Transforming growth factor-alpha prevents detachment-induced inhibition of c-Src kinase activity, Bcl-XL down-regulation, and apoptosis of intestinal epithelial cells

Detachment of epithelial cells from the extracellular matrix (ECM) results in apoptosis, a phenomenon often referred to as anoikis. Acquisition of anoikis resistance is now thought to be a prerequisite for the progression of carcinomas. Colorectal cancer cells frequently secrete epidermal growth factor receptor (EGFR) ligands, which are known to have anti-apoptotic activity. However, whether these ligands have the ability to inhibit anoikis of intestinal epithelial cells is unclear, since at least in some cell types efficient EGFR signaling requires cell-ECM adhesion. Here we report that transforming growth factor-alpha (TGF-alpha), an EGFR ligand that is frequently secreted by colorectal cancer cells, strongly inhibits anoikis of the non-malignant rat intestinal epithelial cell lines, IEC-18 and RIE-1. TGF-alpha exerts its anti-anoikis effect by preventing detachment-induced inhibition of c-Src kinase activity. We also show that Fas activation, a molecular event known to play a critical role in anoikis, is not suppressed by TGF-alpha. On the other hand, this growth factor strongly inhibits the detachment-induced down-regulation of Bcl-X(L), another change that is involved in the induction of anoikis. We further demonstrate that this inhibition occurs in a c-Src-dependent manner. We conclude that TGF-alpha has the ability to suppress anoikis of intestinal epithelial cells, at least in part, by reverting the loss of c-Src activity and Bcl-X(L) expression induced by detachment from the ECM.     

Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.     

Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells.

Normal human mammary epithelial cells (HMECs) proliferate in a serum-free defined growth medium in the absence of epidermal growth factor (Li and Shipley, 1991). Amphiregulin (AR) is a heparin-regulated, EGF-like growth factor. Our observation that one strain of HMECs produce AR mRNA (Cook et al., 1991 a) stimulated us to determine whether AR expression was a common phenomenon in HMECs and whether AR could act as an autocrine growth factor to support the EGF-independent growth of these cells. In this study, we detected high levels of AR expression in four separate HMEC strains while one immortal mammary cell line (HBL-100) and six mammary tumor-derived cell lines had low to undetectable levels of AR. The EGF-independent growth of HMECs was blocked by the addition of heparin or a monoclonal anti-EGF receptor antibody to the culture medium, implicating AR as an autocrine growth mediator. This hypothesis is further supported by the fact that medium conditioned by HMECs contains secreted AR protein. A mammary tumor-derived cell line, Hs578T, which proliferates in an EGF-independent manner, does not express detectable levels of AR and is not growth inhibited by heparin. Examination of the same cell types for expression of transforming growth factor type-alpha (TGF-alpha) mRNA revealed coordinate expression of AR and TGF-alpha in these cells. These data suggest that both AR and TGF-alpha mRNA are produced in much greater abundance by normal HMECs than in tumor-derived cells in culture, and that AR is an important autostimulatory factor for the growth of normal HMECs.     

Involvement of transforming growth factor-alpha secreted by macrophages in metallothionein induction by endotoxin

The mechanism of metallothionein (MT) induction of the liver by endotoxin, which is mediated by a factor secreted by endotoxin-stimulated macrophages, was studied in vitro. MT induction of the liver cells by the endotoxin-stimulated macrophage conditioned medium was inhibited by a monoclonal antiepidermal growth factor (EGF) / transforming growth factor-alpha (TGF-alpha) receptor antibody, which acts as an antagonist of EGF and TGF-alpha. MT was induced by the substance, which was adsorbed by polyclonal antibody to TGF-alpha, but not by a monoclonal antibody to EGF, in the conditioned medium of endotoxin-stimulated macrophages. These results suggest that TGF-alpha secreted by macrophages is involved in MT induction by endotoxin.     

EGF and TGF-alpha in wound healing and repair

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). EGF/TGF-alpha receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-alpha are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-alpha, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-alpha and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-alpha system in chronic wounds, and optimize vehicles for effective delivery of peptide growth factors to wounds.     

Effect of TGF-Alpha on growth of normal human breast epithelial cells in serum-free primary culture using 3-dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.     

FGF9 is an autocrine and paracrine prostatic growth factor expressed by prostatic stromal cells

Polypeptide growth factors, including members of the fibroblast growth factor (FGF) family, play an important role in the growth and maintenance of the normal prostate. We have found that FGF9 is expressed at high levels in the normal peripheral and transition zone of the human prostate. Analysis of FGF9 production by primary cultures of prostatic epithelial and stromal cells has shown that FGF9 is produced and secreted by the prostatic stromal cells. Neither of these processes appears to be modulated by androgens. Production of FGF9 by stromal cells in vivo was confirmed by immunohistochemistry. FGF9 is a potent mitogen for both prostatic epithelial and stromal cells in culture and is a more potent mitogen for these cells than either FGF2 or FGF7, two other FGFs expressed in the human prostate. FGF9 is an abundant secreted growth factor that can act as both a paracrine mitogen for epithelial cells and an autocrine mitogen for stromal cells. Western blot analysis of tissue extracts from the normal and hyperplastic transition zone shows that FGF9 is present at two to threefold higher levels in the hyperplastic transition zone. Overexpression of this paracrine and autocrine growth factor may play an important role in the epithelial and stromal proliferation in benign prostatic hyperplasia. J. Cell. Physiol. 180:53–60, 1999. © 1999 Wiley-Liss, Inc.     

Bombesin-dependent pro-MMP-9 activation in prostatic cancer cells requires beta1 integrin engagement

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates transepithelial electrical resistance (TER), promotes the remodeling of apical junctions, and down-regulates the level of fascin, an actin-bundling protein that can bind to beta-catenin. We have previously shown that ectopic expression of fascin prevented the glucocorticoid-mediated recruitment of tight junction and adherens junction proteins to the site of cell-cell contact. Here we demonstrate that exogenous treatment or constitutive production of transforming growth factor-alpha (TGF-alpha) ablated the dexamethasone down-regulation of the fascin protein level and disrupted the dexamethasone-induced remodeling of the apical junction and stimulation of the monolayer TER. The response to TGF-alpha was polarized in that basolateral, but not apical, exposure to this growth factor coordinately reversed the steroid control of fascin production and tight junction formation. Expression of dominant negative RasN17 or treatment with the PD098059 MEK inhibitor abolished or attenuated the TGF-alpha disruptive effects on TER, junction remodeling, and fascin protein levels. Our results implicate the regulation of fascin protein levels as a target of cross-talk between the Ras-dependent growth factor signaling and glucocorticoid signaling pathways that controls tight junction dynamics in mammary epithelial tumor cells. We propose that reversing the down-regulation of fascin is critical for the ability of TGF-alpha to disrupt the glucocorticoid-induced remodeling of the apical junction that leads to tight junction formation.     

Human transforming growth factor-alpha: precursor structure and expression in E. coli

Transforming growth factor-alpha (TGF-alpha) is secreted by many human tumors and can induce the reversible transformation of nontransformed cell lines. Using long synthetic deoxyoligonucleotides as hybridization probes we isolated an exon coding for a portion of TGF-alpha from a human genomic DNA library. Utilizing this exon as a probe, a cell line derived from a human renal cell carcinoma was identified as a source of TGF-alpha mRNA. A cloned TGF-alpha cDNA was isolated from a cDNA library prepared using RNA from this cell line, and was found to encode a precursor polypeptide of 160 amino acids. The 50 amino acid mature TGF-alpha produced by expression of the appropriate coding sequence in E. coli binds to the epidermal growth factor receptor and induces the anchorage independence of normal mammalian cells in culture.     

Role of epidermal growth factor receptor in basal and stimulated colonic epithelial cell migration in vitro.

Colonic mucosal wounds are repaired, in part, by epithelial migration. Signaling mechanisms regulating this migration are poorly characterized. This study aimed to examine the role that the epidermal growth factor (EGF) receptor (EGF-R) and its ligands, EGF and transforming growth factor-alpha (TGF-alpha), play in migration in wounded in vitro models of colonic epithelium. Migration was assessed over 24 h in circular wounds made in confluent monolayers of LIM1215 human colon cancer cells. EGF and TGF-alpha stimulated migration twofold from 4 h after wounding. Basal migration and the motogenic effects of short chain fatty acids and hepatocyte growth factor were mediated through enhanced binding of TGF-alpha to EGF-R, while trefoil peptide-mediated motogenesis required EGF-R activation independently of TGF-alpha binding. Activation of protein kinase C (PKC) stimulated migration, an effect more potent than, and independent of, EGF-R activation. However, neither inhibition of PKC by Ro 31-8220 nor depletion of PKC by pretreatement with phorbol myristate acetate attenuated EGF-R-mediated motogenesis. In conclusion, EGF-R activation via TGF-alpha binding, or intracellularly, mediates basal LIM1215 migration and the effects of several motogens, with the exception of PKC activators. Since EGF-R and PKC have physiological activators in vivo, they may control colonic mucosal repair processes following injury.     

Induction of transforming growth factor-alpha in activated human alveolar macrophages

The early monocyte infiltration observed in normal wound repair and in a number of pathologic processes precedes the epithelial and connective tissue proliferative responses, suggesting that the monocyte/macrophage may be an important source of growth factors for these tissues. In culture, activated macrophages secrete growth factors active on fibroblasts, smooth muscle, endothelium, and epithelium. This report demonstrates that activated human alveolar macrophages express the gene for transforming growth factor-alpha (TGF-alpha) in an inducible manner and secrete a factor into the culture medium that is functionally and immunologically identical to TGF-alpha. Two different molecular species of TGF-alpha activity (approximately 8,500-12,000 and 28,500 daltons) are identified in macrophage-conditioned medium. These observations establish the macrophage as a diploid human cell capable of synthesizing and secreting TGF-alpha. The activated macrophage therefore represents a cellular source of a mitogenic factor that is potentially important in epithelial proliferation and repair.     

Paracrine action of transforming growth factor-alpha in rectal crypt epithelium of humans

Colon and rectal mucosal crypt epithelium is a rapidly renewing cell population, where cell proliferation is normally balanced by cell loss. This report concerns the putative paracrine action of transforming growth factor alpha(TGF-alpha) in this homeostatic process. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and TGF-alpha was performed on biopsy specimens of rectal mucosa taken from consenting patients. The height of the proliferative compartment in mid-axially sectioned crypts in each individual was determined from the distribution of PCNA stained cells. The number of TGF-alpha stained cells that exhibited intense positive staining in a continuous column from the mouth down the side of the crypt was also scored in each individual patient. There was a significant positive correlation (P=0.05,n =22 patients) between the height of the proliferative compartment and the number of cells staining for TGF-alpha. Non-cellular TGF-alpha reactivity was also observed in the lamina propria adjacent to the TGF-alpha reactive epithelial cells, indicating secretion of TGF-alpha by these epithelial cells. These findings suggest that TGF-alpha is released from epithelial cells in the upper compartment of the crypt into the adjacent lamina propria and then diffuses to the epithelial cells in the lower part of the crypt, resulting in expansion of the proliferative compartment.     

TGF-alpha perturbs surfactant homeostasis in vivo

To determine potential relationships between transforming growth factor (TGF)-alpha and surfactant homeostasis, the metabolism, function, and composition of surfactant phospholipid and proteins were assessed in transgenic mice in which TGF-alpha was expressed in respiratory epithelial cells. Secretion of saturated phosphatidylcholine was decreased 40-60% by expression of TGF-alpha. Although SP-A, SP-B, and SP-C mRNA levels were unchanged by expression of TGF-alpha, SP-A and SP-B content in bronchoalveolar lavage fluid was decreased. The minimum surface tension of surfactant isolated from the transgenic mice was significantly increased. Incubation of cultured normal mice type II cells with TGF-alpha in vitro did not change secretion of surfactant phosphatidylcholine and SP-B, indicating that TGF-alpha does not directly influence surfactant secretion. Expression of a dominant negative (mutant) EGF receptor in the respiratory epithelium blocked the TGF-alpha-induced changes in lung morphology and surfactant secretion, indicating that EGF receptor signaling in distal epithelial cells was required for TGF-alpha effects on surfactant homeostasis. Because many epithelial cells were embedded in fibrotic lesions caused by TGF-alpha, changes in surfactant homeostasis may at least in part be influenced by tissue remodeling that results in decreased surfactant secretion. The number of nonembedded type II cells was decreased 30% when TGF-alpha was expressed during development and was increased threefold by TGF-alpha expression in adulthood, suggesting possible alteration of type II cells on surfactant metabolism in the adult lung. Abnormalities in surfactant function and decreased surfactant level in the airways may contribute to the pathophysiology induced by TGF-alpha in both the developing and adult lung.     

Regulation of ClC-2 chloride channels in T84 cells by TGF-alpha

The almost ubiquitously expressed ClC-2 chloride channel is activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein tyrosine phosphorylation and activation of these channels. This study examines the possibility that transforming growth factor-alpha (TGF-alpha) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-alpha (0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a superimposed reversible activation of the current was observed at 8.3 nM TGF-alpha. Both effects required activation of the intrinsic epidermal growth factor receptor (EGFR) tyrosine kinase activity, of phosphoinositide 3-kinase, and of protein kinase C. With microspectrofluorimetry of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-alpha was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-alpha-induced alkalinization activates ClC-2 current. This study indicates that ClC-2 channels are targets for EGFR signaling in epithelial cells.     

Expression of transforming growth factor alpha in plutonium-239-induced lung neoplasms in dogs: investigations of autocrine mechanisms of growth

We have previously shown that 47% of radiation-induced lung neoplasms in dogs exhibit increased expression of epidermal growth factor receptor (EGFR). In this study, we investigated the expression of transforming growth factor alpha (TGF-alpha), a ligand for EGFR, to determine if an autocrine mechanism for growth stimulation was present in these tumors. As determined by immunohistochemistry, 59% (26/44) of the lung neoplasms examined had increased expression of TGF-alpha. Expression of TGF-alpha was not related to the etiology of the tumor, e.g., spontaneous or plutonium-induced; however, it was related to the phenotype of the tumor. Statistical analysis of the correlation of EGFR and TGF-alpha expression within the same tumor did not show a positive association; however, specific phenotypes did have statistically significant expression of EGFR or TGF-alpha, suggesting that overexpression of either the ligand or its receptor conferred a growth advantage to the neoplasm. Twenty-seven percent (32/117) of radiation-induced proliferative epithelial foci expressed TGF-alpha, and a portion of those foci (8/32) expressed both EGFR and TGF-alpha. This supports the hypothesis that these foci represent preneoplastic lesions, and suggests that those foci exhibiting increased expression of the growth factor or its receptor are at greater risk for progressing to neoplasia.     

Different transforming growth factor-alpha species are derived from a glycosylated and palmitoylated transmembrane precursor

cDNA analysis has revealed that the 50 amino acid transforming growth factor-alpha (TGF-alpha) is derived from a 160 amino acid precursor. Antibodies to TGF-alpha and to a C-terminal portion of the precursor were used to study the biosynthesis and processing of the precursor. CHO cells transfected with a TGF-alpha expression vector secrete high levels of TGF-alpha; a mixture of species of about 18 kd is secreted in addition to the 50 amino acid form. These larger species are N-glycosylated and are derived from the same precursor as the smaller form. The C-terminal segment of the precursor remains anchored in the membrane and has covalently attached palmitate. The newly synthesized TGF-alpha precursor is thus a transmembrane protein that subsequently undergoes external proteolytic cleavages, releasing several TGF-alpha species.     

Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha)

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.