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Transforming growth factor-α
Transforming growth factor-a LISA M. GANGAROSA PETER J. DEMPSEY LARS DAMSTRUP JOHN A. BARNARD ROBERT J. COFFEY Transforming growth factor (TGF) was first isolated as a partially purified active entity from culture medium conditioned by Rous sarcoma virustransformed fibroblasts (Delarco and Todaro, 1978). The addition of this partially purified material to normal fibroblasts caused the reversible appearance of a malignant (transformed) phenotype; hence, the protein was named TGF. Later, it was shown that this material with transforming capability actually consisted of two proteins, now designated TGF-a and TGF-P. TGF-a production was first detected in embryonic and neoplastic cells, resulting in the hypothesis that TGF-a was an embryonic growth factor inappropriately expressed in neoplasia. It was also postulated that TGF-a functioned in an autocrine manner and that over-expression of this protein might contribute to malignant transformation. It will become clear from the ensuing discussion that the early hypotheses regarding the function of TGF-a were too restrictive in their scope. STRUCTURE
OF THE
TGF-a
GENE
AND
PROTEIN
The human TGF-a gene spans 70-100 kilobases (kb) on chromosome 2 and contains six exons (Derynck et al, 1984; Marquardt et al, 1987). The 4.5-4.8 kb TGF-a mRNA transcript encodes a 160 amino acid residue transmembrane precursor protein that contains a mature 50 amino acid residue sequence within the extracellular domain (shown schematically in Figure 1A). Amino acid residues in the mature TGF-a sequence are 35% identical to mature epidermal growth factor (EGF). A signal peptide in the amino terminus is cleaved before insertion of the precursor into the cell membrane. The extracellular N-terminal sequence, which contains both Nand O-linked glycosylation sites, is rapidly cleaved at the alanine/valine site (Bringman et al, 1987). Release of the 50 amino acid residue, mature, soluble polypeptide, results from a slower proteolytic cleavage step that is facilitated by the carboxy-terminal valine in the cytoplasmic tail, permitting BailliZre’s Clinical GastroenterologyVol. 10, No. 1, March 1996 ISBN O-7020-2002-8 095G3528/96/010049 + 15 $12.00/00
49 Copyright 0 1996, by Baillikre Tindall All rights of reproduction in any form reserved
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L. M.
GANGAROSA
ET AL
intracellular control of the extracellular release of TGF-a from its precursor (Pandiella and Massague, 1991a). The identity of the proteolytic enzyme and the precise mechanisms for regulation of cleavage have not yet been determined. It is known that cleavage requires adenosine triphosphate and seems to be dependent on both protein kinase C and G-protein signaling pathways (Pandiella and Massague 1991a,b; Bosenberg et al, 1993). It is not known to what extent these processing steps are important in the regulation of TGF-a bioactivity. The 23 amino acid residue hydrophobic transmembrane region is followed by a 39 amino acid residue intracellular cytoplasmic tail with seven cysteine residues, some of which are covalently linked to palmitate (Bringman et al, 1987). The reported sizes of the TGF-a protein range from 5-30 kDa, a variation that may reflect differential
B TGF-a 39 Amino acid cytoplasmic tail: HCCQVRKHCEWCRALICRHELPSALLKGRTACCHSETVV Figure 1. A, Model finked glycosylation
of TGF-c( site.
with B, the amino
acid composition
of its cytoplasmic
tail. NST,
N-
TRANSFORMING
GROWTH
FACTOR-U
51
glycosylation or proteolytic cleavage. Although the high molecular mass forms of TGF-a bind to the EGF receptor (EGFR), their precise biological relevance is not yet known (Linsley et al, 1985; Bringman et al, 1987). TALE
OF THE
TAIL
The 39 amino acid residue sequence of the TGF-a cytoplasmic tail is highly conserved among species (Figure 1B). Several important regulatory regions have been identified. Massague and coworkers have found that the terminal valine in the cytoplasmic tail confers the ability of the tumour promoter phorbol myristate acetate (PMA) to accelerate the proteolytic cleavage at the C terminus of the mature peptide to release the soluble peptide (Bosenberg et al, 1992). Massague has suggested that PMA alters the conformation of a membrane-spanning elastase-like enzyme and the terminal valine of proTGF-a to produce an active enzyme. Thus, there is an ‘inside-out’ flow of processing information. Derynck and co-workers have observed that activation of membrane-bound TGF-a by TGF-a monoclonal antibody results in a tail-dependent endocytosis of a TGF-a (Shum et al, 1994). Concurrent with antibody binding, two proteins associate with the tail (~86 and p106), the latter of which is tyrosine phosphorylated. Recent work indicates that the regulatory control elements for this process reside in the terminal eight amino acid residues of the tail (R. Derynck, personal communication). These intriguing observations indicate that reverse signalling may occur through a membrane-bound ligand. In addition, preliminary evidence indicates that control of basolateral sorting of TGF-a appears to reside in the proximal eight amino acid residues of the cytoplasmic tail (see below). FAMILY
OF TGF-a
LIGANDS
Understanding the actions of TGF-a requires an appreciation of the full complement of mammalian EGFRs and their cognate ligands (Figure 2). There is increasing evidence of cross-talk among the family of EGFRs, which may reflect heterodimerization and/or transactivation. The mammalian ligands that bind the EGFR include TGF-a, epidermal growth factor (EGF), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC) and possibly epiregulin (Barnard et al, 1995). These ligands are produced as transmembrane glycoproteins that are cleaved by proteolytic enzymes at the cell surface to their mature soluble forms (Figure 3 and Table 1). The mature peptides share the exact spacing of six cysteine residues, which form three disulfide bonds and provide the proper folding of the peptides to allow binding to the EGFR. Although these ligands bind to the same receptor, we have shown that there is extensive auto- and cross-induction among the TGF-a family of ligands (Barnard et al, 1994). It is not clear whether the more recently identified ligands bind additional receptors and/or whether they initiate alternative patterns of cross-talk among the EGFR family.
Very recently described; EGFR; not yet cloned
Only known receptor listed above
Epiregulin
EGFR
molecular
Betacellulin;
BTC
relative
Heparin-binding EGF-like growth factor; found primarily in macrophages and smooth muscle cells; also has nuclear localization signal
HB-EGF
M.W.,
Amphiregulin; commonly overexpressed colon neoplasia; nuclear localization necessary for activity
AR
weight.
limited
so far
for EGF-related
for
peptides
very low affinity
knowledge
in signal
Transforming synthesis intestinal
TGF-u
growth factor; widespread in normal and neoplastic epithelium
Epidermal growth factor; limited production within the GI tract and intestinal lines, production enhanced after injury
comments
1. Characteristic
EGF
General
Table
features
110
170,000
-
No
-6000
10,6,2
Not known
4.2, 3.0, 1.2, 0.9
No
1.4
in small bowel,
Distribution in . small bowel controversial
Not studied
mRNA detected ?locus
Immunostaining in gastric mucosa; mRNA found in small bowel (J Barnard, personal communication)
Differentiated colonocyte; mRNA found in small bowel (.I Barnard, personal communication)
enterocyte
Differentiated
in GI tract
4.5-4.8
of production
Salivary gland; controversial in Brnnners glands and pancreas
Loci
4.1-4.9
32000
22000
14
Yes
ligands. Mature RNA (kb)
2.5
22000
10.2
No
No
mammalian
Yes
5547
221
Heparin binding
and its cognate
Mature peptide (M.W.)
of the EGFR
85
120
Gene size (kb)
TRANSFORMING
GROWTH
53
FACTOR-C’.
TGF-a Amphiregulin HB-EGF Betacellulin
NGF
NGF
Epiregulin (?)
Heregulin
Heregulin
erb&2
erbB-3
erbB-4
HER-2
HER-3
HER-4
EGFR erbB
HER-l Figure EGFR.
2. Family
of EGFRs
and cognate
ligands.
NGF,
nerve
growth
factor;
HER,
homology
to
EGF-like domain N
q
Hydrophilic domain
7
Cleavage site
N
100 amino acid residues
AR HB-EGF N
Figure
TGF-a
3. EGF-like
growth
factor
BTC
precursors.
IN DROSOPHILA
An interesting role for a TGF-a homologue has been identified in Drosophila oogenesis. Drosophila egg chambers are composed of both somatic and germ line-derived cell types (Figure 4) (Neuman-Silberberg and Schupbach, 1993; Gonzalez-Reyes et al, 1995; Roth et al, 1995). A single egg chamber contains one oocyte and 15 nurse cells, all of which are derivatives of a single germ line precursor. The oocyte nurse cell cluster is surrounded by the follicle cells, of somatic origin, which secrete the various
L. M. GANGAROSA Oocyte Cni Grk
Polar
follicle
ET
AL
cell
topLIer
3-4
Anteroposterior I\lllild-typel
Stage
6-7
Grk, fop, Cni mutants
Posterior
Anterior follicle
Bed mRNA I
Ante
Kinesin Figure
4A.
layers of the egg shell. A series of inductive interactions between the oocyte and the follicle cells determine initially anteroposterior (AP) and subsequently dorsoventral (DV) polarity. In both instances, the inductive events are mediated by signalling through TGF-a-EGFR homologues. Gurken (Grk) is the Drosphila homologue of TGF-a and top/Der is the Drosphih homologue of the EGFR. Comichon (Cni) is a novel protein that is found in the cytoplasm of the oocyte. Analysis of females containing mosaic egg chambers have demonstrated that the function of top/DER is required in the somatic follicle cells, whereas Grk and Cni are required in the germ line.
TRANSFORMING
GROWTH
55
FACTOR-a
B
Dorsoventral Stage
8-9 Grk, fop, Cnl mutants
Dorsal
Posterior follicle cells
follicle cells
follicle cells
/
Figure
Dorsal
4. Role of Grk and fop/Der
in anterioposterior
and dorsoventral
polarity
in Drosophila.
AP polarity is established first. Grk in the oocyte induces the adjacent uncommitted polar follicle cells to assume a posterior fate rather than the default anterior fate. The posterior follicle cells exhibit a characteristic aeropyle (Ae) and the anterior follicle cells a micropyle (M). Once cells have adopted a posterior follicle cell fate, they signal back to the oocyte to polarize the microtubule cytoskeleton so the minus ends lie at the anterior pole and the plus ends at the posterior, thereby directing the microtubuledependent localization of bicoid (Bed) and oskar (Osk) mRNAs to opposite poles of the oocyte. Because Bed mRNA encodes the anterior determinant and the localization of Osk mFCNA defines the site of formation of pole plasm (which contains the posterior and germ line determinants), the specification of the posterior follicle cells defines the AP polarity of the resulting embryo. The formation of the DV axis of the embryo involves a similar set of inductive interactions between the germ line and follicle cells. The oocyte nucleus (germinal vesicle) moves along the microtubule cytoskeleton to assume a position at the dorsal posterior margin of the oocyte. The position of the germinal vesicle determines the site of localization of Grk mRNA. Grk acts via top/Der on the adjacent follicle cells to direct a dorsal phenotype, thus determining the embryonic DV axis. The ventral follicle cells subsequently signal back to the germ line to define the high point of dorsal nuclear gradient on the ventral side of the embryo. Thus, Grk signalling
56
L. M.
GANGAROSA
ET
AL
through top/DER appears to set the circuitry for asymmetric spatial localization of key components within the cell by directing AP and DV polarity in Drosophila. SPATIAL
COMPARTMENTALIZATION
OF TGF-a
AND
EGFR
Selected epithelial cell types form tight junctions and become polarized when grown on permeable supports (Transwell filters) in vitro. Madin-Darby canine kidney (MDCK) cells, derived from the distal tubule of the dog kidney, are the most extensively studied polarized epithelial cell line. There is no significant baseline expression of any of the EGF family of ligands in these cells. In MDCK cells stably transfected with a variety of TGF-a constructs, TGF-a is targeted preferentially to the basolateral compartment (Dempsey and Coffey, 1994). Similarly, the EGFR is restricted to the basolateral compartment of polarized epithelial cells in vitro and in vivo, thus establishing an autocrine pathway for TGF-a activity. In MDCK cells, TGF-a is only detected in the basal medium in the presence of an EGFR monoclonal antibody that blocks ligand binding. The 60 000 cell surface EGFRs in these MDCK cells are only downregulated by 9% in the face of sizable TGF-a production (20 ngms/106 cells/24 hours). Thus, this is a dynamic system in which the EGFR has the capacity to accommodate large amounts of ligand (Figure 5). In preliminary results from collaborative studies with HS Wiley (University of Utah), it appears that in epithelial cells the EGFR is recycled lo-20 times compared to 2-3 times in fibroblasts. A corollary point is that TGF-a is unlikely to act as a paracrine factor in this system since the cells producing TGF-a are able to bind it rapidly. In contrast, MDCK cells, in which EGF has been stably transfected, exhibit predominant apical EGF immunofluorescence. Presumably, this apically sorted EGF would not act in an autocrine fashion since tight junctions prevent access to the basolateral EGFR. Proteins targeted directly to the basolateral surface contain sorting signals located in the cytoplasmic domain. Thus, it appears basolateral sorting does not occur by default. Several different cytoplasmic determinants have been identified. These include sequences that correlate or overlap with endocytic signals (Brewer and Roth, 1991; Hunziker et al, 1991; Matter et al, 1992), while others are distinct from these signals and can be either tyrosine dependent (Matter et al, 1992; Thomas et al, 1993) or independent (Casanova et al, 1991; Okamoto et al, 1992). The cytoplasmic sequence of TGF-a does not include tyrosine residues and does not appear to contain recognizable sorting motifs or internalization signals except for a di-leucine motif (Letourneur and Klausner, 1992) located 25 amino acid residues from the transmembrane domain. In addition, no phosphorylation at any residue of proTGF-a has been identified (Pandiella and Massague, 1991b). The lack of clear sequence homology with previously described basolateral sorting signals suggests the possibility of a novel motif or may reflect the view that structural information may be more important for sorting than direct sequence homology (Mostov et al, 1992).
TRANSFORMING
Figure
GROWTH
5. Summary
57
FACTOR-a
of TGF-a
trafficking
and EGFR
kinetics
in polarized
MDCK
cells.
TGF-a IN THE STOMACH TGF-a is produced in the normal gastric mucosa (Beauchamp et al, 1989; Bluth et al, 1995). Steady state n-RNA expression of TGF-a and the EGFR is higher in parietal cell-enriched fractions from guinea pig gastric mucosa compared to unenriched and chief cell-enriched fractions. In humans, TGF-a has been immunolocalized to parietal cells and surface (luminal) foveolar cells (Dempsey et al, 1992). TGF-a inhibits gastric acid secretion both in vitro and in vivo (Lewis et al, 1990; Goldenring et al, 1993; Guglietta et al, 1994). TGF-a also stimulates the growth of cultured guinea pig gastric mucous cells and canine fundic epithelial cells (Rutten et al, 1993). In the latter system, TGF-a is released from parietal cells and appears to act as a paracrine growth factor for mucosal replicating cells. Pre-treatment with systemic TGF-a in rats protects the gastric mucosa against the damaging effects of orogastric administration of ethanol; this protection is associated with a 15-fold increase in gastric mucin (Roman0 et al, 1992). Identification of these roles for TGF-a in the stomach led us to consider that over-production of TGF-a may contribute to the pathogenesis of Menbtrier’s disease (Dempsey et al, 1992). This rare acquired disorder is characterized by foveolar cell hyperplasia with increased mucous production, hypochlorhydria and hypoalbuminaemia. MCnCtrier’s disease is considered a pre-malignant condition (Scharschmidt, 1977). Mayo Clinic investigators have recently subdivided M&r&tier’s disease into two histological categories: massive foveolar hyperplasia (MFH) and hypertrophic
58
L.
M.
GANGAROSA
ET
AL
lymphocytic gastritis (HLG) (Wolfsen et al, 1993). The aetiology of MenCtrier’s disease is unknown in adults, although recent reports have implicated Helicobacter pylori as a causative factor (Bayerdorffer et al, 1994). Since TGF-a stimulates gastric mucous cell proliferation, inhibits gastric acid secretion, stimulates mucous production and since the sustained over-expression of this peptide results in neoplasia, we examined TGF-a production in 15 patients that met clinical and biochemical criteria for both MFH and HLG forms of MCnCtrier’s disease (Dempsey et al, 1992; Bluth et al, 1996). All of these patients had diffuse immunostaining for TGF-a in the gastric mucosa. This circumstantial evidence supporting a role for TGF-a in the pathogenesis of MCnCtrier’s disease was strengthened by the analysis of transgenic mice that over-expressed TGF-a in the stomach (Dempsey et al, 1992). The only genetic abnormality in these mice was targeted over-expression of TGF-a under the control of a heavy metalinducible metallothionein (MT) promoter/enhancer. Many of the features of MCnCtrier’s disease were observed, including foveolar hyperplasia, increased gastric mucus and hypochlorhydria. Expression of other components of the EGFR signaling pathway (additional ligands, receptor and substrates) have not been examined. Nevertheless, it appears that sustained over-expression of TGF-a may contribute to the pathogenesis of MCnCtrier’s disease. Recent studies have demonstrated that TGF-a selectively expands the mucous cell compartment during the ontogeny of the gastric mucosa (Sharp et al, 1995; Goldenring et al, 1996).
LESSONS FROM TRANSGENIC AND NULL MOUSE MODELS The MMTV-TGF-a (mouse mammary tumour virus) transgenic mouse model has provided important insights into the consequences of sustained overexpression of TGF-a in vivo (Matsui et al, 1990; Halter et al, 1992). The TGF-a transgene is expressed predominantly in the small ducts and alveoli of the mammary gland beginning at 5 weeks of age, as determined by in situ hybridization and immunohistochemistry. These mice develop a series of pre-malignant changes in breast tissue (cystic and solid hyperplasia, dysplasia and adenoma) that culminates in mammary adenocarcinoma. Initiating doses of 7, 12 dimethylbenzanthracene (DMBA) result in the dramatic acceleration of mammary tumour formation in MMTV-TGF-a transgenic mice, suggesting that in this model TGF-a acts predominantly as a tumour promoter (Coffey et al, 1994). In contrast, work in E Fuchs’ lab has suggested that the TGF-a transgene acts at the initiation stage in skin carcinogenesis in K14-TGF-a mice (Vassar and Fuchs, 1991; Vassar et al, 1992). G Merlino, N Fausto, and co-workers have suggested that in MT-TGF-a transgenic mice the TGF-a transgene acts at the stages of both promotion and progression in hepatic carcinogenesis (Lee et al, 1992; Takagi et al, 1992). Thus, it appears that TGF-a may play a role in neoplasia that is both tissue and stage-dependent. The MMTV-TGF-a mice continue to be a useful in vivo model to sub-
TRANSFORMING
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FACTOR-U
59
stantiate observations that are made in vitro. As mentioned above, in vitro studies indicate that there is cross-talk among the EGFR family. We have observed increased EGFR expression in tumours and peritumoural tissue in MMTV-TGF-a transgenic mice (Matsui et al, 1990). We have also crossed these mice to MMTV-c-neu mice and found a dramatic synergistic effect on the development of mammary tumours in which there is enhanced expression of TGF-a, EGFR and c-neu. Other studies from our laboratory have demonstrated that TGF-P is able to suppress tumourigenesis in MMTVTGF-a and MMTV-TGF-Pl bigenic mice (Pierce et al, 1995). Targeted disruption of TGF-a by homologous recombination results in a mildly abnormal phenotype. The predominant finding is disordered hair follicle structure resulting in curly whiskers and wavy hair (Luetteke et al, 1993; Mann et al, 1993). These findings may be due to attenuated cell proliferation, but loss of other biological functions of TGF-a such as cell migration and keratinization cannot be excluded. An identical phenotype occurs in waved-l mice in which spontaneous deficiency of TGF-a occurs. It will be informative to further address the surprisingly mild phenotype in TGF-a null mice. Experimental manipulations such as partial hepatectomy and skin carcinogenesis studies have not elicited a phenotype under these pathological conditions (Dlugosz et al, 1995; RJ Coffey and WE Russell, unpublished observations). It will also be of interest to determine if compensatory upregulation of other TGF-a related ligands occurs in the context of TGF-a deficiency. Waved-2 mice exhibit a similar phenotype to waved-l mice (Luetteke et al, 1994; Fowler et al, 1995). These mice have a spontaneous single nucleotide transversion (T to G), which results in a valine to glycine substitution at residue 743 in subdomain III of the EGFR kinase domain. This substitution lies 20 residues C-terminal to the lysine residue that defines the ATP binding site. This substitution occurs at the ATP binding site and results in loss of high-affinity EGFRs, which causes delayed ligand-mediated internalization (Fowler et al, 1995). In vitro, EGFR kinase assays (auto- and transphosphorylation) revealed a non-functioning receptor with the waved-2 mutation. In vivo, EGF-induced receptor phosphorylation and mitogenicity were attenuated but not lost; responses were seen at high doses of EGF. Homozygous waved-2 mothers demonstrated a defect in lactation, in addition to the skin changes seen in waved-l mice and targeted TGF-a null mice (Fowler et al, 1995). The relatively mild phenotype in mice bearing this mutation raises interesting speculation. Does in vivo EGFR signalling occur only at high ligand-receptor concentrations? Is responsiveness to EGF modulated by phosphorylation of the EGFR by other cell surface protein kinases? As mentioned above, there is increasing evidence of interactions among the EGFR family that may take the form of heterodimerization and/or transactivation. Targeted disruption of the EGFR presents more dramatic phenotypes. Recently, three groups (Miettinen et al, 1995; Sibilia and Wagner, 1995; Threadgill et al, 1995) have generated EGFR null mice by targeted disruption using homologous recombination. We have observed three fully penetrant, non-overlapping phenotypes dependent on genetic background
60
L. M. GANGAROSA
ET
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(Threadgill et al, 1995). EGFR deficiency on a CF-1 background resulted in peri-implantation mortality due to degeneration of the inner cell mass; on a 129/Sv background, homozygous mutants died at midgestation due to placental defects. In contrast, the same mutant allele was viable when bred to homozygosity in CD-l mice. These mutants lived for up to three weeks and showed marked abnormalities in skin, kidney and brain with lesser abnormalities in liver and gastrointestinal tract. Derynck and co-workers utilized a similarly designed EGFR targeting construct and generated mice with overlapping yet clearly distinct phenotypes (Miettinen et al, 1995). On a C57 background, Derynck’s group found that the mutant allele was also viable when bred to homozygosity. These mice exhibited changes in the lung as well as the skin, but these investigators did not observe abnormalities in the brain or kidney. Moreover, these mice developed extensive necrotizing enterocolitis which appeared to be the cause of death. The major difference in the approaches used by these two groups was the strain of mice in which the embryonic stem (ES) cells were injected and suggests that there are important modifier genes that determine the action of the EGFR. The importance of modifier genes has been demonstrated in min mice with the MOM1 locus, which recently has been shown to encode secretory type II phospholipase A2 (MacPhee et al, 1995). SUMMARY Major advances in understanding growth factor biology, especially in epithelial cells, have resulted from work with TGF-a over the past decade. It is clear that TGF-a is a potent epithelial oncoprotein, but equally important biological activities in normal epithelial homeostasis have been described. A number of major challenges lie ahead. Foremost is the formidable task of dissecting out the individual contributions of each EGF-related peptide in the biological response to stimulation of the EGFR. Appreciation of the complexity of heterodimerization of receptors within the EGFR family will be equally important in the final analysis. These considerations assure the continued vitality and productivity of investigation of the EGFrelated peptide/EGFR axis. REFERENCES Barnard JA, Graves-Deal R, Pittelkow MR et al (1994) Auto- and cross induction within the mammalian epidermal growth factor-related peptide family. Journal of Biological Chemistry 269: 228 17-22822. Barnard JA, Beauchamp RD, Russell WE, DuBois RN & Coffey RJ (1995) Epidermal growth factorrelated peptides and their relevance to gastrointestinal pathophysiology. Gastroenterology 108: 564-580. Bayerdorffer E, Ritter MM, Hatz R, Brooks W, Ruckdeschel G & Stolte M (1994) Healing of protein losing hypertrophic gastropathy by eradication of Helicobucter yylori-a pathogenic factor in MenCtrier’s disease? Gut 35: 701-704. Beauchamp RD, Barnard JA, McCutchen CM, Chemer JA & Coffey RJ (1989) Localization of
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transforming growth factor CI and its receptor in gastric mucosal cells: implications for a regulatory role in acid secretion and mucosal renewal. Journal of Clinical Investigafion 84: 1017-1023. Bluth RF, Carpenter HA, Pittelkow MR, Page DL & Coffey RJ (1996) Immunolocalization of transforming growth factor-alpha in normal and diseased human gastric mucosa. Human Pathology 26: 1333-1340. Bosenberg MW, Pandiella A & Massague J (1992) The cytoplasmic carboxy-terminal amino acid specifies cleavage of membrane TGFcl into soluble growth factor. Cell 71: 1157-l 165. Bosenberg M, Pandiella A & Massague J (1993) Activated release of membrane-anchored TGF-a in the absence of cytosol. Journal of Biological Chemistry 122: 95-101. Brewer CB & Roth MG (1991) A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemaglutinin. Journal of Cell Biology 114: 413-421. Bringman T, Lindquist P & Derynck R (1987) Different transforming growth factor-a species are derived from a glycosylated and palmitoylated transmembrane precursor. Cell 48: 429-440. Casanova JE, Apodaca G & Mostov KE (1991) An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor. Cell 66: 6.5-75. Coffey RJ, Meise KS, Matsui Y, Hogan BLM, Dempsey PJ & Halter SA (1994) Acceleration of mammary neoplasia in transforming growth factor IX transgenic mice by 7, 12-dimethylbenzanthracene. Cancer Research 54: 1678-1683. Delarco JE & Todaro GJ (1978) Growth factors from murine sarcoma virus-transformed cells. Proceedings of the National Academy of Sciences of the USA 75: 4001-4005. Dempsey PJ & Coffey RJ (1994) Basolateral targeting and efficient consumption of transforming growth factor-a when expressed in Madin-Derby canine kidney cells. Journal of Biological Chemistry 269: 16878-16889. Dempsey PJ, Goldenring JR, Soroka CJ et al (1992) Possible role of transforming growth factor rx in the pathogenesis of Men&tier’s disease: supportive evidence from humans and transgenic mice. Gastroenterology 103: 1950-1963. Derynck R, Roberts A, Winkler M, Chen E & Goeddel D (1984) Human transforming growth factorCL: precursor structure and expression in E. coli. Cell 38: 287-297. Dlugosz AA, Cheng C, Williams EK et al (1995) Keratinocytes from mice with a genetic defect in TGFcx expression are transformed in response to transduction by the v-ras Ii* oncogene and upregulate expression of transcripts encoding other EGF receptor ligands. Cancer Research 55: 1883-1893. Fowler KJ, Walker F, Alexander W et al (1995) A mutation in the epidermal growth factor receptor in waved-2 mice has a profound effect on receptor biochemistry that results in impaired lactation. Proceedings of the National Academy of Sciences of the USA 92: 1465-1469. Goldenring JR, Tsunoda Y, Stoch SA, Coffey RJ & Modlin IM (1993) Transforming growth factoralpha inhibition of parietal cell secretion: structural requirements for activity. Regulatory Peptide 43: 37-47. Goldenring JR, Ray GS, Soroka CJ et al (1996) Overexpression of transforming growth factor-alpha alters the differentiation of gastric cell lineages. Digestive Diseases Science (in press). Gonzalez-Reyes A, Elliott H & Johnston DS (1995) Polarization of both major body axes in Drosophila by gurken-torpedo signalling. Nature 375: 654-658. Guglietta A, Roman0 M, Lesch C, McClure RW & Coffey RJ (1994) Effect of TGFa on gastric acid secretion in rats and monkeys. Digestive Diseases Science 39: 177-182. Halter SA, Dempsey PJ, Matsui Y et al (1992) Distinctive patterns of hyperplasia in MMTV-TGFcr transgenic mice: characterization of mammary gland and skin proliferations. American Journal of Pathology 140: 1131-l 146. Hunziker W, Harter C, Matter K & Mellman I (1991) Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell 66: 907-920. Lee G-H, Merlin0 G & Faust0 N (1992) Development of liver tumors in transforming growth factor M transgenic mice. Cancer Research 52: 5162-5179. Letourneur F & Klausner RD (1992) A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69: 1143-l 157. Lewis JJ, Goldenring JG, Modlin IM & Coffey RJ (1990) Autocrine regulation of parietal cell H’ secretion by transforming growth factor CL. Surgery 108: 220-227. Linsley P, Hargreaves W, Twardzik D & Todaro G (1985) Detection of larger polypeptides structurally and functionally related to type I transforming growth factor. Proceedings of the National Academy of Sciences of the USA 82: 356-360.
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ET
AL
Luetteke NC, Qiu TH, Peiffer RL, Oliver P, Smithies 0 & Lee DC (1993) TGFa deficiency results in hair follicle and eye abnormalities in targeted and waved-l mice. Cell 73: 263-278. Luetteke NC, Phillips HK, Qiu TH et al (1994) The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase. Genes and Developmenr 8: 399-413. MacPhee M, Chepenik KP, Liddell RA, Nelson KK, Siracusa LD & Buchberg AM (1995) The secretory phospholipase A2 gene is a candidate for the Mom1 locus, a major modifier of APC~“induced intestinal neoplasia. Cell 81: 957-966. Mann GB, Fowler KJ, Gabriel A, Nice EC, Williams RL & Dunn AR (1993) Mice with a null mutation of the TGFn gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop comeal inflammation. Cell 73: 249-261. Marquardt H, Hunkapiller M, Hood L & Todaro G (1987) Rat transforming growth factor type 1: structure and relationship to EGF. Science 48: 429-440. Matsui Y, Halter SA, Holt JT, Hogan BLM & Coffey RJ (1990) Development of mammary neoplasia and neoplasia in MMTV-TGFo. transgenic mice. Cell 61: 1147-l 155. Matter K, Hunziker W & Mellman I (1992) Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell 71: 741753. Miettinen PJ, Berger JE, Meneses J et al (1995) EpitheliaI immaturity and multiorgan failure in mice deficient in the epidermal growth factor receptor. Nature 376: 337-341. Mostov K, Apodaca G, Aroeti B & Okamoto C (1992) Plasma membrane protein sorting in polarized epithelial cells. Journal of Cell Biology 116: 577-583. Neuman-Silberberg FS & Schupbach T (1993) The Drosophila dorsoventraI patterning gene gurken produces a dorsally localized RNA and encodes a TGFcr -like protein. Cell 75: 165-174. Okamoto CT, Shia SP, Bird C, Mostov KE & Roth MG (1992) The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal. Journal of Biological Chemistry 267: 9925-9932. Pandiella A & Massague J (1991a) Cleavage of the membrane precursor for transforming growth factor alpha is a regulated process. Proceedings of the National Academy of Sciences of the USA 88: 17261730. Pandiella A & Massague J (1991b) Multiple signals activate cleavage of the membrane transforming growth factor-a precursor. Journal of Biological Chemistry 266: 5769-5773. Pierce DF, Gorska AE, Chytil A et al (1995) Mammary tumor suppression by transforming growth factor j31 transgene expression. Proceedings of the National Academy of Sciences of the USA 92: 4254-4258. Roman0 M, Polk WH, Awad JA et aI (1992) Transforming growth factor CL protects against drug induced injury to rat gastric mucosa in vivo. Journal of Clinical Investigation 90: 2409-2421. Roth S, Neuman-Silberberg FS, Barcelo G & Schupbach T (1995) Cornichon and the EGF receptor signaling process are necessary for both anterior-posterior and dorsal-ventral pattern formation in Drosophila. Cell 81: 967-978. Rutten MJ, Dempsey PJ, Soloman TE & Coffey RJ (1993) Transforming growth factor cx is a potent mitogen for primary cultures of guinea pig gastric mucous epithelial cells. American Journal of Physiology 265: G361-G369. Scharschmidt BF (1977) The natural history of hypertrophic gastropathy (Men&trier’s disease): report of a case with 16 year follow-up and review of 120 cases from the literature. American Journal of Medicine 63: 644-652. Sharp R, Babyatsky MW, Takagi H et aI (1995) Transforming growth factor a disrupts the normal program of cellular differentiation in the gastric mucosa of transgenic mice. Development 121: 149-161. Shum L, Reeves SA, Kuo AC, Fromer ES & Derynck R (1994) Association of the transmembrane TGF-alpha precursor with a protein kinase complex. Journal of Cell Biology 125: 903-916. Sibilia M & Wagner EF (1995) Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269: 234-238. Takagi H, Sharp R, Hammermeister C et aI (1992) Molecular and genetic analysis of liver oncogenesis in transforming growth factor a transgenic mice. Cancer Research 52: 5171-5177. Thomas DC, Brewer CB & Roth MG (1993) Vesicular stomatitis virus glycoprotein contans a dominant cytoplasmic basolateral sorting signal critically dependent upon a tyrosine. Journal of Biological Chemistry 268: 3313-3320. Threadgill DW, Dlugosz AA, Hansen LA et aI (1995) Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269: 230-234.
TRANSFORMING Vassar
GROWTH
FACTOR-61
63
R & Fuchs E (1991) Transgenic mice provide new insights into the role of TGF-a during epidermal development and differentiation. Growth rind Development 5: 714-727. Vassar R, Hutton ME & Fuchs E (1992) Transgenic overexpression of transforming growth factor-a bypasses the need for c-Ha-ras mutations in mouse skin tumorigenesis. Molecular and Cellular Biology 12: 4643-4653. Wolfsen HC, Carpenter HA & Talley NJ (1993) Men&tier’s disease: evidence for pathological heterogeneity. Gastroenterology 104: 1310-1319.
OF THE
TGF-a
GENE
AND
PROTEIN
The human TGF-a gene spans 70-100 kilobases (kb) on chromosome 2 and contains six exons (Derynck et al, 1984; Marquardt et al, 1987). The 4.5-4.8 kb TGF-a mRNA transcript encodes a 160 amino acid residue transmembrane precursor protein that contains a mature 50 amino acid residue sequence within the extracellular domain (shown schematically in Figure 1A). Amino acid residues in the mature TGF-a sequence are 35% identical to mature epidermal growth factor (EGF). A signal peptide in the amino terminus is cleaved before insertion of the precursor into the cell membrane. The extracellular N-terminal sequence, which contains both Nand O-linked glycosylation sites, is rapidly cleaved at the alanine/valine site (Bringman et al, 1987). Release of the 50 amino acid residue, mature, soluble polypeptide, results from a slower proteolytic cleavage step that is facilitated by the carboxy-terminal valine in the cytoplasmic tail, permitting BailliZre’s Clinical GastroenterologyVol. 10, No. 1, March 1996 ISBN O-7020-2002-8 095G3528/96/010049 + 15 $12.00/00
49 Copyright 0 1996, by Baillikre Tindall All rights of reproduction in any form reserved
50
L. M.
GANGAROSA
ET AL
intracellular control of the extracellular release of TGF-a from its precursor (Pandiella and Massague, 1991a). The identity of the proteolytic enzyme and the precise mechanisms for regulation of cleavage have not yet been determined. It is known that cleavage requires adenosine triphosphate and seems to be dependent on both protein kinase C and G-protein signaling pathways (Pandiella and Massague 1991a,b; Bosenberg et al, 1993). It is not known to what extent these processing steps are important in the regulation of TGF-a bioactivity. The 23 amino acid residue hydrophobic transmembrane region is followed by a 39 amino acid residue intracellular cytoplasmic tail with seven cysteine residues, some of which are covalently linked to palmitate (Bringman et al, 1987). The reported sizes of the TGF-a protein range from 5-30 kDa, a variation that may reflect differential
B TGF-a 39 Amino acid cytoplasmic tail: HCCQVRKHCEWCRALICRHELPSALLKGRTACCHSETVV Figure 1. A, Model finked glycosylation
of TGF-c( site.
with B, the amino
acid composition
of its cytoplasmic
tail. NST,
N-
TRANSFORMING
GROWTH
FACTOR-U
51
glycosylation or proteolytic cleavage. Although the high molecular mass forms of TGF-a bind to the EGF receptor (EGFR), their precise biological relevance is not yet known (Linsley et al, 1985; Bringman et al, 1987). TALE
OF THE
TAIL
The 39 amino acid residue sequence of the TGF-a cytoplasmic tail is highly conserved among species (Figure 1B). Several important regulatory regions have been identified. Massague and coworkers have found that the terminal valine in the cytoplasmic tail confers the ability of the tumour promoter phorbol myristate acetate (PMA) to accelerate the proteolytic cleavage at the C terminus of the mature peptide to release the soluble peptide (Bosenberg et al, 1992). Massague has suggested that PMA alters the conformation of a membrane-spanning elastase-like enzyme and the terminal valine of proTGF-a to produce an active enzyme. Thus, there is an ‘inside-out’ flow of processing information. Derynck and co-workers have observed that activation of membrane-bound TGF-a by TGF-a monoclonal antibody results in a tail-dependent endocytosis of a TGF-a (Shum et al, 1994). Concurrent with antibody binding, two proteins associate with the tail (~86 and p106), the latter of which is tyrosine phosphorylated. Recent work indicates that the regulatory control elements for this process reside in the terminal eight amino acid residues of the tail (R. Derynck, personal communication). These intriguing observations indicate that reverse signalling may occur through a membrane-bound ligand. In addition, preliminary evidence indicates that control of basolateral sorting of TGF-a appears to reside in the proximal eight amino acid residues of the cytoplasmic tail (see below). FAMILY
OF TGF-a
LIGANDS
Understanding the actions of TGF-a requires an appreciation of the full complement of mammalian EGFRs and their cognate ligands (Figure 2). There is increasing evidence of cross-talk among the family of EGFRs, which may reflect heterodimerization and/or transactivation. The mammalian ligands that bind the EGFR include TGF-a, epidermal growth factor (EGF), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), betacellulin (BTC) and possibly epiregulin (Barnard et al, 1995). These ligands are produced as transmembrane glycoproteins that are cleaved by proteolytic enzymes at the cell surface to their mature soluble forms (Figure 3 and Table 1). The mature peptides share the exact spacing of six cysteine residues, which form three disulfide bonds and provide the proper folding of the peptides to allow binding to the EGFR. Although these ligands bind to the same receptor, we have shown that there is extensive auto- and cross-induction among the TGF-a family of ligands (Barnard et al, 1994). It is not clear whether the more recently identified ligands bind additional receptors and/or whether they initiate alternative patterns of cross-talk among the EGFR family.
Very recently described; EGFR; not yet cloned
Only known receptor listed above
Epiregulin
EGFR
molecular
Betacellulin;
BTC
relative
Heparin-binding EGF-like growth factor; found primarily in macrophages and smooth muscle cells; also has nuclear localization signal
HB-EGF
M.W.,
Amphiregulin; commonly overexpressed colon neoplasia; nuclear localization necessary for activity
AR
weight.
limited
so far
for EGF-related
for
peptides
very low affinity
knowledge
in signal
Transforming synthesis intestinal
TGF-u
growth factor; widespread in normal and neoplastic epithelium
Epidermal growth factor; limited production within the GI tract and intestinal lines, production enhanced after injury
comments
1. Characteristic
EGF
General
Table
features
110
170,000
-
No
-6000
10,6,2
Not known
4.2, 3.0, 1.2, 0.9
No
1.4
in small bowel,
Distribution in . small bowel controversial
Not studied
mRNA detected ?locus
Immunostaining in gastric mucosa; mRNA found in small bowel (J Barnard, personal communication)
Differentiated colonocyte; mRNA found in small bowel (.I Barnard, personal communication)
enterocyte
Differentiated
in GI tract
4.5-4.8
of production
Salivary gland; controversial in Brnnners glands and pancreas
Loci
4.1-4.9
32000
22000
14
Yes
ligands. Mature RNA (kb)
2.5
22000
10.2
No
No
mammalian
Yes
5547
221
Heparin binding
and its cognate
Mature peptide (M.W.)
of the EGFR
85
120
Gene size (kb)
TRANSFORMING
GROWTH
53
FACTOR-C’.
TGF-a Amphiregulin HB-EGF Betacellulin
NGF
NGF
Epiregulin (?)
Heregulin
Heregulin
erb&2
erbB-3
erbB-4
HER-2
HER-3
HER-4
EGFR erbB
HER-l Figure EGFR.
2. Family
of EGFRs
and cognate
ligands.
NGF,
nerve
growth
factor;
HER,
homology
to
EGF-like domain N
q
Hydrophilic domain
7
Cleavage site
N
100 amino acid residues
AR HB-EGF N
Figure
TGF-a
3. EGF-like
growth
factor
BTC
precursors.
IN DROSOPHILA
An interesting role for a TGF-a homologue has been identified in Drosophila oogenesis. Drosophila egg chambers are composed of both somatic and germ line-derived cell types (Figure 4) (Neuman-Silberberg and Schupbach, 1993; Gonzalez-Reyes et al, 1995; Roth et al, 1995). A single egg chamber contains one oocyte and 15 nurse cells, all of which are derivatives of a single germ line precursor. The oocyte nurse cell cluster is surrounded by the follicle cells, of somatic origin, which secrete the various
L. M. GANGAROSA Oocyte Cni Grk
Polar
follicle
ET
AL
cell
topLIer
3-4
Anteroposterior I\lllild-typel
Stage
6-7
Grk, fop, Cni mutants
Posterior
Anterior follicle
Bed mRNA I
Ante
Kinesin Figure
4A.
layers of the egg shell. A series of inductive interactions between the oocyte and the follicle cells determine initially anteroposterior (AP) and subsequently dorsoventral (DV) polarity. In both instances, the inductive events are mediated by signalling through TGF-a-EGFR homologues. Gurken (Grk) is the Drosphila homologue of TGF-a and top/Der is the Drosphih homologue of the EGFR. Comichon (Cni) is a novel protein that is found in the cytoplasm of the oocyte. Analysis of females containing mosaic egg chambers have demonstrated that the function of top/DER is required in the somatic follicle cells, whereas Grk and Cni are required in the germ line.
TRANSFORMING
GROWTH
55
FACTOR-a
B
Dorsoventral Stage
8-9 Grk, fop, Cnl mutants
Dorsal
Posterior follicle cells
follicle cells
follicle cells
/
Figure
Dorsal
4. Role of Grk and fop/Der
in anterioposterior
and dorsoventral
polarity
in Drosophila.
AP polarity is established first. Grk in the oocyte induces the adjacent uncommitted polar follicle cells to assume a posterior fate rather than the default anterior fate. The posterior follicle cells exhibit a characteristic aeropyle (Ae) and the anterior follicle cells a micropyle (M). Once cells have adopted a posterior follicle cell fate, they signal back to the oocyte to polarize the microtubule cytoskeleton so the minus ends lie at the anterior pole and the plus ends at the posterior, thereby directing the microtubuledependent localization of bicoid (Bed) and oskar (Osk) mRNAs to opposite poles of the oocyte. Because Bed mRNA encodes the anterior determinant and the localization of Osk mFCNA defines the site of formation of pole plasm (which contains the posterior and germ line determinants), the specification of the posterior follicle cells defines the AP polarity of the resulting embryo. The formation of the DV axis of the embryo involves a similar set of inductive interactions between the germ line and follicle cells. The oocyte nucleus (germinal vesicle) moves along the microtubule cytoskeleton to assume a position at the dorsal posterior margin of the oocyte. The position of the germinal vesicle determines the site of localization of Grk mRNA. Grk acts via top/Der on the adjacent follicle cells to direct a dorsal phenotype, thus determining the embryonic DV axis. The ventral follicle cells subsequently signal back to the germ line to define the high point of dorsal nuclear gradient on the ventral side of the embryo. Thus, Grk signalling
56
L. M.
GANGAROSA
ET
AL
through top/DER appears to set the circuitry for asymmetric spatial localization of key components within the cell by directing AP and DV polarity in Drosophila. SPATIAL
COMPARTMENTALIZATION
OF TGF-a
AND
EGFR
Selected epithelial cell types form tight junctions and become polarized when grown on permeable supports (Transwell filters) in vitro. Madin-Darby canine kidney (MDCK) cells, derived from the distal tubule of the dog kidney, are the most extensively studied polarized epithelial cell line. There is no significant baseline expression of any of the EGF family of ligands in these cells. In MDCK cells stably transfected with a variety of TGF-a constructs, TGF-a is targeted preferentially to the basolateral compartment (Dempsey and Coffey, 1994). Similarly, the EGFR is restricted to the basolateral compartment of polarized epithelial cells in vitro and in vivo, thus establishing an autocrine pathway for TGF-a activity. In MDCK cells, TGF-a is only detected in the basal medium in the presence of an EGFR monoclonal antibody that blocks ligand binding. The 60 000 cell surface EGFRs in these MDCK cells are only downregulated by 9% in the face of sizable TGF-a production (20 ngms/106 cells/24 hours). Thus, this is a dynamic system in which the EGFR has the capacity to accommodate large amounts of ligand (Figure 5). In preliminary results from collaborative studies with HS Wiley (University of Utah), it appears that in epithelial cells the EGFR is recycled lo-20 times compared to 2-3 times in fibroblasts. A corollary point is that TGF-a is unlikely to act as a paracrine factor in this system since the cells producing TGF-a are able to bind it rapidly. In contrast, MDCK cells, in which EGF has been stably transfected, exhibit predominant apical EGF immunofluorescence. Presumably, this apically sorted EGF would not act in an autocrine fashion since tight junctions prevent access to the basolateral EGFR. Proteins targeted directly to the basolateral surface contain sorting signals located in the cytoplasmic domain. Thus, it appears basolateral sorting does not occur by default. Several different cytoplasmic determinants have been identified. These include sequences that correlate or overlap with endocytic signals (Brewer and Roth, 1991; Hunziker et al, 1991; Matter et al, 1992), while others are distinct from these signals and can be either tyrosine dependent (Matter et al, 1992; Thomas et al, 1993) or independent (Casanova et al, 1991; Okamoto et al, 1992). The cytoplasmic sequence of TGF-a does not include tyrosine residues and does not appear to contain recognizable sorting motifs or internalization signals except for a di-leucine motif (Letourneur and Klausner, 1992) located 25 amino acid residues from the transmembrane domain. In addition, no phosphorylation at any residue of proTGF-a has been identified (Pandiella and Massague, 1991b). The lack of clear sequence homology with previously described basolateral sorting signals suggests the possibility of a novel motif or may reflect the view that structural information may be more important for sorting than direct sequence homology (Mostov et al, 1992).
TRANSFORMING
Figure
GROWTH
5. Summary
57
FACTOR-a
of TGF-a
trafficking
and EGFR
kinetics
in polarized
MDCK
cells.
TGF-a IN THE STOMACH TGF-a is produced in the normal gastric mucosa (Beauchamp et al, 1989; Bluth et al, 1995). Steady state n-RNA expression of TGF-a and the EGFR is higher in parietal cell-enriched fractions from guinea pig gastric mucosa compared to unenriched and chief cell-enriched fractions. In humans, TGF-a has been immunolocalized to parietal cells and surface (luminal) foveolar cells (Dempsey et al, 1992). TGF-a inhibits gastric acid secretion both in vitro and in vivo (Lewis et al, 1990; Goldenring et al, 1993; Guglietta et al, 1994). TGF-a also stimulates the growth of cultured guinea pig gastric mucous cells and canine fundic epithelial cells (Rutten et al, 1993). In the latter system, TGF-a is released from parietal cells and appears to act as a paracrine growth factor for mucosal replicating cells. Pre-treatment with systemic TGF-a in rats protects the gastric mucosa against the damaging effects of orogastric administration of ethanol; this protection is associated with a 15-fold increase in gastric mucin (Roman0 et al, 1992). Identification of these roles for TGF-a in the stomach led us to consider that over-production of TGF-a may contribute to the pathogenesis of Menbtrier’s disease (Dempsey et al, 1992). This rare acquired disorder is characterized by foveolar cell hyperplasia with increased mucous production, hypochlorhydria and hypoalbuminaemia. MCnCtrier’s disease is considered a pre-malignant condition (Scharschmidt, 1977). Mayo Clinic investigators have recently subdivided M&r&tier’s disease into two histological categories: massive foveolar hyperplasia (MFH) and hypertrophic
58
L.
M.
GANGAROSA
ET
AL
lymphocytic gastritis (HLG) (Wolfsen et al, 1993). The aetiology of MenCtrier’s disease is unknown in adults, although recent reports have implicated Helicobacter pylori as a causative factor (Bayerdorffer et al, 1994). Since TGF-a stimulates gastric mucous cell proliferation, inhibits gastric acid secretion, stimulates mucous production and since the sustained over-expression of this peptide results in neoplasia, we examined TGF-a production in 15 patients that met clinical and biochemical criteria for both MFH and HLG forms of MCnCtrier’s disease (Dempsey et al, 1992; Bluth et al, 1996). All of these patients had diffuse immunostaining for TGF-a in the gastric mucosa. This circumstantial evidence supporting a role for TGF-a in the pathogenesis of MCnCtrier’s disease was strengthened by the analysis of transgenic mice that over-expressed TGF-a in the stomach (Dempsey et al, 1992). The only genetic abnormality in these mice was targeted over-expression of TGF-a under the control of a heavy metalinducible metallothionein (MT) promoter/enhancer. Many of the features of MCnCtrier’s disease were observed, including foveolar hyperplasia, increased gastric mucus and hypochlorhydria. Expression of other components of the EGFR signaling pathway (additional ligands, receptor and substrates) have not been examined. Nevertheless, it appears that sustained over-expression of TGF-a may contribute to the pathogenesis of MCnCtrier’s disease. Recent studies have demonstrated that TGF-a selectively expands the mucous cell compartment during the ontogeny of the gastric mucosa (Sharp et al, 1995; Goldenring et al, 1996).
LESSONS FROM TRANSGENIC AND NULL MOUSE MODELS The MMTV-TGF-a (mouse mammary tumour virus) transgenic mouse model has provided important insights into the consequences of sustained overexpression of TGF-a in vivo (Matsui et al, 1990; Halter et al, 1992). The TGF-a transgene is expressed predominantly in the small ducts and alveoli of the mammary gland beginning at 5 weeks of age, as determined by in situ hybridization and immunohistochemistry. These mice develop a series of pre-malignant changes in breast tissue (cystic and solid hyperplasia, dysplasia and adenoma) that culminates in mammary adenocarcinoma. Initiating doses of 7, 12 dimethylbenzanthracene (DMBA) result in the dramatic acceleration of mammary tumour formation in MMTV-TGF-a transgenic mice, suggesting that in this model TGF-a acts predominantly as a tumour promoter (Coffey et al, 1994). In contrast, work in E Fuchs’ lab has suggested that the TGF-a transgene acts at the initiation stage in skin carcinogenesis in K14-TGF-a mice (Vassar and Fuchs, 1991; Vassar et al, 1992). G Merlino, N Fausto, and co-workers have suggested that in MT-TGF-a transgenic mice the TGF-a transgene acts at the stages of both promotion and progression in hepatic carcinogenesis (Lee et al, 1992; Takagi et al, 1992). Thus, it appears that TGF-a may play a role in neoplasia that is both tissue and stage-dependent. The MMTV-TGF-a mice continue to be a useful in vivo model to sub-
TRANSFORMING
GROWTH
FACTOR-U
59
stantiate observations that are made in vitro. As mentioned above, in vitro studies indicate that there is cross-talk among the EGFR family. We have observed increased EGFR expression in tumours and peritumoural tissue in MMTV-TGF-a transgenic mice (Matsui et al, 1990). We have also crossed these mice to MMTV-c-neu mice and found a dramatic synergistic effect on the development of mammary tumours in which there is enhanced expression of TGF-a, EGFR and c-neu. Other studies from our laboratory have demonstrated that TGF-P is able to suppress tumourigenesis in MMTVTGF-a and MMTV-TGF-Pl bigenic mice (Pierce et al, 1995). Targeted disruption of TGF-a by homologous recombination results in a mildly abnormal phenotype. The predominant finding is disordered hair follicle structure resulting in curly whiskers and wavy hair (Luetteke et al, 1993; Mann et al, 1993). These findings may be due to attenuated cell proliferation, but loss of other biological functions of TGF-a such as cell migration and keratinization cannot be excluded. An identical phenotype occurs in waved-l mice in which spontaneous deficiency of TGF-a occurs. It will be informative to further address the surprisingly mild phenotype in TGF-a null mice. Experimental manipulations such as partial hepatectomy and skin carcinogenesis studies have not elicited a phenotype under these pathological conditions (Dlugosz et al, 1995; RJ Coffey and WE Russell, unpublished observations). It will also be of interest to determine if compensatory upregulation of other TGF-a related ligands occurs in the context of TGF-a deficiency. Waved-2 mice exhibit a similar phenotype to waved-l mice (Luetteke et al, 1994; Fowler et al, 1995). These mice have a spontaneous single nucleotide transversion (T to G), which results in a valine to glycine substitution at residue 743 in subdomain III of the EGFR kinase domain. This substitution lies 20 residues C-terminal to the lysine residue that defines the ATP binding site. This substitution occurs at the ATP binding site and results in loss of high-affinity EGFRs, which causes delayed ligand-mediated internalization (Fowler et al, 1995). In vitro, EGFR kinase assays (auto- and transphosphorylation) revealed a non-functioning receptor with the waved-2 mutation. In vivo, EGF-induced receptor phosphorylation and mitogenicity were attenuated but not lost; responses were seen at high doses of EGF. Homozygous waved-2 mothers demonstrated a defect in lactation, in addition to the skin changes seen in waved-l mice and targeted TGF-a null mice (Fowler et al, 1995). The relatively mild phenotype in mice bearing this mutation raises interesting speculation. Does in vivo EGFR signalling occur only at high ligand-receptor concentrations? Is responsiveness to EGF modulated by phosphorylation of the EGFR by other cell surface protein kinases? As mentioned above, there is increasing evidence of interactions among the EGFR family that may take the form of heterodimerization and/or transactivation. Targeted disruption of the EGFR presents more dramatic phenotypes. Recently, three groups (Miettinen et al, 1995; Sibilia and Wagner, 1995; Threadgill et al, 1995) have generated EGFR null mice by targeted disruption using homologous recombination. We have observed three fully penetrant, non-overlapping phenotypes dependent on genetic background
60
L. M. GANGAROSA
ET
AL
(Threadgill et al, 1995). EGFR deficiency on a CF-1 background resulted in peri-implantation mortality due to degeneration of the inner cell mass; on a 129/Sv background, homozygous mutants died at midgestation due to placental defects. In contrast, the same mutant allele was viable when bred to homozygosity in CD-l mice. These mutants lived for up to three weeks and showed marked abnormalities in skin, kidney and brain with lesser abnormalities in liver and gastrointestinal tract. Derynck and co-workers utilized a similarly designed EGFR targeting construct and generated mice with overlapping yet clearly distinct phenotypes (Miettinen et al, 1995). On a C57 background, Derynck’s group found that the mutant allele was also viable when bred to homozygosity. These mice exhibited changes in the lung as well as the skin, but these investigators did not observe abnormalities in the brain or kidney. Moreover, these mice developed extensive necrotizing enterocolitis which appeared to be the cause of death. The major difference in the approaches used by these two groups was the strain of mice in which the embryonic stem (ES) cells were injected and suggests that there are important modifier genes that determine the action of the EGFR. The importance of modifier genes has been demonstrated in min mice with the MOM1 locus, which recently has been shown to encode secretory type II phospholipase A2 (MacPhee et al, 1995). SUMMARY Major advances in understanding growth factor biology, especially in epithelial cells, have resulted from work with TGF-a over the past decade. It is clear that TGF-a is a potent epithelial oncoprotein, but equally important biological activities in normal epithelial homeostasis have been described. A number of major challenges lie ahead. Foremost is the formidable task of dissecting out the individual contributions of each EGF-related peptide in the biological response to stimulation of the EGFR. Appreciation of the complexity of heterodimerization of receptors within the EGFR family will be equally important in the final analysis. These considerations assure the continued vitality and productivity of investigation of the EGFrelated peptide/EGFR axis. REFERENCES Barnard JA, Graves-Deal R, Pittelkow MR et al (1994) Auto- and cross induction within the mammalian epidermal growth factor-related peptide family. Journal of Biological Chemistry 269: 228 17-22822. Barnard JA, Beauchamp RD, Russell WE, DuBois RN & Coffey RJ (1995) Epidermal growth factorrelated peptides and their relevance to gastrointestinal pathophysiology. Gastroenterology 108: 564-580. Bayerdorffer E, Ritter MM, Hatz R, Brooks W, Ruckdeschel G & Stolte M (1994) Healing of protein losing hypertrophic gastropathy by eradication of Helicobucter yylori-a pathogenic factor in MenCtrier’s disease? Gut 35: 701-704. Beauchamp RD, Barnard JA, McCutchen CM, Chemer JA & Coffey RJ (1989) Localization of
TRANSFORMING
GROWTH
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