kDa Epidermal Growth Factor Research Articles

A (1-->3)-beta-D-glucan recognition protein from the sponge Suberites domuncula. Mediated activation of fibrinogen-like protein and epidermal growth factor gene expression.

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1-->3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1-->3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further genes showed an elevated steady state level of expression; one codes for a fibrinogen-like protein and the other for the epidermal growth factor precursor. Expression of the (1-->3)-beta-D-glucan-binding protein and the fibrinogen-like protein occurred in cells on the sponge surface, in the pinacoderm. By Western blotting, the product of the fibrinogen-like protein gene was identified, the recombinant protein isolated, and antibodies raised to this protein. Their application revealed that a 5 kDa factor is produced, which is apparently processed from the 77 kDa epidermal growth factor precursor. Finally, we provided evidence that a tyrosine kinase pathway is initiated in response to exposure to D-glucan; its phosphorylation activity could be blocked by aeroplysinin. In turn, the increased expression of the downstream genes was suppressed. We conclude that sponges possess a molecular mechanism for recognizing fungi via the d-glucan carbohydrates on their surfaces.

Regulated Cell Surface Pro-EGF Ectodomain Shedding Is a Zinc Metalloprotease-dependent Process

Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.

The vacuolating toxin of Helicobacter pylori: a few answers, many questions

kJanrrr&We: Or V. Ricci, lstituto di Fisiologia Umana, Universitti di Pavia. via Forlanini 6, 27’100 Pavia, italy. Fax: +39-CB92-507SS4. E-meil: wicci@unipv. it Helicobacterpylori (H. pylori) has developed properties that allow it to survive and grow in a hostile environment like the human stomach where it causes an inflammatory reaction and epithelial damage with cellular swelling, cytoplasmic vacuolation, and expansion of endosomal compartments ‘. Bacterial extracts from H. pylori induce cytoplasmic vacuolar degeneration in cultured cells 2-4. A pivotal role in cell damage induced by H. pylori seems to be played by the vacuolating toxin (VacA) 5m7. VacA is a protein toxin (thus far the only protein toxin known to be produced by H. pylori), encoded by the vacA gene, able to induce cytoplasmic vacuoles in eukaryotic cells in culture although sensitivity to VacA differs greatly in the different cell types tested *. When given to mice, VacA causes gastric epithelial damage closely resembling that found in H. pyZori-colonized patients s. VacA toxin is composed of monomers of about 90 kDa synthesized as a 140 kDa precursor with a N-terminal signal sequence of 33 amino acids 5 6 The structure of the vacA gene varies, es. pecially in the region encoding the signal sequence (which may be type s la, s lb or s2) and in the mid-region (which may be type ml or m2) (‘. VacA is released by the bacterium both as soluble secreted protein and as VacA-containing vesicles derived from outer membrane blebs * O. Both soluble secreted VacA and VacA-containing vesicles bind to, and are internalized by, gastric epithelial cells in culture and are detectable in the gastric mucosa from H. pylori-infected patients * 9. Therefore, the release of outer membrane vesicles by H. pylori may represent a mechanism, in addition to secretory pathway, for the delivery of bacterial virulence factors and antigens into the gastric mucosa”. However, the role of VacAcontaining vesicles in VacA-induced cell damage is still unknown. Purified toxin forms high molecular mass (about 1,000 kDa) oligomers which need to be disassembled in monomers by acid or alkaline treatment to become active ‘” ‘I. Nevertheless, the findings that both bacterium-associated toxin and bacterial broth culture filtrates are constitutively very active without requiring acid/alkaline pretreatment ‘O suggest that VacA may be present in the juxtacellular microenvironment of the human gastric mucosa in its active monomeric form. Moreover, it has been suggested that monomers are further processed to produce a 34-37 kDa Nterminal fragment and a 58 kDa C-terminal fragment and that these fragments remain associated after cleavage and may represent two distinct subunits of the toxin (j. This type of cleavage in subunits is characteristic of the A-B family of bacterial toxins in which subunit A exhibits enzymatic activity accounting for the overall toxin action, while subunit B is devoted to cell binding and membrane translocation I*. VacA binds plasma membrane of epithelial cells and is internalized by the cells via an active cellular process ‘j. Some authors have presented data suggesting the existence of a specific, high affinity cell surface receptor for VacA, although they failed to agree on its molecular characteristics. For instance, one group I’ reported that the 250 kDa receptor protein tyrosine phosphatase beta may act as a VacA receptor even if VacA also binds to an unidentified 140 kDa protein on the surface of cultured cells. Another group I4 reported that the 170 kDa epidermal growth factor receptor may

Involvement of prostate specific antigen in the stimulation of LNCaP cell growth

Since its identification in 1979, prostatic specific antigen (PSA) has been used extensively as a serum marker for diagnosis and prognosis of prostate cancer. In addition, PSA is an immunohistochemical marker for the identification of prostatic tissues and cells in histological specimens. PSA is found in normal prostate, benign prostatic hypertrophy (BPH) tissue, in cancer of the prostate, and its metastases as well as in other hormone dependent cancers, such as breast and ovarian carcinoma. However, the importance of PSA as a regulator of cell growth generally has not been appreciated. The role of PSA in the development of prostate or other hormone-dependent cancers has remained unclear. We therefore examined the role of PSA in the control of cell growth using both the PSA positive cell line, LNCaP cells and the PSA negative cell line PC-3 and DU145. LNCaP cell growth was stimulated by the conditioned medium (CM) from LNCaP cells, but not by CM from PC-3 or DU145 cells. No such stimulation was observed when PC-3 or DU145 cells were exposed to CM from LNCaP cells nor from CM produced by their own lines. The stimulation of LNCaP cell growth by its own CM could not be attributed to the high level of insulin-like growth factor binding protein-2 (IGFBP-2) present in the CM since even higher level of IGFBP-2 was also found to be present in CM from both PC-3 and DU145 CMs. High level of PSA and 66 kDa epidermal growth factor (EGF) were present in LNCaP CM as measured by Western blotting. The stimulation of LNCaP cell growth by its own CM was eliminated partially by PSA or EGF antibody. Stimulation of DNA biosynthesis in LNCaP cells by LNCaP CM or pure PSA was also observed. These data indicate that PSA and EGF are involved in the growth regulation of PSA positive LNCaP cell line.

Cleavage of the epidermal growth factor receptor by a membrane‐bound leupeptin‐sensitive protease active in nonionic detergent lysates of senescent but not young human diploid fibroblasts

Numerous studies suggest that epidermal growth factor (EGF) signaling is impaired in nonproliferating senescent human diploid fibroblasts downstream of receptor binding. One possible explanation for these results is that senescent cells possess unique enzymatic activities capable of regulating functional levels of the EGF receptor. To test that hypothesis, nonionic detergent lysates of young and senescent cells were compared for proteolytic activity directed towards the EGF receptor, and a protease that cleaves the 170 kDa EGF receptor was identified in lysates from senescent but not young cells. Although studies presented here were carried out with WI-38 cells, our data indicate that other senescent fibroblasts possess a similar activity. The degradation product immunoprecipitated by a monoclonal antibody specific for an EGF receptor exocytosolic epitope had an approximate molecular weight of 100,000. This product was also detected following cell surface labeling with 125I, and by cross-linking 125I-EGF to intact cells with disuccinimidyl suberate. The proteolytic activity in senescent cell lysates was specifically inhibited by leupeptin and did not require divalent cations; it was also inactivated by aprotic solvents such as dimethylsulfoxide (DMSO) or ethylene carbonate. Interestingly, this protease was not active during ligand-induced intracellular processing of the EGF receptor, suggesting that it does not normally function in endocytic or lysosomal compartments. The susceptibility of the protease to inactivation by cell surface trypsinization is consistent with a plasma membrane localization. Since EGF receptor cleavage is not observed unless senescent cells are solubilized with nonionic detergents, it seems likely that the protease is confined to specialized regions of the plasma membrane. Whether or not the EGF receptor is a physiologic target for this protease is unclear. Its expression at the cell surface is nevertheless significant, since it suggests there are mechanisms for regulating membrane-bound proteins, or biologically active peptides in the extracellular space, in senescent cells that are either absent or inactive in young cells.

Accelerated degradation of 160 kDa epidermal growth factor (EGF) receptor precursor by the tyrosine kinase inhibitor herbimycin A in the endoplasmic reticulum of A431 human epidermoid carcinoma cells

The effect of herbimycin A on the biosynthesis of epidermal growth factor (EGF) receptor was examined in human epidermoid carcinoma A431 cells. Cells were pulse-labelled with [35S]methionine, and EGF receptor biosynthesis was quantified by immunoprecipitation using a monoclonal anti-(EGF receptor) antibody. In the presence of herbimycin A, an immature 160 kDa EGF receptor precursor accumulated in 1 h and disappeared completely in 4 h. Pulse-labelled 160 kDa receptor precursor in the absence of herbimycin A, however, was converted normally into a 170 kDa one by chase with herbimycin A. Herbimycin A affected neither the synthesis of the secreted form of EGF receptor devoid of cytoplasmic domain, nor that of the transferrin receptor in A431 cells. The herbimycin A-induced degradation of 160 kDa EGF receptor precursor was not inhibited by an inhibitor of lysosomal enzymes, NH4Cl. Endoglycosidase H digestion of the 160 kDa precursor converted it into the deglycosylated 130 kDa precursor peptide. These results suggested that herbimycin A selectively acted on the EGF receptor precursor during the synthesis of the 160 kDa form, probably on the cytoplasmic domain, to form an aberrant molecule which was subjected to rapid degradation in the endoplasmic reticulum.

Processing of Epidermal Growth Factor in the Rat Submandibular Gland by Kallikrein-Like Enzymes

Epidermal growth factor (EGF) is synthesized as a precursor which is processed intracellularly to a 6 kDa EGF in the rat submandibular gland. This gland contains very high amounts of kallikrein-like enzymes, and the purpose of the present study was to examine whether any of five such enzymes, rK1, rK2, rK7, rK9 or rK10, can process the rat EGF precursor. Molecular weight forms of EGF, that were N- or C-terminally extended compared to submandibular gland EGF were obtained from rat urine. These extended forms of EGF were incubated with each of the enzymes for 24 h at 37 degrees C. Two enzymes, rK7 and rK10, were able to cleave N- and C-terminally extended EGF, releasing a form of EGF which eluted similarly to submandibular gland EGF upon gel filtration, and which was recognized both by antibodies against rat EGF and by the EGF receptor. One enzyme, rK1, cleaved C- but not N-terminally extended EGF. Neither rK2, nor rK9 cleaved the extended forms of EGF. In previous immunohistochemical studies rK1, rK7 and rK10 have all been demonstrated in the EGF containing cells of the rat submandibular gland. EGF and rK1 are also synthesized in the rat kidney but the present study demonstrated that EGF and rK1 are not colocalized in this organ. Based on the cleavage of the extended forms of rat EGF by rK1, rK7 and rK10 and on the fact that the enzymes are abundant and colocalized with EGF in the rat submandibular gland, we suggest that rK1, rK7 and rK10 can be involved in the processing of the EGF precursor in the rat submandibular gland.

Effect of epidermal growth factor on Cu, Zn-superoxide dismutase expression in cultured fibroblasts from rat skin.

The effects of epidermal growth factor (EGF) on Cu, Zn-superoxide dismutase (SOD) in cultured fibroblasts from rat skin exposed to superoxide anions were studied. Cross-linking of [125I]hEGF using disuccinimidyl suberate and immunoblot analysis using anti-EGF receptor antibody to crude plasma membrane fractions of fibroblasts showed that a 170 kDa EGF receptor protein was present on the membrane, as in A431 cells which over express a specific EGF receptor. The cytosolic SOD enzyme activity in fibroblasts exposed to superoxide anions 24 h after treatment with EGF plus nafamostat (NM), a potent protease inhibitor, was increased 1.6-fold compared to control-treated cells. Treatment with either EGF or NM alone, evoked little increase in SOD enzyme activity. The increase in Cu, Zn-SOD protein levels corresponded to the increase in cytosolic SOD enzyme activity in fibroblasts. The Cu, Zn-SOD mRNA level in fibroblasts treated with EGF plus NM at 3 and 6 h was higher than that of the control. Additionally, levels of [125I]hEGF degradation products released into the medium from fibroblasts exposed to superoxide anions were significantly reduced in the presence of NM. These results suggest that the stabilization of EGF by NM in culture is an important factor in the expression of its effects, and that EGF induces Cu, Zn-SOD expression by accelerating transcription of the Cu, Zn-SOD gene in cells, resulting in their protection from the effects of superoxide anion radicals.

Characterization of the epidermal growth factor receptor from Ehrlich ascites tumor cells.

We have identified the 170 kDa epidermal growth factor (EGF) receptor in crude membrane fractions isolated from Ehrlich ascites tumor cells by its EGF-dependent phosphorylation with [gamma-32P]ATP. An apparent affinity constant for the ligand of 40-50 nM, based on the extent of its EGF-dependent phosphorylation, was calculated. [125I]EGF binds to the 170 kDa receptor and Scatchard plot analysis shows high affinity and low affinity Kds of 1.7 nM and 24 nM, respectively, in whole cells, and 0.2-0.8 nM and 39-116 nM, respectively, in isolated non-phosphorylated membrane fractions. We have estimated the presence of 48 x 10(3) high affinity and 275 x 10(3) low affinity EGF binding sites per tumor cell. Phosphoamino acid analysis shows EGF-dependent phosphorylation of tyrosine and serine residues. A polyclonal antibody to a human EGF receptor/c-erbB-2 product common cytoplasmic domain epitope immunoprecipitates a 45 kDa phosphopolypeptide from the tumor membrane fractions and from whole cell lysates. Phosphoamino acid analysis of the immunoprecipitated 45 kDa phosphopolypeptide shows the presence of phosphoserine. The immunoprecipitated 45 kDa polypeptide is able to undergo EGF-independent phosphorylation, although no significant protein kinase activity towards exogenous substrates is detected.

Human epidermal growth factor—on molecular forms present in urine and blood

A sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of human epidermal growth factor (EGF) was employed to study EGF in urine and blood. The EGF/creatinine ratio in urine was significantly higher for women (range and (median); 0.20–0.83 (0.50) nmol EGF/mmol creatinine) than for men (0.17–0.63 (0.30) nmol EGF/mmol creatinine). We were not able to demonstrate EGF in plasma (median plasma EGF < 0.01 nmol/l) whereas serum contained a range and (median) of 0.02–0.31 (0.12) nmol EGF/l. The amount of EGF in serum showed a weak correlation to the platelet count ( r = 0.327). EGF was partly purified by affinity chromatography from urine (urine EGF) and from activated platelets in platelet rich plasma (blood EGF). Both blood and urine contained a high molecular weight form of EGF (HMW EGF) as well as 6 kDa EGF. HMW EGF from blood was similar to HMW EGF from urine concerning behaviour upon gel filtration, p I and apparent affinity constant for binding to the EGF receptor. However, HMW EGF constituted approx. 40% of blood EGF but only 10% of urinary EGF. The 6 kDa EGF from both blood and urine contained two isopeptides with p I around 4.40 and 4.15 but in various proportions. The apparent affinity constant for binding to the EGF receptor for blood 6 kDa EGF was 1.8·10 10 l/mol compared to 1.0·10 10 l/mol for urinary 6 kDa EGF and 0.8·10 10 l/mol for HMW EGF from both blood and urine. The present study suggests that the processing of the EGF precursor differs in the blood and in the kidneys and that 6 kDa EGF from blood and urine binds to the EGF receptor with a higher apparent affinity constant than does HMW EGF.

The urinary excretion of epidermal growth factor in the rat is reduced by aprotinin, a proteinase inhibitor

The present study on the rat shows that i.v. administration of the proteinase inhibitor aprotinin reduces the urinary output of immunoreactive epidermal growth factor (EGF) while the amount of immunoreactive EGF in the kidneys is increased. This indicates that the EGF-precursor in the rat kidney in vivo is processed by an aprotinin inhibitable proteinase. EGF is produced in the kidneys as a precursor with a molecular weight of approximately 130 kDa. In rat urine, nanomolar amounts of 6 kDa EGF are excreted per 24 h together with small amounts of high molecular weight forms of EGF. During i.v. administration of aprotinin the median urinary output of immunoreactive EGF is reduced to 15% of the excretion of control rats (23 pmol/2 h versus 157 pmol/2 h, P < 0.001). Especially the excretion of 6 kDa EGF is reduced (median excretion 12 pmol/2 h versus 134 pmol/2 h, P < 0.001). The amount of immunoreactive EGF in the kidney tissue is increased after aprotinin administration (median amount 0.11 pmol EGF/mg protein versus < 0.04 pmol EGF/mg protein, P < 0.001). Neither the creatinine clearance, the total urinary protein output, nor the volume of urine produced was affected by aprotinin.

Metabolism of i.v. administered 45 kDa epidermal growth factor in the rat

The 45 kDa epidermal growth factor (EGF-(45 kDa)) has been purified from rat urine. We have investigated the distribution and the processing of i.v. injected 125I-labeled EGF-(45 kDa) in the rat. 2.5 min after the i.v. injection only 12% of the label remained in the blood. Most of the label was found in the liver (54%), in the kidneys (7%) and in the skin (4%). The submandibular glands, stomach, small intestine, colon, spleen and lungs contained 1% or less of the radioactivity. Some of the 125I-EGF-(45 kDa) was processed to 125I-EGF-(6 kDa) immunoreactivity in the liver and in the kidneys. The kidneys excreted 125I-EGF-(45 kDa) in the urine, but we were not able to demonstrate 125I-EGF-(6 kDa) in urine. In conclusion, this study shows that homologous EGF-(45 kDa) is cleared from the circulation of rats within a few minutes, mainly by the liver and the kidneys. In vivo both the liver and the kidneys are able to process some of the EGF-(45 kDa) to EGF-(6 kDa) immunoreactivity.

Saliva from cystic fibrosis patients contains an unusual form of epidermal growth factor

Epidermal Growth Factor (EGF) was assayed in saliva collected from control subjects and cystic fibrosis (CF) patients, using both radioimmuno (RIA) and radioreceptor (RRA) assays. An intriguing finding was that the average ratio of the values found by RRA over those obtained by RIA was of 1.7 for normal subjects and of about 0.4 for CF patients. This observation could be understood following gel filtration analysis of EGF-like material in these salivary fluids. Whereas control saliva contained the expected 6 kDa EGF active peptide, the immunoreactive EGF material from CF patients eluted as a polydisperse macromolecular moiety. The poor biological reactivity of this material as assessed by radioreceptor assay suggests that this EGF anomaly may contribute to the physiopathology of cystic fibrosis, especially as the upper gastrointestinal tract differentiated functions may be the target of normal salivary EGF.

Epidermal growth factor dependent phosphorylation of a 35-kilodalton protein in placental membranes.

In human placental membranes isolated in the presence of ethylenediaminetetraacetic acid (EDTA), epidermal growth factor (EGF) stimulated the [gamma-32P]ATP-dependent phosphorylation of tyrosine residues on the 170-kilodalton (kDa) EGF receptor and on a 35-kDa protein. The initial rate of phosphorylation of these proteins in the presence of EGF was 5.2 and 3.5 nmol of phosphate min-1 (mg of receptor protein)-1, and this was approximately 10- and 6-fold higher than the basal rate, respectively. Half-maximal phosphorylation of both proteins occurred at about 2.5 nM EGF. In the presence of p-nitrophenyl phosphate, EGF stimulated the phosphorylation of the 35-kDa protein but not the EGF receptor, suggesting that hormone-stimulated autophosphorylation of the receptor/kinase was not required for kinase activation. The 35-kDa protein exists in two forms: (1) 35Keluate, which was associated with the membrane in the presence of Ca2+ but was eluted with EDTA, and (2) 35Kmemb, which was not eluted from membranes with EDTA. Both forms were immunologically related to a 35-kDa protein previously isolated from A431 cells. Antiserum against the 35-kDa protein also reacted with a protein with an apparent size of 66 kDa that was phosphorylated in an EGF-dependent manner. In phosphorylation reactions performed in the presence of Mg2+, Ca2+ was required for phosphorylation of the 35Keluate form, but Ca2+ was not required for phosphorylation of the 35Kmemb form. Phosphorylation appears to change the membrane-binding properties of the 35Kmemb form because 32P-labeled 35Kmemb could be eluted from the membrane by EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells.

When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF.