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Published ahead of print on January 10, 2003, doi:10.1165/rcmb.2002-0147OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 48-56, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0147OC

Mucin Biosynthesis

Epidermal Growth Factor Downregulates Core 2 Enzymes in a Human Airway Adenocarcinoma Cell Line

Paul V. Beum, Dhundy R. Bastola and Pi-Wan Cheng

Department of Biochemistry and Molecular Biology, College of Medicine; and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska

Address correspondence to: Pi-Wan Cheng, Ph.D., Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. E-mail: pcheng{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Enzymes which exhibit core 2 ß1,6 N-acetylglucosaminyltransferase (C2GnT) activity play important roles in physiologic processes including the inflammatory response and immune system function, and C2GnT activity is regulated during processes, such as T cell activation and cellular differentiation. In this study, we have examined the regulation of C2GnT activity in the H292 airway epithelial cell line by epidermal growth factor (EGF), which has been previously shown to upregulate expression of the airway mucin MUC5AC in this cell line. We found that EGF suppressed C2GnT activity in a time- and dose-dependent fashion, and also suppressed core 4 ß1,6 N-acetylglucosaminyltransferase (C4GnT) activity. Consistent with the suppression of C4GnT activity, Northern blotting results showed that EGF preferentially inhibited the M isoform of C2GnT, which forms core 2, core 4, and blood group I ß1,6 branched carbohydrate structures, while the L isoform, which forms only the core 2 structure, was only modestly affected. Furthermore, EGF treatment resulted in a shift in the carbohydrate structure of FLAG-tagged MUC1 expressed in the cells from core 2-based toward core 1-based structures, consistent with the inhibitory effects of EGF on C2GnT. Transforming growth factor {alpha} mimicked the effect of EGF on C2GnT, implicating the EGF receptor (EGF-R) in C2GnT suppression, and the EGF-R tyrosine kinase inhibitor AG1478 blocked C2GnT suppression, confirming the role of EGF-R in the inhibition of C2GnT expression. Also, PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 in the Ras–mitogen-activated protein kinase pathway, completely blocked the EGF suppressive effect, suggesting possible involvement of the Ras–mitogen-activated protein kinase pathway in EGF-mediated downregulation of C2GnT. The results of this study suggest that exposure of airway cells to EGF may result in remodeling of mucin carbohydrate structure, potentially altering the biological properties of the cells.

Abbreviations: bovine serum albumin, BSA • UDP-GlcNAc:Galß1-3 GalNAc (GlcNAc to GalNAc) ß1-6 GlcNAc transferase, C2GnT • UDP-GlcNAc:GlcNAcß1-3 GalNAc (GlcNAc to GalNAc) ß1-6 GlcNAc transferase, C4GnT • complimentary DNA, cDNA • cytidine monophosphate, CMP • UDP-Gal:GalNAc ß1-3 Gal transferase, Core 1 Gal TF • epidermal growth factor, EGF • EGF receptor, EGF-R • extracellular signal-regulated kinase, ERK • DYKDDDD epitope, FLAG • galactose, Gal • glyceraldehyde 3-phosphate dehydrogenase, GAPDH • N-acetylgalactosamine, GalNAc • N-acetylglucosamine, GlcNAc • insulin-like growth factor-1, IGF-1 • mitogen-activated protein kinase/extracellular signal-regulated kinase kinase, MEK • messenger RNA, mRNA • platelet-derived growth factor, PDGF • UDP-GalNAc:polypeptide GalNAc transferase, pGalNAc TF • Raf oncogenes-encoded protein-serving/threonine kinases, Raf • Ras oncogenes-encoded small GTP-binding proteins, Ras • transferase, TF • transforming growth factor {alpha}, TGF-{alpha} • 3-[N-morpholino] propane sulfonic acid, MOPS • uridine diphosphate, UDP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Glycoconjugates play vital roles in many biological processes, such as blood group antigenicity, fertilization, cell–cell interactions, immune system function, and tumorigenicity (reviewed in Ref. 1). These functions are dependent on the presence of specific glycan structural units in the glycoconjugate, which are synthesized mainly by Golgi-resident glycosyltransferases. The regulation of these glycosyltransferases is therefore a critical determinant in the types of glycan structures displayed on the cell surface. For example, we recently showed that introduction of a complimentary DNA (cDNA) coding for a core 2 ß1,6 N-acetylglucosaminyltransferase (C2GnT) enzyme into a pancreatic adenocarcinoma cell line resulted in significant changes in the expression of various MUC1 tumor-associated epitopes (2).

The most prominent family of O-linked glycoproteins are mucins, which are high molecular weight glycoproteins produced in the airway, gastrointestinal tract, genitourinary tract, breast and pancreas. Mucin-like proteins, such as the selectin ligands, are also found on the cell surface. Mucin carbohydrate biosynthesis (reviewed in Refs. 3 and 4) begins with the attachment of N-acetylgalactosamine (GalNAc) to a serine or threonine, catalyzed by peptidyl GalNAc transferases. Subsequent attachment of sugar residues to the initial GalNAc results in the formation of the core structures, which can then be elongated to form complex structures. An important determinant of the size, structure, and function of the glycan is its degree of ß1,6 branching, with a greater degree of branching generally leading to larger, more complex chains having greater functional potential.

C2GnT activity is involved in the formation of ß1,6 branch points during mucin glycan biosynthesis. The three enzymes exhibiting C2GnT activity reported to date include leukocyte-type C2GnT (C2GnT-L or C2GnT-1; Ref. 5), mucus-type C2GnT (C2GnT-M or C2GnT-2; Refs. 6–9), and thymus-associated C2GnT (C2GnT-3; Ref. 10). These C2GnT isozymes are distinguished by their nucleotide and amino acid sequences, their tissue distribution, and by the types of structure they are able to form. Both C2GnT-L and C2GnT-3 create only the core 2 ß1,6 N-acetylglucosamine (2 ß1,6 GlcNAc) branched structure (C2GnT activity) (5, 10), whereas C2GnT-M can additionally catalyze the formation of the core 4 structure, GlcNAcß1–3(GlcNAcß1–6) GalNAc {alpha}-ser/thr (C4GnT activity), and the blood group I structure, GlcNAc ß1–3(GlcNAcß1–6)Galß-R (IGnT activity) (6–9). The C2GnT, C4GnT, and IGnT activities exhibited by these C2GnT isozymes are shown below.


The core 2 ß1,6 GlcNAc branch has been implicated in formation of selectin ligands, which are involved in the adhesion of tumor cells (11, 12) and leukocytes (reviewed in Ref. 13). Over-expression (14) or deletion (15) of the C2GnT-L gene in mice caused immune dysfunction, which clearly shows the importance of C2GnT-L in T lymphocyte function and selectin-mediated interactions. C2GnT activity is regulated under certain physiologic conditions (16, 17), and the activity of C2GnT is often altered in cancer such as breast and pancreatic cancer, which may contribute to the altered expression of certain tumor-associated antigens on MUC1 in such tissues (2).

Recently, epidermal growth factor (EGF) was shown to increase expression of MUC5AC in intact airways and in H292 airway epithelial cells (18). In this study, we examined the effects of EGF on C2GnT and several other key mucin glycosyltransferases in the H292 cell line. We found that EGF downregulates C2GnT at the level of messenger RNA (mRNA) and enzyme activity, that the suppression is more pronounced for C2GnT-M than C2GnT-L, and that the suppression of C2GnT activity reduces the degree of MUC1 core 2 carbohydrate structure from 100 to 50% with a concomitant increase of core 1 carbohydrate structure from 0 to 50%.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Materials
The NCI-H292 cell line was purchased from ATCC (Manassas, VA). UDP-[3H]GlcNAc (60 Ci/mmol), UDP-[3H]Gal (60 Ci/mmol), and UDP-[3H]GalNAc (15 Ci/mmol) were obtained from American Radiolabeled Chemicals (St. Louis, MO). Human recombinant EGF, transforming growth factor (TGF)-{alpha}, insulin-like growth factor (IGF)-1 and platelet-derived growth factor (PDGF)–AA were obtained from Gibco Life Technologies (Rockville, MD). Inhibitors PD 98059, SB 202190, PP2, and tyrphostins AG1478 and AG1296 were purchased from Calbiochem (San Diego, CA). Immobilon P polyvinyllidenefluoride membrane was from Millipore (Bedford, MA). X-ray film was from Amersham-Pharmacia Biotech (Arlington Heights, IL). Bond Elut C18 cartridges were from Varian (Sunny Vale, CA). Other chemicals were from Sigma (St. Louis, MO).

Cell Culture
NCI-H292 cells were routinely grown in RPMI 1,640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin). Experiments were conducted on cells between passages 40 and 100, for which reproducible results were obtained. Drug treatments were performed in this same medium, starting at {bsim} 75% confluence, using the following drug concentrations: 25 ng/ml EGF, 25 ng/ml TGF-{alpha}, 20 ng/ml IGF-1, or 10 ng/ml PDGF-AA. Inhibitor treatments were performed by pretreating the cells for 30–45 min with RPMI 1,640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, antibiotics, and inhibitor (10 µM AG1478, 100 µM AG1296, 2 µg/ml PP2, 2 µg/ml SB202190, or 25 µg/ml PD98059), followed by addition of EGF to a final concentration of 25 ng/ml, and incubation of the cells for up to 36 h.

Stable Transfection of MUC1-DYKDDDD (FLAG) Construct into H292 Cells
A DYKDDDD (FLAG)–tagged MUC1 cDNA subcloned into the BamHI site in the expression vector pHß-APr1-neo (2, 19) was transfected into H292 cells using the transferrin-assisted lipofection protocol described previously (20). G418-resistant clones were screened for MUC1-FLAG expression by Western blotting using the M2 anti-FLAG antibody.

Glycosyltransferase Enzyme Assays
Total cell homogenates used in enzyme assays were prepared as follows: cells in T25 flasks were washed twice with cold phosphate-buffered saline, scraped in 200 µl of 0.25 M sucrose, and successively passed through 18-, 20-, and 26-gauge syringe needles to disrupt the cell membrane. Protein concentration was measured by the Bio-Rad assay (Bio-Rad) using bovine serum albumin (BSA) as the standard. All enzyme assays were conducted under conditions in which product formation was linear with respect to time and enzyme amount. An additional reaction without exogenous acceptor was performed to measure endogenous enzyme activity. Enzyme activity was calculated by subtracting endogenous activity from total activity, and was expressed as nmol sugar donor transferred per h per mg total protein. C2GnT activity was assayed essentially as described (6), using Galß1–3GalNAc{alpha}-Benzyl as acceptor. Briefly, the reaction mixture contained 50 mM 3-[N-morpholino] propane sulfonic acid (MOPS) pH 7.5, 5 mM MnCl2, 2% Tween-20, 1 mg/ml BSA, 2 mM UDP-GlcNAc (containing 6,400 dpm/nmol UDP-[3H]GlcNAc), and 1 mM ATP in a final volume of 50 µl. Reactions with acceptor contained 2 mM Galß1–3GalNAc{alpha}-Benzyl, whereas background reactions contained water instead of acceptor. The reaction mixtures were incubated at 37°C for 2 h and then terminated by the addition of 300 µl of 10 mM ZnCl2. A 10-µl aliquot was measured by scintillation counting for determination of total radioactivity in the reaction mixture, then 300 µl of the mixture was applied to a C18 cartridge under house vacuum. The cartridges were washed nine times with 2.5 ml of 0.1 M TRIS-HCl pH 7.5 and then the product was eluted with 2 ml methanol. The methanol was evaporated in a Speed-Vac (Savant Instruments, Holbrook, NY) and the residue was dissolved in 0.5 ml water and measured for radioactivity by scintillation counting. C4GnT activity was measured identically to C2GnT, except that 2 mM GlcNAcß1,3GalNAc{alpha}-p-nitrophenyl was used as acceptor. UDP-GalNAc:polypeptide GalNAc transferase (pGalNAc TF) was assayed as previously described (21), except that the reaction mixture contained 0.1 mM UDP-GalNAc (containing 105 dpm/nmol UDP-[3H]GalNAc), 1 mg/ml BSA, and 0.1 mg/ml acceptor peptide. The acceptor peptide consisted of a synthetic 29–amino acid MUC2 peptide having the sequence PTTTPITTTTTVTPTPTPTGTQTPTTTPI. Although numerous isoforms of pGalNAc TF exist (22) which possess distinct acceptor substrate preferences, the MUC2 tandem repeat sequence has been shown to be utilized efficiently by multiple isoforms of pGalNAc TF (23), and therefore should provide a reasonable measure of overall pGalNAc TF activity. UDP-Gal:GalNAc ß1-3 Gal transferase (core 1 Gal TF), which catalyzes the attachment of galactose in ß1–3 linkage to GalNAc{alpha}-ser/thr, was assayed as previously described (24), except that 2 mM GalNAc{alpha}-benzyl was used as the acceptor and the product was isolated as in the C2GnT assay described above.

Northern Blotting
Total RNA was isolated from harvested cells using Tri reagent (MRC, Cincinnati, OH) according to the manufacturer's protocol. The RNA (10–20 µg) was electrophoresed on formaldehyde–agarose gels and then capillary-blotted to a Nytran membrane (Schleicher and Schuell, Keene, NH). The membrane was crosslinked by ultraviolet and treated with 1% SDS and 0.1 M NaCl at room temperature for 1 h. This pre-hybridization solution was then replaced by 25 ml of hybridization solution HS-114 (MRC) to which {bsim} 1 x 107 dpm of denatured 32P-labeled oligodeoxynucleotide probe against C2GnT-M, C2GnT-L, C2GnT-3, or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) had been added. The probe for C2GnT-M consisted of the sequence from position 383 to 667 in the cDNA sequence (relative to the translation start site; Refs. 7 and 8), whereas that for C2GnT-L ran from 423 to 685 in the C2GnT-L cDNA sequence (5). The C2GnT-3 probe extended from position 223 to 412 in the C2GnT-3 open reading frame (10). These probes were chosen so as to maximize specificity for the desired C2GnT isoform. The GAPDH probe consisted of an 800 bp PstI/XbaI fragment from the 5' end of the human GAPDH cDNA (ATCC). Probe hybridization was performed at 62°C for 24–48 h, followed by washing 5 times, 10 min each, at 55°C in 1x SSC (0.44% sodium citrate, 0.877% sodium chloride). The membrane was then exposed to autoradiography film overnight.

Analysis of MUC1-FLAG O-Glycan Structure
H292 cells expressing a stably transfected FLAG-tagged MUC1 cDNA (2, 19) were grown in T75 flasks for 12 h in the presence or absence of 25 ng/ml EGF. The cells were then metabolically labeled with 10 µCi/ml [3H]glucosamine for 48 h, again in the presence or absence of EGF. Cells from each T75 flask were washed twice with cold phosphate-buffered saline and then harvested by scraping in 1.5 ml lysis buffer (150 mM NaCl, 1.2% Triton X-100, 1 mM phenylmethyl sulfonylfluoride, 2% mammalian protease inhibitor). FLAG-tagged MUC1 was immunoprecipitated by adding an equal volume of a 2:1 mixture of TRIS-buffered saline and M2 anti-FLAG agarose gel suspension (Sigma) and rotating at 4°C for 6 h. The agarose pellet was then washed twice with 600 µL chilled lysis buffer, and the bound FLAG-tagged MUC1 eluted by twice with 600 µL 0.83 nmol/ml FLAG peptide (Sigma) in TRIS-buffered saline, collecting the supernatant each time. The MUC1 O-glycans were then cleaved via ß elimination by treating with fresh 1 M sodium borohydride in 0.1 M NaOH at 42°C for 48 h (25). The mixture was then acidified to pH 5 with 3 M acetic acid, and put through a fresh column of Dowex AG50W-X8 cation resin (200–400 mesh, H+ form) (Bio-Rad, Hercules, CA) equilibrated in water. The sample was eluted from the resin with two 5 ml portions of water, and the eluate was lyophilized overnight. The lyophilizate was dissolved in a minimal amount of methanol and evaporated in a Speed Vac (Savant Instruments). The pale brown residue was then resuspended in 0.5 ml water, and 5 mg each of sialic acid, raffinose, and/or stachyose were added to act as internal elution standards. A few grains of phenol red were added to act as dye marker for monitoring column performance, and the mixture was then loaded onto a Bio-Gel P-4 column (1.5 cm x 115 cm, 200–400 mesh) (Bio-Rad) which had been equilibrated in 0.1 M ammonium bicarbonate and calibrated using BSA to mark the void volume and stachyose, raffinose, and lactose to mark the elution positions for a tetrahexose, trihexose, and dihexose, respectively. The sample was eluted with 0.1 M ammonium bicarbonate at a flow rate of {bsim} 0.25 ml/min, and 40-drop fractions were collected after collecting the void volume. Aliquots of the fractions were measured for radioactivity by scintillation counting and for the presence of the sugar standards, using the resorcinol assay to detect sialic acid standard (26) and the anthrone assay to detect hexose standards (27). Fractions corresponding to a peak of interest were pooled, lyophilized, desalted on a Sephadex G-10 column (1 cm x 50 cm) (Sigma) and lyophilized again. The lyophilizate was resuspended in 100–500 µL water and then digested with a specific exoglycosidase (see EXOGLYCOSIDASE DIGESTION CONDITIONS below). Following exoglycosidase digestion, the Bio-Gel P-4 column analysis procedure described above was repeated, and this digestion-column analysis procedure was repeated until sufficient evidence was available to deduce the structure of the O-glycans.

Exoglycosidase Digestion Conditions
Clostridium Perfringens neuraminidase (Sigma), composed of 50 mM sodium acetate at pH 5.5, 0.1 mU enzyme per µl reaction mixture was incubated at 37°C for 48 h. Jack bean ß hexosaminidase (V labs, Covington, LA), composed of 20 mM sodium citrate at pH 5.0, 1 mU enzyme per µl reaction mixture was incubated at 37°C for 96 h. Bovine testicular ß-galactosidase (Oxford Glycosystems, Rosedale, NY), composed of 20 mM sodium citrate at pH 4.0, 0.2 mU enzyme per µl reaction mixture was incubated at 37° for 96 h. Diplococcus pneumoniae ß galactosidase (Boehringer Mannheim, Indianapolis, IN), composed of 200 mM sodium cacodylate at pH 6.0, 50 µU enzyme per µl reaction mixture was incubated at 37° for 96 h.

Statistical Analysis
The Prism (GraphPad, San Diego, CA) software package was used to compute two-tailed P values using an unpaired t test.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
EGF Downregulates C2GnT and C4GnT Activities in H292 Cells
Takeyama and colleagues (18) recently showed that treatment of H292 cells with EGF or TGF-{alpha} resulted in an {bsim} 2-fold increase in expression of the airway mucin MUC5AC at the mRNA and protein levels. Because of the importance of the carbohydrate portion of mucin in mucin function, we proceeded to determine the effects, if any, of EGF on some glycosyltransferases that participate in the synthesis of mucin carbohydrate. We first confirmed the reported EGF-mediated upregulation of MUC5AC in H292 cells by Western blotting using a monoclonal antibody directed against MUC5AC (data not shown) and then examined the effects of EGF on several key glycosyltransferases in these cells. The enzymes we assayed were: (i) pGalNAc TF, which initiates carbohydrate chain synthesis and thus serves as an indicator of the degree of mucin glycosylation; (ii) core 1 Gal TF, which forms the obligatory acceptor substrate for C2GnT; (iii) C2GnT; and (iv) C4GnT, which synthesizes the core 4 ß1,6 branched structure GlcNAcß1–3(GlcNAcß1–6) GalNAc {alpha}-ser/thr. As shown in Figure 1, treatment with 25 ng/ml human EGF for 36 h had no appreciable effect on pGalNAc TF or core 1 Gal TF. However, EGF caused a definite reduction in C2GnT and C4GnT activities. These findings signal a potential reduction in the amount of ß1,6 branching of mucins caused by EGF in these cells, and also suggests that EGF targets C2GnT-M, because C2GnT-M is the only known enzyme to exhibit C4GnT activity.



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  		Figure 1. EGF downregulates C2GnT and C4GnT activities in H292 cells. H292 cells were grown for 36 h in the presence or absence of 25 ng/ml EGF, and then whole cell extracts were prepared and assayed for (A) peptidyl GalNAc transferase activity, (B) core 1 Gal transferase activity, (C) C2GnT activity, and (D) C4GnT activity. **Indicates a statistically significant difference in means between control and treated samples (P < 0.01), n = 4 independent T25 flasks.

 
We further found that suppression of C2GnT activity by EGF was time- and dose-dependent (Figure 2). The length of time required for the EGF-mediated downregulation of C2GnT activity to occur was relatively long. The observed decrease of enzyme activity may result from inhibition of gene expression, decreased stability of mRNA, or formation of inactive or unstable enzyme due to post-translational modifications. Because these two possibilities depend on the knowledge of the level of gene expression, we decided to examine whether EGF treatment suppressed C2GnT gene expression next.



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  		Figure 2. EGF downregulates C2GnT activity in a time- and dose-dependent fashion. (A) H292 cells were grown for 36 h in the presence or absence of the indicated concentrations of EGF. (B) H292 cells were grown for various lengths of time in the presence or absence of 25 ng/ml EGF. Cell extracts were then assayed for C2GnT activity. *P < 0.05, **P < 0.01, n = 4 independent T25 flasks.

 
EGF Downregulates C2GnT-M to a Greater Extent than C2GnT-L
To date, three glycosyltransferases, C2GnT-L, C2GnT-M, and C2GnT-3, which exhibit C2GnT activity, have been identified. The results of the C2GnT assay shown in Figure 1 potentially reflect contributions from some or all of these C2GnT enzymes. As described above and as shown in Figure 1D, EGF suppressed C4GnT activity, suggesting that C2GnT-M is at least partly responsible for the overall reduction in C2GnT activity caused by EGF treatment because C2GnT-M is the lone enzyme known to exhibit C4GnT activity. To further discriminate between the three distinct C2GnT isozymes, we performed Northern blotting analysis using probes specific for C2GnT-L, C2GnT-M, and C2GnT-3. Northern blotting of total RNA isolated from H292 cells treated with EGF for different lengths of time revealed that C2GnT-M mRNA was strongly downregulated by EGF (87%), while C2GnT-L mRNA levels were only modestly reduced (36%) (Figure 3). No C2GnT-3 message was detected in either the control or EGF-treated cells (data not shown). The Northern blotting results, along with the C4GnT assay results, indicate that the inhibitory effect of EGF is targeted mainly to C2GnT-M.



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  		Figure 3. EGF preferentially downregulates C2GnT-M mRNA levels. H292 cells were exposed to 25 ng/ml EGF for the indicated lengths of time and then total RNA was isolated, fractionated on formaldehyde agarose gels, and blotted to a nylon membrane. The membrane was then probed for (A) C2GnT-L, (B) C2GnT-M, and (C) GAPDH (as an internal control). C2GnT-3 mRNA was not detected in either the control or EGF-treated cells (data not shown). Northern blots against C2GnT-L revealed three bands of approximate sizes 2.4 kD, 4.4 kD, and 5.8 kD, whereas the blots against C2GnT-M revealed three bands of approximate sizes 2.4 kD, 2.8 kD, and 5.1 kD. In both cases, the 2.4 kD band was the most intense band and is the band shown here. The band intensity (after normalization to GAPDH band intensity) of C2GnT-M and C2GnT-L mRNAs in EGF-treated cells relative to that of the corresponding isozymes in the control cells at 4, 12, 24, and 36 h after treatment, respectively, are: C2GnT-M, 1.02, 0.53, 0.32, and 0.13; C2GnT-L, 0.82, 0.64, 0.64, and 0.68.

 
EGF Treatment Increases the Proportion of Core 1-based MUC1 Carbohydrate Structure in H292 Cells
Because EGF suppressed C2GnT activity in H292 cells, we examined whether this treatment resulted in a change in mucin carbohydrate structure in the cells. H292 cells that had been stably transfected with a FLAG-tagged human MUC1 cDNA (H292-MUC1 cells) were left untreated or were treated with EGF for 12 h, then metabolically labeled for 48 h with tritiated glucosamine (still in the presence or absence of EGF), which is incorporated into sialic acid (neuraminic acid), N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc) residues of glycosonjugates. We then immunoprecipitated the FLAG-tagged MUC1 from cell lysates, followed by alkaline borohydride cleavage of the O-glycan chains from the MUC1 peptide (25). The released intact O-glycan chains that contain N-acetylgalactosaminitol (GalNAc-ol) at the reducing termini were resolved by Bio-Gel P-4 column chromatography (Bio-Rad) and analyzed further by digestion with specific exoglycosidases. GalNAc-ol and GlcNAc show characteristic elution behavior on Bio-Gel P-4 (24, 28), with GalNAc-ol eluting approximately as a 3.0-hexose unit and GlcNAc eluting as a 2.5-hexose unit, and the elution behavior of a carbohydrate chain consisting of these residues and neutral sugars behaves approximately as the sum of its parts. Comparison of the elution positions of the exoglycosidase digestion products to those of standard sugars, aided by the knowledge of known mucin oligosaccharide structures and rules of glycosylation (3, 4) allows for deduction of the structure of the carbohydrate chain.

Figure 4 shows the Bio-Gel P-4 column chromatography profiles of the MUC1 O-glycans from untreated H292-MUC1 cells (Figure 4A) and H292-MUC1 cells treated with 25 ng/ml EGF (Figure 4B). Both profiles show a complex, early-eluting peak (peak 1) and a second peak eluting at {bsim} 80 ml (peak 2). We then treated the materials from peaks 1 and 2 with C. perfringens neuraminidase, which cleaves both {alpha}2,6- and {alpha}2,3-linked sialic acid residues. The released sialic acid eluted from Bio-Gel P-4 column as if it were a decahexose under current chromatographic conditions, making sialylated oligosaccharides appear much larger than they actually are, thus complicating its structural analysis. It was noted that the elution properties, such as elution position and recovery, of sialic acid under current chromatographic conditions were not as reproducible as other sugars. This problem was not observed for neutral sugars, GlcNAc, or GalNAc-ol. Peak 1, which eluted at the column void volume, remained at the void volume following neuraminidase digestion (data not shown), suggesting that this peak may contain glycopeptides consisted of N-linked carbohydrates, which were resistant to ß elimination. However, peak 2 in both profiles was amenable for further characterization.



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  		Figure 4. Bio-Gel P-4 column chromatographic profiles of MUC1 mucin-type carbohydrates from H292-MUC1 cells treated without (A) and with (B) EGF. H292-MUC1 cells were grown in the presence of [3H]glucosamine and in the absence (A) or presence (B) of 25 ng/ml EGF, and O-glycans were then cleaved by alkaline borohydride degradation of MUC1 immunoprecipitated from cell lysates and analyzed on a Bio-Gel P-4 column. A, B, and C represent the elution positions of sialic acid, tetrahexose, and trihexose standards, respectively. The volume of each fraction was {bsim} 1.1 ml.

 
Neuraminidase digestion of peak 2 materials (Figure 4) from both untreated (Figure 5A) and EGF-treated H292-MUC1 cells (Figure 5B) yielded three peaks, 2–1, 2–2, and 2–3, as well as one additional peak (peak 2–4) from MUC1 of the EGF-treated cells (Figure 5B). Peak 2–1, which eluted at the same position as the original peak 2 (Figure 4), likely represents incompletely degraded sialylated oligosaccharides. Peak 2–2 co-eluted with the sialic acid internal standard, and the identity of this peak as sialic acid was confirmed by the ability of Dowex-1 anion exchange resin to trap more than 90% of the radioactivity in this peak (data not shown). Peak 2–3 eluted approximately as a 7.5-hexose unit. Cells treated with EGF produced an additional peak (peak 2–4), which co-eluted with tetrahexose and was not seen in the P-4 oligosaccharide profile of MUC1 from the untreated cells.



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  		Figure 5. Bio-Gel P-4 column chromatography profiles of peak 2–2 materials from Figures 4A and 4B following sequential exoglycosidase treatment. (A) C. perfringens neuraminidase-treated peak 2 (see Figure 4A) from MUC 1 of H292-MUC1 cells. (B) C. perfringens neuraminidase-treated peak 2 (see Figure 4B) from MUC 1 of EGF-treated H292-MUC1 cells. (C) D. pneumoniae ß-galactosidase-treated peak 2–3 from Figure 5A. (D) Jack bean ß N-acetylhexosaminidase-treated peak 2–3a from Figure 5C. (E) Bovine testicular ß-galactosidase–treated peak 2–3b from Figure 5D. (F) Bovine testicular ß-galactosidase–treated peak 2–4 in Figure 5B. A, B, and C represent elution positions for sialic acid, tetrahexose, and trihexose internal standards, respectively. GlcNAc and GalNAc-ol behave like 2.5- and 3.0-hexose units on Bio-Gel P4 column chromatography, respectively.

 
To further deduce the structure of the oligosaccharide in peak 2–3, we first digested peak 2–3 material from the untreated H292-MUC1 cells (Figure 5A) with D. pneumoniae ß-galactosidase, which cleaves ß1,4-linked terminal galactose. This treatment resulted in a decrease in the size of the oligosaccharide chain, shifting the peak downward by approximately one hexose unit (peak 2–3 -> 2–3a, Figure 5C), indicating the presence of ß1,4-linked galactose at the non-reducing terminus. Subsequent digestion of peak 2–3a with jack bean ß N-acetylhexosaminidase shifted the peak {bsim} 2.5 hexose units (which would be expected from removal of GlcNAc) to the elution position of the tetrahexose standard (peak 2–3a -> 2–3b, Figure 5D). Digestion of peak 2–3b material with bovine testicular ß-galactosidase, which digests ß1,3-linked galactose residue, produced a peak (peak 2–3b -> 2–3c, Figure 5E), which coincided with the trihexose standard and GalNAc-ol. Similarly, digestion of peak 2–3 material from MUC1 of the EGF-treated cells (Figure 5B) with these glycosidases yielded an identical elution profiles (data not shown). These findings indicate that peak 2–3 (Figures 5A and 5B) represents the structure Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-ol.

The most striking difference in the profiles shown in Figures 5A and 5B was the presence of peak 2–4 in the MUC1 oligosaccharide profile for the EGF-treated cells, which was absent from the profile of the untreated cells. Because this peak co-eluted with the tetrahexose standard, we predicted that the structure represented by peak 2–4 material is Galß1,3GalNAc-ol. To examine this possibility, we digested peak 2–4 material with bovine testicular ß–galactosidase. Figure 5F shows that the digestion resulted in a shift in elution position of the peak from 4.0-hexose to 3.0-hexose units, consistent with the predicted structure for peak 2–4. This result indicates that the level of C2GnT activity (Figure 1) in the untreated H292 cells was sufficient to convert all core 1 structure to core 2 (Figure 5A). Treatment of these cells with EGF decreased the C2GnT activity to ~ 40% of the control (Figure 1C), resulting in the conversion of ~ 50% of the core 2 to core 1 structures (Table 1).


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  		TABLE 1 Molar % Distribution of MUC1 O-Glycan Structures in Untreated and EGF-Treated H292-MUC1 Cells. The molar percentages of observed structures are based on the chromatographic profiles of the MUC1 oligosaccharides before and after exoglycosidase treatment and the knowledge of known mucin oligosaccharide structures and rules for glycosylation (3, 4). The exoglycosidase digestion results and profiles shown in Figures 4 and 5 suggest that in the absence of EGF, all core 1 structure is converted to core 2–based structure, while treatment with EGF reduces C2GnT activity and results in conversion of approximately 50% of the MUC1 core 1 oligosaccharide to core 2 structure

 
Downregulation of C2GnT by EGF Is Blocked by an EGF Receptor Antagonist and by a Mitogen-Activated Protein Kinase/Extracellular Signal-Related Kinase Kinase (MEK)-1/2 Inhibitor
To explore the possible mechanism by which EGF downregulates C2GnT in H292 cells, we first examined whether the effect was mediated via the EGF receptor (EGF-R) by treating H292 cells with EGF in combination with a specific inhibitor of the EGF receptor tyrosine kinase (AG1478) or a negative control inhibitor (AG1296). As shown in Figure 6A, AG1478 completely blocked the suppressive effect of EGF on C2GnT activity, whereas the control inhibitor had no effect, confirming the role of EGF-R in mediating the downregulation of C2GnT. Consistent with these findings, TGF-{alpha}, another EGF-R ligand, suppressed C2GnT activity in H292 cells to nearly an identical extent as EGF, whereas IGF-1 and PDGF, growth factors which do not utilize EGF-R, had no effect on C2GnT (data not shown).



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  		Figure 6. Inhibition of the EGF receptor tyrosine kinase and MEK-1/2 activity blocks the inhibitory effect of EGF on C2GnT activity. (A) H292 cells were grown for 36 h in the presence or absence of 25 ng/ml EGF alone or in combination with EGF-R inhibitor AG1478 or control inhibitor AG1296. Cell extracts were assayed for C2GnT activity. *P < 0.05, **P < 0.01, n = 4 independent T25 flasks. (B) H292 cells were grown for 36 h in the presence or absence of 25 ng/ml EGF alone or in combination with inhibitors PD 98059, SB 202,190 or PP2. Cell extracts were then prepared and assayed for C2GnT activity. **P < 0.01, n = 4 independent T25 flasks, except for untreated control, for which n = 3.

 
Activated EGF-R can transduce extracellular signals to the nucleus via numerous pathways (29). One prominent pathway involves activation of the small GTPase Ras, which in turn can stimulate several downstream pathways (30), including the Raf oncogenes-encoded protein-serine/threonine kinases (Raf) and MEK-extracellular signal-regulated kinase (ERK) pathway and a pathway involving the p38 MAP kinase. Activated EGF-R is also known to act via stimulation of the Src family of tyrosine kinases (31), as in the case of P. aeruginosa–induced upregulation of mucin gene expression in H292 cells and HM3 cells (32). To determine which of these pathways may be involved in the downregulation of C2GnT by EGF, we treated H292 cells with EGF in combination with specific inhibitors that block the activity of MEK1/2 (PD 98,059), Src tyrosine kinase activation (PP2), and p38 MAP kinase activation (SB 202,190). Figure 6B shows that MEK was the only inhibitor which was able to block the effect of EGF on C2GnT activity. Thus, the results shown in Figure 6 suggest that the signaling pathway initiated by EGF involves stimulation of EGF-R, which then leads to activation of the Ras-MEK-ERK pathway and culminates in the C2GnT downregulation in H292 cells. This proposed pathway may be explored in more depth by the use of constitutively active or dominant negative mutants of Ras and MEK.

In conclusion, we have shown that exposure of H292 airway adenocarcinoma cells to EGF results in downregulation of C2GnT at the levels of mRNA and enzyme activity, and that the C2GnT-M isoform is affected to a greater extent than the C2GnT-L, whereas C2GnT-3 was not affected. This is the first report to show differential regulation of the expression of three distinct C2GnT isoforms by a single extracellular stimulus. Furthermore, the results of our carbohydrate structural analysis (summarized in Table 1) indicate that downregulation of C2GnT by EGF resulted in corresponding changes in mucin carbohydrate structure. Taken together, the findings suggest a possible effect of EGF on cell surface carbohydrate structure, acting via modulation of C2GnT activity. Because the EGF receptor is commonly overexpressed in cancer and because Ras is constitutively active in many types of cancer (reviewed in Ref. 33), it is possible that C2GnT activity may be altered in such cancers, with concomitant effects on ß1,6 branching of cell surface O-glycans.

It is of interest to compare the signaling pathways involved in the regulation of C2GnT expression to those involved in the regulation of mucin gene transcription. Several studies examining the effects of environmental factors on mucin gene expression (34) have demonstrated a role for Ras and MEK in the upregulation of mucin gene transcription, similar to our findings for the inhibitory effects of EGF on C2GnT. Thus, it is conceivable that activation of the Ras oncogenes-encoded small GTP-binding proteins (Ras)-Raf-MEK pathway by growth factors such as EGF or by environmental influences may affect cellular mucin content and carbohydrate structure by modulating expression of mucin and glycosyltransferase genes. It appears that at least in H292 cells, EGF has a completely opposite effect on the expression of mucin and glycosyltransferase genes. In this case, EGF enhances MUC5AC expression and Alcian blue-periodic acid Schiff's staining (18), does not affect mucin carbohydrate initiation or core 1 enzymes, but inhibits expression of C2GnT enzymes (Figure 1). Collectively, these results suggest that EGF treatment of H292 cells leads to increased production of MUC5AC carrying predominantly shorter core 1 oligosaccharides. Because in vivo EGF treatment can produce mucus cell metaplasia in rat airway epithelia (18), it will be of interest to examine in this animal model if EGF treatment exerts similar effects on these glycosyltransferases and mucin glycan structure as it does in H292 cells.

To date, considerable evidence exists that suggests that C2GnT activity is regulated during various physiologic processes, such as differentiation (35–36), maturation of granulocytes (16), and T cell activation (17). These findings, along with the differential effects of EGF on mucin C2GnT shown here, suggest that the regulation of C2GnT and the physiologic outcome in the cells expressing mucin and C2GnT warrant further study.


   Acknowledgments
 
The authors thank the National Institutes of Health (RO1 HL48282) and the State of Nebraska (Nebraska Research Initiative–Center of Biomedical Research Excellence in Glycobiology and the LB506 grant) (PWC) and the American Lung Association of Nebraska (PVB) for funding support. They also thank Dr. Michael Hollingsworth for providing the MUC1-FLAG expression construct for transfection, as well as the MUC2 peptide acceptor for assay of pGalNAc TF.

Received in original form August 7, 2002

Received in final form December 30, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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