Increased in vitro activation of EGFR by membrane-bound TGF-alpha from gastric and colonic mucosa of aged rats

Zhi-Qiang Xiao1 and Adhip P. N. Majumdar2,3,4

2 Veterans Affairs Medical Center and 1 Departments of Internal Medicine and 3 Biochemistry and Molecular Biology and 4 Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201


    ABSTRACT
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INTRODUCTION
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DISCUSSION
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Although aging is associated with increased epidermal growth factor receptor (EGFR) tyrosine kinase activity in Fischer 344 rat gastric and colonic mucosa, the regulatory mechanisms for the age-related rise in EGFR tyrosine kinase are poorly understood. Transmembrane transforming growth factor-alpha (TGF-alpha ) may modulate EGFR function through an autocrine/juxtacrine mechanism. The present study aimed to determine the contribution of membrane-bound precursors of TGF-alpha in enhancing EGFR activation in the gastric and colonic mucosa during aging. The extent of EGFR tyrosine phosphorylation, a measure of EGFR activation, was substantially higher (300-350%) in the gastric and colonic mucosa of 23- (aged) vs. 4-mo-old (young) Fischer 344 rats. This was accompanied by an increase (200-1,000%) in the relative concentration of 18- to 20-kDa membrane-bound precursor forms of TGF-alpha . The amount of TGF-alpha bound to EGFR was also higher (150-250%) in the gastric and colonic mucosa of aged vs. young rats. In vitro studies revealed that exposure of HCT 116 cells (a colon cancer cell line) to TGF-alpha from gastric and colonic mucosal membranes of aged rats caused a 200-250% higher activation of EGFR and extracellular signal-related kinases (p42/44) compared with young rats. Our data suggest that the membrane-bound precursor form(s) of TGF-alpha may partly be responsible for enhancing EGFR activation in the gastric and colonic mucosa of aged rats, probably though an autocrine/juxtacrine mechanism(s).

epidermal growth factor receptor signal transduction; tyrosine kinase; autocrine/juxtacrine mechanism; transforming growth factor-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

OVER THE PAST SEVERAL YEARS, results from this and other laboratories (1, 8, 9, 14, 16, 17, 26) have demonstrated that in Fischer 344 rats, aging is associated with increased mucosal proliferative activity in much of the gastrointestinal tract, including the stomach and colon. This has, in part, been attributed (29, 31) to the enhanced transition of mucosal cells through G1 to S phase of the cell cycle. In addition, in the gastric mucosa, aging is found to be associated with increased activation of the p42/44 extracellular signal-related kinases (ERKs) and c-Jun NH2-terminal/stress-activated kinases and transcriptional activity of activator protein-1 and nuclear factor-kappa B (30).

Although the responsible molecular mechanisms for the age-related increase in gastrointestinal mucosal proliferative activity are poorly understood, we (17, 25) have observed that in the gastric mucosa aging is associated with increased expression and activation of certain tyrosine kinases, most notably the epidermal growth factor receptor (EGFR), the common receptor for EGF and transforming growth factor-alpha (TGF-alpha ). Basal EGFR tyrosine kinase activity in the colonic mucosa of aged Fischer 344 rats is also found to be higher than in young animals (19). Numerous studies (11, 23) have demonstrated that the EGF family of peptides, particularly EGF and TGF-alpha , stimulate proliferative activity in much of the gastrointestinal tract, including the stomach and colon.

EGF and TGF-alpha initiate their mitogenic action by activating the intrinsic tyrosine kinase activity of their receptor, triggering the EGFR signaling processes (21, 23, 27). However, TGF-alpha , as opposed to EGF, is synthesized in the mucosa of much of the gastrointestinal tract (2, 5, 23), suggesting that TGF-alpha may be physiologically more important than EGF in modulating mucosal proliferation. Results from cell surface immunocytochemical and biochemical characterization studies (4, 20, 22) have demonstrated that the presence of the transmembrane TGF-alpha at the cell surface is a normal consequence of TGF-alpha synthesis, and in most cases the peptide is present on the cell surface in its precursor form. Moreover, it has been reported (3, 4) that the membrane-bound precursor form(s) of TGF-alpha can also activate the intrinsic tyrosine kinase activity of EGFR, suggesting a role for the precursors of the growth factor in regulating EGFR signal transduction pathways, probably through an autocrine/juxtacrine mechanism (3). Our (25) earlier observation that, in the gastric mucosa, the age-related rise in EGFR tyrosine kinase activity is associated with a concomitant increase in membrane-associated precursor forms of TGF-alpha , suggests a plausible role for the membrane-bound form(s) of TGF-alpha in modulating mucosal EGFR function during aging. Whether the same phenomenon prevails in the colonic mucosa has not been investigated. In addition, no information is available as to whether the membrane-bound precursor form(s) of TGF-alpha from the gastric and/or colonic mucosa will modulate EGFR activation in vitro, and if so, whether this modulation will be affected by aging. Therefore, to determine the role of the membrane-bound precursors of TGF-alpha in regulating EGFR function in the gastric and colonic mucosa during advancing age, the present investigation examines 1) the relationship between EGFR activation and the levels of membrane-associated precursor form(s) of TGF-alpha in the gastric and colonic mucosa during aging and 2) whether aging alters the ability of the membrane-bound precursors form(s) of TGF-alpha to activate EGFR and the signaling pathway.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
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Reagents. Rabbit polyclonal antibodies to EGFR and monoclonal antibody against TGF-alpha were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibody against phosphotyrosine (4G10) was from Upstate Biotechnology (Lake Placid, NY). Polyclonal antibodies to phospho-ERKs [p42/44; (Thr202/Tyr204)] were from New England Biolabs (Beverly, MA). Enhanced chemiluminescence (ECL) protein biotinylation module (RPN2202), goat anti-mouse or rabbit IgG conjugated with horseradish peroxidase (HRP), and the ECL system were products of Amersham (Arlington Heights, IL). Immobilon-P nylon membrane was purchased from Millipore (Bedford, MA). Concentrated protein assay dye reagent was from Bio-Rad (Hercules, CA). Molecular weight marker was obtained from GIBCO-BRL (Grand Island, NY). All other reagents were of molecular biology grade and were purchased from either Sigma or Fisher Scientific.

Animal and collection of tissues. In all experiments, male Fischer 344 rats aged 4 (young) and 23 mo (old) were used. The animals were purchased from the National Institute on Aging (Bethesda, MD) at least 1 mo before the experiment. During this period, the rats had access to Purina rat chow and water ad libitum. Four to five animals from each age group were utilized in this investigation. In all experiments, animals were fasted overnight before being killed. The stomach and entire colon were removed and used immediately for isolation of cells. In experiments in which mucosa was to be used, the stomach and colon were slit open and rinsed thoroughly with cold PBS. Mucosa was obtained by scraping with glass slides. Mucosal scraping were either processed immediately or frozen in small aliquots in liquid nitrogen and stored at -90°C.

Isolation of membrane-bound TGF-alpha . Crude mucosal membranes (30,000 g pellet) were prepared as described previously (26) and subsequently solubilized in lysis buffer (10 mM HEPES, pH 7.2, 150 mM NaCl, 2.5 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM EDTA, 25 µg/ml of aprotinin, leupeptin, and pepstatin A, 0.5% Triton X-100, and 0.5% Nonidet P-40). After clarification at 11,000 g for 15 min at 4°C, the supernatants were used for immunoprecipitation of TGF-alpha . In all immunoprecipitation studies, protein concentration was standardized among the samples.

To immunoprecipitate TGF-alpha , aliquots of mucosal membranes containing 3 mg protein were incubated overnight at 4°C with 1 µg of monoclonal anti-TGF-alpha antibodies and 30 µl of protein A-Sepharose beads. The beads were washed three times with TT buffer (50 mM Tris · HCl, pH 7.6, 0.15 M NaCl, and 0.5% Tween 20). TGF-alpha was then eluted from beads as described previously (28). Briefly, 2 bead volumes (60 µl) of 0.1 M glycine buffer (pH 2.5) were added, and the mixture was incubated at 4°C for 5 min. The reaction mixture was neutralized by adding [1/10] volume (6 µl) of 1 M Tris · HCl, pH 9.0, and then spun at 10,000 g for 10 min to recover TGF-alpha in the supernatant.

Immunoprecipitation and Western immunoblot analysis. This was performed according to our standard protocol (29-31). Briefly, aliquots of the mucosal membranes containing 2 or 3 mg proteins were incubated with either polyclonal anti-EGFR or monoclonal anti-TGF-alpha antibodies and Sepharose-protein G or Sepharose-protein A beads at 4°C for 3 h. Immunoprecipitates were resolved on a 7.5% or 15% SDS-PAGE. The electrophoresed proteins were transferred to Immobilon-P nylon membranes. The membranes containing EGFR were probed with anti-phosphotyrosine, and those containing TGF-alpha were probed with TGF-alpha antibodies. Protein bands were visualized by the ECL detection system and quantitated by densitometry. All Western immunoblots were performed at least three times using different rats from each age group.

Activation of EGFR in vitro. This was performed using the colon cancer cell line HCT 116. Briefly, aliquots of HCT 116 cells were incubated with 20 µl of TGF-alpha eluted from gastric or colonic mucosal membranes (containing 3 mg protein) as described above. Cells were incubated for 8 min at 37°C. After incubation, cells were lysed with 300 µl lysis buffer (50 mM Tris buffer, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 2.5 mM Na3VO4, 25 µg/ml of aprotinin, leupeptin, and soybean trypsin inhibitor A, 0.5% Triton X-100, and 0.5% Nonidet P-40). EGFR was immunoprecipitated from aliquots of cell lysates containing 1 mg protein as described above. The immunprecipitates were washed three times with TT buffer and twice with kinase buffer [25 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MnCl2, 0.5 mM dithiothreitol (DTT), 0.5 mM Na3VO4, 5 mM beta -glycerol phosphate, and 10 mM p-nitrophenyl phosphate], and finally incubated in 25 µl kinase reaction mixture (25 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MnCl2, 0.5 mM DTT, 0.5 mM Na3VO4, 5 mM beta -glycerol phosphate, 10 mM p-nitrophenyl phosphate, and 20 µM ATP) containing 5 µCi [gamma -32P]ATP for 30 min at 30°C. The reactions were stopped by adding 2× loading buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 4% beta -mercaptoethanol, and 0.02% bromophenol blue). The samples were boiled for 4 min and subjected to a 7.5% SDS-PAGE. The gels were dried and autoradiographed. The extent of EGFR phosphorylation was quantitated by densitometry.

In experiments in which ERK (p42/44) activation was measured, cell lysates containing 50 µg of protein were subjected to Western blot analysis using phospho-ERK(p42/44) antibodies as described above.

Biotinylation of cell surface proteins in gastric and colonic mucosal cells. The presence of TGF-alpha on gastric and colonic mucosal cell surface was examined by biotinylation. For this purpose, mucosal cells from all groups of rats were isolated by a slight modification of the procedures described by Kinoshita et al. (12). Briefly, contents of the stomach and colon were washed out by rinsing with PBS. The stomach and colon were transformed into inside-out gastric or colonic bags, respectively, and filled with 3 (stomach) or 1 mg/ml (colon) Pronase solution in buffer A (0.5 mM NaH2PO4, 1 mM Na2HPO4, 70 mM NaCl, 5 mM KCl, 11 mM glucose, 50 mM HEPES, pH 7.2, 20 mM NaHCO3, 2 mM EDTA, and 2% BSA). The filled bags were incubated in Pronase-free buffer A at 37°C for 30 min. The bags were then transferred into buffer B (containing 1 mM CaCl2 and 1.5 mM MgCl2 instead of EDTA in buffer A) and gently agitated by a magnetic stirrer at room temperature for 1 h. The epithelial cells, dispersed in buffer B, were collected by centrifuging at 500 g for 5 min and immediately utilized for biotinylation of protein using a commercial ECL biotinylation kit (Amersham), according to the manufacturer's suggested protocol. Briefly, freshly isolated gastric or colonic mucosal cells were washed twice with cold PBS and resuspended in 1 ml ice-cold 40 mM of bicarbonate buffer at a concentration of 5 × 106 cells/ml. Next, 40 µl of biotinylation reagent (biotinamidocaproate N-hydroxysuccinimide ester) were added per milliliter of cell suspension and subsequently incubated for 30 min at 4°C under constant agitation. The labeled cells were washed three times with cold PBS and lysed in lysis buffer and then centrifuged for 15 min at 4°C. Aliquots of cell lysates containing 500 µg proteins were subjected to immunoprecipitaton with 1 µg monoclonal anti-TGF-alpha antibodies and protein A-Sepharose beads at 4°C for 3 h. The immunoprecipitates were subjected to 15% SDS-PAGE. The electrophoresed proteins were transferred to the Immobilon-P membranes, blocked with 5% blocking reagent in PBS containing Triton X-100 for 1 h and incubated with a 1:1,500 dilution of streptavidin-HRP for 1 h at room temperature. The proteins were visualized with the ECL detection system and quantitated by densitometry.

Statistical analysis. Results were statistically evaluated using Student's t-test for unpaired values.


    RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

Although we (17, 25) have repeatedly demonstrated that aging is associated with increased activation of EGFR in the gastric mucosa, this phenomenon has not been investigated in detail in the colonic mucosa. Therefore, in the current investigation, we examined the changes in EGFR activation in the colonic mucosa of young (4 mo) and aged (23 mo) rats by analyzing the extent of tyrosine phophorylation of the receptor. The gastric mucosa was also included for comparison purposes. Results revealed that in both gastric and colonic mucosa, aging was associated with a marked increase in EGFR activation. The levels of phosphotyrosine EGFR in the gastric and colonic mucosa of 23-mo-old rats were found to be 300-350% higher than the corresponding levels in 4-mo-old animals (Fig. 1).


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Fig. 1.   Western immunoblot showing changes in levels of phosphotyrosine-epidermal growth factor receptor (EGFR) in the gastric and colonic mucosa of 4- and 23-mo-old rats. Relative changes in the levels of phosphotyrosine-EGFR in young and old animals, as determined by densitometric analysis, are shown in the histogram. Values are means ± SE of 3-4 experiments. ** P < 0.01, compared with corresponding 4-mo-old rats.

To determine whether the age-related increase in EGFR activation in the gastric and colonic mucosa was associated with a concomitant increase in membrane-bound precursor form(s) of TGF-alpha , we analyzed the relative concentration of this form(s) in the gastric and colonic mucosa of 4- and 23-mo-old rats by both Western immunoblot and biotinylation of cell surface proteins. The results of Western blot analysis and biotinylation of cell surface proteins revealed the presence of precursors of TGF-alpha in the gastric and colonic mucosal membranes of both age groups; the molecular mass of the membrane-bound forms of TGF-alpha ranged between 18-20 kDa (Fig. 2). The relative concentration of these TGF-alpha forms was found to be substantially higher (200-1,000%) in 23-mo-old rats, compared with the corresponding levels in 4-mo-old animals (Fig. 2). It should also be noted that under both experimental conditions, the TGF-alpha band from gastric and colonic mucosal membranes from aged rats was broader than the corresponding band from young animals (Fig. 2). This could due to the presence of several closely related precursor forms of TGF-alpha in gastric and colonic mucosal membranes of aged rats.


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Fig. 2.   Western immunoblot (A) and cell surface protein biotinylation (B) showing changes in the levels of membrane-bound transforming growth factor-alpha (TGF-alpha ) in the gastric and colonic mucosa of 4- and 23-mo-old rats. Relative changes in membrane-bound TGF-alpha levels in young and old animals, as determined by densitometric analysis, are shown in the histogram. Values are means ± SE of 3-4 experiments. ** P < 0.01, compared with corresponding 4-mo-old rats.

Because ligand binding is one of the primary causes of activation of EGFR, we sought to determine whether a part of the age-related rise in EGFR activation in the gastric and colonic mucosa could be due to a greater amount of TGF-alpha bound to EGFR. Indeed, results of four experiments consistently showed the proportion of TGF-alpha bound to EGFR in the gastric and colonic mucosa of aged rats to be considerably higher than in young animals. The results, depicted in Fig. 3, revealed that the proportion of TGF-alpha bound to EGFR in the gastric and colonic mucosa of aged rats was 150-250% higher than in young animals. The molecular form(s) of TGF-alpha bound to EGFR in all samples was between 18 and 20 kDa, indicating that the major forms of TGF-alpha bound to EGFR are high molecular mass precursor forms of the peptide.


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Fig. 3.   Western immunoblot showing changes in the levels of TGF-alpha bound to EGFR in the gastric and colonic mucosal membranes from 4- and 23-mo-old rats. EGFR immunoprecipitates were subjected to Western blot analysis using anti-TGF-alpha antibodies. Relative changes in the levels of TGF-alpha -bound to EGFR in young and old animals, as determined by densitometric analysis, are shown in the histogram. Values are means ± SE of 3-4 experiments. * P < 0.05, ** P < 0.01, compared with corresponding 4-mo-old rats.

The next set of experiments was performed to compare the ability of TGF-alpha present in gastric and colonic mucosal membranes of young and aged rats to activate EGFR. In vitro studies were performed utilizing the colon cancer cell line HCT 116. We observed that exposure of HCT 116 cells to TGF-alpha , extracted from gastric and colonic mucosal membranes of young and aged rats (TGF-alpha immunoprecipitated from 3 mg membrane and subsequently eluted), stimulated EGFR activation as evidenced by increased phosphorylation of the receptor compared with the control (Fig. 4). However, whereas TGF-alpha from mucosal membranes from 4-mo-old rats produced only a minor (20-30%) increase, the peptide extracted from aged gastric and colonic mucosal membranes caused a marked 175-250% stimulation in EGFR activation compared with the control (Fig. 4). As a positive control, we also measured the magnitude of stimulation of EGFR phosphorylation in HCT 116 cells in response to 1 nM synthetic TGF-alpha , which produced an ~400% stimulation in EGFR activation compared with the control (Fig. 4).


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Fig. 4.   Changes in the extent of EGFR phosphorylation in HCT 116 cells in response to TGF-alpha from the gastric and colonic mucosal membranes of 4- and 23-mo-old rats. Relative changes in phosphorylated EGFR, as determined by densitometric analysis, are shown in the histogram. Values are means ± SE of 3-4 experiments. ** P < 0.01, compared with controls not exposed to TGF-alpha .

The last set of experiments was performed to determine whether the observed induction in EGFR activation in HCT 116 cells by TGF-alpha from aged gastric and colonic mucosal membranes will also result in activation of the downstream events of the EGFR signal transduction pathways. We examined the extent of phosphorylation of ERKs (p42/44), one of the mitogen-activated protein kinases, in HCT 116 cells after an 8-min exposure to TGF-alpha from gastric and colonic mucosal membranes from young and aged rats. As observed for EGFR, TGF-alpha , extracted from gastric and colonic mucosal membranes of aged rats, markedly stimulated (100-200%) the levels of phosphorylated ERKs (p44/42), compared with the control (Fig. 5). On the other hand, TGF-alpha from gastric and colonic mucosal membranes of young rats caused only a small (20-40%) increase in phosphorylation of ERKs over the control (Fig. 5).


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Fig. 5.   Western immunoblot showing changes in the levels of phosphorylated extracellular signal-related kinases (ERKs; p42/44) in HCT 116 cells in response to TGF-alpha from the gastric and colonic mucosal membranes of 4- and 23-mo-old rats. Relative changes in phosphorylated ERKs, as determined by densitometric analysis, are shown in the histogram. Values represent means ± SE of 3-4 experiments. * P < 0.05, ** P < 0.01, compared with controls not exposed to TGF-alpha .


    DISCUSSION
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INTRODUCTION
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Accumulating evidence suggests that many gastrointestinal hormones and growth factors, most notably EGF and its structural and functional homologue TGF-alpha , are critically involved in regulating mucosal growth in various parts of the gastrointestinal tract, including the stomach and colon (2, 11, 23). However, mucosal responsiveness to different gastrointestinal hormones and growth factors changes with aging. We (15, 18) have reported that doses of gastrin and bombesin, which stimulate gastric mucosal proliferative activity in young rats, have no effect on mucosal proliferation in aged rats. On the other hand, pharmacological doses of either EGF or TGF-alpha have been found to inhibit gastric mucosal proliferation in aged rats (14, 26). This has, however, been attributed to increased sensitivity of the aged gastric mucosa to EGF and TGF-alpha , so that low doses are stimulatory, whereas high doses inhibit proliferative processes (26).

EGF and TGF-alpha exert their mitogenic action by binding to their common receptor, the EGFR, a 170-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity (21, 27). On binding to ligands, EGFR undergoes dimerization followed by activation of the intrinsic tyrosine kinase with subsequent auto- and transphosphorylation of the receptor leading to the induction of the signal transduction pathways, resulting in stimulation in cell proliferation (7, 21, 27). Our current observation that activation of EGFR, as assessed by the extent of tyrosine phosphorylaton of the receptor, is higher in the gastric and colonic mucosa of aged than in young rats supports our contention that stimulation of EGFR signaling pathways is partly responsible for the age-related increase in gastric and colonic mucosal proliferation. Although the regulatory mechanisms for EGFR activation in the gastric and colonic mucosa of aged rats remain to be elucidated, we postulated that TGF-alpha , one of the primary ligands for EGFR, plays a key role in regulating this process. The basis for this postulation comes from the fact that TGF-alpha , but not EGF, is synthesized in much of the gastrointestinal tract (2, 5, 23), and specific binding sites for TGF-alpha are particularly abundant in the stomach and colon (24). Additionally, we (25) have reported that the age-associated rise in tyrosine kinase activity and tyrosine phosphorylation of EGFR in the gastric mucosa is also accompanied by a concomitant increase in the levels of membrane-bound precursor form(s) of TGF-alpha . Our current observation that the levels of membrane-bound precursors form(s) of TGF-alpha are also higher in the gastric and colonic mucosa of aged than in young rats suggests that the membrane-bound TGF-alpha may partly be responsible for activating EGFR in these tissues, probably through an autocrine/juxtacrine mechanism(s). Additional support comes from the observation that the fraction of TGF-alpha bound to gastric and colonic mucosal EGFR is also higher in aged than in young rats. Because aging is also associated with increased expression of EGFR protein in the gastric mucosa (25), our finding of a greater amount of TGF-alpha bound to EGFR in the gastric and colonic mucosa of aged than in young rats could partly be due to an increased number of EGFR molecules.

TGF-alpha is derived from a larger 20- to 22-kDa transmembrane precursor (4, 6), and the presence of the transmembrane TGF-alpha at the cell surface is a normal consequence of TGF-alpha synthesis (4, 20, 22). Although the precise mechanism(s) for accumulation of precursor form(s) of TGF-alpha in cellular membranes has not been fully elucidated, it has been demonstrated that release of mature TGF-alpha from pro-TGF-alpha is inefficient in most cells, which may cause accumulation of pro-TGF-alpha on the cell surface (20). However, studies (4, 13) utilizing different cell lines have demonstrated that the membrane-associated precursors of TGF-alpha are biologically active in that they activate intrinsic tyrosine kinases on adjacent cells. Our current data are in complete agreement with these observations. The results of our biotinylation of cell surface proteins show the presence of 18- to 20-kDa transmembrane precursor forms of TGF-alpha in both gastric and colonic mucosa, indicating that normal gastrointestinal mucosal membranes, as has been observed in malignant cells (20), also possess precursors of TGF-alpha . The fact that the relative concentration of these forms of TGF-alpha in gastric and colonic mucosal membranes is higher in aged than in young rats suggests that they are partly responsible for modulating the age-related increase in EGFR activation in the gastric and colonic mucosa. In support of this postulation, we have observed that exposure of HCT 116 cells to TGF-alpha , extracted from gastric and colonic mucosal membranes of aged rats, causes a substantially greater stimulation in EGFR phosphorylation, compared with the levels achieved with the corresponding TGF-alpha from young rats. Further support comes from the observation that, in HCT 116 cells, TGF-alpha from gastric and colonic mucosal membranes of aged rats produces a comparatively greater stimulation in ERK activation than that achieved with the membrane-associated peptide from young rats. ERKs, which are known to be activated by several growth factors, including the EGF family of peptides, are linked to cell proliferation (21). Our observation of increased activation of ERKs (p42/44) in HCT 116 cells by TGF-alpha from gastric and colonic mucosal membranes of aged rats further suggests participation of the transmembrane TGF-alpha in enhancing the EGFR signaling pathways, leading to induction of mucosal proliferative activity during aging.

In conclusion, our current data demonstrate that aging is associated with increased activation of EGFR in both gastric and colonic mucosa. This is also accompanied by a concomitant increase in membrane-bound precursor forms of TGF-alpha . Moreover, the membrane-associated precursors of TGF-alpha from gastric and colonic mucosa of aged rats are more active than those from young rats in inducing the EGFR signal transduction processes.


    ACKNOWLEDGEMENTS

This study was supported by National Institute on Aging Grant AG-14343 and by the Department of Veterans Affairs.


    FOOTNOTES

Z.-Q. Xiao is a visiting scientist from Hunan Medical University, Changsha, People's Republic of China

Address for reprint requests and other correspondence: A. P. N. Majumdar, Research Service-151, Veterans Affairs Medical Center, 4646 John R, Detroit, MI 48201 (E-mail: a.majumdar{at}wayne.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 August 2000; accepted in final form 31 January 2001.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 281(1):G111-G116