Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization.

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

Heparin-binding epidermal-growth-factor-like growth factor gene expression is induced by scrape-wounding epithelial cell monolayers: involvement of mitogen-activated protein kinase cascades.

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.

Cyclic AMP inhibits agonist-induced heparin-binding EGF gene expression independently of effects on p42/p44 MAPK activation.

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.

Characterisation of the rat heparin-binding epidermal growth factor-like growth factor gene promoter.

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.

Quantitative analysis reveals differential expression of mucin (MUC2) and intestinal trefoil factor mRNAs along the longitudinal axis of rat intestine.

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 coordinately regulated in intestinal cells.

Characterization of a mammalian cDNA encoding a protein with high sequence similarity to the Drosophila regulatory protein Rhomboid.

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.

Identification of a minimal promoter element of the mouse epidermal growth factor gene.

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.

Identification of a functional promoter element in the 5'-flanking region of the rat cMG1/TIS11b gene.

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.

Cloning and characterization of ERF-1, a human member of the Tis11 family of early-response genes.

Members of the Tis11 family of early-response genes are characterized by a high degree of sequence similarity around a putative zinc finger motif. They are induced by a variety of cell agonists and polypeptide mitogens, including 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF). We describe the cloning and sequencing of a human member of this gene family, EGF-response factor 1 (ERF-1), the homolog of the mouse Tis11b/rat cMG1 genes. The human and rodent genes are similar, with 5' UTR, coding sequence, and 3' UTR highly conserved. The promoter/enhancer region and intron sequences contain multiple putative transcription factor binding motifs characteristic of early-response genes. Amino acid sequence comparison of the seven members of the Tis11 family cloned so far identifies a repeated consensus motif of (x+)YKTELC(x+)x5GxCxYGx(x+)CxFxH involving the potential zinc finger. Toward the carboxyterminal end is a region with a high percentage of prolines (15/73) and, partially overlapping, a serine-rich domain (20/54). These may be important as trans-activation and phosphorylation sites. The 3' untranslated region is unusually long, extending over 1,860 bp. The sequence immediately downstream from the translational stop codon has extensive secondary structure potential. The 3' UTR is 60% AT rich, but contains two GC rich (> 70%) regions. In addition there are multiple reiterations of a destabilization sequence, as well as a single UUAUUUAU motif characteristic of mRNAs specifying proteins involved in the inflammatory response. The mRNA contains a consensus polyadenylation signal.

Molecular cloning and tissue distribution of pig transforming growth factor alpha.

Transforming growth factor alpha (TGF alpha) 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 TGF alpha in normal tissues. Therefore, an amplified DNA fragment encoding the pig TGF alpha 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 TGF alpha precursors at 14, 15 and six sites respectively. The distribution of TGF alpha mRNA in a wide variety of pig tissues was analysed by RT-PCR, using oligonucleotide primers based on the pig TGF alpha cDNA sequence. TGF alpha 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 TGF alpha peptide, we have immunolocalized TGF alpha protein to cells within the red pulp of the spleen and to the distal convoluted tubules of the kidney.

Tissue distribution of mRNA for heparin-binding epidermal growth factor.

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 the genes for TGF alpha, EGF and the EGF receptor during early pig development.

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 TGF alpha 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, TGF alpha 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 TGF alpha mRNA during early pig development is coincident with the onset of blastocyst elongation and suggests a possible role for TGF alpha 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.

Nucleotide sequence and tissue distribution of mouse transforming growth factor-alpha.

Transforming growth factor-alpha (TGF alpha) is secreted into the medium of the Moloney murine sarcoma virus-transformed 3T3 cell line, 3B11-1C. Using reverse transcription-polymerase chain reaction (RT-PCR), we have amplified, cloned and sequenced a cDNA fragment from this cell line which encodes the full protein-coding region of the mouse TGF alpha precursor. The deduced amino acid sequence (159 residues) differs from that of human TGF alpha at 13 sites and from that of rat at 3 sites. In the mouse, TGF alpha transcripts were detected in a variety of normal adult tissues including two previously unreported sites: the preputial glands and the bladder.

Epidermal growth factor concentrations in pig tissues and body fluids measured using a homologous radioimmunoassay.

A homologous radioimmunoassay for the measurement of epidermal growth factor (EGF) levels in pig tissues and body fluids has been developed using an antiserum to recombinant porcine EGF. The assay is highly specific, showing no cross-reactivity with a variety of other polypeptides including the structurally related protein, transforming growth factor-alpha. Furthermore, < 1% cross-reactivity was observed with mouse EGF emphasizing the necessity for homologous assays for EGF measurement. Immunoreactive EGF was present in extracts of pig kidney and pancreas (3.44 +/- 0.43 and 0.76 +/- 0.13 (S.E.M.) pmol/g wet weight respectively), but was not detected in extracts of submaxillary gland or liver. Although immunoreactive EGF was not detectable in uterine, allantoic or ovarian follicular fluids, colostrum contained EGF at biologically active concentrations (0.84 +/- 0.15 nmol/l). Immunoreactive EGF was also present in pig urine, with similar concentrations in samples from male or female animals. In addition, pig urine inhibited the binding of 125I-labelled EGF to 3T3 fibroblasts and stimulated DNA synthesis in quiescent monolayers of these cells, indicating that the immunoreactive material in urine is biologically active. Quantitative comparisons of the data presented here with that published previously indicate considerable species variation in the EGF levels of various tissues and body fluids.

Mitogen-induced expression of the primary response gene cMG1 in a rat intestinal epithelial cell-line (RIE-1).

cMG1 is a primary response gene first identified in a rat intestinal epithelial (RIE-1) cell-line [(1990) Oncogene 5, 1081-1083]. A number of mitogens, including epidermal growth factor (EGF), angiotensin II (AII), serum and insulin rapidly induced 2- to 6-fold increases of cMG1 mRNA in RIE-1 cells, while transforming growth factor-beta caused a small reduction. Cyclic AMP-elevating agents blocked the increase of cMG1 mRNA induced by EGF. The AII-stimulated increase of cMG1 mRNA was blocked by the depletion of protein kinase C, whereas the EGF-stimulated increase was not affected, indicating that protein kinase C-dependent and -independent signalling pathways stimulate cMG1 expression.

Expression of epidermal growth factor and its mRNA in pig kidney, pancreas and other tissues.

The levels of epidermal growth factor (EGF) mRNA in a wide range of pig tissues were measured by a RNAase-protection assay. The highest levels were found in kidney and pancreas, with lower levels in submaxillary gland, prostate gland and seminal vesicles. Immunocytochemical staining using an antiserum raised against recombinant pig EGF localized expression of the factor to the distal convoluted tubules of the kidney and to the epithelial cells lining the pancreatic and salivary ducts. The observed expression of EGF mRNA and EGF in pig tissues is consistent with a possible role for EGF in the maintenance and repair of the epithelial lining of various ductal systems.

Cloning and characterization of a gene encoding pig epidermal growth factor.

A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron-exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5' end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5' end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.