Regulation of TGF-{alpha}-induced activation of AP-1 in the aging gastric mucosa

Zhi-Qiang Xiao,2 Jianling Li,2 and Adhip P. N. Majumdar1,2,3

1Veterans Affairs Medical Center, 2Department of Internal Medicine, and 3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201

Submitted 19 February 2003 ; accepted in final form 28 March 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Although the age-related activation of EGF receptor (EGFR) in the gastric mucosa of Fischer 344 rats is associated with increased DNA binding activity of activator protein-1 (AP-1), little is known about the EGFR signaling cascades that regulate this process. The primary objective of this investigation was to determine the role of signaling pathways initiated by EGFR in regulating the transforming growth factor-{alpha} (TGF-{alpha})-induced activation of AP-1 in the gastric mucosa in aged rats. Freshly isolated gastric mucosal cells from male young (4–5 mo) and aged (22–24 mo) rats were used. We have observed that although exposure of mucosal cells from young (4–5 mo) and old (22–24 mo) rats to 1 nM TGF-{alpha} for 20 min stimulates the DNA binding activity of AP-1 in both age groups, the magnitude of stimulation is substantially higher in aged (131%) than in young (35%) rats. This stimulation in the aged is associated with a concomitant activation of MEKs and ERKs, but not JNKs and p38. The TGF-{alpha} induction of AP-1 transcriptional activity in gastric mucosal cells from aged rats could be totally abrogated by either PD153035, a specific inhibitor of EGFR tyrosine kinase, or PD98059, a specific inhibitor of MEKs, but not by Wortmannin, which inhibits phosphatidylinositol kinase. PP2, a specific inhibitor of Src kinase, produces a 50% inhibition of the TGF-{alpha}-induced activation of AP-1 transcriptional activity. Our results suggest that the TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in the gastric mucosa of aged rats is primarily through a signaling pathway involving MEKs and ERKs, whereas Src kinase pathways play a minor role.

epidermal growth factor receptor signaling processes; phosphatidylinositol kinase; Src kinase; transcription factor


OVER THE PAST SEVERAL YEARS, results from this and other laboratories have demonstrated that in barrier-reared Fischer 344 rats, aging is associated with increased mucosal proliferative activity in various tissues of the gastrointestinal tract, including the oxyntic gland area of the stomach (2, 11, 12, 21, 19, 22). In the oxyntic gland area (referred to as gastric mucosa), the age-related rise in mucosal proliferative activity was demonstrated by increased labeling index, DNA synthesis, and thymidine kinase and orinithine decarcoxylase (ODC) activities (6, 1921, 23) Moreover, progression of gastric mucosal cells through G1 into S phase was also shown to be increased in aged rats, as reflected by increased levels of cyclin E and the activity of cyclin-dependent kinase (Cdk2) (35)

Although the responsible mechanisms for the age-related rise in gastric mucosal proliferative activity are poorly understood, we have suggested that EGF receptor (EGFR)-induced signaling pathways may play a critical role in regulating this process (22, 30). The basis for this postulation comes from our observation that increased gastric mucosal proliferative activity during aging in Fischer 344 rats is associated with a concomitant activation of EGFR, as evidenced by increased tyrosine kinase activity and tyrosine phosphorylation of the receptor (30, 34). Aging is also found to be associated with increased levels of membrane-bound precursor forms of transforming growth factor-{alpha} (TGF-{alpha}), one of the primary ligands of EGFR in the gastric mucosa (34). In addition, sensitivity of EGFR in the gastric mucosa to TGF-{alpha} and EGF is found to increase with advancing age (31).

Activation of EGFR triggers a complex array of enzymatic events through activation of Ras, converging on MAPKs, which, after translocation to the nucleus, activate transcription factors and produce activation of genes that promote growth (24, 27, 28, 32). We have demonstrated that with aging, there is a marked activation of the ERKs and JNKs, and these changes are accompanied by a concomitant stimulation of the DNA binding activity of activator protein-1 (AP-1) and NF-{kappa}B (33). AP-1, which is involved in regulating cell proliferation, responds to many stimuli including growth factors and cytokines (1, 14). In view of this, we also examined the responsiveness of AP-1 to TGF-{alpha} in the gastric mucosa of young and aged rats and demonstrated a comparatively greater activation of AP-1 in aged than in young rats (33). However, the intracellular signaling events involved in mediating this process in the gastric mucosa of aged rats remains to be delineated.

Numerous studies have demonstrated that activation of EGFR not only stimulates Ras/Raf/MAP kinase signaling cascade but also phosphatidylinositol (PI3) kinase and Src kinase pathways (3, 13, 24), each of which is capable of affecting transcriptional activity of AP-1. However, the signaling pathway(s) involved in mediating the TGF-{alpha}-induced activation of AP-1 in the gastric mucosa during aging remain to be determined. The present investigation was, therefore, undertaken to examine the involvement of different EGFR signaling pathways in regulating the TGF-{alpha}-induced activation of AP-1 in the gastric mucosa of aged rats.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Reagents. Double-stranded oligonucleotide probe containing the consensus sequence of AP-1 (5'-CGC TTF ATG AGT CAG CCG GAA-3') and the T4 polynucleotide kinase were purchased from Promega (Madison, WI), Poly (dI-dC). Poly (dI-dC) was obtained from Pharmacia Biotech (Piscataway, NJ). [{gamma}-32P]ATP (3,000 Ci/mmol) was from New England Nuclear Life Science (Boston, MA). Polyclonal rabbit antibodies to EGFR, JNK, and ERK1/2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit antibodies to p38, phospho-p38 (Thr-180/Tyr-182), phospho-ERK1/2 (Thr-202/Tyr-204), phospho-JNK1/2 (Thr-183/Tyr-185), MEK1/2, and phospho-MEK1/2 (Ser-212/221) were purchased from New England Biolaboratories (Beverly, MA). Recombinant human TGF-{alpha}, PD153035, PD98059, PP2, and Wortmaninn were obtained from Calbiochem (La Jolla, CA). Goat anti-rabbit IgG conjugated with horseradish peroxidase and enhanced chemiluminescence (ECL) were obtained from Amersham (Arlington Heights, IL). Immobilon-P nylon membrane was from Millipore (Bedford, MA). Concentrated protein assay dye reagent was from Bio-Rad Laboratories (Hercules, CA). Molecular weight marker and DMEM medium were from GIBCO-BRL (Grand Island, NY). All other reagents were of molecular biology grade and were purchased from Sigma or Fisher Scientific.

Animals and isolation of gastric mucosal epithelial cells. Male Fischer 344 rats aged 4–5 (young) and 22–24 (aged) mo were used. The animals were purchased from the National Institute on Aging (Bethesda, MD) 2 mo before the experiment. During this period, they had access to Purina rat chow and water ad libitum. Animals were fasted overnight before the experiments.

All experiments were performed using freshly isolated gastric mucosal cells. Cells were isolated from overnight fasted rats by a slight modification (33) of the procedure described by Kinoshita et al. (16). Briefly, the contents of the stomach were washed out with PBS. The stomach was then ligated at the base of the forestomach and the proximal end of antrum to obtain mucosal cells primarily from the oxyntic gland area of the stomach. After being transformed into inside-out gastric bags, they were filled with 5 ml of 3 mg/ml pronase solution in buffer A (in mM: 0.5 NaH2PO4, 1.0 Na2HPO4, 70 NaCl, 5.0 KCl, 11 glucose, 50 HEPES, pH 7.2, 20 NaHCO3, and 2 EDTA and 2% BSA). The filled gastric bags were incubated in pronase-free buffer A at 37°C for 30 min. The gastric bags were then transferred into buffer B (containing 1.0 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 subsequently were resuspended in 4–5 ml serum-free DMEM. The yield ranged between 50 and 70 x 106 cells/stomach. After aliquots of cell suspension in 1 ml of serum-free DMEM were washed twice with serum-free DMEM, they were exposed to TGF-{alpha} for 20 min at 37°C. In experiments to study the effect of inhibitors of EGFR signaling pathways, aliquots of cells in 1 ml serum-free DMEM containing ~10 x 106 cells were pretreated with different concentrations of PD153035, PD98059, Wortmanin, or PP2 for 10 min at 37°C. After treatment of mucosal cells with TGF-{alpha} or different inhibitors, nuclear extracts were immediately prepared and used for assessing AP-1 activity by EMSA as described in EMSA. At the beginning and end of each experiment, cell viability was monitored by the trypan blue exclusion test.

Preparation of nuclear extracts. Nuclear extracts were prepared by a slight modification (33) of the method described by others (4, 10). Briefly, freshly isolated mucosal cells were resuspended in ice-cold hypotonic buffer (in mM: 10 HEPES, pH 7.9, 1.5 MgCl2, 10 KCl, 0.2 PMSF, and 1 DTT, with 5 µg/ml of aprotinin, pepstatin A, and leupeptin) and were incubated for 10 min at 4°C. Swollen cells were homogenized with 10 or more slow up and down strokes in a glass Dounce homogenizer and centrifuged at 3,300 g for 15 min at 4°C. The pelleted nuclei were washed once with ice-cold low-salt buffer (in mM: 20 HEPES, pH 7.9, 1.5 MgCl2, 20 KCl, 0.2 PMSF, 1.0 DTT, and 0.2 EDTA with 25% glycerol and 5 µg/ml of aprotinin, pepstatin A, and leupeptin) by centrifuging at 10,000 g for 15 min at 4°C. The nuclei were resuspended in ice-cold low-salt buffer, and nuclear protein was released by adding an ice-cold high-salt buffer (same as the low-salt buffer, except that it contained 1.2 M KCl) drop by drop to a final concentration of 0.4 M KCl. The samples were rotated at 4°C for 30 min. The nuclear extracts were recovered by centrifugation at 25,000 g for 30 min at 4°C and stored at -80°C in small aliquots.

EMSA. EMSA was used to determine the DNA binding activity of AP-1 by assaying the extent of binding of nuclear extracts to AP-1 consensus sequence as described by Gupta et al. (10). Briefly, probes containing the consensus sequences of AP-1 were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase according to the protocol provided by Promega. Labeled oligonucleotides were purified by chromatography through a Sephadex G-25 spin column. For DNA-protein binding reactions, 5 or 10 µg of nuclear protein and 2 µg of Poly (dI-dC) were preincubated in 20 µl binding buffer (10 mM HEPES, pH 7.5, 4% glycerol, 1.0 mM MgCl2, 50 mM KCl, 0.5 mM EDTA, and 1.0 mM DTT) for 15 min at 4°C, and then 300,000 counts/min (cpm) of radiolabeled probe was added. Reactions were further incubated for 20 min at room temperature. The resulting products were separated by 6% native polyacrylamide gel containing 0.25x 89 mM Tris, pH 8.3, 89 mM boric acid, and 2 mM EDTA (TBE) with 0.25x TBE as the running buffer. Gels were dried and exposed to film at -80°C with intensifying screens. Signals on the film were quantitated by densitometry using ImageQuant Image Analysis System (Storm Optical Scanner, Molecular Dynamics, Sunnyvale, CA). Competition was performed by adding the respective nonradioactive oligonucleotide probes to the reaction mixture in 50-fold molar excess. Signals on the blots were visualized by autoradiography and quantitated by densitometry using ImageQuant image-analysis system. All assays were repeated at least three times using nuclear extracts from different rats for each age group.

Cell lysis and Western blot analysis. Freshly isolated gastric mucosal cells from each rat were lysed in 0.2 ml lysis buffer (50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 2.5 mM EDTA, 2.5 mM Na3VO4, 0.5% Triton X-100, 0.5% Nonidet P-40, 5 µg/ml of aprotinin, pepstatin, and leupeptin). Western blotting was performed according to our standard protocol (34, 35). In all Western blot analyses, protein concentration was standardized among the samples. Briefly, 100 µg proteins were separated on a 10% SDS-PAGE and subsequently electroblotted to Immobilon-P nylon membranes. The membranes were blocked overnight with 5% BSA or nonfat dried milk in buffer containing 20 mM Tris·HCl, pH 7.6, 100 mM NaCl, and 0.01% Tween-20 (TBS-T) followed by 3 h of incubation with the primary antibodies (phospho-ERK1/2, phospho-MEK1/2, phospho-JNK1/2, or phosphop38, each at 1:1,000 final dilution) in TBS-T buffer containing 5% BSA at room temperature. After the membranes were washed three times with TBS-T buffer, they were incubated with a horseradish peroxide-conjugated goat anti-rabbit IgG (1:5,000 final dilution) as a second antibody for 1 h at room temperature. Protein band(s) were visualized using ECL detection system and quantitated by densitometry. After detection of phospho-ERK1/2, MEK1/2, JNK1/2, or p38, the membranes were treated with SDS/2-mercaptoethanol stripping buffer and reprobed with the corresponding nonphosphorylated antibodies (1:1,000 final dilution). All Western blots were performed at least three times using total cell extracts from different rats.

EGFR tyrosine kinase activity. Immunocomplex assays were performed as described previously (31). Briefly, freshly isolated gastric mucosal cells were washed with ice-cold PBS and lysed in lysis buffer. Lysates were clarified by centrifuging at 11,000 g for 15 min at 4°C. After determination of protein by the Bio-Rad protein assay kit, aliquots of cell lysates containing 1.5 mg protein were subjected to immunoprecipitation with EGFR antibodies and Sepharose G under constant stirring at 4°C for 3 h. Immunocomplexes were washed three times with TT buffer (50 mM Tris·HCl, pH 7.6, 0.15 M NaCl, and 0.5% Tween-20) and twice with kinase buffer [20 mM HEPES, pH 7.5, 20 mM {beta}-glycerol phosphate, 10 mM p-nitrophenyl phosphate (PNPP), 10 mM MnCl2, 1 mM DTT, and 0.5 mM Na3VO4]. The kinase reactions were performed by incubating the immunoprecipitates with 25 µl kinase reaction buffer (20 mM HEPES, pH 7.5, 20 mM {beta}-glycerol phosphate, 10 mM PNPP, 10 mM MnCl2, 1 mM DTT, 0.5 mM Na3VO4, and 20 µM unlabeled ATP containing 5 µCi [{gamma}-32P]ATP). After 30 min at 30°C, the reactions were stopped by adding 2x loading buffer (125 mM Tris·HCl, pH 6.8, 4% SDS, 10% glycerol, 4% {beta}-mercaptoethanol, and 0.02% bromophenol blue). The samples were boiled for 4 min and resolved by 7.5% SDS-PAGE. The gels were dried and subjected to autoradiography. The extent of EGFR phosphorylation was quantitated by densitometry as described in EMSA. The kinase assays were performed three times using cell extracts from different rats.

Statistical analysis. Where applicable, results were analyzed using ANOVA followed by Fischer's protected least-significant differences or Scheffé's test. A P value of <0.05 was designated as the level of significance.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
TGF-{alpha}-induced activation of AP-1 is greater in the gastric mucosa of aged than in young rats. Our previous observation that the age-related rise in EGFR activation in the gastric mucosa was accompanied by a concomitant increase in membrane-bound precursor form(s) of TGF-{alpha} led us to postulate that TGF-{alpha} might play a critical role in regulating gastric mucosal EGFR function during aging through an autocrine/juxtacrine mechanism (34). To further determine whether conditions that activate EGFR in aged gastric mucosa will also stimulate the transcriptional activity of AP-1, we compared the effect of TGF-{alpha} on DNA binding activity of AP-1 in freshly isolated gastric mucosal cells from young (4–5 mo) and aged (22–24 mo) rats. In agreement with our earlier observation (33), we noted that although TGF-{alpha} stimulated DNA binding activity of AP-1 in both age groups, the magnitude of stimulation was substantially higher in aged (130%) than in young (35%) rats when compared with the corresponding controls (Fig. 1). In this and all subsequent experiments, no appreciable binding of nuclear extracts to the AP-1 consensus sequence was detected in the presence of 50-fold molar excess of unlabeled oligonucleotide probe, indicating specificity of binding of AP-1 to oligonucleotide probe containing the consensus sequence of the transcription factor. Further support for this was derived from our earlier experiment, which demonstrated that antibodies to either Jun or Fos completely supershifted AP-1/DNA complex formed with these proteins from rat gastric mucosal nuclear extracts (33).



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Fig. 1. Representative autoradiograph of EMSA showing changes in DNA binding activity of activator protein-1 (AP-1) in freshly isolated gastric mucosal cells from 4 (young)- and 23 (aged)-mo-old rats in response to transforming growth factor-{alpha} (TGF-{alpha}). Aliquots of gastric mucosal cells from young and aged rats were incubated at 37°C for 20 min in the absence (controls) or presence of 1 nM TGF-{alpha}. After incubation, aliquots of nuclear extracts (5 µg protein) from mucosal cells were subjected to EMSA analysis with 32P-labeled oligonucleotide probe containing consensus sequence of AP-1. Histogram shows the relative changes in DNA binding activity in response to TGF-{alpha}, as determined by densitometric analysis. The DNA binding activity of AP-1 of nuclear extracts from gastric mucosal cells of young rats was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.01 compared with the control.

 

TGF-{alpha}-induced stimulation of AP-1 activity in aged gastric mucosa is dependent on EGFR activation. To determine the role EGFR plays in regulating the TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in the gastric mucosa of aged rats, we exposed freshly isolated gastric mucosal cells from 22- to 24-mo-old (aged) rats to either TGF-{alpha} or PD153035, a specific inhibitor of EGFR (8), or a combination of both for 10 min and subsequently examined for changes in EGFR tyrosine kinase activity and DNA binding activity of AP-1. As shown in Fig. 2, whereas TGF-{alpha} by itself caused a significant three- to fourfold stimulation in EGFR phosphorylation, addition of PD153035, at a dose of either 1 or 5 µM, totally abrogated this stimulation. PD153035 by itself caused no apparent change in EGFR phosphorylation when compared with the control (Fig. 2). A similar phenomenon was also observed for the DNA binding activity of AP-1 in that TGF-{alpha} stimulated the DNA binding activity of AP-1, and this stimulation could be completely blocked by the EGFR inhibitor PD153035 (Fig. 3). Taken together, the data suggest that the TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in the gastric mucosa of aged rats is through the EGFR signaling cascades.



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Fig. 2. Effect of EGF receptor (EGFR) tyrosine kinase inhibitor PD153035 on TGF-{alpha}-induced stimulation of tyrosine kinase activity of EGFR in freshly isolated gastric mucosal cells from 23-mo-old rats, as determined by the levels of tyrosine phosphorylated EGFR. After pretreatment with 1 and 5 µM PD153035 for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor PD153035. After incubation, aliquots of mucosal cell extracts containing 1.5 mg protein were subjected to immunoprecipitation with EGFR antibodies, and immunoprecipitates were assayed for EGFR tyrosine kinase activity. Histogram shows the relative changes in the levels of phosphorylated EGFR, as determined by densitometric analysis. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.01 compared with the control.

 


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Fig. 3. Effect of EGFR tyrosine kinase inhibitor PD153035 on TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in gastric mucosal cells from 23-mo-old rats. After pretreatment with 1 and 5 µM PD153035 for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor PD153035. After incubations, aliquots of nuclear extracts containing 5 µg protein were subjected to EMSA analysis as described above in Fig. 1. Five right lanes show extent of AP-1 binding activity in the presence of 50-fold molar excess of unlabeled oligonucleotide probe. Relative changes in DNA binding activity of AP-1, as determined by densitometric analysis, are depicted in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.01 compared with the control.

 

TGF-{alpha} activates ERKs but not JNKs and p38. Activation by TGF-{alpha} of EGFR initiates a series of signaling events through phosphorylation of interacting proteins, including ERKs, JNKs, and p38, which, in turn, transmit signals to the nucleus by activating transcriptional factors. To determine the involvement of MAPK signaling processes in regulating TGF-{alpha}-induced activation of AP-1 in the aged gastric mucosa, we examined the effect of TGF-{alpha} or PD153035, alone or in combination, on activation of ERK1/2, JNK1/2, or p38 in freshly isolated gastric mucosal cells from 22- to 24-mo-old rats. We observed that whereas TGF-{alpha} caused a marked stimulation in ERK1/2 activation, as evidenced by a close to threefold increase in the levels of phosphorylated ERK1/2 (Fig. 4), it produced no significant change in activation of either JNK1/2 or p38 (Fig. 5). However, the TGF-{alpha}-induced stimulation of ERK1/2 phosphorylation was totally abrogated by 5 µM PD153035, a specific inhibitor of EGFR, and by ~60% with a 1 µM dose of the same inhibitor (Fig. 4). PD153035 by itself produced no apparent change in the levels of phosphorylated ERK1/2, JNK1/2, or p38 (Figs. 4 and 5). The observed changes in the levels of phosphorylated ERKs in response to TGF-{alpha}, PD153035, or TGF-{alpha} plus PD153035 could not be attributed to differences in loading, because the levels of total ERK1/2 were found to be the same among the samples (Fig. 4).



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Fig. 4. Effect of EGFR tyrosine kinase inhibitor PD153035 on TGF-{alpha}-induced changes in the levels of total and phosphorylated forms of ERK1/2 in freshly isolated gastric mucosal cells from 23-mo-old rats. After pretreatment with 1 and 5 µM of PD153035 for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor PD153035. After incubations, aliquots of mucosal cell extracts containing 100 µg protein were subjected to Western blot analysis with polyclonal antibodies to phosphorylated ERK1/2. The membranes were stripped and probed again with nonphosphorylated (total) ERK1/2. Relative changes in phosphorylated over nonphosphorylated ERK1/2, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.01 compared with the control.

 


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Fig. 5. Effect of EGFR tyrosine kinase inhibitor PD153035 on TGF-{alpha}-induced changes in the levels of total and phosphorylated forms of JNK1/2 and p38 in freshly isolated gastric mucosal cells from 23-mo-old rats. The experimental protocol was the same as that described in legend to Fig. 4 with the exception that polyclonal antibodies to phosphorylated and nonphosphorylated JNK1/2 or p38 were used. Relative changes in phosphorylated over the corresponding nonphosphorylated JNK1/2 or p38, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations.

 

TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 is dependent on MEK1/2 and ERK1/2. The data from the foregoing experiments (Figs. 3 and 4) demonstrate that the PD153035-induced inhibition of TGF-{alpha} induction of EGFR phosphorylation in gastric mucosal cells from aged rats leads not only to attenuation of ERK activation but also DNA binding activity of AP-1. We hypothesize that the ERK signaling pathway plays a key role in regulating the TGF-{alpha}-induced activation of AP-1 in aged gastric mucosa. To test this hypothesis, we examined the role of MEK1/2, an upstream regulator of ERK1/2, in modulating ERKs and AP-1 activity in gastric mucosal cells from aged rats. We have observed that exposure of freshly isolated gastric mucosal cells from 22- to 24-mo-old (aged) rats to TGF-{alpha}, which stimulates ERK1/2 activation (Fig. 4), also causes a significant twofold stimulation of activation of MEKs (as evidenced by the increased levels of phosphorylated MEK1/2) without affecting the levels of total MEK1/2 (Fig. 6). Again, this induction could be completely inhibited by 5 µM PD153035 and by ~36% with 1 µM of the same compound, indicating that inhibition of EGFR activation leads to decreased activation of MEKs.



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Fig. 6. Effect of EGFR tyrosine kinase inhibitor PD153035 on TGF-{alpha}-induced changes in the levels of total and phosphorylated forms of MEK1/2 in freshly isolated gastric mucosal cells from 23-mo-old rats. After pretreatment with 1 and 5 µM of PD153035 for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor PD153035. After incubations, aliquots of mucosal cell extracts containing 100 µg protein were subjected to Western blot analysis with polyclonal antibodies to phosphorylated MEK1/2. The membranes were stripped and probed again with nonphosphorylated (total) MEK1/2. Relative changes in phosphorylated over the corresponding nonphosphorylated MEK1/2, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.01 compared with the control.

 

To determine whether inhibition of TGF-{alpha}-induced activation of MEKs would lead to attenuation of ERK activation and, in turn, the DNA binding activity of AP-1 in the gastric mucosa, isolated gastric mucosal cells from aged rats were exposed to TGF-{alpha} or PD98053, a specific inhibitor of MEKs, alone or in combination. The cells were assayed for the levels of phosphorylated ERKs and DNA binding activity of AP-1. As expected, the levels of phosphorylated ERK1/2 in gastric mucosal cells were greatly increased (~2-fold) in response TGF-{alpha} when compared with the controls, and this induction was completely inhibited by 5 µM PD98059 (Fig. 7). Neither TGF-{alpha} nor PD98059 caused any significant change in the levels of total ERK1/2 (Fig. 7). Similar to what has been observed for ERKs, the DNA binding activity of AP-1 was also greatly stimulated (~3-fold) by TGF-{alpha} over the corresponding control (Fig. 8). Again, this stimulation was totally abrogated by 5 or 25 µM PD98059 (Fig. 8).



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Fig. 7. Effect of MEK1/2 kinase inhibitor PD98059 on TGF-{alpha}-induced changes in the levels of total and phosphorylated forms of ERK1/2 in freshly isolated gastric mucosal cells from 23-mo-old rats. After pretreatment with 5 µM of PD98059 for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor PD98059. The rest of the experimental protocol was the same as described in legend to Fig. 6. Relative changes in phosphorylated over the corresponding nonphosphorylated ERK1/2, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.01 compared with the control.

 


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Fig. 8. Effect of MEK1/2 kinase inhibitor PD98059 on TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in gastric mucosal cells from 23-mo-old rats. After pretreatment with 5 and 25 µM PD98059 for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor PD98059. After incubations, aliquots of nuclear extracts containing 5 µg protein were subjected to EMSA analysis as described in Fig. 1. Four extreme right lanes show extent of AP-1 binding activity in the presence of 50-fold molar excess of unlabeled oligonucleotide probe. Relative changes in DNA binding activity of AP-1, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. *P < 0.025 compared with the control.

 

TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 involves Src kinase but not PI3 kinase signaling pathway. Activation of EGFR not only stimulates Ras-MAPK signaling pathway but also PI3 and Src kinase signaling processes. The last set of experiments were therefore performed to determine the involvement of PI3 and Src kinases in mediating the TGF-{alpha}-induced activation of AP-1 in the gastric mucosa of aged rats. The experimental protocol was the same as stated above, with the exception that Wortmannin, a specific inhibitor of PI3 kinase, or PP2, a specific inhibitor of Src kinase, was used either alone or in combination with TGF-{alpha}. The cells were assayed for the DNA binding activity of AP-1. As expected, TGF-{alpha} caused a significant three- to four-fold stimulation in DNA binding activity of AP-1 (Fig. 9). However, Wortmannin at a dose of either 20 or 100 nM produced no significant inhibition of the TGF-{alpha}-induced increase in DNA binding activity of AP-1 (Fig. 9). In contrast, the threefold stimulation of the DNA binding activity of AP-1 by TGF-{alpha} was inhibited by ~50% with 50 or 250 nM PP2 (Fig. 10).



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Fig. 9. Effect of Wortmannin, a phospatidylinositol (PI3) kinase inhibitor, on TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in gastric mucosal cells from 23-mo-old rats. After pretreatment with 20 and 100 nM Wortmannin for 10 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 20 min. Control incubations contained neither TGF-{alpha} nor Wortmannin. The rest of the experiment was the same as described in the legend to Fig. 1. Relative changes in DNA binding activity of AP-1, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. **P < 0.025 compared with the control.

 


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Fig. 10. Effect of PP2, an inhibitor of Src kinase, on TGF-{alpha}-induced stimulation of DNA binding activity of AP-1 in gastric mucosal cells from 23-mo-old rats. After pretreatment with 50 and 250 nM PP2 for 15 min, mucosal cells were stimulated with 1 nM TGF-{alpha} at 37°C for 30 min. Control incubations contained neither TGF-{alpha} nor Wortmannin. The rest of the experiment was the same as described in the legend to Fig. 1. The four extreme right lanes show extent of AP-1 binding activity in the presence of 50-fold molar excess of unlabeled oligonucleotide probe. Relative changes in DNA binding activity of AP-1, as determined by densitometric analysis, are shown in the histogram. Densitometric value from control cells was taken as 1. Values represent the means ± SE of 4 observations. **P < 0.025 and *P < 0.05 compared with the control.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The structural and functional integrity of different tissues of the gastrointestinal tract, including that of the stomach, are maintained by constant renewal of cells. Therefore, a detailed knowledge of mucosal cell proliferation and regulation of this process is essential for a better understanding of the normal aging process as well as many gastrointestinal diseases that arise from dysregulation of growth. Although earlier observations in the mouse suggest that with aging, proliferative activity of the small intestine either decreases (7, 18) or remains unchanged (9), recent morphological and biochemical studies from our own and other laboratories have demonstrated that in barrier-reared Fischer 344 rats, aging is associated with increased mucosal proliferative activity in various parts of the gastrointestinal tract, including that of the stomach (2, 11, 12, 19, 21, 22).

Although the responsible mechanism(s) for the age-related rise in gastrointestinal mucosal proliferative activity in Fischer 344 rats still remains to be elucidated, results from this laboratory suggest a role for EGFR signaling pathways in regulating this process. The basis for this postulation comes from the observation that increased gastric mucosal proliferative activity during aging is accompanied by a concomitant increase in tyrosine kinase activity and tyrosine phosphorylation of EGFR, leading to a marked activation of ERKs and JNKs and stimulation of DNA binding activity of AP-1 and NF-{kappa}B (33). Moreover, the fact that these changes are also associated with a marked rise in the levels of membrane-bound precursor form(s) of TGF-{alpha}, one of the primary ligands of EGFR that is synthesized in the gastric mucosa, suggests a role for TGF-{alpha} in regulating the EGFR signaling processes during aging (30, 34). Our current observation, which is similar to what we noted earlier (33), provides further support for this postulation. We have observed that although TGF-{alpha} stimulates the DNA binding activity of AP-1 in gastric mucosal cells from both young and aged rats, the magnitude of this stimulation is considerably greater in aged than in young rats. Whether this could partly be the result of increased sensitivity of the gastric mucosa of aged rats to TGF-{alpha} remains to be determined. This possibility arises from our earlier observation that lower concentrations of EGF or TGF, which are ineffective in stimulating tyrosine kinase activity of EGFR in the gastric mucosa of young rats, induce activation of the same in aged rats (31).

Induction of tyrosine kinase activity of EGFR is one of the essential events for activating the receptor signaling processes. It is generally accepted that after ligand binding, EGFR undergoes dimerization resulting in activation of the intrinsic tyrosine kinase via auto- and transphosphorylation. The receptor phosphorylation, in turn, leads to activation of a number of signaling pathways, including PI3 and Src kinases (13, 27, 32). Subsequently, the activated signaling pathways converge on the nuclear transcription factor AP-1 as shown schematically in Fig. 11. Although the intracellular events regulating the TGF-{alpha}-induced activation of AP-1 transcriptional activity in aged gastric mucosa have not been fully elucidated, our observation that doses of PD153035, which completely inhibited the TGF-{alpha}-induced phosphorylation of EGFR in gastric mucosal cells from aged rats and also produced a similar inhibition of DNA binding activity of AP-1, suggests that EGFR-dependent signaling pathways are important in regulating the TGF-{alpha}-induced stimulation of AP-1 transcriptional activity in the gastric mucosa of the aged.



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Fig. 11. Schematic representation of ligand activation of EGFR signaling pathways leading to stimulation in transcriptional activity of AP-1. SOS, son of sevenless; SHC, Src homology collagen; GRB, growth factor receptor-binding.

 

Numerous studies have demonstrated that the MAPK signaling pathways, which regulate cellular growth and differentiation, respond to EGFR activation (24, 26, 29). At least three distinct families of MAPKs are present in mammalian cells that include p42/44 ERKs, JNK/SAPKs, and p38 (25). It has been suggested that ERKs are primarily responsive to cell proliferation signals, whereas JNKs and p38 respond to cellular stress (5, 15, 17). Our current observation that, in gastric mucosal cells from aged rats, TGF-{alpha} induces activation of ERKs but not JNKs or p38 and that the activation of ERKs by TGF-{alpha} can be totally abrogated by the EGFR tyrosine kinase inhibitor PD153035 suggests that the downstream events of EGFR activation leading to stimulation of DNA binding activity of AP-1 are through the ERKs signaling pathway. Further support for this postulation comes from the observation that inhibition of MAP kinase kinase (MEK1/2), an upstream regulator of MAPKs, by PD98059, a specific inhibitor of MEKs, not only inhibits TGF-{alpha}-induced activation of ERKs but also the DNA binding activity of AP-1 in gastric mucosal cells from aged rats. Taken together, the results suggest that in aged gastric mucosa, TGF-{alpha}-induced activation of the DNA binding activity of AP-1 is through a signaling pathway involving MEK1/2 and ERK1/2.

EGFR activation is known to induce a number of signaling pathways, including PI3 and Src kinases. As depicted in Fig. 11, whereas activation of Src kinase can stimulate transcriptional activity of AP-1 either directly or through activation of the Ras-MAPK pathway, PI3 kinase-mediated activation of AP-1 can occur independently of MAPK. Our current data suggest that the PI3 kinase pathway is not involved in TGF-{alpha}-induced activation of AP-1 in the gastric mucosa of aged rats, because Wortmannin, a specific inhibitor of PI3 kinase, failed to block the TGF-{alpha}-dependent AP-1 activation. In contrast, inhibition of Src kinase by PP2 caused a partial inhibition of the TGF-{alpha}-induced stimulation of the DNA binding activity of AP-1 in the gastric mucosa of aged rats, indicating a minor role of this signaling pathway in regulating AP-1 activation.

In conclusion, our data demonstrate that aging is associated with a marked induction of the DNA binding activity of AP-1 in the gastric mucosa. This could be further activated by TGF-{alpha}, one of the primary ligands of EGFR. This stimulation of the DNA binding activity of AP-1 in the gastric mucosa of aged rats is found to be primarily through a signaling pathway involving MEKs and ERKs, whereas Src kinase pathways play a minor role in this process. The PI3-kinase signaling pathway does not appear to be involved in regulating the TGF-{alpha}-induced stimulation of AP-1 activation in the gastric mucosa during aging.


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

Z.-Q. Xiao is a visiting scientist from Human Medical University, Changsha, Peoples Republic of China.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. P. N. Majumdar, Research Service, 151 VA 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.


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