Increased Epidermal Growth Factor Receptor Research Articles

Gintonin-Induced Wound-Healing-Related Responses Involve Epidermal-Growth-Factor-like Effects in Keratinocytes.

Epidermal growth factor (EGF) receptor activation and related downstream signaling pathways are known to be one of the major mechanisms of the proliferation and migration of keratinocytes. The heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors and stimulates keratinocyte proliferation and migration. Gintonin, a novel ginseng compound, is a lysophosphatidic acid (LPA) receptor ligand. Gintonin has skin-wound-healing effects. However, the underlying mechanisms for these gintonin actions remain unclear. In this study, we aimed to elucidate the involvement of EGFRs in gintonin-induced wound repair in HaCaT keratinocytes. In this study, a water-soluble tetrazolium salt-based assay, a modified Boyden chamber migration assay, and immunoblotting were performed. Gintonin increased EGF receptor activation in HaCaT cells. However, the gintonin-induced phosphorylation of the EGF receptor was markedly reduced via treatment with the LPA inhibitor Ki16425 or the EGF receptor inhibitor erlotinib. Gintonin-enhanced proliferation and migration were blocked by the EGF receptor inhibitors (erlotinib and AG1478). Additionally, gintonin stimulated the expression and release of HB-EGF in HaCaT cells. EGF receptor inhibitors blocked gintonin-enhanced HB-EGF expression. These results indicate that the wound-healing effects of gintonin are closely related to the collaboration between EGF receptor activation and HB-EGF release-mediated downstream signaling pathways.

Synergistic effect between LH and estrogen in the acceleration of cumulus expansion via GPR30 and EGFR pathways.

The estrogen membrane receptor GPR30 (also known as G-protein coupled receptor 30) has recently been shown to be involved in the regulation of oocyte maturation and cumulus expansion. However, whether GPR30 expression is regulated by gonadotropin stimulation and how it participates in the regulation of the maturation process is still not clear. In this study, we explored the mechanism underlying the synergy between luteinizing hormone and 17β-estradiol (17β-E2) to improve the epidermal growth factor (EGF) response in cumulus oocyte complexes (COCs) during oocyte maturation in mice. The expression and distribution of GPR30, EGFR, and EGF-like growth factors were examined by real-time quantitative PCR, western blot, and immunofluorescence staining. Lyso-Tracker Red labeling was performed to detect the lysosomal activity in follicle granular cells (FGCs). Cumulus expansion of COCs was evaluated after in vitro maturation for 16 h. We found that EGF-like growth factors transmit LH signals to increase GRP30 levels by inhibiting protein degradation in lysosomes. Meanwhile, 17β-E2 stimulates the GPR30 signaling pathway to increase EGF receptor levels, enhancing the response ability of EGF signaling in COCs and thus promoting cumulus expansion. In conclusion, our study reveals the synergistic mechanism between LH and estrogen in the regulation of cumulus expansion during oocyte maturation process.

A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth

BackgroundHomeobox A5 (HOXA5), a member of the HOX family, plays an important role in tumor development and morphogenesis, although opposite effects on tumorigenesis have been observed, depending on the tissue type. In this study, we aimed to investigate the role of a novel transcript from the HOXA6-HOXA5 locus in colon cancer tumorigenesis.MethodsHuman colon cancer cell lines were analyzed using next generation sequencing-based targeted mRNA capture. The effects of overexpression and silencing of HOXA5 transcripts were evaluated in vitro and using a xenograft nude mouse model.ResultsWe identified three novel transcripts (HOXA5 short, long 1, and long 2) transcribed from the HOXA6-HOXA5 locus in HCT116 colon cancer cells using next generation sequencing-based targeted mRNA capture. Knockdown of HOXA5 long 1 and long 2 transcripts did not affect cell growth, while selective silencing of HOXA5 short RNA inhibited cell growth independent of HOXA5 expression. Stable overexpression of HOXA5 short RNA promoted proliferation and migration of colon cancer cell lines HCT116, DLD1, and HT-29 and accelerated tumor growth in the xenograft mouse model. In vitro translation assays suggested HOXA5 short RNA was a functional long non-coding RNA (lncRNA). Consistent with these observations, expression of HOXA5 short RNA was upregulated in advanced colon cancer tissues. Ingenuity Pathway Analysis of differentially expressed genes between HOXA5 short RNA overexpressed and silenced HCT116 cells revealed that HOXA5 short RNA preferentially modified expression of epidermal growth factor (EGF) signal-related genes. Western blot analysis demonstrated that stable overexpression of HOXA5 short RNA increased EGF receptor levels and facilitated its phosphorylation in both HCT116 cells and xenograft tumors.ConclusionsOur results suggested that HOXA5 short RNA, a novel lncRNA, may play a crucial role in colon tumor growth through activation of EGF signaling.

Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation.

We previously demonstrated that non-toxic doses of Celecoxib induced the immediate phosphorylation of Erk1-2 in colon tumor associated fibroblasts (TAFs), increasing their responsiveness to epidermal growth factor (EGF). We have now identified two concomitant mechanisms explaining the EGF-Celecoxib cooperation. We found that a 24-48h Celecoxib priming increased EGF receptor (EGFR) mRNA and protein levels in colon TAFs, promoting EGF binding and internalization. Celecoxib-primed TAFs showed a reduced EGFR degradation after EGF challenge. This delay corresponded to a deferred dissociation of EEA1 from EGFR positive endosomes and the accumulation of Rab7, pro Cathepsin-D and SQSTM1/p62, suggesting a shared bottleneck in the pathways of late-endosomes/autophagosomes maturation. Celecoxib modulated the levels of target proteins similarly to the inhibitors of endosome/lysosome acidification Bafilomycin-A1 and NH(4)Cl. Cytoplasmic vesicles fractionation showed a reduced maturation of Cathepsin-D in late endosomes and an increased content of EGFR and Rab7 in lysosomes of Celecoxib-treated TAFs.Our data indicate a double mechanism mediating the increased response to EGF of colon TAFs treated with Celecoxib. While EGFR overexpression could be targeted using anti EGFR drugs, the effects on endosome trafficking and protein turnover represents a more elusive target and should be taken into account for any long-term therapy with Celecoxib.

Targeting cancer stem cell plasticity through modulation of epidermal growth factor and insulin-like growth factor receptor signaling in head and neck squamous cell cancer.

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.

Fibroblast growth factor, estrogen, and prolactin receptor features in different grades of prostatic adenocarcinoma in elderly men

The objective was to characterize and associate the receptor reactivities of fibroblastic growth factor (FGF)-2, FGF-7, FGF-8, epidermal growth factor (EGF), α-actin and vimentin in relation to the androgen receptor (AR), α and β estrogen receptors (ERα and ERβ), and prolactin receptor in the prostate of elderly men showing low- and high-grade adenocarcinoma. Thirty prostatic samples were taken from 60- to 90-year-old patients without prostatic lesions and with low-grade cancer and high-grade cancer, from the University Hospital, School of Medicine, the State University of Campinas. The results showed that increased FGF-2, FGF-7, and FGF-8 receptor reactivities and decreased AR reactivity were verified in both high- and low-grade cancer. However, the FGF-8 receptor showed greater involvement at the beginning of the malignancy alterations. Increased EGF receptor (EGFR) reactivity and diminished α-actin immunohistochemistry were identified in both cancer groups. Also, increased ERα, PR, and vimentin receptors were verified in both cancer groups. To conclude, the ERα involvement in the reactive stroma activation led to a microenvironment, which was favorable to cancer progression, due to maximizing stromal imbalance. The prolactin could be related to cancer progression due to its interaction with ERα action, indicating that this hormone could be a relevant target to prevent the estrogenic effects in the prostatic lesions. Both FGF receptor (FGFR)-2 and FGFR-8 play a fundamental role in the early stages of prostate cancer, suggesting that these molecules could be a promising therapeutic target. The differential localization of the fibroblastic factors between the prostatic epithelium and stroma of elderly men, who presented prostate cancer, could indicate a favorable distinction for tumoral progression.

Clozapine-Induced ERK1 and ERK2 Signaling in Prefrontal Cortex Is Mediated by the EGF Receptor

The atypical antipsychotic drug clozapine is effective in treatment-refractory schizophrenia. The intracellular signaling pathways that mediate clozapine action remain unknown. A potential candidate is the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK-ERK) cascade that links G-protein-coupled receptor and ErbB growth factor signaling systems, thereby regulating synaptic plasticity and connectivity, processes impaired in schizophrenia. Here, we examined how clozapine differentially modulated phosphorylation of the MAPK isoforms, ERK1/ERK2 in primary murine prefrontal cortical neurons compared to the typical antipsychotic drug haloperidol. While clozapine and haloperidol acutely decreased cortical pERK1 activation, only clozapine but not haloperidol stimulated pERK1 and pERK2 with continued drug exposure. This delayed ERK increase however, did not occur via the canonical dopamine D(2)-Gi/o-PKA or serotonin 5HT(2A)-Gq-phospholipase-C-linked signaling pathways. Rather, epidermal growth factor (EGF) receptor signaling mediated clozapine-induced ERK activation, given dose-dependent reduction of pERK1 and pERK2 stimulation with the EGF receptor inhibitor, AG1478. Immunocytochemical studies indicated that clozapine treatment increased EGF receptor (Tyr1068) phosphorylation. In vivo mouse treatment studies supported the in vitro findings with initial blockade, subsequent activation, and normalization of the cortical ERK response over 24 h. Furthermore, in vivo clozapine-induced ERK activation was significantly reduced by AG1478. This is the first report that clozapine action on prefrontal cortical neurons involves the EGF signaling system. Since EGF receptor signaling has not been previously linked to antipsychotic drug action, our findings may implicate the EGF system as a molecular substrate in treatment-resistant schizophrenia.

Phospholipase C-ϵ Augments Epidermal Growth Factor-dependent Cell Growth by Inhibiting Epidermal Growth Factor Receptor Down-regulation

The down-regulation of the epidermal growth factor (EGF) receptor is critical for the termination of EGF-dependent signaling, and the dysregulation of this process can lead to oncogenesis. In the present study, we suggest a novel mechanism for the regulation of EGF receptor down-regulation by phospholipase C-epsilon. The overexpression of PLC-epsilon led to an increase in receptor recycling and decreased the down-regulation of the EGF receptor in COS-7 cells. Adaptor protein complex 2 (AP2) was identified as a novel binding protein that associates with the PLC-epsilon RA2 domain independently of Ras. The interaction of PLC-epsilon with AP2 was responsible for the suppression of EGF receptor down-regulation, since a perturbation in this interaction abolished this effect. Enhanced EGF receptor stability by PLC-epsilon led to the potentiation of EGF-dependent growth in COS-7 cells. Finally, the knockdown of PLC-epsilon in mouse embryo fibroblast cells elicited a severe defect in EGF-dependent growth. Our results indicated that PLC-epsilon could promote EGF-dependent cell growth by suppressing receptor down-regulation.

CIN85 associates with multiple effectors controlling intracellular trafficking of epidermal growth factor receptors.

CIN85 is a multidomain adaptor protein involved in Cbl-mediated down-regulation of epidermal growth factor (EGF) receptors. CIN85 src homology 3 domains specifically bind to a proline-arginine (PxxxPR) motif in Cbl, and this association seems to be important for EGF receptor endocytosis. Here, we report identification of novel CIN85 effectors, all containing one or more PxxxPR motifs, that are indispensable for their mutual interactions. These effectors include phosphatidyl-inositol phosphatases SHIP-1 and synaptojanin 2B1, Arf GTPase-activating proteins ASAP1 and ARAP3, adaptor proteins Hip1R and STAP1, and a Rho exchange factor, p115Rho GEF. Acting as a molecular scaffold, CIN85 clusters its effectors and recruits them to high-molecular-weight complexes in cytosolic extracts of cells. Further characterization of CIN85 binding to ASAP1 revealed that formation of the complex is independent on cell stimulation. Overexpression of ASAP1 increased EGF receptor recycling, whereas ASAP1 containing mutated PxxxPR motif failed to promote this event. We propose that CIN85 functions as a scaffold molecule that binds to numerous endocytic accessory proteins, thus controlling distinct steps in trafficking of EGF receptors along the endocytic and recycling pathways.

Insulin-like Growth Factor-binding Protein-3 Potentiates Epidermal Growth Factor Action in MCF-10A Mammary Epithelial Cells: INVOLVEMENT OF p44/42 AND p38 MITOGEN-ACTIVATED PROTEIN KINASES

Insulin-like growth factor-binding protein-3 (IGFBP-3) is inhibitory to the growth of many breast cancer cells in vitro; however, a high level of expression of IGFBP-3 in breast tumors correlates with poor prognosis, suggesting that IGFBP-3 may be associated with growth stimulation in some breast cancers. We have shown previously in MCF-10A breast epithelial cells that chronic activation of Ras-p44/42 mitogen-activated protein (MAP) kinase confers resistance to the growth-inhibitory effects of IGFBP-3 (Martin, J. L., and Baxter, R. C. (1999) J. Biol. Chem. 274, 16407-16411). Here we show that, in the same cell line, IGFBP-3 potentiates DNA synthesis and cell proliferation stimulated by epidermal growth factor (EGF), a potent activator of Ras. A mutant of IGFBP-3, which fails to translocate to the nucleus and has reduced ability to cell-associate, similarly enhanced EGF action in these cells. By contrast, the structurally related IGFBP-5, which shares many functional features with IGFBP-3, was slightly inhibitory to DNA synthesis in the presence of EGF. IGFBP-3 primes MCF-10A cells to respond to EGF because pre-incubation caused a similar degree of EGF potentiation as co-incubation. In IGFBP-3-primed cells, EGF-stimulated EGF receptor phosphorylation at Tyr-1068 was increased relative to unprimed cells, as was phosphorylation and activity of p44/42 and p38 MAP kinases, but not Akt/PKB. Partial blockade of the p44/42 and p38 MAP kinase pathways abolished the potentiation by IGFBP-3 of EGF-stimulated DNA synthesis. Collectively, these findings indicate that IGFBP-3 enhances EGF signaling and proliferative effects in breast epithelial cells via increased EGF receptor phosphorylation and activation of p44/42 and p38 MAP kinase signaling pathways.

The invasive phenotype in HMT-3522 cells requires increased EGF receptor signaling through both PI 3-kinase and ERK 1,2 pathways.

We studied the invasion of HMT-3522 breast epithelial cells in response to epidermal growth factor (EGF), and the associated signaling pathways. HMT-3522 T4-2 cells were shown to invade Matrigel-coated Transwell membranes in response to EGF while HMT-3522 S-1 cells failed to invade when treated with EGF. Studies utilizing specific molecular inhibitors showed the importance of beta1 integrin, phosphatidylinositol 3 kinase (PI 3-kinase), p38, extracellular regulated kinase 1, 2 (Erk 1,2) MAP kinases, and metalloproteinases in invasion and motility. T4-2 cell invasion was shown to be time-dependent and also gene transcription-dependent as shown by inhibition with Actinomycin D. T4-2 cells exhibited an increased activation of MAP kinases Erk 1,2 (2-fold), EGF receptor (3-fold), and PI 3-kinase (3- to 4-fold) when compared to the S-1 cells. In response to EGF, T4-2 cells showed a 5-fold greater secretion of matrix metalloproteinase-9 (MMP-9) as compared to S-1 cells, and this increase was largely dependent on the activity of PI 3-kinase. These findings indicate that expression of the invasive phenotype in these breast epithelial cells requires increased EGF receptor signaling, involving both PI 3-kinase and Erk 1,2 activities, which leads to multiple downstream effects, including enhanced secretion of MMP-9 and transcription of invasion-related genes.

Modulation of EGF Receptor Activity by Changes in the GM3 Content in a Human Epidermoid Carcinoma Cell Line, A431

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 μM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.

Effect of epidermal growth factor on enzymes of phospholipid biosynthesis in lung and liver of fetal rat in vivo and in vitro.

Epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, is an important regulator of cell differentiation and fetal lung surfactant synthesis, and may be used as a potential novel therapeutic agent in prematurity. Nevertheless, the distinct role in lung development and its mechanisms of action are not well understood. We investigated in vivo the systemic effect of intrafetally administered EGF (200 ng/g fetal body weight) and maternally administered dexamethasone (DEXA; 0.2 and 2.0mg/kg maternal body weight) on the activity of important enzymes of the phospholipid synthesis in the fetal rat lung and liver: choline kinase (EC 2.7.1.32), cholinephosphate cytidyltransferase (EC 2.7.7.15), choline phosphotransferase (EC 2.7.8.2), lysolecithin acyltransferase (EC 2.3.1.23) and glycerolphosphate phosphatidyltransferase (EC 2.7.8.5). Additionally, in vivo and in vitro effects of DEXA on EGF receptor synthesis, and the effects of EGF on protein content and morphogenesis of the fetal rat lung organoid culture, were evaluated. Whereas DEXA induced the activity of all investigated enzymes of phospholipid synthesis and increased EGF receptor synthesis, EGF has no effects on the enzymes, either in vivo or in vitro. EGF enhanced protein synthesis and morphogenesis in vitro. With respect to our data and the literature, we hypothesize that DEXA and EGF may act on different cellular sides. Whereas glucocorticoids induce surfactant phospholipid synthesis, EGF should be more involved in cell proliferation and morphogenesis.

Regulation of mammary hormone receptor metabolism by a retroviral envelope protein.

In a previous study, the envelope protein (gp52) of the mouse mammary tumour virus (MMTV) was shown to facilitate mammary gland differentiation by increasing prolactin (PRL) receptors via increased receptor synthesis and via the redistribution of existing receptors from an internal pool. In this study, receptors for other hormones known to affect mammary gland metabolism were investigated. Epidermal growth factor (EGF) stimulates mammary epithelial growth and inhibits differentiation; its receptor is rapidly and dramatically down-regulated by gp52. This is accomplished by its internalization and by decreasing its half-life from 27 h to 2.4 h. Surprisingly, it also increased EGF receptor synthesis, although this effect was not great enough to overcome receptor down-regulation. In contrast, gp52 did not affect the distribution, half-life or synthesis of the insulin receptor. These results demonstrate that MMTV can enhance mammary differentiation by coordinately regulating several hormone receptors: specifically, it can increase the number of receptors for PRL, a differentiative hormone, while decreasing the number of receptors for EGF, a growth/anti-differentiative hormone.

Regulation of epidermal growth factor receptor by androgens in human endometrial cells in culture.

Women with polycystic ovaries (PCO) have a thicker endometrium than women with normal ovaries. This cannot be due to unopposed oestrogen, as it occurs in ovulatory cycles. Androgens may be involved, as these are raised in women with PCO. The effects of steroids are partly mediated by growth factors and their receptors. The aim of this study was to investigate the effect of androgens on epidermal growth factor (EGF) receptor in human endometrium. Endometrium was enzymatically dispersed and glands and stromal cells separated. Cells were incubated in Ham's F10 medium supplemented with 5% charcoal-stripped fetal calf serum and either androgens or vehicle. Specific binding of [125I]-labelled EGF was measured. Testosterone and dihydrotestosterone (DHT) (10 micromol/l) increased EGF receptor concentration (control 100 +/- 9%, testosterone 196 +/- 23% control; control 100 +/- 1%, DHT 244 +/- 6% control) but did not alter receptor affinity. The effect of testosterone was inhibited by the anti-androgen hydroxyflutamide, but not by the antioestrogen ICI182780 nor the aromatase inhibitor 4-hydroxyandrostenedione. EGF receptor levels were increased by androstenediol (control 100 +/- 2%, androstenediol 120 +/- 10% control) but not by androstanediol, dehydroepiandrosterone (DHA), DHA sulphate or androstenedione. Testosterone and DHT increased EGF receptor concentrations in glandular epithelium (control 100 +/- 24%, testosterone 147 +/- 5%, DHT 185 +/- 30% control). These data suggest that androgens may have an effect on the endometrium via an increase in EGF receptor concentrations.

Epidermal growth factor upregulates the expression of its own intestinal receptor after small bowel resection

Background/Purpose: Epidermal growth factor (EGF) binds to its enterocyte receptor and enhances intestinal adaptation after massive small bowel resection (SBR). To ascertain the mechanism for enhanced adaptation by EGF, we sought to determine the effect of EGF administration on in vivo expression of the intestinal EGF receptor after SBR. Methods : Male ICR mice underwent a 50% proximal SBR and then were assigned randomly to EGF (150 μg/kg/d) or saline by twice daily intraperitoneal injection. After 3 days, the ileum was harvested and total protein and DNA content were measured. Northern hybridization and a ribonuclease protection assay were used to detect qualitative and quantitative expression of EGF receptor mRNA. The remaining ileum was pooled for each group and Western blotting used to determine expression of EGF receptor protein. Results : EGF augmented adaptation after SBR as monitored by significant increases in ileal protein (2.7 ± 0.08 saline versus 3.9 ± 0.17 mg/cm EGF; P < .001) and DNA (55.8 ± 1.6 saline versus 104 ± 8.4 μm/cm EGF; P < .001) content. Northern blotting results showed a marked (>fivefold) increase in ileal EGF receptor mRNA, which was confirmed with the ribonuclease protection assay. Administration of EGF after SBR induced a similar expression of EGF receptor protein. Conclusions : EGF enhanced intestinal adaptation after SBR. This augmented response is associated with increased ileal expression of EGF receptor mRNA and protein. Increased EGF receptor expression and subsequent enhanced ligand/receptor activity may be one important mechanism for the beneficial effect of EGF administration during intestinal adaptation.

Epidermal growth factor (EGF) receptor density controls mitogenic activation of normal rat kidney (NRK) cells by EGF.

Normal rat kidney (NRK) fibroblasts are immortalized cells that are strictly dependent on externally added growth factors for proliferation. When cultured in the presence of epidermal growth factor (EGF) as the only growth stimulating hormone, these cells have a normal phenotype and undergo density-dependent growth inhibition. It has been postulated that this density-arrest results from a decrease of EGF receptor levels below a threshold level which makes these cells unresponsive to stimulation by EGF. In the present study, we show that NRK cells, made quiescent by serum-deprivation at submaximum density, are mitogenically still responsive to EGF, but show enhanced mitogenic stimulation after 8 hr pre-treatment with either transforming growth factor beta (TGF beta) or retinoic acid (RA), while prostaglandin F2 alpha (PGF2 alpha) and bradykinin (BK) enhance the mitogenic stimulation by EGF only slightly under these conditions. Addition of TGF beta or RA results in an increase of both 125I-EGF-binding capacity and EGF receptor mRNA levels. Using flow cytometric analysis, we show that pre-treatment with TGF beta or RA increases the percentage of cells entering the cell cycle as a function of time. Furthermore, pre-treatment of the cells with TGF beta or RA increases the rate of mitogen-activated protein kinase (MAPK) phosphorylation by EGF. PGF2 alpha and BK also increase EGF receptor levels, but only with delayed kinetics. These results show that already in serum-deprived quiescent NRK cells, EGF receptor levels limit EGF-induced mitogenic stimulation. This observation provides further evidence for the regulating role of the EGF receptor in density-dependent growth control of NRK cells.

Gβγ Subunits Mediate Src-dependent Phosphorylation of the Epidermal Growth Factor Receptor: A SCAFFOLD FOR G PROTEIN-COUPLED RECEPTOR-MEDIATED Ras ACTIVATION

In many cells, stimulation of mitogen-activated protein kinases by both receptor tyrosine kinases and receptors that couple to pertussis toxin-sensitive heterotrimeric G proteins proceed via convergent signaling pathways. Both signals are sensitive to inhibitors of tyrosine protein kinases and require Ras activation via phosphotyrosine-dependent recruitment of Ras guanine nucleotide exchange factors. Receptor tyrosine kinase stimulation mediates ligand-induced receptor autophosphorylation, which creates the initial binding sites for SH2 domain-containing docking proteins. However, the mechanism whereby G protein-coupled receptors mediate the phosphotyrosine-dependent assembly of a mitogenic signaling complex is poorly understood. We have studied the role of Src family nonreceptor tyrosine kinases in G protein-coupled receptor-mediated tyrosine phosphorylation in a transiently transfected COS-7 cell system. Stimulation of Gi-coupled lysophosphatidic acid and alpha2A adrenergic receptors or overexpression of Gbeta1gamma2 subunits leads to tyrosine phosphorylation of the Shc adapter protein, which then associates with tyrosine phosphoproteins of approximately 130 and 180 kDa, as well as Grb2. The 180-kDa Shc-associated tyrosine phosphoprotein band contains both epidermal growth factor (EGF) receptor and p185(neu). 3-5-fold increases in EGF receptor but not p185(neu) tyrosine phosphorylation occur following Gi-coupled receptor stimulation. Inhibition of endogenous Src family kinase activity by cellular expression of a dominant negative kinase-inactive mutant of c-Src inhibits Gbeta1gamma2 subunit-mediated and Gi-coupled receptor-mediated phosphorylation of both EGF receptor and Shc. Expression of Csk, which inactivates Src family kinases by phosphorylating the regulatory carboxyl-terminal tyrosine residue, has the same effect. The Gi-coupled receptor-mediated increase in EGF receptor phosphorylation does not reflect increased EGF receptor autophosphorylation, assayed using an autophosphorylation-specific EGF receptor monoclonal antibody. Lysophosphatidic acid stimulates binding of EGF receptor to a GST fusion protein containing the c-Src SH2 domain, and this too is blocked by Csk expression. These data suggest that Gbetagamma subunit-mediated activation of Src family nonreceptor tyrosine kinases can account for the Gi-coupled receptor-mediated tyrosine phosphorylation events that direct recruitment of the Shc and Grb2 adapter proteins to the membrane.

Efficient Gene Transfer to EGF Receptor Overexpressing Cancer Cells by Means of EGF-Labeled Cationic Liposomes

Epidermal growth factor (EGF)-labeled cationic liposomes (EGF-liposomes) were prepared for efficient gene transfer vector to EGF receptor expressing cells. Transfection activity of EGF-liposomes associating plasmid PGV-C which encodes luciferase showed a 2-fold increase in EGF receptor expressing cells, HEC-1-A, compared to that of EGF-non-labeled liposomes (N-liposomes). In EGF receptor deficient HRA cells, however, both EGF- and N-liposomes exhibited low transfection efficiency and no difference was observed between them. Furthermore, by the addition of anti EGF receptor antibody, transfection efficiency of EGF-liposomes was suppressed, suggesting EGF receptor-mediated endocytosis of EGF-liposomes. Transfection activity of EGF-liposomes was strongly dependent on the concentrations of fusogenic lipid, dioleoylphosphatidylethanolamine in liposomes. By X-gal staining 6–8 % of GCH-1(m) cells which also had EGF receptor expressed β-galactosidase activity following the transfection by EGF-liposomes associating pSV-β-galactosidase. These findings indicate that EGF-liposomes could be a preferable vector for EGF-receptor expressing cells.

Activation of Epidermal Growth Factor Receptor Gene Transcription by Phorbol 12-Myristate 13-Acetate Is Mediated by Activator Protein 2

The response of the epidermal growth factor (EGF) receptor gene to phorbol 12-myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run-off experiments showed that PMA increased EGF receptor gene transcription. Cell-free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene.