Expression of epidermal growth factor in the rat kidney. An immunocytochemical and in situ hybridization study

The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Expression of epidermal growth factor in the kidney and submandibular gland during mouse postnatal development

Earlier work has demonstrated that the salivary glands and kidneys are the major sites of epidermal growth factor (EGF) synthesis in adult mice. The precise timing of the onset of endogenous EGF synthesis in these tissues is not yet clear. In the present study we assessed the ontogenesis of EGF expression in the Swiss-Webster mouse. Paraformaldehyde-fixed frozen sections of neonatal kidneys and salivary glands were probed with proEGF cRNA labelled with 35S for in situ hybridization and with rabbit antisera to mouse EGF for immunocytochemistry. Both EGF mRNA and immunoreactivity were first detected in the developing distal nephron between days 3 and 5 postpartum. Juxtamedullary nephrons underlying the superficial nephrogenic zone were the first to express EGF. During the 2nd week after birth, EGF-expressing tubules became more abundant and distributed to medullary as well as cortical regions, corresponding to the thick ascending limb of Henle and distal convoluted tubule. Initial EGF mRNA and immunoreactivity in the submandibular gland were first detected between days 18 and 20 postpartum and increased notably during the following weeks.     

Biochemical properties of epidermal growth factor in the mouse kidney

Epidermal growth factor (EGF) concentration in the mouse kidney was exceedingly low when compared with the submandibular gland level. Gel filtration of kidney extract showed that kidney EGF had the same molecular weight as the submandibular gland peptide. The isoelectric point of kidney EGF was between pH 4.3 and 4.6. From reversed phase HPLC, two species of EGF, alpha-EGF and beta-EGF, were clearly detected in the kidney sample.     

Immunocytochemical localization of epidermal growth factor in mouse kidney

Epidermal growth factor (EGF) was originally isolated from mouse submandibular glands (SMG). However, SMG removal failed to lower circulating EGF, and large amounts of EGF have been found in mouse urine. In addition, the presence of pre-pro-EGF mRNA in mouse kidney has recently been reported by others. Kidneys may therefore represent an alternate source of EGF. In the present study, we investigated the immunocytochemical localization of EGF in mouse kidney. Male and female adult Swiss Webster mice were fixed by perfusion with 4% paraformaldehyde or Zamboni's fixative, the kidneys were frozen, and serial sections were obtained. Rabbit EGF antiserum was used for the primary incubation and the avidin-biotin complex immunoperoxidase procedure was utilized for immunostaining. EGF was immunolocalized in the apical portion of the cells lining the thick ascending limb of Henle (TALH) and the distal convoluted tubule (DCT). The macula densa, in contrast, lacked EGF immunoreactivity. No sex differences were observed in the distribution pattern or intensity of immunostaining. Infusion of EGF into sheep renal artery has been reported to induce changes in urine flow and ionic composition. Immunolocalization of EGF in the TALH and DCT documented here supports a regulatory role for EGF in the function of the mouse distal nephron.     

The membrane fraction of homogenized rat kidney contains an enzyme that releases epidermal growth factor from the kidney membranes

High levels of epidermal growth factor (EGF) are excreted in the urine and high levels of mRNA for the EGF-precursor have been demonstrated in the kidney. The EGF-precursor is a membrane bound peptide in the kidney, but little is known about the renal processing of the precursor. The present study shows that the membrane fraction of homogenized rat kidney contains an enzyme that releases immuno and receptor reactive EGF from the kidney membranes when incubated at 37 degrees C. Gel filtration shows that the EGF reactivity released from the membranes is similar to the EGF reactivity in rat urine. The EGF releasing enzyme is inhibited by the serine proteinase inhibitor aprotinin and by low temperatures (4 degrees C). The pH optimum of the reaction is pH 7.5-8.0.     

Expression of epidermal growth factor in transgenic mice causes growth retardation

The epidermal growth factor (EGF) family of peptides signals through the erbB family of receptor tyrosine kinases and plays important roles in development and tumorigenesis. Both EGF and transforming growth factor (TGF)-alpha only bind to erbB1 and activate it. The precursor of EGF is distinct from that of TGF-alpha in having eight additional EGF-like repeats. We have recently shown that the EGF precursor without these repeats is biologically active and leads to hypospermatogenesis in transgenic mice. Here we present evidence that the growth of transgenic mice widely expressing this engineered EGF precursor is also stunted. These mice were consistently born at half the normal weight and reached almost 80% of normal weight at adulthood. The mechanism involved a reduction of serum insulin-like growth factor-binding protein-3. Chondrocyte development in the growth plate was affected, and osteoblasts accumulated in the endosteum and periosteum. Besides these novel findings on the in vivo effects of EGF on bone development, we observed no sign of tumor formation in our transgenic animals. In contrast to previous reports on TGF-alpha transgenic mice, we show that the biological functions of EGF and TGF-alpha are clearly distinct.     

Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor

Human, rat and mouse epidermal growth factors (EGF) bind to the same receptor on human placenta, but the binding characteristics differ. The apparent affinity constant (KA) for human EGF is higher (15 X 10(9) l/mol) than KA for rat EGF (10 X 10(9) l/mol). Mouse EGF binds with the lowest KA (5 X 10(9) l/mol). The pH optimum differs so that human and rat EGF bind with a pH optimum of 8.0, whereas mouse EGF binds with an optimum of pH 7.4. Half maximal dissociation is 130, 50 and 25 min for human, rat and mouse EGF, respectively. The structures of human, rat and mouse EGF differ somewhat. At least 11 of the first 24 residues differ. The N-terminal sequence of rat EGF is: Ala/Ser-Gly-X-Pro-Pro-Ser-Tyr-Asp-Gly-Tyr-X-Lys-Asp-Gly-Gly-Val-X-Met-Ty r-Val -Glu.     

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture.

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.     

Binding of epidermal growth factor in rat pancreatic acini

Specific, saturable EGF receptors were demonstrated in isolated rat pancreatic acini. Binding of EGF to these receptors was one-half maximal at 20 min and maximal at 120 min. Scatchard analyses revealed a single order of binding sites with a Kd of 4.90 nM. Following binding, EGF was rapidly internalized and converted to two acidic species. EGF did not alter either basal amylase release or the rate of [3H]phenylalanine incorporation into TCA-precipitable protein. The finding of high affinity EGF receptors in pancreatic acinar cells supports the hypothesis that EGF participates in the long-term regulation of pancreatic exocrine function.     

Central role of epidermal growth factor (EGF) receptor density in anchorage-independent growth of normal rat kidney cells

The gamma-carboxyglutamic acid (Gla) content of several variants of human prothrombin has been measured by using capillary electrophoresis and laser-induced fluorescence (CE-LIF). Both plasma-derived prothrombin and recombinant prothrombin contain ten residues of Gla per molecule of protein. In contrast, a variant of human prothrombin (containing the second kringle domain of bovine prothrombin) was separated into two populations that differed in their Gla content. Direct measurement of the Gla content showed an association with the presence or absence of the calcium-dependent conformational change that is required for prothombinase function. Thus, the CE-LIF assay is useful in determining the carboxylation status of recombinant proteins.     

Renal origin of rat urinary epidermal growth factor

The origin of rat urinary epidermal growth factor (EGF) has been investigated. Unilateral nephrectomy decreased the concentration, total output of EGF and EGF/creatinine ratio by approximately 50%, while the output of creatinine was unchanged. Removal of the submandibular glands and duodenal Brunner's glands, organs known to produce EGF, had no influence on the output of EGF in urine. Renal clearance of EGF exceeded that of creatinine, and after bilateral nephrectomy or bilateral ligation of the ureters, the concentration of creatinine in serum increased, while the concentration of EGF was below the detection limit of the assay. Renal production of EGF was confirmed by immunohistochemistry demonstrating EGF immunoreactivity in the afferent arteriole of the juxtaglomerular apparatus. EGF in the submandibular glands and in urine was found to differ with chromatofocusing and reverse-phase HPLC. At isoelectric focusing the pI of submandibular EGF was 4.8 and 5.4 while that of urinary EGF was 5.3 and 6.4.In conclusion, this study demonstrates that urinary EGF mainly originates from the kidneys and is localized to the renal juxtaglomerular apparatus.     

Expression of epidermal growth factor and platelet-derived growth factor receptors during cervical carcinogenesis

Summary Altered expression of epidermal growth factor receptor (EGFR) is common in a variety of epithelial malignancies, including cervical cancer. However, the prognostic significance of EGFR expression is controversial for cervical cancer. Platelet-derived growth factor receptor (PDGFR) expression status is unknown in cervical cancer. Our results demonstrated that expression of EGFR and PDGFR was greatly enhanced in vivo and in organotypic cultures of low-grade cervical dysplastic tissues, but levels were decreased in high-grade lesions. To our knowledge, this is the first report identifying the expression of PDGFR in human epithelium. When low-grade dysplastic organotypic culture tissues were induced to differentiate more completely, EGFR expression, but not PDGFR expression, was relocalized to the basal layer as seen in normal tissues. Differentiation also induced phosphorylation of EGFR but not PDGFR. Our results suggest a role for EGFR and PDGFR during the early stages of cervical carcinogensis, and demonstrate the facility of organotypic cultures to study the role of these growth factors in the development of cervical cancer.     

Expression of epidermal growth factor in salivary adenoid cystic carcinoma

This study used an immunohistochemical technique to assess the expression of epidermal growth factor (EGF) in 40 specimens of salivary adenoid cystic carcinoma (ACC), 7 specimens of labial glands adjacent to mucocele, and 5 specimens of normal submandibular glands. In normal submandibular glands, immunohistochemically detectable EGF was demonstrated in all ductal segments, including intercalated, striated, and excretory duct cells. No EGF positive staining was found in acinar compartments. including serous and mucous acinar cells. In degenerated labial glands adjacent to mucocele, no EGF staining was detected in the remaining acinar and ductal cells. In salivary ACCs, positive EGF immunostaining was observed in one of the 5 (20%) ACCs with a solid pattern and in 13 of the 35 (37.1%) ACCs with a tubular-cribriform pattern. The overall EGF expression rate in 40 salivary ACCs was 35%. Positive EGF staining was predominantly found in tubular structures in the tubular ACCs and in duct-like structures in large cribriform patterns or in the stroma of the cribriform ACCs. There was no significant correlation between EGF expression in salivary ACCs and any of the clinicopathological parameters including patient age and sex, cancer location, TNM status, clinical stage, histologic type, perivascular or perineural invasion, focal necrosis of tumor, and cellular atypia. We conclude that the duct segments of the normal submandibular gland are the sites of EGF synthesis and secretion. In degenerated labial glands adjacent to mucocele, EGF synthesis is completely inhibited. Furthermore, EGF is mainly biosynthesized in cells forming tubular or duct-like structures in tubular or cribriform salivary ACCs, and EGF may play a biologic role, particularly as a mitogen in salivary ACC growth.     

Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Characterization of hepatic epidermal growth factor receptors in the developing rat

Binding of 125I-labeled epidermal growth factor (EGF) was characterized in basolateral plasma membranes prepared from the livers of 21-day gestation fetuses, 14-day-old sucklings and adult Sprague-Dawley rats using a self-generating Percoll gradient method. The membrane preparations employed have been previously assayed in terms of plasma membrane protein yield, enrichment of various marker enzymes and sodium-dependent bile acid and amino acid transport. 125I-EGF binding was saturable and time and temperature dependent. Equilibrium analyses showed that the suckling period is characterized by a marked decrease in overall hepatic EGF binding capacity (460 +/- 50 fmol/mg protein) compared to either the fetal period (1290 +/- 160 fmol/mg) or to adults of either sex (males = 1540 +/- 230, females 1010 +/- 130 fmol/mg). Treatment of the suckling rat with parenteral EGF resulted in a 78% reduction in the observed binding capacity when assessed 2 h after growth factor administration. Comparison of binding affinities revealed no significant difference between the suckling and adult preparations (Kd = 0.40 +/- 0.03 vs. 0.39 +/- 0.02 nM, respectively); however, both preparations differed significantly from the fetal group which exhibited a decreased affinity of binding with a higher overall dissociation constant (Kd = 0.68 +/- 0.06 nM). Thus, it appears that major ontogenetic changes occur in the rat hepatic ligand/receptor system for epidermal growth factor. These changes are discussed in the context of transitional events in mammalian development such as birth and weaning.     

Expression of recombinant epidermal growth factor inE. coli

Epidermal growth factor (EGF) known as a urgastrone is a powerful mitogen with a wide variety of possibilities for medical usages. A mature EGF coding region was isolated from human prepro-EGF sequence by a conventional PCR and cloned into pQE vector in which the gene product was supposed to be expressed with 6×His tag for the subsequent purification. The recombinant mature EGF was expressed in M15[Rep4], anEscherichia coli host strain, in amount of 30–40% of total proteins present inE. coli extract by the addition of isopropylthio-β-galactopyranoside (IPTG). The recombinant EGF purified using a Ni²⁺-NTA affinity column chromatography was active in its ability to induce phosphorylation on tyrosine residues of several substrate proteins when murine NIH3T3 and human MRC-5 fibroblast cells were stimulated with it. This work may provide the basic technology and information for the production of recombinant EGF.