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Virology |
Department of Otolaryngology, Long Island Jewish Medical Center, The Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, New York 11040
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The EGFR is a member of the erbB family of receptor tyrosine kinases. erbB receptors and EGFR in particular are often overexpressed in malignancies and derivative cell lines compared to normal epithelium. The EGFR responds to EGF and other cognate ligands by activating an intrinsic tyrosine kinase that generates a signaling complex by creating phosphotyrosine nucleation sites for SH2-containing proteins. However, ligands that bind EGFR at its external binding site are not the only mechanism through which the EGFR may become activated. Activation of G protein-coupled receptors may also stimulate phosphorylation of the EGFR (6) . Activation of the EGFR results in a cascade of phosphorylations through the canonical ras-MAPK pathway affecting cell cycle regulation and differentiation (7) . A key step in this process is the activation of ERK 1 and 2 (referred to as MAPK) by dual phosphorylation on threonine and tyrosine (8 , 9) .
Immunohistochemical analysis suggests that the EGFR is overexpressed in laryngeal papillomas (5)
. Moreover, removal of EGF from the medium of cultured papilloma cells provokes morphological and biochemical differentiation (5)
. However, the mechanism of this overexpression and its consequent effects on MAPK have not been addressed. Here, we quantitated overexpression of the EGFR and showed that it is a consequence of receptor recycling rather than elevated gene expression. We also demonstrated that the EGFR tyrosine kinase is hyperresponsive to EGF stimulation in cultured papilloma cells and that the basal activation of MAPK is completely dependent on EGFR kinase. The synthesis of the endogenous ligand TGF-
by these cells could account for basal levels of MAPK activity. The overexpression of the EGFR, enhanced phosphotyrosine content, and elevated activated MAPK were all consistent features of papilloma tissue, confirming that these differences in cultured cells reflected in vivo alterations in signaling.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Scatchard Analysis.
Analysis was modified from Santos-Buch et al. (10)
. Briefly, cells were seeded at 50,000–80,000 per well in 96-well microculture dishes in KGM, cultured until they were 95% confluent, washed twice with ice-cold Krebs-Ringer binding buffer containing 10 mg/ml BSA, and then incubated for 1 h at 4°C with binding buffer containing 2 mg/ml BSA and increasing concentrations of [125I]EGF (Amersham, Arlington Heights, IL), ranging from 0.2 to 5 ng/ml. To control for nonspecific binding, parallel wells were incubated with 500-fold excess unlabeled EGF as well as iodinated EGF. Cells were washed twice with ice-cold PBS, lysed with 100 µl of 1 g/100 ml SDS, and lysates were counted in a scintillation counter. Specific binding was determined by correcting total binding for nonspecific binding, and results were analyzed as per Scatchard (11)
. Results are the average of three experiments with each cell type, with each experiment done in duplicate.
Southern Blot Assessment of EGFR Gene Amplification.
DNA from laryngeal papillomas and normal laryngeal tissues (9 µg each) was digested with EcoRI, electrophoresed through 8 mg/ml agarose gels, and transferred to nylon membranes. Blots were hybridized at 42°C with a 1000-bp EcoRI fragment of the human EGFR cDNA (pCO12-EGFR; Ref. 12
), labeled by random priming with 32P. To ensure that equal amounts of DNA were loaded in each lane, blots were probed simultaneously for the pro2(I) gene of human type I collagen (plasmid NJ3 3.55; Ref. 13
), a single-copy gene. Blots were washed stringently in 0.1x SSC/1% SDS at 68°C. Autoradiograms were prepared at -70°C using intensifying screens and Kodak X-Omat AR film.
Slot Blot Analysis of EGFR mRNA.
Normal and papilloma-derived laryngeal epithelial cells were seeded in KGM and mRNA isolated when the cells neared confluence (
4 days). mRNA (1 µg, on average) was slot-blotted onto membranes, hybridized at 42°C with a 32P-labeled 1838-bp EcoRI fragment of the EGFR cDNA, washed stringently (Tm, -20), autoradiographed, and quantified by densitometry. The EGFR probe was removed by boiling for 20 min in 10 mM Tris (pH 8), 1 mM EDTA, and 10 mg/ml SDS, and the blot was reprobed with a 32P-labeled 196-bp PstI fragment of the 36B4 cDNA, a housekeeping gene encoding the acidic ribosomal phosphoprotein P0 (14)
. mRNA levels of 36B4 are constant within cells, with the protein levels regulated posttranscriptionally (15)
. The EGFR mRNA values were normalized to the 36B4 values to compare the different samples. The EGFR mRNA autoradiogram needed to be exposed 20 times longer than the 36B4 autoradiogram.
Recycling of the EGFR.
Cells were plated in 16-mm wells, cultured until they were 95% confluent, incubated overnight in KGM without EGF, and then incubated for 90 min at 37°C in EGF-free KGM containing 100 ng/ml cycloheximide to prevent new EGFR synthesis. Initial binding assays were performed as described for Scatchard analysis, using 4 ng/ml [125I]EGF, to determine initial receptor levels. Parallel wells were then incubated with supplemented keratinocyte basal medium plus 100 ng/ml cycloheximide and 50 ng/ml EGF for 1 h and washed with PBS, and the noninternalized receptor was quantified in a subset of wells by [125I]EGF binding. After internalization, a wash, and 5 h of incubation in EGF-free KGM containing 100 ng/ml cycloheximide, recycled receptor was measured on cells in remaining wells by [125I]EGF binding. Internalization was defined as the reciprocal of the percentage of initial specific binding remaining at 1 h. Recycling of receptor was defined as the percentage of internalized receptor available for binding after 5 h incubation. Results are the means and SDs of two experiments with normal cells and three with papilloma cells, all from different patients. All experiments were performed in duplicate.
Immunofluorescent Detection of EGF.
Normal and papilloma-derived laryngeal epithelial cells were grown on glass coverslips in KGM. To monitor the internalization of EGFRs into endocytic vesicles, cells were incubated for 24 h in medium lacking EGF, 100 ng/ml EGF was then added, and EGFR internalization was monitored at 0 time and 1 h later using indirect immunofluorescence. Cells were fixed with cold acetone for 5 min at -20°C, incubated with a 1:10 dilution of a mouse monoclonal antibody that reacts with the extracellular domain of the human EGFR (clone 2E9; Caltag Laboratories, San Francisco, CA), and then incubated with fluorescein-conjugated second antibody.
Western Blots.
Cells were lysed by three cycles of dry ice freeze/ice thaw in lysis buffer [20 mM HEPES (pH 7.4), 137 mM NaCl, 2 mM EDTA, 10 µl/ml Triton X-100, 5 mg/ml deoxycholate, 1 mg/ml SDS, 0.1 ml/ml glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1.0 µM pepstatin, 50 µM NaF, 25 mM ß-glycerophosphate, and 1 mM NaVO4]. Nuclei remaining on the plate were lysed with Laemmli sample buffer (16)
to yield triton-insoluble extracts. Biopsy tissues were extracted by thawing snap-frozen specimens and sonicating in lysis buffer. Protein concentrations were determined by the bicinchoninic acid method (Pierce, Rockford, IL), and 20 µg of total protein were loaded per lane on polyacrylamide gels containing 0.1 g/ml SDS. Gels were electroblotted onto polyvinylidene difluoride membranes, and blots were stained with fast green (17)
to confirm equivalence in loading and transfer. Nonspecific binding to the filters was blocked by preincubating in TBS buffer [20 mM Tris (pH 7.4) and 9 mg/ml NaCl] plus 5% BSA, and the filters were incubated with primary antibodies for 1 h in TBS plus 0.05% Tween 20 and 1% BSA with shaking at room temperature, washed, and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Following extensive washing in TBS plus 0.05% Tween 20, reactive antigens were detected using chemiluminescence. Monoclonal antiphosphotyrosine, anti-EGFR (LA1), and anti-ERK-1 (all from Upstate Biotechnology Inc., Lake Placid, NY) were used at 1:1000 dilution. Polyclonal rabbit antibodies anti-EGFR (Calbiochem, San Diego, CA) and ERK-1 NT (Transduction Labs, Lexington, KY) were used at 1 µg/ml. Anti-activated MAPK (Promega, Madison, WI) was used at 1:10,000 dilution. Anti-TGF- (Santa Cruz Biotechnology, Santa Cruz, CA) was used at 200 ng/ml.
Inhibition by PD153035 and LA1.
Confluent monolayer cultures were incubated in KGM without EGF for 3 days and then incubated in the same medium containing either 1 µM PD153035 (Tocris Cookson, St. Louis, MO) for 30 min or 5 µg/ml LA1 (Upstate Biotechnologies Inc.) for 2 h. Control cultures were incubated with an irrelevant monoclonal antibody that recognizes TGF- (Santa Cruz) but does not inhibit binding of TGF- to the EGFR, at 5 µg/ml. Cultures were lysed as above for Western blot analysis.
EGFR Thresholds for Tyrosine Phosphorylation and MAPK Activation.
Cells were cultured in KGM until they were subconfluent; incubated in KGM without EGF for 3 days; pulsed for 10 min with 0, 0.1, 1, 10, or 100 ng/ml EGF; and analyzed by Western blot as described above.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Of a total of nine papilloma extracts probed for phosphotyrosine (data not shown), all had elevated levels when compared to extracts of normal laryngeal tissues. To determine whether the levels of phosphotyrosine found in papillomas correlated with changes in the abundance of the EGFR and the activation of MAPK, the Western blot was sequentially reprobed for the EGFR, for activated MAPK, and for total ERK-1 (Fig. 1)
. The responses indicated that, for a fixed level of ERK-1, the EGFR was more highly expressed in papillomas, phosphotyrosine content was radically elevated, and MAPK was activated to a greater extent than in normal laryngeal tissue. The apparent difference in molecular weight of the EGFR in lysates of laryngeal papillomas (Fig. 1
, Lanes 3 and 4) may reflect variation in phosphorylation or posttranslational modification of the EGFR among tissues from different patients.
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. Similar to the results found in tissue, the levels of the EGFR in four independent papilloma cultures were consistently higher than those found in uninfected laryngeal cells. We then asked whether the overexpression of the EGFR on papilloma cells could be confirmed with EGF binding studies, using monolayer cultures of normal and papilloma cells. This approach allowed quantitative determination of both receptor number and ligand affinity on the proliferative basal cell component. Scatchard analysis of the binding studies (Fig. 3)
confirmed that the papilloma cells expressed 3 times more high-affinity receptors on their surface than the cultured normal laryngeal keratinocytes but that the affinity of the receptors for EGF was the same (Kd, 1 nM).
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. Another possible explanation for EGFR overexpression was a defect in internalization or degradation of the EGFR in response to ligand binding, similar to that reported for HPV 16 E5-transfected keratinocytes (18)
. To test for a block in initial internalization, normal, and papilloma cells were starved of EGF to maximize surface receptor number. EGF was then replaced and internalization of receptor into endocytic vesicles was monitored by indirect immunofluorescence (Fig. 4)
. EGFRs were internalized into punctate intracytoplasmic organelles in both normal and papilloma-derived cells within 1 h of addition of ligand.
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. These experiments were performed in the presence of cycloheximide to prevent de novo receptor synthesis. They confirmed that both types of cells had essentially internalized all receptor 1 h after the EGF pulse but that HPV 11-infected papilloma cells recycled at least 20% of the EGFR within 5 h following ligand binding. Normal laryngeal cells recycled minimal EGFR. From these studies, we concluded that receptor recycling accounted for the increased number of EGFR on papilloma cells.
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. The response of primary uninfected laryngeal cells to 10 ng/ml EGF varied somewhat in intensity among different cultures, but the patterns were consistent. Evidence of EGF response in these cells was most apparent with phosphorylation of the Mr 180,000 protein and barely detectable phosphorylation of the Mr 65,000 protein. There was little or no change in the tyrosine phosphorylation of the Mr 42,000 protein in this culture. In contrast, the papilloma cells showed strong phosphorylation of the Mr 180,000, 65,000, and 55,000 proteins, and an increase in phosphorylation of the Mr 42,000 protein with 10 ng/ml EGF. They showed clearly detectable phosphorylation of the Mr 180,000 band at 1 ng/ml EGF, and phosphorylation of the Mr 65,000 band at 0.1 ng/ml.
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The papilloma tissues (see Fig. 1
) had shown an increase in both phosphotyrosine and activated MAPK when compared to normal tissue, suggesting a possible difference in threshold for MAPK activation by the in vivo endogenous ligands for the EGFR. Moreover, the increased tyrosine phosphorylation of the Mr 42,000 MAPK in papilloma cells, shown in Fig. 5
, supported this possibility. We, therefore, asked what concentration of EGF was required to activate MAPK in normal and papilloma cells (Fig. 6)
. Papilloma and normal cells were starved of exogenous growth factors for 3 days and then pulsed with increasing amounts of EGF. At 0.1 and 1 ng/ml EGF, the normal cells showed no increase in phosphorylation of the Mr 180,000 erbB family of proteins. In contrast, tyrosine phosphorylation was detectable in papilloma cells with 0.1 and 1 ng/ml EGF. Papilloma and normal cells both exhibited tyrosine phosphorylation with 10 ng/ml EGF.
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MAPK Activation Depends on the EGFR Kinase.
A basal level of MAPK activation was seen in both cell types when starved of EGF for 3 days. We, therefore, asked whether the basal levels were a result of EGFR activation. To address this question, we used PD153035, a specific inhibitor of the EGFR tyrosine kinase (20)
. This drug completely eliminated the basal level of activated MAPK in papilloma cells (Fig. 7)
. This was consistent with a dependence of basal activation of MAPK on the EGFR tyrosine kinase.
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, indicating that basal MAPK activation depends upon ligand occupancy of the EGFR.
Because LA1 blocked MAPK activation in both cell types, we asked whether ligands of the EGFR were produced by the cells. Western blots of Triton X-100-soluble extracts detected bands at the molecular weight of the TGF- precursor (Mr 8,000–12,000, depending on extent of posttranslational modification and partial cleavage) in both papilloma and normal tissues and cultured cells (Fig. 8)
. This was consistent with an autocrine mechanism for basal MAPK activation. There were interesting differences in the expression of TGF- between biopsy lysates and cultured cells. Although there was obvious quantitative variation on this blot, these were due, at least in part, to differences in protein loaded on the gel. We have presented these data as evidence of the presence of an endogenous EGFR ligand that could account for the LA1-inhibitable activity. They were not intended to be evidence of quantitative expression differences among these sample types. The variation in molecular weights of TGF- between biopsy lysates and cultured cells was, however, consistent enough to advance the speculation that growth of cells in culture affects the synthesis or processing of this protein. TGF- undergoes multiple modifications during its maturation, including glycosylation and proteolysis. It is possible that the reduced molecular weights of the TGF- forms seen in cultured cells are the result of a failure to glycosylate the peptide or a facile cleavage not catalyzed in intact tissues.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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as an endogenous product of laryngeal epithelium. Our cell culture experiments explain the elevation of the EGFR and the increased levels of phosphotyrosine and MAPK activation that are apparent in papillomas. The overexpression of the EGFR was due to posttranslational recycling of the EGFR and not to an enhancement of transcription. This response to HPV 6/11 infection is similar to the previously described effect of HPV 16 E5 protein on EGFR recycling in foreskin epithelial cells (17) . Cells transfected with HPV 16 E5 and HPV 6 E5a respond to EGF in clonogenic assays for transformation, and HPV 6 E5a has been shown to associate with both the EGFR and erbB-2 (22 , 23) . The low-risk HPV genomes in laryngeal papillomas exist as unintegrated episomes, and transcripts arising from the viral promoter region contain E5a-encoding sequences (24) . Thus, our data are consistent with elevation of the EGFR via an HPV 6- or HPV 11 E5a-mediated effect.
The status of epithelial differentiation may affect the EGFR, independent of HPV infection. Therefore, because HPV infection affects the state of differentiation (4) , the function and abundance of the EGFR could be a secondary effect of infection rather than a direct result of one or more HPV gene products. EGFR expression diminishes in suprabasal layers of normal human epithelium. Inhibition of the completion of terminal differentiation by HPV infection could, therefore, contribute to an overexpression of the EGFR. However, cells cultured from papillomas are necessarily from the proliferative compartment of the laryngeal epithelium. We would therefore expect that cultured cells would retain the same level of the EGFR found in uninfected cells, if the state of differentiation were the only factor in determining receptor expression. This is not the case. Instead, we find enhanced expression of the EGFR in both the papillomas and in their cultured cells. This is more consistent with a direct effect of HPV gene expression on EGFR levels than with a change in EGFR levels as a secondary result of infection altering differentiation.
The EGFR kinase was hyperresponsive to EGF in papilloma cells. This responsiveness was reflected in the abundance of phosphotyrosine incorporated into proteins at Mr 180,000, 65,000, 55,000, and 42,000–44,000. However, the largest of these proteins was not the EGFR because, in the 10-min treatment we used, the EGFR was completely lost from the Triton-soluble fraction (data not shown). Similarly, Daub et al. (6) reported that a 5-min treatment of COS-7 cells with 30 ng/ml EGF resulted in the disappearance of Triton-soluble EGFR but an increase in phosphotyrosine at Mr 170,000. We have ascribed this phosphorylation target to erbB-2 because it has been shown that erbB-2 is a target for hyperresponsive EGFR in HPV 16 E5 transfection experiments (19) . erbB-2, erbB-3, and erbB-4 were not overexpressed in laryngeal papillomas, although all were present (data not shown). This was consistent with specific enhancement of EGFR abundance due to receptor recycling following ligand mediated internalization because none of remaining erbB family members undergo ligand-mediated internalization and, therefore, would not increase by that recycling mechanism (25) . The phosphorylated bands at Mr 55,000 and 65,000 are presently unidentified. However, EGFR targets at these approximate molecular weights have been ascribed to isoforms of shc. The band at Mr 65,000 was highly sensitive to EGF-stimulated phosphorylation in papilloma cells, acquiring phosphotyrosine at 10 pg/ml EGF. Hyperphosphorylation at low concentrations of EGF was less evident in the bands that we ascribed to ERK-1 and -2, because these were already highly phosphorylated on tyrosine in both normal and papilloma cells. It is important not to construe this as evidence of a comparable enhancement of basal activated (as opposed to tyrosine-phosphorylated) MAPK in papilloma cells compared to normal cells because activation requires both threonine and tyrosine phosphorylation. Our data suggests that this was not the case in EGF-starved cells in monolayer cultures (data not shown).
The heightened response of papilloma cells to EGF could be a simple result of mass action because, for a given level of ligand, a greater number of receptors would yield a greater activation of tyrosine kinases. However, the lack of any observable change in phosphorylation at Mr 180,000 in normal cells treated with low concentrations of EGF argues for a qualitative difference between infected and uninfected cells. This difference may reflect the observation that HPV E5 proteins not only inhibit receptor degradation by binding the vacuolar (H+)-ATPase, but also bind the EGFR and erbB-2 (23) . The nature of the direct effect of HPV 11 E5a on EGFR-dependent signaling is currently under study in our laboratory.
Sustained MAPK activation has been described as necessary for onset of differentiation in PC12 cells following transfection of oncogenes or treatment with nerve growth factor (26 , 27) . However, by itself, sustained activation of MAPK is insufficient for PC12 differentiation (28) . In contrast, MAPK activation inhibits murine adipocyte differentiation, indicating that the effect of MAPK activation on differentiation is cell type specific (29) . Our previous data indicated that a prolonged withdrawal of EGF from cell culture media induced morphological and biochemical markers of differentiation in papilloma cells growing on organotypic rafts (5) . Thus, MAPK activation in papilloma cells does not provoke morphological differentiation and may be a mechanism to inhibit the differentiation of these cells. This hypothesis is in agreement with the observation of elevated MAPK in papilloma tissues that are characterized by a block in differentiation.
Here, we demonstrated a basal activation of MAPK in monolayer cultures of papilloma cells following EGF withdrawal, dependent on both tyrosine kinase activity and ligand occupancy of the EGFR. Thus, it might be expected that removal of EGF would not permit differentiation of either cell type. However, our previous studies (5) showed that expression of the EGFR was limited to basal cells in the normal cell raft cultures, although it extended throughout most of the spinous layer in the papilloma cell raft cultures. Thus, the increased sensitivity of suprabasal papilloma cells to MAPK activation by EGF could explain the ability of normal cells on raft cultures to differentiate in the presence of EGF, although papilloma cells were blocked.
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FOOTNOTES |
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1 This study was supported by National Institute on Deafness and Other Communication Disorders Grant P50-DC00203 and grants from the Morris S. and Florence H. Bender Foundation and the Irving and Helen Schneider Family Foundation. 
2 Present address: Saunders Trades and Technical High School, Yonkers, NY 10701.
3 Present address: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461.
4 To whom requests for reprints should be addressed, at Department of Otolaryngology, Long Island Jewish Medical Center, 270-05 76th Avenue, New Hyde Park, NY 11040. Phone: (718) 470-7553; Fax: (718) 347-2320; E-mail: bsteinbe{at}aecom.yu.edu
5 The abbreviations used are: HPV, human papillomavirus; EGF, epidermal growth factor; EGFR, EGF receptor; MAPK, mitogen-activated protein kinase; ERK, extracellular regulated kinase; TGF-, transforming growth factor ; KGM, keratinocyte growth medium; TBS, Tris-buffered saline.
Received 8/14/98. Accepted 12/18/98.
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