Increased production of transforming growth factor α following acute gastric injury

GASTROENTEROLOGY

1992;102:1467-1474

Increased Production of Transforming Growth Factor a Following Acute Gastric Injury WILLIAM H. POLK, Jr., PETER J. DEMPSEY, WILLIAM E. RUSSELL, PAMELA I. BROWN, R. DANIEL BEAUCHAMP, JOHN A. BARNARD, and

ROBERT

J, COFFEY,

Jr.

Departments of Surgery, Medicine, Pediatrics, and Cell Biology, Vanderbilt University, Nashville, Tennessee; Department of Surgery, University of Texas Medical Branch, Galveston, Texas; and Children’s Service, Massachusetts General Hospital, Boston, Massachusetts

Transforming growth factor a (TGF-a) production recently has been found in normal mammalian gastric mucosa. Inasmuch as TGF-a and epidermal growth factor (EGF) both stimulate epithelial cell migration and proliferation and suppress gastric acid secretion, the authors of the current study proposed that these growth factors may participate in tissue repair after acute gastric mucosal injury. Consequently, TGF-a and EGF production were examined after orogastric administration of either acidified taurocholate or 0.6 mol/L HCl to rats. TGF-a messenger RNA (mRNA) expression increased in a dose- and time-dependent manner after administration of taurocholate, whereas EGF mRNA expression was not detected. Radioimmunoassay of gastric mucosal scrapings obtained 6 hours after gastric injury induced by 0.6 mol/L HCl showed a 2.1-fold increase in immunoreactive TGF-a hut no increase in immunoreactive EGF. In addition, there was a 68-fold increase in immunoreactive TGF-a in gastric juice within 30 minutes of gastric instillation of HCl and, again, no increase in immunoreactive EGF. There is a rapid appearance of TGF-a in the gastric juice within 30 minutes of injury, which is followed by increased expression of TGF-a mRNA and protein in the gastric mucosa. These studies suggest that locally produced TGF-a may participate in gastric mucosal repair following acute gastric injury to rats. he reparative events following acute gastric mucosal injury have been studied extensively using many in vitro and in vivo models.14 Repair of superficial epithelial cell loss, a process that is dependent on cell migration, begins within 5 minutes of acute injury and is nearly complete within 1 hour. Deeper mucosal erosions may persist for 5 days after acute injury and require DNA synthesis for repair.’ A variety of factors may contribute to repair of acute gastric mucosal injury. Prostaglandins may

T

limit the initial injury to the mucosa and lamina propria6 but are not required for epithelial cell migration.2 An intact lamina propria with injury limited to the superficial mucosa is a likely prerequisite for rapid early epithelial repair,’ suggesting that the epithelium interacts with factors in the underlying connective tissue stroma. The presence of a “mucoid cap” promotes restitution, probably by protecting the lamina propria from luminal acid, thus limiting the extent of the initial injury.’ Epidermal growth factor (EGF) has been considered an attractive candidate to participate in gastric repair, because it is acid stable,g stimulates epithelial cell migration” and DNA synthesis,“-‘3 suppresses acid production,‘4-16 and stimulates gastric mucus production.17*le EGF is a X&amino acid polypeptidelg that is produced in the submandibular gland, Brunner’s glands of the duodenum, and the distal tubule of the kidney.” EGF messenger RNA (mRNA) expression is not detected in normal adult human gastric mucosa,21 but EGF is produced in the salivary glands and delivered to the stomach in saliva. The EGF receptor is found in normal adult gastric mucosa.22 The hypothesis that EGF participates in the repair of gastric mucosal injury is suggested by studies in sialoadenectomized rats. These rats have atrophic gastric mucosa23 and delayed healing of gastric and duodenal ulceration that is reversed by oral or parenteral EGF.24 Transforming growth factor a (TGF-a) is an acidstable protein that shares 35% sequence homology, a common receptor (EGFr), and a nearly identical spectrum of biological activities2’ with EGF. TGF-a exists in the mature form as a 50-amino acid polypeptide. Recently, TGF-(x. mRNA expression and protein production were shown in normal gastric mucosa.21,26 Similar to EGF, TGF-a suppresses acid 0 1992 by the American Gastroenterological 0016-5065/92/$3.00

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production,27 stimulates epithelial cell migration,28 and promotes epithelial cell proliferation.2Q Therefore, we postulated that increased TGF-a production within the gastric mucosa after injury may contribute to the processes of cellular migration and proliferation that are important in repair. In the present study, acidified sodium taurocholate (TC) or 0.6 mol/L HCl were instilled into the stomach to induce acute injury in rats.30 TGF-a mRNA expression was increased in a time- and dose-dependent manner after injury induced by TC. A 68-fold increase in TGF-a immunoreactivity was detected in the gastric juice 30 minutes after 0.6 mol/L HCl-induced injury, with a gradual return to baseline levels at 4 hours. A 2.1-fold increase in immunoreactive (IR)-TGF-a was detected in the gastric mucosa 6 hours after this injury. In contrast, IR-EGF did not change in gastric juice or in gastric mucosa under these experimental conditions. Materials and Methods

Determination of DNA Synthesis

Reagents The following chemicals, reagents, and equipment were purchased: TC (Sigma Chemical Co., St. Louis, MO): [3H]thymidine (50-80Ci/mmol; New England Nuclear, Boston, MA]; Ilford K-5 nuclear tracking emulsion (Polysciences Inc., Worthington, CA); Microcomp Integrated Analysis System and Olympus Vanox Microscope (Southern Micro Inc., Atlanta, GA); RiaCalc Data Analysis System (Pharmacia, Turku, Finland); synthetic rat TGF-a (Peninsula Laboratories Inc., Belmont, CA); goat antirabbit immunoglobulin G serum (Calbiochem Corp., San Diego, CA); and Hybridization Transfer Membrane [Micron Separations Inc. (MSI), Westboro, MA]. The following chemicals and reagents were gifts: rat EGF (purified from rat salivary glands) and rabbit antiserum to mouse EGF were kindly provided by Dr. David Orth, Vanderbilt University, Nashville, TN. Rat EGF and TGF-a were labeled with lz51by the chloramine T method. Induction

fixed overnight in 10% buffered formalin for histological examination. Sections were stained with H&E and examined to determine the depth and extent of mucosal injury. The remaining mucosa was scraped using glass slides. Mucosa from each treatment group was pooled and frozen in liquid nitrogen. Histology was obtained to assure consistent scraping in each treatment group. In separate experiments, 1 mL of 37°C NS or 0.6 mol/L HCI was instilled into the stomach. IR-TGF-a and IR-EGF levels in gastric mucosa were examined 1,6,and 12 hours after orogastric administration of NS or 0.6 mol/L HCl. At these times, rats were killed, and the gastric mucosa was scraped and frozen in liquid nitrogen. IR-TGF-a and IREGF levels were also measured in gastric juice after HClinduced gastric injury. Anesthesia was induced with intramuscular ketamine (83 mg/kg) and xylazine (17mg/kg) before performing a midline laparotomy. The pylorus was ligated, and 1 mL of NS or 0.6 mol/L HCl was instilled through the nonglandular portion of the stomach using a 22-gauge needle. The stomach was gently flushed with 0.5 mL of NS 30 minutes after the initial instillation and at l-hour intervals thereafter.

of Mucosal

Injury

Male Sprague-Dawley rats, 150-175 g, were fasted in wire-bottom cages for 24 hours before each experiment. Water was provided ad libitum. Three rats were included per treatment group in each experiment. TC was dissolved in normal saline (NS) with 0.2 mol/L HCl (final pH 1.4). In dose-response experiments, rats were given 1 mL of NS (37°C) or 5, 15, and 30 mmol/L of TC; they were killed 4 hours later. In time course experiments, 1 mL of NS or 30 mmol/L TC were instilled; the animals were killed 1, 6, and 24 hours later. In both experiments, the abdomen was opened immediately, and the stomach was removed. After excising the rumen, the remaining stomach was opened along the lesser curvature and gently washed with NS. The mucosal surface was assessed for the presence or absence of hyperemia and hemorrhagic streaks. A single 3 X 5-mm section of proximal fundus immediately adjacent to the rumen was obtained from each treatment group and

In preliminary experiments, DNA synthesis was determined in four groups of rats. Twenty-one hours after orogastric instillation of 1 mL of NS or 5, 15, or 30 mmol/L TC, three rats in each group were injected IP with 500 pCi/kg [3H]thymidine, and the rats were killed 3 hours later. A 3 X 5-mm section of proximal fundus was fixed overnight in buffered formalin and embedded in paraffin for subsequent autoradiography. The remaining stomach was frozen in liquid nitrogen and homogenized in trichloroacetic acid, and acid precipitable counts were determined as previously described.31 The formalin-fixed specimens were embedded in paraffin using standard methods. Seven-micrometer sections were cut, and autoradiographs were prepared with Ilford K-5 emulsion, exposed for 4 weeks at 4”C, developed with Kodak D19 (Eastman-Kodak, Rochester, NY), and stained with 0.02% toluidine blue. Nuclear labeling was measured in each stomach in three well-oriented, random fields. The number of labeled cells per unit area was determined by planar morphometry using the Microcomp Integrated Analysis System and an Olympus Vanox Microscope; results were expressed as the number of labeled nuclei per square millimeter of mucosa -t SD. Northern

Blot Analysis

Gastric mucosa was scraped from the underlying submucosa by glass slides and homogenized in 4 mol/L guanidine isothiocyanate. Total cellular RNA was pelleted through a cesium chloride cushion by ultracentrifugation3’ Poly (A) RNA was extracted using oligo (dT) cellulose as previously described. ” Five micrograms of poly (A) RNA per lane was separated by electrophoresis in 1% agarose/formaldehyde gels.33 RNA was transferred to MS1 paper3*; these filters were hybridized to a 32P-labeled cRNA probe for human TGF-u~~ and complementary DNA (cDNA) probes for human EGF,36 rat EGF receptor,37 and

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the constitutively expressed rat D5 genee3’ Hybridization and posthybridization washes were performed at 65’ for the cRNA probe and 43’C for the cDNA probes as previously describede3’ For all Northern blots, hybridization with a cDNA probe for D5 showed that the RNA was intact, allowing comparison of relative loadingby laser densitometry on autoradiograms. Sample Preparation Radioimmunoassays

for TGF-a and

EGF

Scraped gastric mucosa was lysed in 50 mmol/L NaCl and 25 mmol/L Tris HCl (pH 8.1)containing 0.5% Nonidet P40, 0.5% sodium deoxycholate, and 0.02% (wt/ vol) sodium azide. Lysates were centrifuged at 10,000 X g, and supernatants were stored at -20°C before analysis. Protein determinations were performed using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Richmond, CA). For analysis of IR-TGF-a and EGF in gastric juice, the aspirated gastric contents were titrated to pH 7 with 1 mol/L NaOH, centrifuged at 1000 X g for 5 minutes, and supernatants were stored at -20aC before analysis.

TGF-a and EGF Radioimmunoassays A sensitive homologous radioimmunoassay (RIA) for rat TGF-a was used as previously described4’ with the following modifications. Synthetic rat TGF-a was used both as a standard and as ‘251-labeled tracer. Briefly, 100 pL of synthetic TGF-a standard or 100 pL of sample diluted in RIA buffer [62.5 mmol/L Na,HPO,, 12.7mmol/L ethylenediaminetetraacetic acid (EDTA), and 3 mmol/L NaN,, pH 7.41 containing 0.1% Triton X-100 were mixed with 100 pL of rabbit anti-rat TGF-a, diluted l:lO,OOO in 1% normal rabbit serum in RIA buffer containing 0.1% Triton X-100 and incubated for 3 days at 4’C. One hundred microliters of ‘2SI-labeled rat TGF-a diluted in RIA buffer was added and incubated for 24 hours at 4’C. The bound ligand was precipitated by the addition of 100 pL of goat anti-rabbit immunoglobulin G serum diluted 1:40 in RIA buffer. After a &hour incubation at 4”C, 1.5 mL of RIA buffer containing 2.5% bovine serum albumin and 2% polyethylene glyco1 8000 was added and immediately spun at 3000 x g (Beckman TJ-6) for 30 minutes. Bound and free label were separated by pouring off the supernatant. The pellets (bound label) were counted for 5 minutes in a gamma counter. Results were calculated using linear regression of a log-logit transformation of the data. The assay was highly specific for rat TGF-a with no displacement of ‘251-labeled trace observed with rat, mouse, or human EGF up to 1 pg/mL. The sensitivity of the assay was 5 pg TGF-a per tube. Dose-response curves of IR-TGF-a found in gastric mucosa and juice were parallel to standard (synthetic rat TGF-a) in the TGF-a RIA. A heterologous RIA for rat EGF was performed using highly purified rat EGF (purified from rat salivary glands) both as standard and as “‘I-labeled tracer. The RIA was performed essentially as described above for the TGF-a RIA except that a rabbit antiserum to mouse EGF, which showed strong cross-reactivity with rat EGF, was used as the primary antibody. The antibody was used at a dilution of 1:20,000. The rat EGF RIA was very sensitive with 4 pg

EGF per tube required for 10% displacement of ‘251-labeled tracer. The rat EGF RIA showed some cross-reactivity with mouse EGF, but no displacement of ‘251-labeled tracer was observed with recombinant human EGF, TGF-a, or synthetic rat TGF-a up to 1 pg/mL. Dose-response curves of IR-EGF detected in gastric juice were parallel to standard (purified rat EGF); insufficient amounts of IR-EGF were detected in gastric mucosa to determine parallelism.

Results Gross and Microscopic

Appearance

No gross or microscopic injury occurred after NS or 5 mmol/L TC. Scattered hemorrhagic streaks were present I hour after 15 mmol/L TC; multiple hemorrhagic streaks were present after 30 mmol/L TC and 0.6 mol/L HCl (data not shown). These streaks persisted during the 24 hours following administration of 15 and 30 mmol/L TC and 0.6 mol/L HCl. Light microscopy showed widespread sloughing of the superficial mucous cells and formation of scattered erosions extending to half the depth of the gastric glands 30 minutes after 15 mmol/L TC (data not shown). After 30 mmol/L TC, there was widespread loss of superficial epithelial cells with erosions into the base of the glands (data not shown). Restitution of the superficial epithelial cell loss was often completed by 1 hour, whereas many of the deeper erosions persisted throughout the 24-hour test period. Enhanced

DNA Synthesis After Acute Gastric

Injury A dose-dependent increase in [3H]thymidine incorporation was observed 24 hours after TC-induced injury; the greatest increase was Z&fold after the 30 mmol/L dose (Figure 1). Autoradiography

200

T

CONTROL

5mMTC

15

mid

TC

30

mM

TC

Figure 1. Effect of TC on DNA synthesis in rat stomach. TC (5, 15,and 30 mmol/L) was administered by orogastric tube. Twenty-one hours later, a s-hour pulse of [W]thymidine (500 pCi/kg) was delivered IP. There were three rats in each treatment group: the data are expressed as the mean counts per minute + SE per microgram of DNA.

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showed 22 + 13 labeled nuclei/mm2 in the control group compared with 130 f 58 labeled nuclei/mm2 in the 30-mmol/L TC group (P < 0.001,unpaired t test)41 with labeling limited to the proliferative zone in both treatment groups. These findings confirmed that increased DNA synthesis was responsible for the increased [3H]thymidine incorporation and are consistent with the results of other investigators.5 Increased TGF-a mRNA Expression and Protein Immunoreactivity in Gastric Mucosa After Acute Gastric Injury TGF-a mRNA expression was examined after TC-induced gastric injury. Two to three animals were included in each treatment group, and the mucosal scrapings within each group were pooled before processing. The data presented are representative of replicate experiments. As previously reported,‘* the 4.8-kilobase TGF-a transcript was detected in normal rat gastric mucosa. A dose-dependent increase in the intensity of the TGF-a mRNA transcript was seen 4 hours after TC, with a 2.9-fold increase produced by the 30 mmol/L dose (Figure 2). In time-course experiments, a 1.3-fold increase in

TC (mM1 CTL

=

28 S

the TGF-a mRNA signal was detected 1 hour after injury with 30 mmol/L TC. This increased to 2.6fold 6 hours after injury and returned to baseline 24 hours after TC (Figure 3A and B). Thus, there is a dose- and time-dependent increase in gastric TGF-a, mRNA expression after TC-induced injury. A weak EGF receptor mRNA signal was detected in rat gastric mucosa of both the NS- and TC-treated animals, but no significant changes in its intensity were shown at any of the time points (data not shown). The EGF mRNA transcript was detected in rat kidney and submandibular gland but not in control or TC-treated gastric mucosa even after prolonged autoradiographic exposure (data not shown). Injury was also induced by the intragastric administration of 0.6 mol/L HCI. A 4.2fold increase in TGF-a expression in poly (A) RNA isolated from the scraped gastric mucosa was observed 6 hours after HCl administration (Figure 4). Thus, upregulation of TGF-a mRNA expression is not limited to TC-induced acute gastric injury and may represent a generalized response to acute gastric injury. Because administration of 0.6 mol/L HCI resulted in a greater increase in TGF-a mRNA expression than TC, mucosal IR-TGF-a and IR-EGF were quantified by RIA 1,6, and 12 hours after orogastric administration of 0.6mol/L HCI or NS. Examination of EGF production was considered important because of its relevant biologic activities and because of a recent study showing EGF immunostaining in chronically injured human gastric mucosa.42 A statistically significant 2.1-fold increase in TGF-a immunoreactivity was observed at 6 hours in the gastric mucosa of rats treated with 0.6mol/L HCl (Figure 5); however, no significant differences in TGF-a levels were observed between injured stomachs and controls 1 and 12 hours after administration of test substance. In contrast, mucosal EGF immunoreactivity was found in relatively small amounts and was not increased 1, 6, or 12 hours after acute gastric injury (Figure 5).

18 S Increased Levels of TGF-a (But Not EGF) in Gastric Juice After Acute Gastric injury

D5 . Figure 2. TGF-a mRNA expression after increasing doses of TC. Four hours after orogastric administration of normal saline (CTL) or TC (5,15, or 30 mmol/L), gastric mucosa was obtained and RNA extracted as described in Materials and Methods. Northern blot analysis was performed with 5 pg of poly (A) RNA per lane using a “P-labeled TGF-a cRNA probe. Equivalent loading was verified using a 32P-labeled D5 cDNA probe.=

The effect of intragastric 0.6mol/L HCl on the levels of IR-TGF-a and IR-EGF in gastric juice was also investigated. Thirty minutes after instillation of 1 mL of 0.6mol/L HCl, a 68-fold increase in IR-TGFa was detected. This increase gradually declined over the next 4 hours until TGF-a levels returned to baseline levels 4.5 hours after treatment (Figure 6A). IR-EGF in gastric juice was not significantly altered after acute gastric injury. The highest levels of IREGF were detected within 30 minutes of injury for both the HCl- and NS-treated animals, with a grad-

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May 1992

1H A

w/--h_7 NS

6H TC

NS

24 H TC

-\

NS

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INjURY

1471

3.0

TC

5

25

2

28

s

ia s 14

D5 -

0.0

lh

6h

r-I_ 2411

Figure 3. Time course of TGF-a mRNA expression after TC-induced gastric injury. Gastric mucosa was obtained 1,6, and 24 hours after orogastric administration of NS or 30 mmol/L TC. RNA was extracted as described in Materials and Methods. (A) Northern blot analysis was performed with 5 pg of poly (A) RNA per lane using a “P-labeled TGF-a cRNA probe. Once again, equivalent loading was verified with the “P-labeled D5 cDNA probe. (B) Graphic representation of relative expression of TGF mRNA after normalization to D5 expression.

ual decrease ure 6B).

during the remainder

of the study (Fig-

Discussion EGF-like peptides may play a role in the reparative events following upper gastrointestinal injury. Reduction of salivary EGF by excision of the submandibular glands results in delayed healing of experimentally induced gastric and duodenal ulcers that is corrected by parenteral or enteral administration of exogenous EGF.43 EGF immunostaining has recently been reported in gastric mucosa surrounding areas of chronic u1ceration4’; however, we have not detected EGF mRNA expression in normal or acutely injured rat gastric mucosa. On the other hand, TGF-a is synthesized in normal gastric mucosal cells; also, its effective local concentration may be greater than that of EGF in gastric fluids, because TGF-a does not have to cross physical barriers such as the mucous coat to reach receptors in gastric mucosa. The gross and microscopic injury observed in the current experiments is consistent with the findings of other investigators. Two types of mucosal injury were observed after intraluminal TC or HCl, i.e., superficial epithelial cell loss and deeper erosions involving X1%-100% of the depth of the gastric glands.5*30 The superficial injury is rapidly repaired by epithelial cell migration beginning within minutes of injury, with restitution potentially completed as early as 1 hour later.3 Cell division does not play a major role at this point, because a 16-hour delay occurs after injury before the peak of DNA synthesis.’ In the present study, a dose- and time-dependent

increase in TGF-a mRNA expression in the gastric mucosa was detected after acute gastric injury with TC. Studies using 0.6mol/L HCl to induce gastric injury also showed an increase in TGF-a mRNA expression and a time-dependent increase of TGF-a immunoreactivity in the gastric mucosa. Furthermore, an increase in levels of IR-TGF-a in gastric juice was detected 30 minutes after intragastric administration of HCl; this elevation persisted for 4 hours after injury. In contrast, EGF protein in gastric mucosa was found in low levels and did not increase after acute mucosal injury. Likewise, the amount of EGF in gastric juice following injury was not increased. Although there was an absence of EGF mRNA expression in both the uninjured and injured stomach, low levels of gastric mucosal EGF protein production and release in gastric juice cannot be ruled out. A more probable source of the IR-EGF detected in gastric mucosa and juice is swallowed saliva. Other investigators have shown 39 ng/mL of IR-EGF in rat saliva,44 an amount that could easily explain the amount of IR-EGF that we detected in gastric juice and gastric mucosa. Although a moderate degree of intragastric hemorrhage occurs after HCl-inducedinjury, this did not appear to contribute significantly to the amount of TGF-a or EGF found in gastric contents because RIA of rat serum showed only 88 5 39 pg/mL of TGF- c1 and -450pg/mL of EGF (Dempsey and Coffey, personal communication, October 1990). The low level of EGF detected in rat serum is consistent with the findings of other investigators.44 In light of the known effects of TGF-a on gastric mucosa, these observations suggest that enhanced levels of IR-TGF-a may participate in the reparative events within the gastric mucosa following acute injury.

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CTL

HCI

28 S

8s

Figure 4. TGF-a mRNA expression after HCl-induced gastric injury. Gastric mucosa was obtained 6 hours after oragastric instillation of 0.6 mol/L HCl. RNA was extracted as described in Materials and Methods. Northern blot analysis was performed with 5 pg of poly (A) RNA per lane using a “P-labeled TGF-a cRNA probe. Equivalent loading was verified using a SzP-labeled D5 cDNA probe.

It is important to consider the origin of increased levels of TGF-a in the gastric juice immediately after injury. The rapidity with which levels are increased argues against de novo protein synthesis. Proteolytic cleavage of a biologically active transmembrane form of TGF-a has been shown in certain cell types45,46 and may occur under the experimental conditions of the current study. Lysis of injured gastric mucosal cells may release TGF-a, and recruitment of inflammatory cells into the area of injury may also result in increased levels of TGF-cz.~~ It is difficult to assess the relative significance of these potential sources for TGF-a, but the paucity of inflammatory cells in histological sections taken early after injury and the low levels of TGF-a in serum (see above) suggest that the TGF-a most likely originates

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within the gastric mucosa. The loss of mucosal integrity associated with cell lysis could provide access to the EGF receptor, which is probably located on the basolateral membrane,” and enhance the effects of luminal TGF-a. Furthermore, the pH microenvironment for TGF-a binding to its receptor is probably favorable because of the flux of HCO, to the luminal surface as well as the presence of the mucoid cap. Regardless of the form or mechanism of its release, TGF-a in gastric juice is probably biologically active.45*4sThis leads us to speculate that increased levels of TGF-a in the gastric juice participate in the reparative events that occur after acute injury. Some of the deeper erosions evident after the higher doses of TC persisted throughout the X-hour test period. As previously mentioned, TGF-a stimulates epithelial cell proliferation, a process required for the healing of these deep erosions. Because a peak in both gastric mucosal TGF-a mRNA and TGF-a immunoreactivity is seen 6 hours after injury and because DNA synthesis does not increase significantly until 16 hours after mucosal injury, the timing of this increase is consistent with participation by TGF-ct in the subsequent epithelial cell proliferation. In summary, a dose- and time-dependent increase in TGF-a mRNA expression and TGF-a immunoreactivity was observed following acute gastric mucosal injury. This increase is correlated with the gross and histological degree of injury as well as the subsequent proliferative response. These findings, together with the rapid appearance of increased IRTGF-a in gastric juice and enhanced IR-TGF-a within gastric mucosa, are consistent with an important role for TGF-a acting in an autocrine/paracrine

1000

lh

6h

TGFa

lh

6h

EGF

Figure 5. TGF-a and EGF immunoreactivity in gastric mucosa after HCI-induced gastric injury. Gastric mucosa was obtained 1 and 6 hours after orogastric instillation of NS [O) or 0.6 mol/L HCI (H). Protein was extracted as described in Materials and Methods, and RIA was performed for either TGF-a or EGF. Data were analyzed using the unpaired t test. *P < 0.05.

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Figure 6. TGF-a and EGF immunoreactivity in gastric juice after HCl-induced gastric injury. Gastric contents were removed at l-hour intervals beginning 30 minutes after intragastric instillation of NS (A) or 0.6 mol/L HCl (0). Samples were pH-corrected to 7 and examined by TGF-a (A) or EGF (B) RIA as described in Materials and Methods. The levels are expressed as total nanograms.

manner to stimulate epithelial cell migration and cell division following gastric injury. A causal relationship between increased levels of TGF-a and subsequent reparative events after acute gastric injury is by no means proven by these studies. One approach to establish causality is to determine whether a neutralizing antibody to TGF-(r or its receptor would delay or retard the reparative process. An alternative approach is to examine whether transgenic mice overproducing TGF-a in the gastric mucosa are resistant to acute gastric injury. Such experimental approaches are underway in the laboratory. References I. Svanes K. Ito S. Takeuchi K. Silen W. Restitution of the sur-

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contribution of the C-terminal undecapeptide sequence of urogastrone-epidermal growth factor to its biological action. Regul Pep 1988;22:217-226. 14. Gregory H. Isolation and structure of urogastrone and its relationship to epidermal growth factor. Nature 1975;257:325327. 15. Konturek

SJ, Cieszkowski M, Jaworek J, Konturek J, Brzozowski T, Gregory H. Effects of epidermal growth factor on gastrointestinal secretions. Am J Physiol 1984;246:G580G586. 16. Gonzalez A, Garrido J, Vial JD. Epidermal growth factor inhibits cytoskeleton-related changes in the surface of parietal cells. J Cell Biol 19al;aa:loa-114, 17. Sarosiek J, Bilski J, Murty VLN, Slomiany A, Slomiany BL. Role of salivary epidermal growth factor in the maintenance of physiochemical characteristics of oral and gastric mucosal mucus coat. Biochem Biophys Res Comm 19aa;152:14211427. ia. Kelly SM, Hunter JO. Epidermal

growth factor stimulates synthesis and secretion of mucus glycoproteins in human gastric mucosa. Clin Sci 1990;79:425-427. 19. Cohen S, Carpenter G. Human epidermal growth factor: isolation and chemical and biological activities. Proc Nat1 Acad Sci USA 1975;72:1317-1321. 20. Kasselberg AG, Orth DN, Gray ME, Stahlman MT. Immunocytochemical localization of human epidermal growth factor/ urogastrone in several human tissues. J Histochem Cytochem 2985;35:315-322. 21. Beauchamp RD, Barnard

JA, McCutchen CM, Cherner JA, Coffey RJ. Localization of TGFa and its receptor in gastric mucosal cells: implications for a regulatory role in acid secretion and mucosal renewal. J Clin Invest 1990:84:1017-1023.

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22. Forgue-Lafitte ME, Kobari L, Gespach C, Chamblier ME, Rosselin G. Characterization and repartition of epidermal growth factor-urogastrone receptors in gastric glands from young and adult guinea pigs. Biochem Biophys Acta 1984;798:192-198. 23. Skinner KA, Soper BD, Tepperman BL. Effect of sialoadenectomy and salivary gland extracts on gastrointestinal mucosal growth and gastrin levels in the rat. J Physiol 1984;351:1-12. 24. Konturek SJ, Demhinski A, Warzecha Z, Brzozowski T, Gregory H. Role of epidermal growth factor in healing of chronic gastroduodenal ulcers in rats. Gastroenterology 1988;

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Received May 15, 1991. Accepted September 17, 1991. Address requests for reprints to: Robert J. Coffey, Jr., M.D., Departments of Medicine and Cell Biology, Vanderbilt University, Nashville, Tennessee 37232. Supported by National Cancer Institute (NCI) CA 46413; Veterans Administration and Culpeper Foundation (R.J.C.); NC1 CA 08815-02 (W.H.P.); NC1 CA 01309 (R.D.B.); and DK 44557 (W.E.R.). The authors thank Mary Katherine Stokes, Mary McKissack, and Ramona Graves-Deal for excellent technical assistance: Robert McClure for statistical support; and Wendell Nicholson for radiolabeling of peptides.