Apicomplexan parasites secrete transmembrane (TM) adhesive proteins as part of the process leading to host cell attachment and invasion. These microneme proteins are cleaved in their TM domains by an unidentified protease termed microneme protein protease 1 (MPP1). The cleavage site sequence (IA↓GG), mapped in the Toxoplasma gondii microneme proteins TgMIC2 and TgMIC6, is conserved in microneme proteins of other apicomplexans including Plasmodium species. We report here the characterisation of novel T. gondii proteins belonging to the rhomboid family of intramembrane-cleaving serine proteases. T. gondii possesses six genes encoding rhomboid-like proteins. Four are localised along the secretory pathway and therefore constitute possible candidates for MPP1 activity. Toxoplasma rhomboids TgROM1, TgROM2 and TgROM5 cleave the TM domain of Drosophila Spitz, an established substrate for rhomboids from several species, demonstrating that they are active proteases. In addition, TgROM2 cleaves chimeric proteins that contain the TM domains of TgMIC2 and TgMIC12.

Rhomboid-1 is a serine protease that cleaves the membrane domain of the Drosophila EGF-family protein, Spitz, to release a soluble growth factor. Several vertebrate rhomboid-like proteins have been identified, although their substrates and functions remain unknown. The human rhomboid, RHBDL2, cleaves the membrane domain of Drosophila Spitz when the proteins are co-expressed in mammalian cells. However, the membrane domains of several mammalian EGF-family proteins were not cleaved by RHBDL2, suggesting that the endogenous targets of the human protease are not EGF-related factors. We demonstrate that the amino acid sequence at the luminal face of the membrane domain of a substrate protein determines whether it is cleaved by RHBDL2. Based on this finding, we predicted B-type ephrins as potential RHBDL2 substrates. We found that one of these, ephrinB3, was cleaved so efficiently by the protease that little ephrinB3 was detected on the surface of cells co-expressing RHBDL2. These results raise the possibility that RHBDL2-mediated proteolytic processing may regulate intercellular interactions between ephrinB3 and eph receptors.

We report here distinct interdependent functions for two proteins, Star and Rhomboid, that are key determinants of the epidermal-growth-factor (EGF)-receptor signalling pathway in Drosophila. When we expressed the Drosophila EGF-receptor ligand Spitz in mammalian cells, the protein failed to traffic to the plasma membrane, as assessed by either cell-surface protein biotinylation or immunocytochemical staining. However, when we co-expressed Star with Spitz, trafficking of Spitz to the cell surface could be demonstrated. Only when we co-expressed Spitz, Star and Rhomboid could the release of soluble Spitz protein into the medium be shown. Taken together, our results indicate that Star is required for the intracellular trafficking of Spitz, and that Rhomboid is essential for the release of soluble Spitz protein from cells.

Peptide growth factors can promote the cell migration and proliferation that is needed to repair epithelia after mechanical or chemical injury. We report here that scrape-wounding rat intestinal epithelial (RIE-1) cell monolayers caused a rapid increase in levels of heparin-binding epidermal-growth-factor-like growth factor (HB-EGF) mRNA, with a maximal response at approx. 1 h. Hybridization in situ showed that transcript induction occurred primarily in cells at or near wound borders. The increase in HB-EGF mRNA was preceded by activation of the p42 mitogen-activated protein kinase (MAPK) in the wounded cell cultures. Moreover, the induction of HB-EGF mRNA was blocked by PD098059 and U0126, inhibitors that prevent the activation of p42/p44 MAPKs and extracellular signal-regulated protein kinase 5 (ERK5). Both p42 MAPK activation and HB-EGF mRNA induction were inhibited by genistein, indicating a requirement for an upstream tyrosine kinase activity. In contrast, neither response was affected by inhibition of phosphoinositide 3-kinase activity, down-regulation of protein kinase C, or disruption of the actin cytoskeleton with cytochalasin B. We conclude that scrape-wounding epithelial cell monolayers induces HB-EGF mRNA expression by a mechanism that most probably requires p42/p44 MAPK activation, although we cannot exclude a role for ERK5. Our results suggest a physiological role for locally synthesized HB-EGF in promoting epithelial repair after injury.

Heparin-binding EGF-like growth factor (HB-EGF) mRNA levels are increased up to 20-fold in RIE-1 cells by two agonists that act through distinct receptor types. We demonstrated a common requirement for p42/p44 mitogen-activated protein kinase (MAPK) in this response using the selective MAPK kinase (MEK) inhibitor, PD 098059. Agonist-mediated induction of HB-EGF mRNA was markedly suppressed in cells that had been treated with cyclic AMP-elevating agents. In contrast, the activation of p42 MAPK in response to agonists was not affected by raising cellular cyclic AMP levels. We conclude that cyclic AMP negatively regulates the HB-EGF gene, but that the inhibitory action is either independent of the p42/p44 MAPK pathway or the site of action is distal to MAPK activation.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene expression is strongly activated by a variety of extracellular stimuli, acting through the Raf/MEK/MAP kinase pathway. To study the elements that respond to this pathway, we have isolated and sequenced a fragment of the rat HB-EGF gene promoter. By transfection of a series of promoter/reporter constructs into cells, a minimal promoter element was demonstrated to lie between 448 bp upstream of the transcriptional start site and 103 bp into the first exon of the gene. However co-transfection of the promoter constructs with a plasmid directing expression of RafCAAX, an activated c-Raf-1 protein, gave a fold-stimulation of activity no greater than that seen for the parental pGL3-Basic plasmid alone. In addition, agonist stimulation of cell lines stably transfected with a HB-EGF promoter/luciferase construct produced little or no increase in reporter enzyme activity. These results suggest that the c-Raf-1 responsive elements lie outside the tested region of the rat HB-EGF gene. However, it has been reported that a c-Raf-1 responsive element is present within the equivalent region of the mouse gene. A comparison of the 5′-flanking regions of the mouse, rat and human HB-EGF genes indicated that the mouse sequence diverges abruptly from that of the other two species approximately 260 bp upstream of the transcriptional start site. PCR analysis of mouse genomic DNA suggests that this sequence divergence is due to DNA rearrangement during the cloning of the mouse gene. Additional studies are therefore required to identify Raf/MAP kinase responsive elements in the HB-EGF gene.

MUC2 and intestinal trefoil factor (ITF) are considered to have important roles in intestinal mucosal protection and epithelial repair. In order to investigate whether these genes are co-ordinately expressed, we have used competitive reverse transcription-polymerase chain reaction assays to measure MUC2 and ITF mRNA levels in human intestinal cell lines and along the longitudinal axis of rat intestine. ITF mRNA was expressed in several intestinal cell lines. However, MUC2 mRNA was detected only in LS174T cells where it was present at approx. 25-fold lower levels than the ITF transcript. In contrast, in rat intestinal tissues, MUC2 mRNA levels were generally higher than ITF mRNA levels. The levels of both transcripts increased markedly during postnatal development. In adult rats, the expression patterns of MUC2 and ITF mRNAs along the longitudinal axis of the small intestine were similar, with lowest levels in the proximal duodenum and relatively constant levels in the other regions assayed. In contrast, the expression patterns of MUC2 and ITF in different regions of the large intestine showed a marked divergence. Our results strongly suggest that expression of the MUC2 and ITF genes is not co-ordinately regulated in intestinal cells.

The Drosophila regulatory protein Rhomboid has been demonstrated genetically to facilitate signalling within the Spitz/epidermal growth factor receptor/mitogen-activated protein kinase pathway. Using a polymerase chain reaction (PCR)-based strategy, we have cloned a human cDNA which encodes a protein that has high sequence similarity to Rhomboid. The encoded protein, termed rhomboid-related protein (RRP), is predicted to contain seven transmembrane domains. Northern analysis indicates that RRP mRNA is expressed at highest levels in brain and kidney.

We have previously generated a transgenic mouse line (EGF/Tag) in which simian virus 40 (SV40) T-antigen expression is directed by the mouse epidermal growth factor (EGF) gene promoter. In these mice, cellular hyperproliferation is observed in the submaxillary gland associated with SV40 T-antigen expression. In addition, SV40 T-antigen-expressing tumours of prostatic origin are seen. We have now derived immortalized cell lines from these tissues and have used the cells to perform a functional analysis of the EGF gene promoter. Cells were transfected with EGF promoter/reporter constructs, and an element located between 51 and 35 bases upstream of the EGF mRNA start site required for basal activity of the promoter was identified. Electrophoretic mobility-shift analysis suggests that three proteins bind to this region, one of which is either Sp1 or a closely related protein.

Fluids that accumulate at wound sites may be an important reservoir of growth factors that promote the normal wound healing response. The presence of heparin-binding growth factors was studied in burn wound fluid (BWF) from 45 pediatric patients who had sustained partial thickness burns. One of the growth factors present was similar to platelet-derived growth factor (PDGF) based on its heparin affinity, inhibition of bioactivity by a PDGF antiserum, and detection in a PDGF-AB enzyme-linked immunosorbent assay. A second growth factor was identified as heparin-binding epidermal growth factor-like growth factor (HB-EGF) based on its heparin affinity, competition with 125I-labeled epidermal growth factor (EGF) for EGF receptor binding, and recognition in biological assays and Western blots by two HB-EGF antisera. Amino acid sequence analysis of one form of this second growth factor verified its identity as an N-terminally truncated form of HB-EGF. Immunohistochemical analysis of partial thickness burns demonstrated the presence of HB-EGF in the advancing epithelial margin, islands of regenerating epithelium within the burn wound, and in the duct and proximal tubules of eccrine sweat glands. HB-EGF in the surface epithelium of burn wounds was uniformally distributed, whereas it was restricted to the basal epithelium in nonburned skin. These data support a role for PDGF and HB-EGF in burn wound healing and suggest that the response to injury includes deposition of HB-EGF and PDGF into blister fluid and a redistribution of HB-EGF in the surface epithelium near the wound site.

The cMG1 gene was originally identified as a mitogen-stimulated primary response gene. However, in contrast to genes such as c-fos and TIS11, cMG1 is also expressed at significant levels before and after the transient elevation induced by agonists. We have sequenced a 1.3 kb rat genomic cMG1 clone, which includes 931 bp upstream of the transcription start site identified by primer-extension analysis. A 1033 bp fragment, including this 5'-flanking sequence, directed the expression of the reporter gene chloramphenicol acetyltransferase (CAT) in transfected NIH-3T3 cells. Progressive 5'-to-3' deletion indicated that an element located between -138 and -114 was responsible for most of this basal CAT expression. DNA mobility-shift assays showed that the sequence between -143 and -105 contained binding sites for cellular proteins, the principal complexes involving nucleotides between -119 and - 105. We conclude that these complexes may represent the transcription factor-DNA element interactions that determine basal cMG1 expression.

A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5.5 kb fragment of the 5'-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5.5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.

Proliferation of the rat intestinal epithelial cell-line, RIE-1, has previously been shown to be stimulated by certain polypeptide growth factors acting via receptors that possess intrinsic tyrosine kinase activity. In this study, we show that the octapeptide hormone angiotensin II (AII), apparently acting through the AT1 G-protein-coupled receptor, is also a mitogen for RIE-1 cells. Maximal stimulation of DNA synthesis and cellular proliferation occurred at an AII concentration of 10-100 nM, with half-maximal stimulation at 1 nM. The mitogenic response to AII was completely inhibited by the AT1 angiotensin-receptor antagonist, DuP753, but not by the AT2-receptor antagonist, PD123319. The early signalling responses activated by AII in RIE-1 cells include increased production of inositol phosphates, a transient increase in the intracellular concentration of free calcium, an activation of protein kinase C, and a rapid change in the pattern of cellular protein-tyrosine phosphorylation. These results implicate an activation of the inositol lipid signalling pathway via the AT1 receptor subtype in the AII-stimulated mitogenic response of this normal epithelial cell line.

Transforming growth factor alpha (TGFalpha) was originally identified as a product of tumour tissues and transformed cells in culture. Although it is now clear that expression of this factor is not restricted to neoplastic cells, there remains relatively little information about the sites of expression of TGFalpha in normal tissues. Therefore, an amplified DNA fragment encoding the pig TGFalpha precursor was cloned by reverse transcription-PCR (RT-PCR) using RNA isolated from normal skin tissue as the template. Nucleotide sequence analysis predicts a 160-residue transmembrane polypeptide that differs from the rat, mouse and human TGFalpha precursors at 14, 15 and six sites respectively. The distribution of TGFalpha mRNA in a wide variety of pig tissues was analysed by RT-PCR, using oligonucleotide primers based on the pig TGFalpha cDNA sequence. TGFalpha transcripts were detected in RNA isolated from 17 of the 22 tissues analysed, including four previously unreported sites. Using an antibody raised against a synthetic TGFalpha peptide, we have immunolocalized TGFalpha protein to cells within the red pulp of the spleen and to the distal convoluted tubules of the kidney.

Members ofthe TisI 1 family of early response genes are rapidly and transiently induced by the treatment of cells with agonists such as the tumour promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) and polypeptide mitogens such as epidermal growth factor (EGF) or fibroblast growth factor (FGF) (reviewed in 1). Several members of this family have been cloned, including mouse genes (Tisl 1, Tisl lb and Tisl ld) (2, 3) and a gene isolated from an EGF-stimulated rat intestinal epithelial cell line (cMG1) (4). The cloning of the first human member of this gene family, GOS24, the homologue of the mouse Tisi 1 gene, has recently been reported (5, 6). All family members contain a putative zinc finger motif, and the gene specifying the mouse Tisl 1 gene has been labelled Zfp-36 by the Committee on Standardised Genetic Nomenclature for Mice. Here we describe the coding sequence of the second human family member, the homologue of the Tisllb/cMG1 genes, which we have called EGF-response factor 1 (ERF-1). The human ERF-1, rat cMGl and mouse Tis llb coding sequences are strongly conserved (94.5% and 93.7% respectively at the nucleic acid level, which translates to 99% sequence similarity at the amino acid level). By comparing the sequence from

We have used the reverse transcription-polymerase chain reaction (RT-PCR) to determine whether transcripts for the epidermal growth factor (EGF) receptor and its four known ligands-EGF, transforming growth factor alpha (TGFalpha), amphiregulin (Ar), and heparin-binding EGF (HB-EGF)-are expressed in porcine oviduct and endometrium. We were able to detect mRNA for the EGF receptor, EGF, TGFalpha, and Ar in both the oviduct and endometrium, whereas HB-EGF mRNA was not detectable in either tissue. Through use of an antiserum raised against recombinant pig EGF, expression of EGF was found to be localized to the columnar epithelial cells of the oviduct and to the glandular epithelial cells of the endometrium. The possible physiological roles of the EGF family in the reproductive tract are discussed.

Heparin-binding epidermal growth factor (HB-EGF) is a recently identified member of the EGF family. Mature HB-EGF is processed from a larger transmembrane precursor which can itself act as a cell-surface receptor for the internalization of diphtheria toxin into eukaryotic cells. However, to date there is no information available on the distribution of HB-EGF in mammalian tissues. We have therefore used reverse-transcription PCR to analyse the expression of HB-EGF mRNA in a wide range of tissues. HB-EGF transcripts were detected in RNA isolated from 15 of the 22 tissues obtained from adult pigs, which is consistent with the ability of diphtheria toxin to affect many body tissues.

Expression of mRNA for transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF) and the epidermal growth factor receptor (EGF-R) during early pig development was evaluated by reverse transcription-PCR. In the unfertilised pig oocyte, maternal transcripts for EGF, but not for TGFalpha or the EGF-R, were detected. Pig conceptuses were analysed at days 7, 8, 10, 12, 15, 17, 18 and 22 of pregnancy. EGF-R mRNA was detected at all stages of conceptus development analysed. Interestingly, TGFalpha mRNA was expressed by the developing blastocyst only at days 8, 10 and 12 of pregnancy, with the highest levels apparent at day 10. In contrast, EGF mRNA was first expressed by the post-elongation conceptus at around day 15 of pregnancy with levels continuing to increase up to day 22. In the day-18 and day-22 conceptuses, this EGF message was shown to be primarily a product of the embryo-amnion and not the placental membranes. Furthermore, EGF was immunolocalised in the day-22 embryo to the developing lung bud, gut loop and amnion. In summary, the expression pattern of TGFalpha mRNA during early pig development is coincident with the onset of blastocyst elongation and suggests a possible role for TGFalpha during this period of cellular remodelling. The temporal and spatial expression of EGF mRNA and protein suggests a possible involvement for EGF in the establishment of the early organ systems.