Stimulatory Effect Of Epidermal Growth Factor Research Articles

Epidermal growth factor synergistically enhances interleukin-8 production in human gingival fibroblasts stimulated with interleukin-1β

The chemokine interleukin-8 (IL-8) has been implicated in inflammatory diseases including periodontitis. In this study the effect of epidermal growth factor (EGF) on the production and regulation of interleukin-8 (IL-8) in human gingival fibroblasts challenged with interleukin-1beta (IL-1beta) was investigated. EGF, in comparison to the effect of IL-1beta, weakly increased the mRNA and protein expression of IL-8 in gingival fibroblasts. When the cells were treated simultaneously with EGF and IL-1beta, however, EGF synergistically enhanced the mRNA expression and production of IL-8. The stimulatory effect of EGF on IL-1beta-induced IL-8 production was completely abolished by the broad range tyrosine kinase inhibitor Herbimycin A, and considerably reduced by the receptor tyrosine kinase specific inhibitor PD 153035. Herbimycin A abolished IL-8 production induced by IL-1beta, whereas PD 153035 had no effect on the cytokine-induced IL-8 production. Furthermore, the p38 mitogen-activated protein (MAP) kinase inhibitor SB 203580 reduced IL-8 production induced by IL-1beta as well as by the combination of EGF and IL-1beta but had no effect on EGF-induced IL-8 production. In conclusion, the study demonstrates that EGF synergistically stimulates IL-8 production in the presence of IL-1beta and that tyrosine kinase(s) seem to be involved in the signalling pathway of IL-1beta and EGF. The synergistic interactions between EGF and IL-1beta on IL-8 production may play an essential role in the pathogenesis of the inflammatory disease periodontitis.

Acute regulation of OAT3-mediated estrone sulfate transport in isolated rabbit renal proximal tubules.

We investigated the regulation of organic anion transport driven by the organic anion transporter 3 (OAT3), a multispecific OAT localized at the basolateral membrane of the renal proximal tubule. PMA, a PKC activator, inhibited uptake of estrone sulfate (ES), a prototypic substrate for OAT3, in a dose- and time-dependent manner. This inhibition was reduced by 100 nM bisindoylmaleimide I (BIM), a specific PKC inhibitor. The alpha(1)-adrenergic receptor agonist phenylephrine also inhibited ES uptake, and this effect was reduced by BIM. These results suggest that PKC activation downregulates OAT3-mediated organic anion transport. In contrast, epidermal growth factor (EGF) increased ES uptake following activation of MAPK. Exposure to PGE(2) or dibutyryl (db)-cAMP also enhanced ES uptake. Stimulation produced by PGE(2) and db-cAMP was prevented by the PKA inhibitor H-89, indicating that this stimulation required PKA activation. In addition, inhibition of cyclooxygenase 1 (COX1) (but not COX2) inhibited ES uptake. Furthermore, the stimulatory effect of EGF was eliminated by inhibition of either COX1 or PKA. These data suggest that EGF stimulates ES uptake by a process in which MAPK activation results in increased PGE(2) production that, in turn, activates PKA and subsequently stimulates ES uptake. Interestingly, EGF did not induce upregulation immediately following phenylephrine-induced downregulation; and phenylephrine did not induce downregulation immediately after EGF-induced upregulation. These data are the first to show the regulatory response of organic anion transport driven by OAT3 in intact renal proximal tubules.

Epidermal Growth Factor Potentiates Cholecystokinin/Gastrin Receptor-mediated Ca2+ Release by Activation of Mitogen-activated Protein Kinases

Small differences in amplitude, duration, and temporal patterns of change in the concentration of free intracellular Ca2+ ([Ca2+](i)) can profoundly affect cell physiology, altering programs of gene expression, cell proliferation, secretory activity, and cell survival. We report a novel mechanism for amplitude modulation of [Ca2+](i) that involves mitogen-activated protein kinase (MAPK). We show that epidermal growth factor (EGF) potentiates gastrin-(1-17) (G17)-stimulated Ca2+ release from intracellular Ca2+ stores through a MAPK-dependent pathway. G17 activation of the cholecystokinin/gastrin receptor (CCK(2)R), a G protein-coupled receptor, stimulates release of Ca2+ from inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Pretreating rat intestinal epithelial cells expressing CCK(2)R with EGF increased the level of G17-stimulated Ca2+ release from intracellular stores. The stimulatory effect of EGF on CCK(2)R-mediated Ca2+ release requires activation of the MAPK kinase (MEK)1,2/extracellular signal-regulated kinase (ERK)1,2 pathway. Inhibition of the MEK1,2/ERK1,2 pathway by either serum starvation or treatment with selective MEK1,2 inhibitors PD98059 and U0126 or expression of a dominant-negative mutant form of MEK1 decreased the amplitude of the G17-stimulated Ca2+ release response. Activation of the MEK1,2/ERK1,2 pathway either by pretreating cells with EGF or by expression of constitutively active K-ras (K-rasV12G) or MEK1 (MEK1*) increased the amplitude of G17-stimulated Ca2+ release. Although EGF, MEK1*, and K-rasV12G activated the MEK1,2/ERK1,2 pathway, they did not increase [Ca2+](i) in the absence of G17. These data demonstrate that the activation state of the MEK1,2/ERK1,2 pathway can modulate the amplitude of the CCK(2)R-mediated Ca2+ release response and identify a novel mechanism for cross-talk between EGF receptor- and CCK(2)R-regulated signaling pathways.

Epidermal growth factor inhibits 14C-alpha-methyl-D-glucopyranoside uptake in renal proximal tubule cells: involvement of PLC/PKC, p44/42 MAPK, and cPLA2.

The effect of EGF on (14)C-alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signaling pathways were examined in primary cultured rabbit renal proximal tubule cells (PTCs). Epidermal growth factor (EGF) (50 ng/ml) was found to inhibit alpha-MG uptake, a distinctive proximal tubule marker. The EGF effect was blocked by AG1478 (an EGF receptor antagonist) or genistein and herbimycin (tyrosine kinase inhibitors), respectively. In addition, the EGF-induced inhibition of alpha-MG uptake was blocked by neomycin and U73122 (phospholipase C inhibitors) as well as staurosporine, H-7, and bisindolylmaleimide I (protein kinase C inhibitors). EGF was also observed to increase inositol phosphate formation. Furthermore, both the EGF-induced inhibition of alpha-MG uptake and increase of arachidonic acid (AA) release were blocked by AACOCF(3) (a cytosolic phospholipase A(2) inhibitor), indomethacin (a cyclooxygenase inhibitor), and econazole (a cytochrome P-450 epoxygenase inhibitor). We examined the involvement of mitogen-activated protein kinases (MAPKs) in mediating the effect of EGF on alpha-MG uptake. Indeed, EGF increased phosphorylation of p44/p42 MAPK and the EGF-induced inhibition of alpha-MG uptake as well as the stimulatory effect of EGF on AA release was blocked by PD 98059 (a p44/42 MAPK inhibitor), suggesting a causal relationship. However, inhibitors of PKC also prevented the EGF-induced increase of AA release. In conclusion, EGF partially inhibited alpha-MG uptake via PLC/PKC, p44/42 MAPK, and PLA(2) signaling pathways.

Epidermal growth factor and transforming growth factor-alpha stimulate the proliferation of mouse uterine stromal cells.

Growth factors produced in the uterine endometrium are considered to be involved in the proliferation of the mouse uterine stromal cells induced by estradiol-17beta (E(2)) and progesterone (P). The effect of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), one of EGF-related growth factors, on the proliferation of mouse uterine stromal cells was studied in a serum-free culture. The growth of the uterine stromal cells was measured by MTT assay. EGF was found to increase the number of uterine stromal cells in a dose-dependent manner. The DNA-replicating cells were investigated using the immunocytochemical detection of bromodeoxyuridine (BrdU)-labeled cells. EGF and TGF-alpha increased the percentage of BrdU-labeled cells in a dose-dependent manner. Administration of the combination of E(2) (10(-9) M) and P (10(-7) M) for 2 days increased the percentage of BrdU-labeled cells 2.3-fold. The stimulatory effect of EGF, TGF-alpha and the combination of E(2) and P on DNA replication in the uterine stromal cells was repressed by RG-13022 (10(-5) M, the inhibitor of the EGF receptor tyrosine kinase). RT-PCR analysis of EGF-receptor-, TGF-alpha-, and EGF-mRNA was carried out in the cultured uterine stromal cells, and revealed the expression of those mRNAs. These data supported the hypothesis that uterine endometrial stromal growth induced by sex steroids required the EGF family of ligands such as EGF and TGF-alpha, both produced in the stromal cells, acting for DNA synthesis through EGF receptors.

Epidermal growth factor upregulates beta-adrenergic receptor signaling in a human salivary cell line.

The effects of epidermal growth factor (EGF) on the beta-adrenergic receptor-coupled adenylyl cyclase system were studied in a human salivary cell line (HSY). The beta-adrenergic agonist isoproterenol (10(-5) M) stimulated adenylyl cyclase activity by approximately 2-fold, and the isoproterenol response was increased 1.8-fold after prolonged (48 h) exposure to EGF (5 x 10(-10) M). In contrast, enzyme activation via stimulatory prostaglandin receptors and by agents acting on nonreceptor components of the adenylyl cyclase system was not enhanced by EGF. beta-Adrenergic receptor density, assessed by binding of the beta-adrenergic receptor antagonist (-)-[(125)I]iodopindolol, was increased threefold after EGF treatment. Competition binding studies with unlabeled antagonists selective for beta(1)- and beta(2)-adrenergic receptor subtypes indicated that the increase in (-)-[(125)I]iodopindolol binding sites induced by EGF reflected an increased number of beta(2)-adrenergic receptors. Likewise, Northern blot analysis of RNA from EGF-treated cells revealed selective induction of beta(2)-adrenergic receptor mRNA, which was blocked by the RNA synthesis inhibitor actinomycin D. The increase in beta-adrenergic receptor density produced by EGF was unaltered after phorbol ester-induced downregulation of protein kinase C (PKC). Enhancement of isoproterenol-responsive adenylyl cyclase activity and phosphorylation of mitogen-activated protein kinase (MAPK) by EGF were both blocked by the MAPK pathway inhibitor PD-98059. The results suggest that in HSY cells EGF enhances beta-adrenergic responsiveness by upregulating beta(2)-adrenergic receptor expression at the transcriptional level. Moreover, the stimulatory effect of EGF on beta(2)-adrenergic receptor signaling appears to be mediated by the MAPK pathway and independent of PKC activation.

Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D 3 is mediated by mutual modulation of receptor expression

The human colon adenocarcinoma-derived cell line Caco-2 was used as a model system to study the interaction of epidermal growth factor (EGF) and 1,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) in control of colorectal cancer cell growth. The mitogenic stimulus of EGF was rapidly transduced via apical and basal membrane receptors alike into elevation of c- myc expression, causing a shift of Caco-2 cells from the G 0/G 1 into the S phase of the cell cycle. The stimulatory effect of EGF on cell division was effectively counteracted by 1,25(OH) 2D 3: the presence of the steroid hormone prevents the negative effect of EGF on vitamin D receptor abundance and concurrently minimises ligand-occupied EGF receptor numbers on both sides of Caco-2 cell monolayers. Our data suggest that EGF and 1,25-(OH) 2D 3 actions on mutual receptor levels represent a specific feature of the potent antimitogenic effect of the steroid hormone on colon cancer cells.

PKC activation is required by EGF-stimulated Na(+)-H+ exchanger in human pleural mesothelial cells.

Epidermal growth factor (EGF) stimulates the Na(+)-H+ exchanger, leading to enhanced cell proliferation. In human pleural mesothelial cells (PMCs), the intracellular signaling mechanism mediating the EGF-induced stimulation of the Na(+)-H+ exchanger has not yet been identified. Using a pH-sensitive fluorescent probe, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, to measure changes in intracellular pH (pHi), we found that 1) EGF and 12-O-tetradecanoylphorbol 13-acetate (TPA; a phorbol ester) both stimulate the ethylisopropyl amiloride-sensitive Na(+)-H+ exchanger; 2) TPA-induced alkalosis can be blocked by protein kinase C (PKC) inhibitors (chelerythrine and staurosporine) or by PKC down-regulation, indicating that PKC activation is involved in the stimulation of the Na(+)-H+ exchanger. However, TPA-induced alkalosis is not blocked by tyrosine kinase inhibitors; and 3) the stimulatory effect of EGF on the Na(+)-H+ exchanger acts via stimulation of tyrosine kinase-receptor activity because it is inhibited by tyrosine kinase inhibitors (genistein, lavendustin A, and herbimycin A). It also involves PKC activation because EGF-induced alkalosis was blocked by PKC inhibitors. These results suggest that PKC activation is one of the downstream signals for EGF-induced activation of the Na(+)-H+ exchanger in primary cultures of human pleural mesothelial cells.

Stimulation of the preprothyrotropin-releasing hormone gene by epidermal growth factor.

Regulation of the expression of the prepro-TRH (ppTRH) gene by epidermal growth factor (EGF) was investigated. The i.p. injection of EGF significantly stimulated hypothalamic ppTRH messenger RNA levels in rats. To clarify whether this stimulatory effect of EGF could be exerted at the level of gene transcription, the 5'-flanking region (-1893/+127) of the mouse ppTRH gene fused to a luciferase reporter gene was transiently transfected into pituitary GH4C1 cells, and the effect of EGF on gene transcription was measured by a luciferase assay. EGF stimulated ppTRH gene promoter activity in a time- and dose-dependent manner. Deletion analysis revealed that two different regions of the promoter, between -254 and -218 [EGF response element-1 (EGFRE1)] and between -130 and -84 (EGFRE2) were required for full stimulation by EGF. The two EGFREs possessed putative binding sequences for the transcription factor Sp1, and they functioned cooperatively in heterologous promoters. Nuclear extracts from GH4C1 cells specifically bound those two EGFREs in gel retardation assays. Two protein-DNA complexes were found on EGFRE1, whereas four complexes were observed on EGFRE2. Although the binding of nuclear extracts to EGFRE1 was competed for by the consensus Sp1 binding sequence, the complexes on EGFRE1 were not supershifted by an Sp1 antibody. Formation of the slower migrating protein complex on EGFRE1 was prevented by EDTA, suggesting that one of the EGFRE1-binding proteins might be an Sp1-related zinc finger protein. Competition and supershift experiments demonstrated that the EGFRE2-binding protein showing that the slowest migration possessed a characteristic similar to that of Sp1. Selective mutations of the Sp1-binding site in EGFRE2 markedly diminished the EGF-induced stimulation. These results suggest that EGF may function as a positive regulator of ppTRH gene expression, and that the stimulatory effect may be mediated through a cooperative interaction between Sp1 or Sp1-related proteins and additional factors that bind to two separate DNA regions.

Synergistic Effect of Substance P with Epidermal Growth Factor on Epithelial Migration in Rabbit Cornea

In order to investigate the role of neural regulation in corneal epithelial healing, we examined the effect of substance P (SP) on corneal epithelial migration using an organ culture system of rabbit corneas. We investigated the synergistic effects of SP with (1) growth factors: epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-β(TGF-β); (2) extracellular matrix proteins: fibronectin, vitronectin, laminin, and collagen type IV; and (3) cytokines: interleukin-1α (IL-1α), IL-1β, and interleukin-6 (IL-6). Rabbit corneal blocks were cultured in the absence or presence of various reagents for 24 hr. The corneal blocks were then fixed, dehydrated, embedded in paraffin and stained by hematoxylin-eosin, and the length of the path of epithelial migration was measured. The addition of SP alone, at concentrations up to 50 μg ml−1, did not affect epithelial migration. EGF, fibronectin, vitronectin, collagen type IV, and IL-6 stimulated epithelial migration, but bFGF, TGF-β, laminin, IL-1α, and IL-1βdid not. The stimulatory effect of EGF on the epithelial migration was enhanced by the presence of SP. This synergistic effect of SP and EGF on corneal epithelial migration was abolished by the addition of an SP antagonist or enkephalinase. Other neurotransmitters (vasoactive intestinal peptide, calcitonin gene-related peptide, acetylcholine chloride, norepinephrine, serotonin) and tachykinins (neurokinin A, neurokinin B, kassinin, eledoisin, physalaemin) were examined, but none exhibited a synergistic effect with EGF. Interestingly, EGF alone stimulated the incorporation of3H-thymidine into corneal epithelial cells, but the addition of SP with EGF did not enhance this effect. These results demonstrate that SP enhanced the EGF stimulation of corneal epithelial migration in vitro in a specific manner, suggesting a possible role of SP as a modulator of epithelial wound healing.

Regulation of transforming growth factor beta 1 messenger ribonucleic acid expression in porcine thyroid follicles in vitro by growth factors, iodine, or delta-iodolactone.

Transforming growth factor beta 1 (TGF beta 1) is an autocrine growth factor for thyrocytes and is supposed to be the mediator of iodine-induced growth inhibition of thyroid epithelial cells, but this is still controversial. We further investigated this hypothesis using intact porcine thyroid follicles ex vivo in a three-dimensional culture system. In this culture system it has been shown previously that both iodide as well as delta-iodolactone, the putative iodocompound mediating thyroid cell proliferation, inhibit growth of these follicles. We measured the amount of TGF beta 1 mRNA expression in these follicles after treatment either with thyrotropin (TSH), epidermal growth factor (EGF), or transforming growth factor alpha (TGF alpha) for growth stimulation or with inorganic iodine or delta-iodolactone in concentrations known to inhibit growth. TGF beta 1-mRNA was detected by Northern blot analysis. The known major transcript of 2.5 kb was detected in a steady state level up to 48 hours in untreated thyroid follicles. EGF and TGF alpha (5 ng/mL each) enhanced TGF beta 1 mRNA about threefold within 4 and 8 hours. This increase of TGF beta 1 mRNA was slightly decreased by simultaneous incubation with delta-iodolactone (1 microM) or iodide (40 microM KI). In contrast, both TSH (1 mU/mL) and forskolin (16 microM) decreased TGF beta 1 mRNA expression to about 70%, and this effect was abolished when follicles were pretreated with iodide (40 microM KI) in a concentration known to inhibit TSH action on cyclic adenosine monophosphate (cAMP) formation and proliferation. Iodide or delta-iodolactone alone had no significant effect on basal TGF beta 1 mRNA expression. We conclude that the growth inhibitory effect of iodide as well as of delta-iodolactone is not mediated through TGF beta 1 in intact porcine thyroid follicles ex vivo. The stimulatory effect of EGF and TGF alpha on TGF beta 1 expression might be related to extracellular matrix modulation during proliferation.

Effect of Sialoadenectomy and Epidermal Growth Factor Administration on Liver Regeneration After Partial Hepatectomy

Epidermal growth factor (EGF), a mitogen in vitro for hepatocytes, produces in various cell lines changes similar to those observed very rapidly in hepatocytes after partial hepatectomy (PH). These changes include ion movements, membrane hyperpolarization and proto-oncogene expression. A stimulatory effect of EGF on liver regeneration can therefore tentatively be associated with the events occurring within the first 3 hours after a PH, sometimes referred to as the "priming phase." To assess this hypothesis, we examined in Wistar rats the effect of EGF deprivation produced by sialoadenectomy (SX) performed before or after a PH of 70%. SX at the time of PH significantly decreased the 3H-thymidine uptake in the DNA 24 hours later (147 +/- 14 DPM per microgram of DNA, mean +/- SE) compared with a simple PH (322 +/- 16; P < .01), but also compared with results obtained when PH is combined with a sham sialoadenectomy (SSX) or in rats pair-fed with the sialoadenectomized rats. This incomplete inhibition was confirmed by a decreased rise in thymidine kinase (TK) activity and by reduced proliferating cell nuclear antigen (PCNA) labeling and mitotic indices 30 hours after PH. By contrast, SX did not inhibit the early expression of c-jun and c-fos, or of c-myc, 30 or 120 minutes after PH, respectively. A reduction of DNA synthesis was also obtained when SX was performed 3 hours after PH (127 +/- 15 DPM per microgram of DNA vs. 350 +/- 21 in SSX; P < .001) but not when SX was delayed until the 6th or the 17th hour after PH. It was sufficient to administer EGF (40 microg) from the third to the ninth hour to correct the reduction of [3H]thymidine uptake in rats sialoadenectomized before PH. These results indicate that the diminished EGF availability following SX decreases or at least delays liver regeneration, and that the effect of EGF on liver regeneration does not seem related to the early changes of proto-oncogene expression, but rather to events occuring later, at the time of reported internalization and binding of EGF to its nuclear receptors.

Hypoxia and proliferation are primarily responsible for induction of lactate dehydrogenase activity in cultured cells.

In vitro to in vivo extrapolations require cellular models that closely mimic cells in vivo. Typically, cultured cells revert to glycolysis, have increased lactate dehydrogenase (LDH) activity, have decreased oxidative metabolism, and dedifferentiate. This study examined the role of hypoxia and proliferation in the regulation of LDH activity, and the temporal relationships among induction of glycolysis, LDH activity, and proliferation in primary cultures of rabbit renal proximal tubular cells (RPTC). LDH activity in RPTC grown under standard conditions (STILL) did not increase during the first 3 d of culture, but increased 18-fold by d 7. LDH activity in RPTC grown under conditions of increased oxygen supply (SHAKE) did not increase during the first 3 d of culture, but increased 5.5-fold by d 7. Hypoxia and proliferation were responsible for 73% and 27%, respectively, of the increase in LDH activity in STILL RPTC. Hypoxia had no effect on RPTC proliferation. Neither medium glucose nor insulin concentrations had any effect on LDH activity in SHAKE RPTC. Supplementation of the culture medium with ribose 5-phosphate or ribose diminished the increase in LDH activity in SHAKE RPTC to 62% and 52% of controls but had no effect on monolayer DNA content. Two-day treatment of confluent SHAKE RPTC with epidermal growth factor (EGF) resulted in 1.6-, 1.4-, and 1.9-fold increases in LDH and G6PDH activities and monolayer DNA content, respectively. The stimulatory effect of EGF on LDH and G6PDH activities, but not monolayer DNA content, was abolished by ribose or ribose 5-phosphate. In contrast, transforming growth factor-beta 1 (TGF-beta 1) treatment stimulated lactate production but had no effect on LDH and G6PDH activities, or proliferation of SHAKE RPTC. These results show that (1) both hypoxia and proliferation are primarily responsible for the induction of LDH activity in cultured cells, (2) LDH activity is not an accurate indicator of glycolysis, (3) induction of LDH activity is secondary and due, in part, to the induction of the pentose phosphate pathway, (4) EGF stimulates glycolysis, LDH activity, and proliferation, and (5) TGF-beta 1 stimulates glycolysis but has no effect on LDH activity or proliferation.

Presence and regulation of Raf-1-K (Kinase), MAPK-K, MAP-K, and S6-K in rat nephron segments.

Renal nephron segments are heterogeneous, and receptors for endothelin (ET)-1, ET-3, Angiotensin II (AII), epidermal growth factor (EGF), and insulin-like growth factor I distribute differently along the nephron segments. Recently, growth factors and vasoactive substances are reported to stimulate mitogen-activated protein kinase (MAP-K). In this study, we showed that mRNA and proteins of MEK-K, Raf-1-K, MAPK-K, MAP-K (p42 and p44), and S6-K are expressed ubiquitously in intact nephron segment. We demonstrated that four tiers of a cascade composed of the Raf-1-K, MAP-K, MAP-K, and S6-K are stimulated by ET-1 and ET-3 in rat intact glomeruli (Glm) via primarily B-type ET receptors and PKC. The stimulatory effect of EGF and IGF-I to MAP-K activity is inhibited by a tyrosine kinase inhibitor in Glm. IGF-I significantly stimulates MAP-K activity and EGF and All moderately stimulate MAP-K activity in the proximal convoluted tubule (PCT). EGF significantly increased MAP-K cascades and ET-1 and ET-3 slightly increased MAP-K cascades in the medullary thick ascending limb (MTAL). EGF significantly stimulated MAP-K cascades, and ET-1 and ET-3 moderately stimulate MAP-K cascades in the outer medullary collecting duct (OMCD) and the inner medullary collecting duct (IMCD). MAPK-K and S6-K are similarly stimulated by these agonists in each segment. This study shows that MAP-K cascades are expressed in every nephron segment. ET-1, ET-3, All, EGF, and IGF-I stimulate MAP-K cascades heterogeneously along the nephron segment. It was concluded that MAP-K cascades play an important role in the regulation of renal function.

Stimulation of proximal convoluted tubule phosphate transport by epidermal growth factor: signal transduction.

The present study investigated the signal-transduction pathway responsible for the epidermal growth factor (EGF) stimulation of phosphate transport (JPhos) in the rabbit proximal convoluted tubule (PCT). Genistein, 10(-4) M, bath and lumen, an inhibitor of EGF receptor tyrosine kinase activity, blocked the EGF effect on JPhos, consistent with a role for tyrosine kinase in the signal-transduction pathway. Both staurosporine (5 x 10(-8) M) and calphostin C (10(-8) M), inhibitors of protein kinase C, blocked the EGF stimulation of JPhos, indicating that protein kinase C is involved in EGF signaling. Intracellular calcium (Ca2+i) concentrations were measured in perfused tubules using fura PE3 to determine whether changes in Ca2+i were also part of the signaling pathway. After addition of 3 nM EGF, there was no change in Ca2+i, suggesting that stimulation of protein kinase C is not from phosphatidylinositol hydrolysis by phospholipase C-gamma. To determine whether phospholipase A2 (PLA2) is involved, the inhibitor mepacrine was used. Mepacrine (5 x 10(-5) M) had no direct effect on PCT transport but blocked the stimulatory effect of EGF on JPhos. PLA2 activity, assessed as free arachidonic acid release from proximal tubules in suspension, increased by 18.8% with 3 nM EGF. Thus the stimulation of JPhos by EGF is mediated via a signal-transduction pathway involving tyrosine kinase, protein kinase C, and PLA2.

EGF stimulates polyamine uptake in Caco-2 cells

We investigated the effects of epidermal growth factor (EGF) on polyamine uptake in Caco-2 cell monolayers. Cells were grown until confluence (day 7) or until differentiation (day 14). Polyamine uptake into Caco-2 cells was stimulated by EGF in a dose-dependent manner. Both basal and EGF-stimulated uptake rates were higher in 7- than in 14-day-old Caco-2 cells. Stimulation with EGF resulted in a significant increase in Vmax and an increased affinity for putrescine and spermine. Polyamine uptake was not inhibited when protein synthesis was blocked by cycloheximide, implying that no additional protein synthesis occured for stimulatory effect of EGF on polyamine uptake. The tyrosine kinase inhibitor, genistein, completely inhibited EGF-stimulated polyamine uptake, indicating that tyrosine phosphorylation plays a role in EGF-stimulated polyamine uptake in Caco-2 cells. The effect of EGF on polyamine uptake into Caco-2 cells, therefore, could be due to translocation of intracellular proteins which were not previously incorporated into the membrane, or direct alteration of polyamine transporter.

Regulation of 17 beta-hydroxysteroid dehydrogenase type 1 by epidermal growth factor and transforming growth factor-alpha in choriocarcinoma cells.

17 beta-Hydroxysteroid dehydrogenase type 1 (17HSD type 1) is a steroidogenic enzyme that catalyzes the reversible interconversion of estrone and estradiol. In this study, we investigated the roles of epidermal growth factor (EGF) and tumor growth factor-alpha (TGF alpha) in the regulation of 17HSD type 1 gene expression and catalytic activity in cultured JAR, JEG-3, and BeWo choriocarcinoma cells. EGF and TGF alpha increased 17HSD type 1 protein concentrations in JAR and JEG-3 cells, as measured by time-resolved immunofluorometric assay, and 17HSD catalytic activity, as determined by production of estradiol from estrone. These increases were accompanied by parallel increases in concentrations of the 1.3-kilobase messenger RNA coding for 17HSD type 1 in these cells. EGF receptor tyrosine kinase activity inhibitors, tyrphostins, inhibited EGF action in JEG-3 cells, indicating that tyrosine kinase activity is needed for stimulation of the 17HSD type 1 gene. Treatment with 8-bromo-cAMP or phorbol 12-myristate 13-acetate increased the amount of 17HSD type 1 protein. Furthermore, phorbol 12-myristate 13-acetate potentiated the stimulatory effect of EGF. These results suggest that EGF and/or TGF alpha may play an important role in 17HSD type 1 regulation and, consequently, in estrogen production in the human placenta.

Effect of epidermal growth factor on mouse sperm acrosome reaction induced by zona pellucida.

The effect of epidermal growth factor (EGF) on the acid-solubilized zona pellucida (ZP)-induced acrosome reaction was investigated in mouse sperm. Mouse epididymal sperm were capacitated in modified Krebs-Ringer bicarbonate buffer (m-KRB) for 120 min and further treated with acid-solubilized ZP(4 zona/microliters) for an additional 60 min to induce the acrosome reaction. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. The acrosome reacted sperm featured the acrosome reacted (AR) pattern, which demonstrates the lack of fluorescence on the head and bright midpiece. EGF caused an early increase in the AR pattern in response to the acid-solubilized ZP in a dose-dependent manner. The EGF-dependent stimulation of the ZP-induced acrosome reaction was inhibited by an inhibitor of protein tyrosine kinases, genistein or activators of Ca++ and phospholipid-dependent protein kinase (protein kinase C). Furthermore, the stimulatory effect of EGF was not attenuated when sperm were capacitated in the presence of islet-activating protein, an inactivator of inhibitory guanine nucleotide-binding regulatory protein (Gi protein). EGF stimulates the ZP-induced acrosome reaction in a manner that is independent of the Gi protein. The EGF action is regulated by protein tyrosine kinase and protein kinase C.

Effects of Epidermal Growth Factor on Invasiveness through the Extracellular Matrix in High‐ and Low‐metastatic Clones of RCT Sarcoma in vitro

We investigated the invasiveness of tumor cells through the extracellular matrix and the influence of epidermal growth factor (EGF) on tumor cell invasion using in vitro systems in high‐[RCT(+)] and low‐metastatic [RCT(–)] clones established from poorly differentiated murine RCT sarcoma in C3H/He mice. In the invasion assay using a filter coated with reconstituted basement membrane (Matrigel) in a Boyden chamber, RCT(+) cells were more invasive than RCT(‐) cells. The attachment of RCT(+) cells to extracellular matrix components and the degradation of type IV collagen by the cells were significantly greater than with RCT(–) cells. However, there was no significant difference in the migration of cells to the extracellular matrix components between cultured RCT(+) and RCT(‐) cells. These findings suggested that the different invasiveness of these clone cells was associated with the difference in the ability of attachment to and degradation of the matrix. The level of laminin receptor expression in RCT(+) cells was about four‐fold that in RCT(–) cells and laminin stimulated the type IV collagenolytic activity of RCT(+) cells, suggesting that RCT(+) cell attachment to laminin via laminin receptor on the cell surface induced the production of type IV collagenase by the tumor cells. EGF did not affect the invasiveness of RCT(–) cells. In RCT(+) cells, EGF stimulated the invasiveness through Matrigel, the attachment to extracellular matrix components and the degradation of type IV collagen through high‐affinity EGF receptors (EGFR), with Kt of pM order, while the migration to the matrix was not influenced by EGF. These findings suggest that the stimulatory effect of EGF on invasion is related to the acceleration of cell adhesion, and the degradative cascade of the extracellular matrix and high‐affinity EGFRs play an important role in the effect of EGF on in vitro invasiveness in this tumor.

Epidermal growth factor receptor expression in three different human endometrial cancer cell lines.

The aim of the current study was to establish the characteristics of the epidermal growth factor (EGF) receptor in endometrial cell lines to determine the possible relation of EGF to endometrial cancer. Three different cell lines were used: RL95-2 (derived from a moderately differentiated adenosquamous carcinoma), HEC-I-A (from a moderately differentiated adenocarcinoma), and KLE (from a poorly differentiated adenocarcinoma). The binding of (125-I) EGF to these cell lines and the stimulatory effect of EGF on (3H) thymidine incorporation into DNA were examined. EGF receptor was present in all three cell lines. The binding of 125-I-labeled EGF was saturable and of high affinity. Scatchard analysis of the competitive binding data for KLE, HEC-I-A, and RL95-2 revealed linear plots, indicating a single class of binding sites with almost identical equilibrium dissociation constants (0.34 nM, 0.23 nM, and 0.20 nM, respectively). Other peptides, such as insulin-like growth factor (IGF) I and II and insulin, did not compete for the receptor. RL95-2 cells bound significantly more EGF (P < 0.005) than did the HEC-I-A and KLE cell lines. EGF increased 3H-thymidine incorporation in all three cell lines. Because EGF receptors are expressed by all three cell lines at markedly different levels and because EGF stimulates 3H-thymidine incorporation into DNA in the three cell lines, the current study suggests that EGF may play a role in the promotion of growth endometrial adenocarcinoma.