Localization of transforming growth factor alpha and its receptor in gastric mucosal cells. Implications for a regulatory role in acid secretion and mucosal renewal. R D Beauchamp, … , J A Cherner, R J Coffey Jr J Clin Invest. 1989;84(3):1017-1023. https://doi.org/10.1172/JCI114223. Research Article Transforming growth factor alpha (TGF alpha) shares with epidermal growth factor (EGF) structural homology (35%), a common cell-surface membrane receptor (TGF alpha/EGF receptor), and a nearly identical spectrum of biological activity, including inhibition of gastric acid secretion. Herein, we report expression of TGF alpha mRNA in normal gastric mucosa of the adult guinea pig, rat, and dog. TGF alpha mRNA was also detected in matched surgically resected gastric mucosa and adjacent gastric carcinoma from 10 patients, and in gastric mucosa adjacent to a benign ulcer from an additional patient. TGF alpha protein was quantitated by radioimmunoassay and was present in tumor and adjacent mucosa. TGF alpha/EGF receptor mRNA was also detected in gastric mucosa from all species studied. Localization of TGF alpha and TGF alpha/EGF receptor mRNA expression was examined in samples of unfractionated guinea pig gastric mucosa and from chief cell-enriched and parietal cell-enriched fractions. All samples exhibited TGF alpha and TGF alpha/EGF receptor expression. The TGF alpha signal was greatest in the parietal cell fraction (5.8-fold increase), but was also enhanced in the chief cell fraction (1.9-fold increase) relative to the unfractionated gastric mucosa. Like TGF alpha expression, TGF alpha/EGF receptor mRNA expression was most intense in the parietal cell-enriched fraction (7.8-fold increase), but was also increased in the chief cell-enriched fraction (2.7-fold increase) relative to the unfractionated […] Find the latest version: https://jci.me/114223/pdf Localization of Transforming Growth Factor a and Its Receptor in Gastric Mucosal Cells Implications for a Regulatory Role in Acid Secretion and Mucosal Renewal R. D. Beauchamp,** J. A. Bamard,*9 C. M. McCutchen,11 J. A. Chemer,11 and R. J. Coffey, Jr.*,' *Departments ofCell Biology, $Surgery, §Pediatrics, and IIMedicine, Vanderbilt University School ofMedicine, Nashville, Tennessee 37232 Abstract pression has been detected in a number of nontransformed cells and tissues (7-9). The spectrum of biological activity for Transforming growth factor a (TGFa) shares with epidermal TGFa and EGF are qualitatively similar, although several growth factor (EGF) structural homology (35%), a common quantitative differences have been observed (10, 1 1). All cell-surface membrane receptor (TGFa/EGF receptor), and a known actions of both ligands appear to be mediated via bind- nearly identical spectrum of biological activity, including inhi- ing to the TGFa/EGF receptor; no discrete TGFa receptor has bition of gastric acid secretion. Herein, we report expression of been identified (12). TGFa mRNA in normal gastric mucosa of the adult guinea pig, Both EGF and TGFa have been shown to have potent rat, and dog. TGFa mRNA was also detected in matched sur- effects on the gastric mucosa, a complex epithelium composed gically resected gastric mucosa and adjacent gastric carcinoma of multiple cell types. Surface epithelial cells, which line the from 10 patients, and in gastric mucosa adjacent to a benign mucosa of all regions of the stomach, secrete mucus and bi- ulcer from an additional patient. TGFa protein was quantitated carbonate. Parietal cells, which secrete hydrochloric acid, and by radioimmunoassay and was present in tumor and adjacent chief cells, which secrete pepsinogen, are found primarily in mucosa. TGFa/EGF receptor mRNA was also detected in gastric glands in the fundus and body. Neuroendocrine cells gastric mucosa from all species studied. Localization of TGFa consist of gastrin-producing G-cells, somatostatin-producing and TGFa/EGF receptor mRNA expression was examined in D-cells, enterochromaffin cells and enterochromaffin-like samples of unfractionated guinea pig gastric mucosa and from cells; their distribution varies with the particular cell type, e.g., chief cell-enriched and parietal cell-enriched fractions. All the stomach's G-cells are confined to the antrum (13). samples exhibited TGFa and TGFa/EGF receptor expression. EGF stimulates DNA synthesis in the gastric mucosa of The TGFa signal was greatest in the parietal cell fraction rodents (14-16) and protects against gastric ulcer formation in (5.8-fold increase), but was also enhanced in the chief cell have no effect on acid secretion gas- rats and cats at doses that ( 16). fraction (1.9-fold increase) relative to the unfractionated Removal of salivary glands (the major intraluminal source of tric mucosa. Like TGFa expression, TGFa/EGF receptor in rats results in of mucosa and a mRNA expression was most intense in the parietal cell- EGF) atrophy gastric (17) delay in healing of gastric and duodenal ulcerations that can be enriched fraction (7.8-fold increase), but was also increased in reversed by treating the animals with either oral or parenteral the chief cell-enriched fraction (2.7-fold increase) relative to EGF (18). These studies imply an important physiological role the unfractionated guinea pig gastric mucosa. We conclude in Of in that and for EGF gastric mucosal growth. potential importance TGFa TGFa/EGF receptor genes are expressed in TGFa and EGF are acid stable normal adult mammalian gastric mucosa. These findings, when the stomach, both (19) (20) in TGFa and EGF, proteins. interpreted light of described actions of Extracts from human urine have long been known to in- provide evidence that local production of TGFa could play an acid secretion The acid role in the and mucosal hibit gastric (21, 22). putative gastric important regulation of acid secretion inhibitory agent was named urogastrone. Subsequent purifica- renewal in the stomach. tion and characterization of human EGF (20, 21) and human Introduction f3-urogastrone (24) revealed that they were identical. In con- scious dogs, EGF inhibits gastric acid secretion stimulated by Epidermal growth factor (EGF)' and transforming growth pentagastrin, histamine, and urecholine, as well as that stimu- factor a (TGFa) are homologous polypeptides (1, 2), both of lated by meal and sham feeding (25). In anesthetized rats, EGF which bind to the EGF receptor (3-5). TGFa was initially inhibits gastric acid secretion stimulated by carbachol, hista- postulated to be an embryonic growth factor that was inappro- mine, and pentagastrin (26). EGF (27) and TGFa (28) inhibit priately expressed in neoplasia (6). More recently, TGFa ex- histamine-stimulated gastric acid secretion from isolated guinea pig gastric mucosa when delivered to the serosal, but Address reprint requests to Dr. Coffey, Jr., Department of Medicine, not luminal surface; intravenous delivery of both ligands in- Gastroenterology Division, Vanderbilt University, Nashville, TN hibits acid secretion in rats (29). In addition to inhibiting acid 37232. secretion, EGF inhibits pepsinogen secretion in rabbit gastric Received for publication 8 November 1988 and in revised form 4 mucosa stimulated by agonists that increase intracellular May 1989. cAMP (30). High affinity TGFa/EGF binding sites have been 1. Abbreviations used in this paper: EGF, epidermal growth factor; demonstrated on both fundic and antral gastric glands from TGFa, transforming growth factor a. the guinea pig (31). TGFa is no longer regarded simply as an embryonic J. Clin. Invest. growth factor that is inappropriately expressed in neoplasia, © The American Society for Clinical Investigation, Inc. but also as an integral physiological regulator of growth in 3021-9738/89/09/1017/07 $2.00 normal tissues ( 12, 32, 33). In screening gastrointestinal tissues Volume 84, September 1989, 1017-1023 for TGFa expression, we found relatively abundant levels in Transforming Growth Factor a in the Stomach 1017 the gastric mucosa. Given the aforementioned effects of TGFa RNA was quantitated by ultraviolet spectroscopy at 260 nM. Relative and EGF on gastric mucosal growth, repair, and acid secretion, loading of lanes and the integrity of RNA was verified by ethidium we examined gastric mucosa from four mammalian species for bromide-stained ribosomal RNA band intensity where precise quanti- TGFa, EGF, and TGFa/EGF receptor expression and at- tation was necessary. Laser densitometry was performed for quantita- tempted to localize expression to a subpopulation of gastric tion of 28S ribosomal RNA on a photographic negative of gels and for mucosal signal intensity on autoradiograms of Northern blots. cells. Protein extraction for TGFa. Extraction of gastric mucosal TGFa protein was performed using a modification of a previously described Methods method (19). One to two grams of previously frozen (-80°C) human gastric mucosa or carcinoma was homogenized and stirred overnight at Processing oftissue. Gastric mucosa was obtained from normal adult 4°C in 4 ml/g of acid-ethanol solution containing 90% ethanol, 0.2 M mongrel dogs, guinea pigs, and rats at sacrifice and kidneys were ob- HCI, pepstatin (5 Mg/ml), leupeptin (5 gg/ml), and PMSF (0.5 mM). tained from rats and dogs. Human gastric and kidney samples were The homogenate was clarified by centrifugation and the supernatant obtained fresh from the Department of Surgical Pathology. Human stored while the pellet was resuspended and stirred 4 h at 4°C in a gastric carcinoma and adjacent uninvolved gastric mucosa were also second acid-ethanol solution containing 77% ethanol and 0.2 M HCI, obtained from the Cooperative Human Tissue Network of the Na- after which the suspension was centrifuged. Both supernatants were tional Cancer Institute and from the Mayo Clinic (Rochester, MN). then combined, extensively dialyzed using Spectrapor 3 dialysis tubing The gastric mucosa was washed with cold water and then rapidly (3,500 mol wt cut-off) against 0.1 M acetic acid, and lyophilized. dissected from the muscularis. The samples were rapidly frozen in Before the TGFa assay, the extracts were resuspended in a minimal liquid nitrogen and stored at -80°C until processing.