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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 306-313, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.4850

Neuregulin-1 Activates the JAK-STAT Pathway and Regulates Lung Epithelial Cell Proliferation

Jinbo Liu and Jeffrey A. Kern

Department of Internal Medicine, Pulmonary and Critical Care Division, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, Ohio

Address correspondence to: Jeffrey A. Kern, M.D., Department of Medicine, Pulmonary and Critical Care Division, University Hospital of Cleveland, Wearn 610, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail: Jeffrey.Kern{at}UHHS.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuregulin-1 (NRG-1) is part of a family of proteins whose members are structurally related to epidermal growth factor. NRG-1 induces cell proliferation through a high-affinity receptor complex composed of a heterodimer of human epidermal growth factor–like receptor (HER) 2 and 3. In this study, we show that NRG-1 activates the Janus kinases (JAK) and signal transducer and activator of transcription proteins (STAT). NRG-1 induced a rapid and transient increase in tyrosine phosphorylation of TYK2 and JAK3, but not JAK1 or JAK2, and induced STAT3 and STAT5 tyrosine phosphorylation. Upon phosphorylation, STAT3 translocated to the nucleus within 1 h. Activation of the JAK-STAT pathway was dependent on HER2/HER3 heterodimerization and was necessary for NRG-1–induced proliferation. Inhibition of HER2's ability to dimerize using the HER2-specific antibody 2C4 completely blocked NRG-1–induced JAK3, TYK2, STAT3, and STAT5 tyrosine phosphorylation. Blocking the JAK-STAT pathway with a specific JAK-STAT pathway inhibitor, AG490, inhibited NRG-1–induced JAK and STAT phosphorylation and cell proliferation. These data suggest that NRG-1 activates the JAK-STAT signal transduction pathway through its high-affinity receptor, the HER2/HER3 heterodimer. This pathway plays an important role in NRG-1–stimulated proliferation of pulmonary epithelial cells.

Abbreviations: human anti-HER2 antibody, 2C4 • Dulbecco's modified Eagle's medium, DMEM • dimethyl sulfoxide, DMSO • epidermal growth factor, EGF • extracellular-regulated kinase, ERK • fetal calf serum, FCS • human EGF-like receptor, HER • Janus kinase, JAK • neu differentiation factor, NDF • neureglin-1, NRG-1 • phosphate-buffered saline, PBS • platelet-derived growth factor receptor, PDGFR • phosphoinositide 3-kinase, PI3K • receptor tyrosine kinase, RTK • signal transducer and activator of transcription protein, STAT • sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE • transforming growth factor, TGF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
There are four members of the Neuregulin (NRG) family: NRG-1 (1), NRG-2 (2), NRG-3 (3), and NRG-4 (4), which are all structurally related to epidermal growth factor (EGF) (5). NRGs act as specific activating ligands for the human EGF-like receptor (HER) family. NRG-induced receptor activation results in multiple biologic activities in epithelial cells, and NRGs act as mitogens, differentiation agents, and transforming agents (6–8). The epidermal growth factor receptor (EGFR or HER1) is the prototypical member of this family of receptors, which also includes HER2 (neu), HER3, and HER4 (9–12). Structurally, the HER proteins consist of an extracellular ligand-binding domain, a transmembrane-spanning segment, an intracellular tyrosine kinase (TK) domain, and a C-terminal region. When comparing all members of the HER family, the C-terminus is the most divergent region, suggesting that it encodes signaling information unique to each receptor protein. This region contains multiple tyrosines, which upon phosphorylation provides docking sites for signal transducer proteins such as GRB2 and SHC (13, 14). The HER receptor complex is activated via a process of receptor homo- and heterodimerization, which is initiated by engagement of ligand with the HER extracellular domain. Receptor activation results in auto- and transphosphorylation, with subsequent signaling through the phosphoinositide 3-kinase (PI3K) (15) and extracellular-regulated kinase (ERK) (16) pathways. With multiple activating ligands and dimeric receptor protein combinations possible, signaling through this receptor/ligand family is diverse (17).

Growth factor receptor tyrosine kinases have also been shown to couple to the Janus kinase (JAK) and signal transducer and activators of transcription (STAT) pathway (18). JAKs are one of eleven mammalian nonreceptor tyrosine kinase families that are essential mediators of cellular signaling through cytokine receptors (19). There are four members of the JAK family: JAK1, JAK2, JAK3, and TYK2, whereas seven STAT proteins have been identified in mammalian cells. Following receptor activation by ligand engagement, the receptor-associated JAKs are induced to autophosphorylate and transphosphorylate tyrosine residues within the receptor's cytoplasmic domain. The newly phosphorylated receptor sites now serve as docking sites for the recruitment of inactive cytoplasmic STAT monomers through interaction with a STAT's SH2 domain. JAK-mediated phosphorylation of an invariant tyrosine residue on the receptor-bound STAT monomer induces STAT homodimerization, with resultant STAT activation. The activated STAT dimers then translocate to the nucleus, where they bind to specific DNA-response elements in the promoters of target genes and thereby induce unique gene expression programs (20). In contrast to typical cytokine receptor signaling, activated growth factor receptors such as EGFR and platelet-derived growth factor receptor (PDGFR) with intrinsic tyrosine kinase activity may bypass JAK activation and directly phosphorylate STAT proteins (18). The functional consequence of activation of the STAT pathway varies. STAT1 plays an important role in growth arrest and in promoting apoptosis. As such, it has been implicated as a tumor suppressor protein (21–23). The activation of STAT3 and STAT5 contributes to malignant progression by stimulating cell proliferation (24, 25) and regulating the expression of genes known to be involved in cell cycle control (26) and preventing apoptosis (27, 28).

Previous work from our laboratory has identified HER proteins in the human lung epithelium (29) and epithelial-derived lung cancers (30–34). In the human lung, NRG-1–induced signal transduction appears to occur mainly through HER2/HER3 heterodimer formation. In addition, NRG-1 has also been identified in lung epithelium, suggesting autocrine regulation of epithelial cell growth and differentiation. With the known ability of STATs to be activated by receptor tyrosine kinases (35–37) and their role in regulating cellular proliferation, we hypothesized that NRG-1 would activate the JAK-STAT pathway through its high-affinity receptor and regulate pulmonary epithelial cell growth.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
AG490 was purchased from Calbiochem (La Jolla, CA). JAK1, JAK2, JAK3, TYK2, STAT1, STAT3, STAT5, and STAT6 polyclonal antibodies, and anti-phosphotyrosine monoclonal antibody were purchased from Santa Cruz Biotechnology (San Diego, CA). CellTiter 96 Aqueous One Solution Cell Proliferation Assay was obtained from Promega (Madison, WI). Sepharose 4B protein A beads, and BrdU labeling agent and staining kits were obtained from Zymed (San Francisco, CA). NRG-1177–244, antibody 2C4, and an isotype-matched human monoclonal control antibody (hmAb) were gifts from Dr. Mark Sliwkowski (Genentech, Inc., South San Francisco, CA).

Cell Culture
NCI-H441 and NCI-H520 cell lines were obtained from American Type Culture Collection (Manassas, VA). NCI-H441 (ATCC HTB-174) was derived from a human lung epithelial adenocarcinoma. NCI-H520 (ATCC HTB-182) was derived from a human lung squamous cancer. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and HAM's F-12 (1:1) supplemented with 10% (vol/vol) fetal calf serum (FCS), 1% glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified 95% air/5% CO2 atmosphere. All experiments were conducted using cells growth-arrested by incubation in media without FCS for 24 h.

Cell Proliferation Assay
Cells were plated in 96-well plates (5,000 cells/well) and grown in DMEM/HAM's F-12 supplemented with 10% FCS for 24 h. Cells were growth-arrested by incubation in serum-free media for 24 h, NRG-1177–244 (10-8 M), and/or AG490 (10–100 µM) was added and the culture was incubated for 3 d. CellTiter 96 Aqueous One Solution Reagent (20 µl) was added to each well. The plates were incubated at 37°C for 1–4 h and absorbance recorded at 490 nm using a 96-well plate reader.

Fluorescence Microscopy
Cells were grown in DMEM/HAM's F-12 medium on 60-mm2 dishes with sterile glass microscope coverslips. After 24 h, cells were growth-arrested by incubation in serum-free media for 48 h. Cells were stimulated with 10-8 M NRG-1177–244 for the indicated time points, washed three times with phosphate-buffered saline (PBS), and fixed in methanol (-10°C; 5 min). Cells were then blocked in 1% bovine serum albumin/PBS for 1 h followed by primary antibody (1 µg/ml) for 2 h. Cells were washed three times in PBS and incubated with fluorescein-conjugated secondary antibody (1:1,000; 1 h), followed by extensive washing with PBS. Nonquenching mounting medium (Vector, Burlingame, CA) was applied to the slide and the coverslip mounted.

Immunoprecipitation and Western Blotting
Growth-arrested NCI-H441 or NCI-H520 cells were treated with 10-8 M NRG-1177–244 in the presence or absence of 2C4 antibody (50 µg/ml) at 10-7 M, an isotype matched control antibody (50 µg/ml), or dimethyl sulfoxide (DMSO) (1:1,000 final dilution) for indicated time points. Cells were lysed in buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml pepstatin, 0.4 mM EDTA, 0.4 mM NaVO4, 10 mM NaFl, and 10 mM Na pyrophosphate). For immunoprecipitation, cell lysates containing 200 µg of protein were incubated with antibody (2 µg, 30 min, 4°C). The antibody–protein complexes were then incubated with protein A beads for 1 h at 4°C with mixing. The beads were washed three times in PBS and resuspended in Laemmli sample buffer. Samples were loaded on a 4–20% or a 7.5% SDS-PAGE gel. In experiments using the JAK inhibitor AG490, growth-arrested NCI-H520 cells were pretreated with AG490 for 16 h, then NRG-1177–244 (10-8 M) added. Cell lysis, immunoprecipitation, and Western Blot analysis were as described above. AG490 was dissolved in DMSO. Images were digitized and quantified using NIH Image software.

BrdU Labeling
NCI H441 and NCI H520 cells were cultured overnight on 2-well chamber slides. Cells were growth-arrested by incubation in serum-free media for 24 h. Arrested cells were treated with NRG-1 (10-8 M) and/or AG490 (50 µM) for 24 h. BrdU labeling and staining was performed according to the manufacturer's recommendation (Zymed). Cells were incubated at 37°C for 2 h in BrdU labeling reagent (1:100 dilution), fixed in 70% ETOH (4°C; 30 min), denatured, and nonspecific binding sites blocked. Anti-BrdU antibody was applied, followed by streptavidin-peroxidase, and visualized with diaminobenzidine. Cells were counterstained with Hematoxylin and coverslips applied with Histomount medium.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NRG-1177–244 Stimulates JAK-STAT Tyrosine Phosphorylation in Human Lung Epithelial Cells
Our first experiment sought to determine if NRG-1 would activate the JAK-STAT pathway. Growth-arrested pulmonary epithelial cell lines NCI-H520 and NCI-H441 were treated with NRG-1177–244 (10-8 M) for 10 min. NRG-1177–244 is a recombinant protein consisting of the 50 amino acids spanning the NRG-1 EGF-like domain. All bioactivities of NRG-1 are a function of this domain (1, 38, 39). JAK1, JAK2, JAK3, TYK2, STAT1, STAT3, STAT5, and STAT6 were individually immunoprecipitated from cell lysates. Immunoprecipitated protein was analyzed by Western blotting for changes in phosphotyrosine content as a measure of activation. As shown in Figures 1 and 2 , NRG-1 177–244 resulted in increased tyrosine phosphorylation of specific JAKs and STATs in both cell lines. Tyrosine phosphorylation of JAK3 and TYK2 was significantly elevated by NRG-1177–244. Basal tyrosine phosphorylation of JAK1 and JAK2 was present, but no changes in JAK1 or JAK2 phosphorylation were induced by NRG-1177–244. Along with activation of JAK3 and TYK2, activation of STAT3 and STAT5 was also seen. Basal activation of STAT1 and STAT6 was present, but STAT1 and STAT6 phosphorylation did not increase with NRG-1177–244 exposure. Basal JAK-STAT activation has been reported in lung, head, neck, and breast carcinoma, and most likely effects the high proliferation rates of these cell lines (40–42). STAT6 phosphorylation decreased slightly with NRG-1177–244 exposure in H520 but not H441. As this result was not present across all cell lines, it was not studied further. In both cell lines, the pattern and degree of JAK and STAT activation were similar. The blots were stripped or duplicate blots were probed for JAK and STAT expression to ensure equal loading of protein (Figures 1 and 2). JAK-STAT protein expression was not changed by NRG-1177–244 during the 10-min exposure.



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  		Figure 1. NRG-1177–244 induces JAK-STAT phosphorylation in the human lung epithelial cell NCI-H520. Cells were growth-arrested for 24 h and stimulated with 10-8 M NRG-1177–244 for 10 min. Cells were lysed and equal amounts of protein were immunoprecipitated with JAK1, JAK2, JAK3, TYK2, STAT1, STAT3, STAT5, and STAT6. Western blot analysis of immunoprecipitated protein was performed with an antiphosphotyrosine antibody. Duplicate blots or identical blots were stripped and probed for JAK or STAT to ensure equal protein loading. The blot is representative of three experiments.

 


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  		Figure 2. NRG-1177–244 induces JAK-STAT phosphorylation in the human lung epithelial line NCI-H441. Cells were growth-arrested for 24 h and stimulated with 10-8 M NRG-1177–244 for 10 min. Cells were lysed and equal amounts of protein were immunoprecipitated with JAK1, JAK2, JAK3, TYK2, STAT1, STAT3, STAT5, and STAT6. Western blot analysis of immunoprecipitated protein was performed with an antiphosphotyrosine antibody. Duplicate blots or identical blots were stripped and probed for JAK or STAT to ensure equal protein loading. The blot is representative of three experiments.

 
NRG-1177–244–Induced Tyrosine Phosphorylation of JAK and STAT Is Time-Dependent
To confirm the kinetics of NRG-1177–244–induced JAK-STAT tyrosine phosphorylation, we exposed cell line NCI-H520 cells to NRG-1177–244 (10-8 M), harvested the cells at various time points, and examined JAK3, TYK2, STAT3, and STAT5 for changes in phosphorylation. Maximal phosphorylation of JAK3 occurred 10 min after NRG-1177–244 exposure, resulting in a 7-fold increase in phosphotyrosine content. The phosphorylation rapidly decreased to basal levels (Figures 3A and 3B) . Tyrosine phosphorylation of TYK2 followed a similar pattern, with peak phosphorylation occurring 10 min after NRG-1177–244 exposure, resulting in a 14-fold increase in phosphotyrosine content. This decreased to baseline over the next 20 min (Figures 3C and 3D). Maximal tyrosine phosphorylation occurred at 5 min after NRG-1177–244 exposure for both STAT3 and STAT5. NRG-1177–244 stimulation resulted in a 13-fold increase in STAT3 phosphorylation, which returned to near baseline within 15 min (Figures 4A and 4B) . STAT5 phosphorylation was also maximal at 5 min, and increased 8-fold from resting levels. STAT5 phosphorylation remained elevated 4- to 5-fold over resting level throughout the 2-h NRG-1177–244 exposure (Figures 4C and 4D). These results indicate that NRG-1–induced activation of JAK and STAT is time-dependent and transient.



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  		Figure 3. Kinetics of NRG-1177–244–induced JAK3 and TYK2 tyrosine phosphorylation. The NCI-H520 cell line was growth-arrested in serum-free medium for 24 h and exposed to NRG-1177–244 (10-8 M) for the indicated times. Cells were harvested, lysed, and equal amounts of protein were immunoprecipitated with JAK3 (A) or TYK2 (C) antibody. Immunoprecipitated protein was subjected to Western blot analysis with an antiphosphotyrosine antibody. The blots were stripped or duplicate blots were probed for JAK3 or TYK2 to verify equal protein loading. Phosphorylation content was quantified and plotted versus time for JAK3 (B) and TYK2 (D). Results of the blots are graphed as mean ± SD. Results are representative of two experiments. The single asterisk represents a significant difference when compared with untreated cells (Time = 0) (P < 0.01), and the double asterisk represents a significant difference compared with NRG-1 treatment at 10 min (Time = 10) (P < 0.01).

 


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  		Figure 4. NRG-1177–244–induced STAT3 and STAT5 tyrosine phosphorylation is time-dependent. The NCI-520 cell line was growth-arrested in serum-free medium for 24 h and exposed to NRG-1177–244 (10-8 M) for the indicated times. Cells were harvested, lysed, and equal amounts of protein were immunoprecipitated with STAT3 (A) or STAT5 (C) antibody. Immunoprecipitated protein was subjected to Western blot analysis with an antiphosphotyrosine antibody. The blots were stripped or duplicate blots were probed for STAT3 or STAT5 to verify equal protein loading. Phosphorylation content was quantified and plotted versus time for STAT3 (B) and STAT5 (D). Results are graphed as mean ± SD. Results are representative of two experiments. The single asterisk represents a significant difference when compared with untreated cells (Time = 0) (P < 0.01), and the double asterisk represents a significant difference compared with NRG-1 treatment at 5 min (Time = 5) (P < 0.01).

 
HER2 Is Required for NRG-1177–244 Activation of the JAK-STAT Pathway
The high-affinity NRG-1 receptor consists of an HER2/HER3 heterodimer. To determine if the NRG-1–induced changes were mediated though the HER2/HER3 complex, we reasoned that inhibition of HER2/HER3 dimerization would inhibit NRG-1177–244–induced JAK-STAT activation. To inhibit HER2/HER3 dimerization, we used the antibody 2C4. 2C4 is a humanized antibody, derived from the monoclonal antibody 4D5 (43). This antibody is HER2-specific, inhibits HER2 dimerization with any other HER member, and abrogates NRG-1177–244–induced receptor activation. Both cell lines were exposed to 2C4 followed by NRG-1177–244. JAK3, TYK2, STAT3, and STAT5 were then immunoprecipitated and analyzed for tyrosine phosphorylation. As anticipated, 2C4 completely blocked NRG-1–induced phosphorylation of JAK3, TYK2, STAT3, and STAT5 (Figures 5A and 6) , whereas the control antibody had no effect (Figure 5B). These results confirmed that HER2 is required for NRG-1177–244 activation of the JAK-STAT pathway.



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  		Figure 5. HER2 is required for NRG-1177–244 activation of the JAK-STAT pathway in the NCI-H520 cell line. The growth-arrested cell line NCI-H520 was pretreated with (A) 2C4 (50 µg/ml for 30 min) or (B) an isotype-matched control antibody (50 µg/ml for 30 min) and then stimulated with NRG-1177–244 (10-8 M) for 10 min. JAK3, TYK2, STAT3, and STAT5 were immunoprecipitated from cell lysates containing equal amounts of protein and analyzed by Western blotting for phosphotyrosine content. Blots were stripped or duplicate blots were probed for JAK3, TYK2, STAT3, or STAT5 to ensure equal amount of protein. Results are representative of two experiments.

 


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  		Figure 6. HER2 is required for NRG-1177–244 activation of the JAK-STAT pathway in the NCI-H441 cell line. The growth-arrested human lung epithelial cell line NCI-H441 was pretreated with 2C4 (10-7M) for 30 min and then stimulated with NRG-1177–244 (10-8M) for 10 min. JAK 3, TYK2, STAT 3 and STAT5 were immunoprecipitated from cell lysates containing equal amounts of protein and analyzed by Western blotting for phosphotyrosine content. Blots were stripped or duplicate blots were probed for JAK3, TYK2, STAT3 or STAT5 to ensure equal amount of protein. Results are representative of two experiments.

 
NRG-1 Induces Nuclear Translocation of STAT3 and STAT5
Upon STAT activation and dimerization, the phosphorylated homodimer translocates to the nucleus and induces transcription. To confirm STAT3 and STAT5 translocation after NRG-1177–244 stimulation, we followed their cellular location by immunofluoresence. NCI-H441 and NCI-H520 cells were stimulated with NRG-1177–244 (10-8 M), and stained with corresponding STAT antibodies, followed by a FITC-conjugated secondary antibody (Figure 7) . Translocation of STAT3 and STAT5 in response to NRG-1177–244 was seen at 10 min. STAT3 began to move to the nucleus and cytoplasmic staining became less dense. By 30 min, STAT3 was aggregated in the cytoplasm, giving a granular appearance. At 60 min, nuclear staining for STAT3 was more prominent than cytoplasmic staining. Changes in the STAT5 staining pattern were similar, but less pronounced than STAT3 (data not shown). These data are consistent with our Western blot data, which showed tyrosine phosphorylation changes in STAT5 that were less remarkable than that of STAT3. The translocation kinetics of STAT3 are consistent with the results of Fernandes and coworkers, with nuclear accumulation occurring 2 h after a rapid cytoplasmic phosphorylation event (40).



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  		Figure 7. NRG-1177–244 induces nuclear translocation of STAT3. NCI-H520 (A) and NCI-H441 (B) cells were grown on glass coverslips and growth-arrested for 48 h. The cells were stimulated with NRG-1177–244 (10-8 M) at 10 min, 30 min, and 1 h, and stained for STAT3, following a FITC-conjugated secondary antibody (green). Nuclei were stained with DAPI.

 
AG490 Inhibits NRG-1177–244–Induced Proliferation
In other epithelial cell lines, activation of the JAK-STAT pathway results in cell proliferation and differentiation (15, 16, 44). We hypothesized that inhibition of NRG-1177–244–induced activation of the JAK-STAT pathway would result in decreased cell proliferation. To test this hypothesis, we used AG490, which has been reported to be a JAK-STAT pathway inhibitor (45). Growth-arrested NCI-H520 cells were cultured with 50 µM AG490 for 16 h, then exposed to 10-8 M NRG-1177–244 for 10 min. AG490 completely inhibited NRG-1177–244–induced tyrosine phosphorylation of TYK2 and JAK3, as well as STAT3 and STAT5 (Figure 8) . No effect was seen in cells pretreated with DMSO (AG490 diluent). With the definition of AG490's inhibition of the JAK-STAT pathway, we next determined its effect on cell proliferation. Growth-arrested NCI-H520 and NCI-H441 cells were cultured with 10-8 M NRG-1177–244 with or without AG490 at 10 µM, 50 µM, and 100 µM. AG490 significantly inhibited the NRG-1177–244–induced proliferation of NCI-H520 and NCI-H441 in a dose-dependent manner (Figure 9) . AG490 at 10 µM significantly decreased NRG-induced proliferation at 72 h by 26% in the NCI-H520 cells (P < 0.05) and 27% in NCI-H441 cells (P < 0.05). AG490 (50 µM) exposure decreased NRG-1–induced proliferation at 72 h by 38% (P < 0.01) in both cell lines. Exposure to 100 µM AG490 did not significantly decrease proliferation further. DMSO did not inhibit proliferation of NCI-H520 and NCI-H441 cell lines. Cell viability remained greater than 95% by Trypan blue exclusion, confirming that AG490 was not cytotoxic at the concentrations used in this experiment. These data confirm that cell proliferation is regulated in part through activation of the JAK-STAT pathway. To confirm the AG490 effect, proliferation studies were also performed using BrdU labeling. As shown in Table 1, NRG-1 significantly increased BrdU labeling in NCI-H520 and NCI-H441 (P < 0.05). Exposure to AG490 resulted in a significant inhibition of the NRG-1–induced BrdU labeling (P < 0.05).



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  		Figure 8. AG490 blocks NRG-1177–244–induced JAK-STAT activation. Growth-arrested NCI-H520 cells were pretreated with 50 µM AG490 in DMSO or DMSO alone for 16 h, and stimulated with 10-8 M NRG-1177–244 for 10 min. Cells were harvested, lysed, and lysates containing an equal amount of protein were immunoprecipitated with JAK3, TYK2, STAT3, and STAT5. Western blot analysis was performed with a phosphotyrosine antibody. The blots were stripped or duplicate blots were probed for JAK3, TYK2, STAT3, and STAT5 to ensure equal amounts of protein. Results shown are representative of two experiments.

 


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  		Figure 9. Blockade of the JAK-STAT pathway inhibits NRG-1177–244–induced proliferation. Growth-arrested NCI-H520 (A) and NCI-H441 (B) cell lines were stimulated withNRG-1 177–244 (10-8 M) with or without AG490 at 10 µM or 50 µM or DMSO (1:1,000 dilution). Cell proliferation was analyzed at 72 h with a modified MTT assay. Data represent the mean ± SD of three experiments, each with six replicates. The asterisk represents significant differences when compared with NRG-1177–244 treatment alone (P < 0.05).

 

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  		TABLE 1 Effect of NRG-1 and AG490 on proliferation

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A growth factor's interaction with its cell surface receptor activates several intracellular signaling pathways. The growth factor NRG-1 is known to activate the MAPK (16) and the PI3K (15) pathways through its high-affinity receptor, a HER2/HER3 heterodimer, affecting cell proliferation and survival. Recently growth factors such as EGF and PDGF have been also shown to activate JAK and STAT signaling molecules (36, 46–48). We show in these studies that NRG-1 also activates the JAK-STAT signal pathway as determined by increased JAK3, TYK2, STAT3, and STAT5 tyrosine phosphorylation, translocation of STAT3 and STAT5 to the nucleus, and increased cell proliferation dependent on JAK-STAT activation. NRG-1 had inconsistent effects on STAT6 phosphorylation in pulmonary epithelial cells. STAT6 role in regulation of proliferation, if any, is not clear but does not appear to be a universal process in pulmonary epithelial cells.

Olayioye and coworkers have reported on a preliminary evaluation of the JAK-STAT pathway by neu differentiation factor (NDF) (36). Analysis of an NIH3T3 cell line model transfected with specific HER proteins found that JAK and STAT proteins were complexed with the HER receptors, suggesting that activation of HER2, HER3, or HER4 may activate the JAK-STAT pathway. In their fibroblast cell line, exposure to NDF activated STAT5a and JAK2 and required HER4 expression. Our work identifies a different spectrum of JAK-STAT activation by NRG-1 in pulmonary epithelial cells and further shows the functional consequences of STAT pathway activation. NRG-1 activated JAK3, TYK2, STAT3, and STAT5 in the pulmonary epithelial cell line studied. In addition, activation of the JAK-STAT pathway was required for NRG-1–induced proliferation. Our identification of a prolonged activation of STAT5 in these cell lines mimics what Oliayioye and colleagues reported in their model. However, our work differs in their requirement of HER4 expression for STAT5 activation, as the cell lines studied in this report do not express HER4. Our studies have identified a central role for HER2 in JAK-STAT activation in pulmonary epithelial cells. Blocking the ability of HER2 to participate in NRG-1 receptor formation by inhibiting its ability to dimerize using the antibody 2C4 resulted in the loss of NRG-1–induced JAK-STAT activation. Fernandez and coworkers (40) also recognized the importance of HER2 in STAT activation. In transformed pulmonary epithelial cells with an EGFR/transforming growth factor (TGF)-{alpha} autocrine loop–driving proliferation, HER2 was required for TGF-{alpha}–induced activation of STAT3. Though HER2 is required for both TGF-{alpha} and NRG-1, the different ligands activate a different spectrum of JAK-STAT proteins in pulmonary epithelial cells. For example, EGF has been reported to activate STAT1, STAT3, and STAT5 (46, 49) as well as JAK2 (50), whereas NRG-1 activates STAT3, STAT5, JAK3, and TYK2. The difference in specific JAK and STAT activation and the different receptors required for activation suggests that there are both ligand- and tissue-specific requirements for JAK-STAT activation.

The mechanism of NRG-1–induced JAK-STAT tyrosine phosphorylation in these cells is not clear. The kinetics of JAK-STAT phosphorylation shown in our work does not suggest a linear JAK to STAT progression of phosphorylation. Receptor tyrosine kinases (TKs) have the ability to directly activate STATS or indirectly activate them through nonreceptor TKs such as c-src. This also occurs with HER proteins as exposure of HER2/HER4-transfected NIH 3T3 cells to NDF results in an increase in c-src complexed with the receptor (36). Thus, NRG-1–induced STAT activation may occur through any of these pathways: JAK activation, direct receptor phosphorylation, or indirectly through nonreceptor TKs.

The functional result of NRG-1–induced JAK-STAT activation in these cells is proliferation. Tyrphostin AG490 is a JAK-STAT pathway inhibitor (45). Regardless of the level of inhibition (JAK, STAT, or both), the use of this inhibitor clearly shows the importance of the pathway in NRG-1–induced proliferation. Exposure of the studied cell lines to AG490 reduced NRG-1–induced JAK as well as STAT activation, and had a profound effect on proliferation (51). AG490 abrogated the NRG-1–induced increase in proliferation, suggesting JAK-STAT is a significant regulatory pathway of NRG-1–induced proliferation in pulmonary epithelial cells. JAK-STAT activation has been reported in unregulated growth states such as lung cancer, and this is supported by the basal JAK-STAT tyrosine phosphorylation found in NCI-H520 and NCI-H441. In addition, the JAK-STAT pathway has been implicated in human lung fibroblast growth, as interferon-{alpha}–stimulated growth inhibition is mediated by activation of this pathway (52). The effects of this pathway on proliferation suggest that targeting the JAK-STAT pathway may be an effective strategy to control abnormal cell proliferation in lung cancer or pulmonary fibrosis.


   Acknowledgments
 
This work was supported by National Institutes of Health Grant HL-60156.

Received in original form February 25, 2002

Received in final form April 30, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Holmes, W. E., M. X. Sliwkowski, R. W. Akita, R.,W. J. Henzel, J. Lee, J. W. Park, D. Yansura, N. Abadi, H. Raab, and G. D. Lewis. 1992. Identification of heregulin, a specific activator of p185erbB2. Science 256:1205–1210.[Abstract/Free Full Text]
  2. Carraway, K. L., 3rd, J. L. Weber, M. J. Unger, J. Ledesma, N. Yu, M. Gassmann, and C. Lai. 1997. Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases. Nature 387:512–516.
  3. Zhang, D., M. X. Sliwkowski, M. Mark, G. Frantz, R. Akita, Y. Sun, K. Hillan, C. Crowley, J. Brush, and P. J. Godowski. 1997. Neuregulin-3 (NRG3): a novel neural tissue-enriched protein that binds and activates ErbB4. Proc. Natl. Acad. Sci. USA 94:9562–9567.[Abstract/Free Full Text]
  4. Harari, D., E. Tzahar, J. Romano, M. Shelly, J. H. Pierce, G. C. Andrews, and Y. Yarden. 1999. Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase. Oncogene 18:2681–2689.[Medline]
  5. Prigent, S. A., and N. R. Lemoine. 1992. The type 1 (EGFR-related) family of growth factor receptors and their ligands. Prog. Growth Factor Res. 4:1–24.[Medline]
  6. Hynes, N. E., and D. F. Stern. 1994. The biology of erbB-2/neu/HER-2 and its role in cancer. Biochim. Biophys. Acta 1198:165–184.[Medline]
  7. Dougall, W. C., X. Qian, N. C. Peterson, M. J. Miller, A. Samanta, and M. I. Greene. 1994. The neu-oncogene: signal transduction pathways, transformation mechanisms and evolving therapies. Oncogene 9:2109–2123.[Medline]
  8. Earp, H. S., T. L. Dawson, X. Li, and H. Yu. 1995. Heterodimerization and functional interaction between EGF receptor family members: a new signaling paradigm with implications for breast cancer research. Breast Cancer Res. Treat. 35:115–132.[Medline]
  9. Carraway, K. L. iii, and L. C. Cantley. 1994. A neu acquaintance for erbB3 and erbB4: a role for receptor heterodimerization in growth signaling. Cell 78:5–8.[Medline]
  10. Riese, D. J. ii, T. Komurasaki, G. D. Plowman, and D. F. Stern. 1998. Activation of ErbB4 by the bifunctional epidermal growth factor family hormone epiregulin is regulated by ErbB2. J. Biol. Chem. 273:11288–11294.[Abstract/Free Full Text]
  11. Riese, D. J. ii, and D. F. Stern. 1998. Specificity within the EGF family/ErbB receptor family signaling network. Bioessays 20:41–48.[Medline]
  12. Tzahar, E., J. D. Moyer, H. Waterman, E. G. Barbacci, J. Bao, G. Levkowitz, M. Shelly, S. Strano, R. Pinkas-Kramarski, J. H. Pierce, G. C. Andrews, and Y. Yarden. 1998. Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network. EMBO J. 17:5948–5963.[Medline]
  13. Schlessinger, J. 1993. How receptor tyrosine kinases activate Ras. Trends Biochem. Sci. 18:273–275.[Medline]
  14. Guy, P. M., J. V. Platko, L. C. Cantley, R. A. Cerione, and K. L. Carraway. 1994. Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. Proc. Natl. Acad. Sci. USA 91:8132–8136.[Abstract/Free Full Text]
  15. Tan, M., R. Grijalva, and D. Yu. 1999. Heregulin beta1-activated phosphatidylinositol 3-kinase enhances aggregation of MCF-7 breast cancer cells independent of extracellular signal-regulated kinase. Cancer Res. 59:1620–1625.[Abstract/Free Full Text]
  16. Lessor, T., J. Y. Yoo, M. Davis, and A. W. Hamburger. 1998. Regulation of heregulin beta1-induced differentiation in a human breast carcinoma cell line by the extracellular-regulated kinase (ERK) pathway. J. Cell. Biochem. 70:587–595.[Medline]
  17. Brennan, P. J., T. Kumogai, A. Berezov, R. Murali, and M. I. Greene. 2000. HER2/neu: mechanisms of dimerization/oligomerization. Oncogene 19: 6093–6101.[Medline]
  18. Bowman, T., R. Garcia, J. Turkson, and R. Jove. 2000. STATs in oncogenesis. Oncogene 19:2474–2488.[Medline]
  19. Aringer, M., A. Cheng, J. W. Nelson, M. Chen, C. Sudarshan, Y. J. Zhou, and J. O'Shea. 1999. Janus kinases and their role in growth and disease. Life Sci. 64:2173–2186.[Medline]
  20. Seidel, H. M., L. H. Milocco, P. Lamb, J. E. Darnell, Jr., R. B. Stein, and J. Rosen. 1995. Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity. Proc. Natl. Acad. Sci. USA 92: 3041–3045.[Abstract/Free Full Text]
  21. Bromberg, J. F., C. M. Horvath, Z. Wen, R. D. Schreiber, and J. E. Darnell, Jr. 1996. Transcriptionally active Stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma. Proc. Natl. Acad. Sci. USA 93:7673–7678.[Abstract/Free Full Text]
  22. Bromberg, J. F., Z. Fan, C. Brown, J. Mendelsohn, and J. E. Darnell, Jr. 1998. Epidermal growth factor-induced growth inhibition requires Stat1 activation. Cell Growth Differ. 9:505–512.[Abstract]
  23. Kaplan, D. H., V. Shankaran, A. S. Dighe, E. Stockert, M. Aguet, L. J. Old, and R. D. Schreiber. 1998. Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice. Proc. Natl. Acad. Sci. USA 95:7556–7561.[Abstract/Free Full Text]
  24. Bromberg, J. F., M. H. Wrzeszczynska, G. Devgan, Y. Zhao, R. G. Pestell, C. Albanese, and J. E. Darnell, Jr. 1999. Stat3 as an oncogene. Cell 98:295–303.[Medline]
  25. Matsumura, I., T. Kitamura, H. Wakao, H. Tanaka, K. Hashimoto, C. Albanese, J. Downward, R. G. Pestell, and Y. Kanakura. 1999. Transcriptional regulation of the cyclin D1 promoter by STAT5: its involvement in cytokine-dependent growth of hematopoietic cells. EMBO J. 18:1367–1377.[Medline]
  26. Levy, D. E., and D. G. Gilliland. 2000. Divergent roles of STAT1 and STAT5 in malignancy as revealed by gene disruptions in mice. Oncogene 19:2505–2510.[Medline]
  27. Karni, R., R. Jove, and A. Levitzki. 1999. Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors. Oncogene 18:4654–4662.[Medline]
  28. Dumon, S., S. C. Santos, F. Debierre-Grockiego, V. Gouilleux-Gruart, L. Cocault, C. Boucheron, P. Mollat, S. Gisselbrecht, and F. Gouilleux. 1999. IL-3 dependent regulation of Bcl-xL gene expression by STAT5 in a bone marrow derived cell line. Oncogene 18:4191–4199.[Medline]
  29. Patel, N. V., M. J. Acarregui, J. M. Snyder, J. M. Klein, M. X. Sliwkowski, and J. A. Kern. 2000. Neuregulin-1 and human epidermal growth factor receptors 2 and 3 play a role in human lung development in vitro. Am. J. Respir. Cell Mol. Biol. 22:432–440.[Abstract/Free Full Text]
  30. Weiner, D. B., J. Nordberg, R. Robinson, P. C. Nowell, A. Gazdar, M. I. Greene, W. V. Williams, J. A. Cohen, and J. A. Kern. 1990. Expression of the neu gene-encoded protein (P185neu) in human non-small cell carcinomas of the lung. Cancer Res. 50:421–425.[Abstract/Free Full Text]
  31. Kern, J. A., D. A. Schwartz, J. E. Nordberg, D. B. Weiner, M. I. Greene, L. Torney, and R. A. Robinson. 1990. p185neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res. 50:5184–5187.[Abstract/Free Full Text]
  32. Kern, J. A., R. A. Robinson, A. Gazdar, L. Torney, and D. B. Weiner. 1992. Mechanisms of p185HER2 expression in human non-small cell lung cancer cell lines. Am. J. Respir. Cell Mol. Biol. 6:359–363.
  33. Kern, J. A., L. Torney, D. Weiner, A. Gazdar, H. M. Shepard, and B. Fendly. 1993. Inhibition of human lung cancer cell line growth by an anti-p185HER2 antibody. Am. J. Respir. Cell Mol. Biol. 9:448–454.
  34. Kern, J. A., R. J. Slebos, B. Top, S. Rodenhuis, D. Lager, R. A. Robinson, D. Weiner, and D. A. Schwartz. 1994. C-erbB-2 expression and codon 12 K-ras mutations both predict shortened survival for patients with pulmonary adenocarcinomas. J. Clin. Invest. 93:516–520.
  35. Chaturvedi, P., S. Sharma, and E. P. Reddy. 1997. Abrogation of interleukin-3 dependence of myeloid cells by the v-src oncogene requires SH2 and SH3 domains which specify activation of STATs. Mol. Cell. Biol. 17:3295–3304.[Abstract]
  36. Olayioye, M. A., I. Beuvink, K. Horsch, J. M. Daly, and N. E. Hynes. 1999. ErbB receptor-induced activation of stat transcription factors is mediated by Src tyrosine kinases. J. Biol. Chem. 274:17209–17218.[Abstract/Free Full Text]
  37. Wang, Y. Z., W. Wharton, R. Garcia, A. Kraker, R. Jove, and W. J. Pledger. 2000. Activation of Stat3 preassembled with platelet-derived growth factor beta receptors requires Src kinase activity. Oncogene 19:2075–2085.[Medline]
  38. Carraway, K. L. iii, M. X. Sliwkowski, R. Akita, J. V. Platko, P. M. Guy, A. Nuijens, A. J. Diamonti, R. L. Vandlen, L. C. Cantley, and R. A. Cerione. 1994. The erbB3 gene product is a receptor for heregulin. J. Biol. Chem. 269:14303–14306.[Abstract/Free Full Text]
  39. Kita, Y., J. Mayer, T. Zamborelli, S. Hara, M. Rohde, E. Watson, R. Koski, B. Ratzkin, and M. Nicolson. 1995. Bioactive synthetic peptide of NDF/heregulin. Biochem. Biophys. Res. Commun. 210:441–451.[Medline]
  40. Fernandes, A., A. W. Hamburger, and B. I. Gerwin. 1999. ErbB-2 kinase is required for constitutive stat 3 activation in malignant human lung epithelial cells. Int. J. Cancer 83:564–570.[Medline]
  41. Grandis, J. R., S. D. Drenning, Q. Zeng, S. C. Watkins, M. F. Melhem, S. Endo, D. E. Johnson, L. Huang, Y. He, and J. D. Kim. 2000. Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proc. Natl. Acad. Sci. USA 97:4227–4232.[Abstract/Free Full Text]
  42. Garcia, R., C. L. Yu, A. Hudnall, R. Catlett, K. L. Nelson, T. Smithgall, D. J. Fujita, S. P. Ethier, and R. Jove. 1997. Constitutive activation of Stat3 in fibroblasts transformed by diverse oncoproteins and in breast carcinoma cells. Cell Growth Differ. 8:1267–1276.[Abstract]
  43. Carter, P., L. Presta, C. M. Gorman, J. B. Ridgway, D. Henner, W. L. Wong, A. M. Rowland, C. Kotts, M. E. Carver, and H. M. Shepard. 1992. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc. Natl. Acad. Sci. USA 89:4285–4289.[Abstract/Free Full Text]
  44. Xiong, S., R. Grijalva, L. Zhang, N. T. Nguyen, P. W. Pisters, R. E. Pollock, and D. Yu. 2001. Up-regulation of vascular endothelial growth factor in breast cancer cells by the heregulin-beta1-activated p38 signaling pathway enhances endothelial cell migration. Cancer Res. 61:1727–1732.[Abstract/Free Full Text]
  45. Nielsen, M., K. Kaltoft, M. Nordahl, C. Ropke, C. Geisler, T. Mustelin, P. Dobson, A. Svejgaard, and N. Odum. 1997. Constitutive activation of a slowly migrating isoform of Stat3 in mycosis fungoides: tyrphostin AG490 inhibits Stat3 activation and growth of mycosis fungoides tumor cell lines. Proc. Natl. Acad. Sci. USA 94:6764–6769.[Abstract/Free Full Text]
  46. David, M., L. Wong, R. Flavell, S. A. Thompson, A. Wells, A. C. Larner, and G. R. Johnson. 1996. STAT activation by epidermal growth factor (EGF) and amphiregulin: requirement for the EGF receptor kinase but not for tyrosine phosphorylation sites or JAK1. J. Biol. Chem. 271:9185–9188.[Abstract/Free Full Text]
  47. Ruff-Jamison, S., K. Chen, and S. Cohen. 1995. Epidermal growth factor induces the tyrosine phosphorylation and nuclear translocation of Stat 5 in mouse liver. Proc. Natl. Acad. Sci. USA 92:4215–4218.[Abstract/Free Full Text]
  48. Zhong, Z., Z. Wen, and J. E. Darnell, Jr. 1994. Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 264:95–98.[Abstract/Free Full Text]
  49. Deb, T. B., L. Su, L. Wong, E. Bonvini, A. Wells, M. David, and G. R. Johnson. 2001. Epidermal growth factor (EGF) receptor kinase-independent signaling by EGF. J. Biol. Chem. 276:15554–15560.[Abstract/Free Full Text]
  50. Nakamura, N., H. Chin, N. Miyasaka, and O. Miura. 1996. An epidermal growth factor receptor/Jak2 tyrosine kinase domain chimera induces tyrosine phosphorylation of Stat5 and transduces a growth signal in hematopoietic cells. J. Biol. Chem. 271:19483–19488.[Abstract/Free Full Text]
  51. Madamanchi, N. R., S. Li, C. Patterson, and M. S. Runge. 2001. Thrombin regulates vascular smooth muscle cell growth and heat shock proteins via the JAK-STAT pathway. J. Biol. Chem. 276:18915–18924.[Abstract/Free Full Text]
  52. Pansky, A., P. Hildebrand, M. H. Heim, M. Eberhard, T. Kissel, and C. Beglinger. 1998. Growth effects of alpha-interferon but not of bombesin or angiotensin II are mediated by activation of STAT proteins. Eur. J. Clin. Invest. 28:398–406.[Medline]



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