Decreased expression of epidermal growth factor receptor and mRNA of its ligands in Helicobacter pylori-infected gastric mucosa

BACKGROUND: Epidermal growth factor (EGF) and TGF-alpha play a central role in maintaining gastric mucosal integrity. Little is known about the regulative role of the four other widely expressed epidermal growth factor receptor ligands, heparin-binding EGF, amphiregulin, betacellulin and cripto in the gastric mucosa. METHODS: Nineteen patients with Helicobacter pylori-positive gastritis and 32 healthy controls were investigated. Mucosal mRNA expression of EGF receptor ligands was determined by quantitative PCR before and after H. pylori eradication. PCR products were analyzed by soft laser scanning densitometry. Moreover, the effect of chronic active gastritis on EGF receptor expression was assessed by [125I] EGF receptor autoradiography. Immunohistochemistry was performed for TGF-alpha to localize growth factor expression. RESULTS: Antral and oxyntic biopsies showed strong mRNA expressions for TGF-alpha, amphiregulin and heparin binding EGF, but not for EGF, cripto and betacellulin. mRNA expression was significantly reduced down to 50% in H. pylori infection, significantly lower compared to normal gastric mucosa, and increased after eradication therapy. Moreover, chronic gastritis was associated with decreased antral EGF receptor binding compared to healthy controls, possibly reflecting reduced autoinduction. Immunohistochemical analyses localized TGF-alpha in the cytoplasma of gastric epithelial cells and revealed its increased expression after H. pylori eradication. CONCLUSIONS: The data presented suggest that amphiregulin, heparin binding EGF and TGF-alpha are important EGF receptor ligands in the gastric mucosa. H. pylori infection apparently suppresses their mRNA as well as receptor expression that is reversed by H. pylori eradication. This deficiency of the gastroprotective EGF system may contribute to the gastric pathogenicity of H. pylori infection.     

A variant epidermal growth factor receptor exhibits altered type alpha transforming growth factor binding and transmembrane signaling.

Epidermal growth factor (EGF) and type alpha transforming growth factor (TGF-alpha) bind to a specific region in subdomain III of the extracellular portion of the EGF receptor (EGFR). Binding leads to receptor dimerization, auto-and transphosphorylation on intracellular tyrosine residues, and activation of signal transduction pathways. We compared the binding and biological actions of EGF and TGF-alpha in Chinese hamster ovary (CHO) cells expressing either wild-type human EGFR (HER497R) or a variant EGFR that has an arginine-to-lysine substitution in the extracellular domain at codon 497 (HER497K) within subdomain IV of EGFR. Both receptors exhibited two orders of binding sites with radioiodinated EGF (125I-EGF). Similar results were obtained with 125I-TGF-alpha in cells expressing HER497R. In contrast, only one order of low-affinity binding sites was seen with 125I-TGF-alpha in the case of HER497K. Although EGF and TGF-alpha enhanced tyrosine phosphorylation of both receptors, CHO cells expressing HER497K exhibited an attenuated growth response to EGF and TGF-alpha and a reduced induction of the protooncogenes FOS, JUN, and MYC. Moreover, high concentrations of TGF-alpha (5 nM) inhibited growth in these cells but not in cells expressing HER497R. These findings indicate that a region in subdomain IV of EGFR regulates signal transduction across the cell membrane and selectively modulates that binding characteristics of TGF-alpha.     

Mice harboring a defective epidermal growth factor receptor (waved-2) have an increased susceptibility to acute dextran sulfate-induced colitis

BACKGROUND: It has been reported that epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) play an important role in colonic mucosal defense and repair. Waved-2 (wa-2) mice harboring a defect EGF-R and phenotypically similar to TGF-alpha knockout mice provide a novel approach to study the role of EGF-R ligands in the maintenance and repair of colonic mucosa. METHODS: Acute colonic mucosal injury was induced by oral administration of dextran sodium sulfate (DSS: 5 g%) given for 6 days ad libitum to wa-2 homozygotes and their genetic controls (n = 10, each group), as well as to wa-2 mice with and without exogenous EGF administration. Severity of colonic injury was assessed histologically of the entire colon and graded. A crypt damage score (CDS) reflecting all three grades of mucosal pathology was calculated. Decrease in total body weight, colon length and colonic blood content was determined for all groups. RESULTS: Thirty-eight percent of the entire colonic mucosa was destroyed in wa-2 animals compared to 15% in control mice. The CDS was 16.0 +/- 1.4 and 9.6 +/- 0.8 in wa-2 and control mice, respectively. EGF application to wa-2 mice did not reduce the severity of mucosal injury (CDS: 18.9 +/- 1.7 and 19.4 +/- 2.1 in EGF and vehicle injected mice, respectively). CONCLUSIONS: The increased susceptibility of wa-2 mice to DSS demonstrates the pivotal role of EGF-R ligands such as EGF and TGF-alpha in preserving the integrity of the colonic mucosa against mucosal injury. The missing beneficial effect of exogenous EGF administration in these mice further underlines the importance of an intact ligand/EGF-R pathway.     

Stimulatory actions of insulin-like growth factor-I and transforming growth factor-alpha on intestinal neurotensin and peptide YY.

Proliferation of the gastrointestinal mucosa is stimulated by the growth factors, insulin-like growth factor-I (IGF-I) and transforming growth factor-alpha (TGF-alpha), or the closely related epidermal growth factor (EGF), as well as the gastrointestinal hormones, gastrin, neurotensin (NT), and peptide YY (PYY). The stimulatory actions of these growth factors or gastrointestinal hormones on the gastrointestinal mucosa may be direct or mediated in part by gastrointestinal peptides or the growth factors, respectively. The purpose of these studies therefore was to examine the effects of IGF-I and TGF-alpha on stomach gastrin and intestinal NT and PYY gene expression [i.e. messenger RNA (mRNA), peptide levels] and secretion. Mice were given recombinant human IGF-I (3, 6 mg/kg BW/day x 14 days). Transgenic mice with the rat TGF-alpha gene linked to a metallothionein promoter were used as a model of chronic TGF-alpha excess. IGF-I and TGF-alpha did not affect gastrin gene expression. Steady-state intestinal NT and PYY mRNA and peptide levels were elevated in a dose-related manner by IGF. TGF-alpha also increased intestinal expression of NT and PYY peptide, but not mRNA levels. Basal serum levels of PYY were elevated by IGF-I and TGF-alpha. IGF-I and TGF-alpha did not increase intestinal chromogranin A (CGA) gene expression, a marker of endocrine cells, or the density of PYY-containing cells in the colon, indicating that the elevations in intestinal gut peptide gene expression by IGF-I and TGF-alpha are not due simply to an increased number of enteroendocrine cells. IV infusion of EGF also stimulated release of PYY in the dog. Together, these findings indicate that IGF-I and TGF-alpha may cause secretion of gut hormones and exert a major upregulatory influence on the regulation of intestinal peptide hormone homeostasis.     

Low diacylglycerol values in colonic adenomas and colorectal cancer.

The biochemical events that make colonic epithelial cells proceed along the adenoma-carcinoma sequence are not well understood. The phosphoinositol signal transduction pathway is involved in the regulation of cell growth and differentiation. To determine its role in colonic neoplasias we performed mass measurements of its second messenger sn-1,2-diacylglycerol in biopsy specimens from normal mucosa and neoplasias of the colon. Normal colonic mucosa was also investigated in patients without colonic abnormalities (n = 10). Compared with pooled diacylglycerol values from five colonic sites (100%), values in patients with a normal colon were highest in the ascending colon (120 (5)%, p less than 0.05) and lowest in the rectum (81 (5)%, p less than 0.01). Absolute diacylglycerol values in patients with normal colons (2.62 (0.16) nmol/mg protein) were not significantly different from those found in the normal mucosa of patients with colorectal neoplasias (2.45 (0.17) nmol/mg protein). Both colonic adenomas (n = 15) and colorectal carcinomas (n = 14) showed significantly decreased diacylglycerol values compared with the adjacent normal mucosa of each patient (72 (4)%, p less than 0.001, and 71 (4)%, p less than 0.001 respectively). The appreciable decrease in mass diacylglycerol values clearly distinguishes adenomas and carcinomas of the colon from the surrounding normal mucosa. This finding suggests that profound metabolic changes of the phosphoinositol signal transduction pathway occur early in the adenoma-carcinoma sequence and may be important in colonic carcinogenesis.     

Epidermal growth factor partially restores colonic ion transport responses in mouse models of chronic colitis

BACKGROUND & AIMS: Epidermal growth factor receptor (EGFR) activation, which plays an important role in regulating intestinal ion transport, can alleviate clinical symptoms such as diarrhea in patients with ulcerative colitis and promote mucosal restitution in animal models of colitis. Here, we investigate whether EGFR can regulate colonic ion transport in the setting of colitis. METHODS: Distal colon from control mice and mice with colitis was retained for immunohistochemistry or mounted in Ussing chambers. Ion transport responses across the tissues to the calcium agonist carbachol and the adenosine 3',5'-cyclic monophosphate agonist forskolin were measured with or without epidermal growth factor (EGF) pretreatment. RESULTS: EGF pretreatment of normal colonic mucosa inhibited ion transport responses to carbachol and forskolin but potentiated the reduced ion transport responses seen in dextran sulfate sodium (DSS)-treated and mdr1a knockout mouse colon. Ion substitution studies and the sodium transport inhibitor amiloride showed that sodium movement primarily accounted for the potentiating effect of EGF in DSS-treated tissues, despite decreased sodium channel expression. EGF potentiation of transport responses in DSS-treated colon was completely blocked by the cytoskeletal disruptor cytochalasin D and the phosphatidylinositol 3-kinase inhibitor wortmannin, whereas the novel and conventional protein kinase C isoform inhibitor G?6850 and the extracellular signal-regulated kinase inhibitor PD98059 partially reduced EGF effects. EGFR epithelial distribution and transforming growth factor alpha expression were also altered in DSS-treated tissues. CONCLUSIONS: Chronic inflammation uncovers a potentiating effect of EGFR activation on epithelial electrogenic sodium absorption that would be expected to ameliorate diarrheal symptoms associated with colitis.     

Autonomous proliferation of colon cancer cells that coexpress transforming growth factor alpha and its receptor. Variable effects of receptor-blocking antibody.

Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.     

An immunohistochemical study of the distribution of blood group substances and related antigens in primary colorectal carcinomas and metastatic lymph node and liver lesions,using monoclonal antibodies against A,B, H type 2, Lea,and Lex antigens

Using indirect immunoperoxidase staining, we examined the distribution of blood group substances and related antigens, including Le^a, A, B, H type 2, and Le^x, in 87 carcinomas and 42 normal mucosal specimens of colon and rectum, as well as in metastatic lesions of 32 lymph nodes and 9 liver specimens. The compatible expression rate of A/B/H type 2 antigens was 33.3% (3/9) in proximal normal colon, but only 6.1% (2/33) in distal normal colon. Compatible expression was observed in 14 of 25 carcinomas in the proximal colon (56.0%), but these antigens in distal colon cancers reappeared with a high positive rate, 62.9% (39/62). The rate of H type 2 accumulation with the deletion of A and/or B antigens was 6.9% (6/87). Incompatible expression was not observed in colorectal cancer. Le^a and Le^x antigens were expressed in normal mucosa and primary carcinoma throughout the colon. Le^a and Le^x in primary carcinoma that showed A/B deletion with/without H type 2 accumulation was expressed more than in carcinoma with A/B/H type 2 compatible expression (88.2% vs 71.7% for Le^a; 94.1% vs 88.7% for Le^x, respectively). In 16 of 32 patients (50.0%), A/B/H type 2 antigen expression in metastatic lesions had disappeared or was decreased compared with that in primary carcinoma, followed by metastasis to regional lymph nodes. These results suggested that: (a) A/B/H type 2 blood group substances in the distal colon behave as tumor-associated antigens in colorectal carcinoma. (b) Most frequently, A/B/H type 2 antigens expressed in primary carcinoma were weakened or had disappeared in metastatic lymph nodes. Further investigation of the biological function of carbohydrate chains, such as those of blood group substances and related antigens on cancer cell surfaces may lead to a solution of the problem of the metastatic behavior of tumor cells.     

Escherichia coli heat-stable enterotoxin receptors

PURPOSE: Receptors for Escherichia coli heat-stable toxin (ST) are selectively expressed in membranes of intestinal mucosa cells and colon carcinoma cells in vitro,suggesting their use as a marker for colorectal tumors in vivo.The present studies examined the expression and function of ST receptors in normal human tissues and primary and metastatic colorectal tumors obtained from patients at surgery. METHODS: Surgical specimens were obtained as follows: from normal colon; from primary adenocarcinomas from all anatomic divisions of the colon and rectum; from gallbladder, kidney, liver, lung, lymph node, ovary, peritoneum, stomach; and from colon carcinomas metastatic to liver, lung, lymph node, ovary, and peritoneum. Membranes prepared from these specimens were assessed for the presence and functional characteristics of ST receptors. RESULTS: ST bound specifically to membranes from each division of normal colon and rectum and all primary and metastatic colorectal tumors examined. The affinity and density of ST receptors were similar in tumors of different grades and from various metastatic sites. ST-receptor interaction was coupled to activation of guanylyl cyclase in all normal samples of colon and rectum and all primary and metastatic colorectal tumors examined. In contrast, neither ST binding nor ST activation of guanylyl cyclase was detected in any extraintestinal tissues examined. CONCLUSIONS: Functional ST receptors are expressed in normal colonic tissue and primary and metastatic colorectal tumors but not by extraintestinal tissues in humans. Expression of ST receptors does not vary as a function of the metastatic site or grade of these tumors. Receptors expressed by colorectal tumors retain their characteristic function, with binding of ST coupled to activation of guanylyl cyclase. These studies support the suggestion that ST receptors represent a specific marker for human colorectal tumors that may have use as a target for directing diagnostics and therapeutics to these tumors in vivo.Supported, in part, by grants from the National Science Foundation (IBN-9205717), the National Institutes of Health (1 R55 DK43805), the Elsa U. Pardee Foundation, and Targeted Diagnostics and Therapeutics, Inc. Stephen L. Carrithers was the recipient of a National Institutes of Health Postdoctoral Fellowship (1 F32 CA63764-01).Poster presentation at the meeting of The American Society of Colon and Rectal Surgeons, Montreal, Quebec, Canada, May 7 to 12, 1995.     

Immunohistochemical study on the expression of epidermal growth factor receptor (EGFR) in human colorectal carcinomas

The expression of epidermal growth factor receptor (EGFR) was studied immunohistochemically in 92 colorectal carcinomas: 21 early and 71 advanced. EGFR immunoreactivity was detected in 15 cases (16.3%) of colorectal carcinomas. All EGFR-positive cases was advanced carcinomas, while no EGFR immunoreactivity was found in early carcinomas. EGFR-positive cases were macroscopically 3.4 type and more than 2 cm in diameter. No significant correlation of EGFR expression with tumor location, stage, lymph node metastasis, degree of differentiation, and prognosis was found. All EGFR-positive cases was synchronous positive for the expression of EGF. These results suggest that EGFR may play an important role in tumor progression in cooperation with EGF.     

Transforming growth factor-alpha in vivo stimulates epithelial cell proliferation in digestive tissues of suckling rats.

BACKGROUND: The role that exogenous transforming growth factor-alpha (TGF-alpha) may exert on cell proliferation in vivo is poorly understood. AIM: To investigate the effect of rat TGF-alpha on epithelial cell proliferation in all suckling rat digestive tissues and to compare it with that of rat epidermal growth factor (EGF). ANIMAL AND METHODS: TGF-alpha and EGF were given three times daily either subcutaneously (10 or 20 micrograms/kg) or intraperitoneally (100 micrograms/kg) to rats from the ninth postnatal day. Cell proliferation was assessed through 5-bromo- 2-deoxyuridine incorporation and estimation of labelling indices. RESULTS: For both growth factors, the highest dose given for only two days significantly increased stomach and intestinal weights compared with controls (p < 0.05 to p < 0.001). The proliferative responded depended on the dose given, colonic mucosa being the most sensitive whereas oxyntic mucosa remained unresponsive. TGF-alpha was as potent as EGF in stimulating epithelial cell proliferation in antral, duodenal, and colonic mucosae. However, EGF was more active on oesophageal and jejunal cell proliferation whereas TGF-alpha was more active on pancreatic exocrine cell proliferation and the differences between the two growth factor treated groups were significant. CONCLUSIONS: These results prove for the first time the stimulating effect in vivo of exogenous rat TGF-alpha on epithelial cell proliferation in rat digestive tissues during the developmental period and support a functional role for TGF-alpha at that time.     

Nuclear localization of epidermal growth factor and epidermal growth factor receptors in human thyroid tissues.

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.     

Increased mRNA expression of a laminin-binding protein in human colon carcinoma: complete sequence of a full-length cDNA encoding the protein.

Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers we constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here we report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is approximately equal to 9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. Containing only two cysteine residues, the protein does not have consensus sequences for asparagine-linked glycosylation, amphipathic alpha-helix, or the N-terminal leader signal sequences for entry into endoplasmic reticulum, although hydrophobic segments for potential membrane associations exist. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant (no lysine or arginine) and highly negatively charged (13 aspartic plus glutamic residues). Within this segment are five repeats of (Asp/Glu)-Trp-(Ser/Thr); two of these are nearly tandem repeats of Thr-Glu-Asp-Trp-Ser-Ala-Xaa-Pro.