC-6 cells with its effect on these responses in HeLa cells. As has been reported by Chin et al. (3), EGF did not induce the tyrosine phosphorylation of either STAT1 or STAT2 in HeLa cells. Surprisingly, however, although EGF had no effect on STAT1 localization, it had a pronounced effect on the localization of STAT2. Within 2 min of exposure to EGF, there was a dramatic relocalization of STAT2 to the nucleus and an intensification in the cytoplasm of HeLa cells (Fig. 2). By 30 min STAT2 staining had returned to near-control levels, although the nuclei retained an increased level of staining. Thus in both cell types EGF caused the translocation of STAT2 to the nucleus without inducing its tyrosine phosphorylation. In both cell types, IFN-alpha and -beta caused the tyrosine phosphorylation of STAT2 and STAT1 (Figs. 5 and 6). As has previously been shown for HeLa cells (12), IFN treatment resulted in the translocation of both STAT1 and STAT2 to the nuclei of IEC-6 cells (Fig. 4).

These data raise the question of whether STAT2 can act in a transcription factor complex for EGF-induced gene expression without being tyrosine phosphorylated. STAT2 is the only member of the STAT family of transcription factors without a DNA binding domain and, therefore, can act as a transcription factor only when complexed to another STAT or protein. Kumar et al. (9) have recently answered this question for STAT1 by using human fibrosarcoma cells and mutant cells that lack STAT1 expression. STAT1 is necessary for tumor necrosis factor-induced expression of caspase and for apoptosis. The formation of STAT1 dimers requires the phosphorylation of Tyr-701. Cells that expressed a mutated form of STAT1 in which Tyr-701 was converted to Phe-701, and thus that were unable to form STAT1 homodimers, nevertheless were sensitive to apoptosis stimulated by tumor necrosis factor and actinomycin D (9). Whereas STAT1 is activated by a variety of agents (IFNs, cytokines, and growth factors), the only known activators of STAT2 are IFN-alpha and -beta (12). Whether the translocation of STAT2 to the nucleus in IEC-6 and HeLa cells induced by EGF is true activation in the absence of tyrosine phosphorylation is not known, but in any case, this is a clear effect of a ligand other than IFN-alpha and -beta on STAT2.

Another interesting result from our study was the colocalization of STAT1 and STAT2 in the Golgi apparatus of IEC-6 cells but not HeLa cells. To our knowledge this is the first report of the localization of STATs to the Golgi. This finding was confirmed by showing that STAT2 colocalized with beta -COP. beta -COP has been localized to coated Golgi-derived vesicles and is necessary for vesicular transport between Golgi compartments (16). The significance of finding STAT1 and STAT2 in the Golgi apparatus of IEC-6 cells remains to be determined.

The studies reported here are the first to describe 1) the immunocytochemical localization of STATs in intestinal epithelial cells and 2) the effects of EGF on the tyrosine phosphorylation and localization of STAT1 and STAT2 in these cells. Each of our novel findings raises an important question for further investigation. First, since STAT1 was tyrosine phosphorylated in IEC-6 cells after exposure to EGF, does STAT1 have a signaling function in these cells? Second, the localization of both STAT1 and STAT2 to the Golgi apparatus of IEC-6, but not HeLa, cells emphasizes the variability of STATs in different cell types. More important, however, is the question of whether these STATs have a function in the Golgi. Third, the dramatic translocation of STAT2 to the nuclei of cells of both cell types stimulated by EGF is the first effect on STAT2 to be described for any ligand other than IFN-alpha and -beta . This raises the question of whether STAT2 mediates a nuclear response to EGF. Fourth, since EGF caused the translocation of STAT2 to the nucleus without its tyrosine phosphorylation, what pathway is involved in moving the STAT from the cytoplasm to the nucleus?


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 (L. R. Johnson) and National Cancer Institute Grant CA-73753 (L. M. Pfeffer).


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests: L. R. Johnson, Dept. of Physiology and Biophysics, Univ. of Tennessee, 894 Union Ave., Memphis, TN 38163.

Received 20 July 1998; accepted in final form 26 October 1998.


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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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