Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis

In the last few decades, several growth factors were identified in the testis of various mammalian species. Growth factors are shown to promote cell proliferation, regulate tissue differentiation, and modulate organogenesis. In the present investigation we have studied the localization of EGF and EGFR in the adult bovine testis by means of immunohistochemical method. Our results demonstrated that EGF and EGFR were localized solely to the bovine testicular germ cells (spermatogonia, spermatocytes, and round spermatids). In contrast, the somatic testicular cells (i.e., Sertoli, Leydig, and myofibroblast cells) exhibited no staining affinity. EGF and EGFR were additionally detected in the epithelial lining of straight tubules and rete testis. Interestingly, the distribution of EGF and EGFR in the germ cells was mainly dependent upon the cycle of the seminiferous epithelium since their localization appeared to be preponderant during the spermatogonia proliferation and during the meiotic and spermiogenic processes. In conclusion, such findings may suggest that EGF and EGFR are important paracrine and/or autocrine regulators of spermatogenesis in bovine.     

GPR48-Induced keratinocyte proliferation occurs through HB-EGF mediated EGFR transactivation

GPR48 can mediate keratinocyte proliferation and migration. Our investigations showed that AG1478, an inhibitor of EGFR tyrosine kinase, could block GPR48-mediated cellular processes. AG1478 treatment of Gpr48^+/+ cells also decreased phosphorylation of EGFR, ERK and STAT3. Subsequent screening using conditioned media immunodepleted of EGFR ligands identified HB-EGF as the ligand responsible for phosphorylation of EGFR, ERK and STAT3. HB-EGF was reduced in Gpr48^−/− cell culture medium, but its addition restored the phosphorylation of EGFR, ERK, STAT3, as well as cell proliferation. Confirmation that GPR48 mediates EGFR signaling pathway through HB-EGF was subsequently performed using an inhibitor of HB-EGF.     

Basic fibroblast growth factor promotes epidermal growth factor responsiveness and survival of mesencephalic neural precursor cells

Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induce proliferation of neural precursor cells from several central nervous system regions in vitro. We have previously described two neural precursor cell populations from 13.5 days postcoitium (dpc) mesencephalon, one forming colonies in response to EGF, present in the ventral mesencephalon, and other forming colonies in response to EGF + bFGF, mainly present in the dorsal mesencephalon. In the present work, we show that 13.5 dpc dorsal mesencephalic cells required bFGF only for 1 h to form colonies in response to EGF alone, indicating that these two growth factors act in sequence rather than simultaneously. Absence of bFGF at the beginning of the culture gave rise to very few colonies, even after the addition of EGF + bFGF, suggesting that cells responsive to bFGF were very labile in the primary culture condition. This result is in contrast with cells pretreated with bFGF, which could survive for up to 5 days in the absence of bFGF or EGF, and then were capable of efficiently forming colonies in response to EGF. Basic FGF was also able to support survival of EGF‐responsive neural precursors from both ventral and dorsal mesencephalon. The population requiring bFGF to form colonies in response to EGF was identified at different developmental stages (11.5–15.5 dpc), with higher contribution to the total number of neural precursors cells detected (EGF‐responsive plus bFGF‐responsive) at early stages and in the dorsal region. We show that the differentiation effect of bFGF resulted in the appearance of the mRNA coding for the EGF receptor. Our data suggest that bFGF‐responsive neural precursors are the source of EGF‐responsive neural precursors. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 14–27, 1999     

Hepatocyte growth factor induces epithelial cell motility through transactivation of the epidermal growth factor receptor

Hepatocyte growth factor (HGF) is a potent inducer of motility in epithelial cells. Since we have previously found that activation of the epidermal growth factor receptor (EGFR) is an absolute prerequisite for induction of motility of corneal epithelial cells after wounding, we investigated whether induction of motility in response to HGF is also dependent on activation of the EGFR. We now report that HGF induces transactivation of the EGFR in an immortalized line of corneal epithelial cells, in human skin keratinocytes, and in Madin-Darby canine kidney cells. EGFR activation is unconditionally required for induction of motility in corneal epithelial cells, and for induction of a fully motile phenotype in Madin-Darby canine kidney cells. Activation of the EGFR occurs through amphiregulin and heparin-binding epidermal growth factor-like growth factor. Early after HGF stimulation, blocking EGFR activation does not inhibit extracellular-signal regulated kinase 1/2 (ERK1/2) activation by HGF, but the converse is seen after approximately 1 h, indicating the existence of EGFR-dependent and -independent routes of ERK1/2 activation. In summary, HGF induces transactivation of the EGFR in epithelial cells, and this is a prerequisite for induction of full motility.     

Photonic modulation of epidermal growth factor receptor halts receptor activation and cancer cell migration

Epidermal growth factor receptor (EGFR) plays a key role in regulating cell survival, proliferation and migration, and its overexpression and activation has been correlated with cancer progression. Cancer therapies targeting EGFR have been applied in the clinic with some success. We show, by confocal microscopy analysis, that illumination of adenocarcinomic human alveolar basal epithelial cells (Human A549—EGFR biosensor cell line) with 280 nm at irradiance levels up to 20 times weaker than the Ultraviolet B (UVB) solar output for short periods of time (15‐45 minutes) prevents epidermal growth factor‐mediated activation of EGFR located on the cell membrane, preventing or reducing cellular disaggregation, formation of filopodia and cell migration. This effect of Ultraviolet (UV) light illumination was confirmed further in a functional scratch assay, and shown to be more effective than that of a specific EGFR‐signaling inhibitor. This new photonic approach may be applicable to the treatment of various types of cancer, alone or in combination with other therapies.      

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.     

Growth factors modify the epidermal growth factor receptor through multiple pathways

Previous results have shown that tumor promoters modify the properties of the epidermal growth factor (EGF) receptor through the activation of protein kinase C. Diacylglycerol-generating factors such as platelet-derived growth factor (PDGF) and p28sis should activate protein kinase C and alter EGF receptor properties in a similar manner. To test directly the involvement of protein kinase C in the action of media from v-sis-transformed cells on the EGF receptor, Swiss 3T3 cells were first extensively treated with various concentrations of the tumor-promoter phorbol dibutyrate (PDBu) This treatment reduced levels of active protein kinase C in the cells, making them less responsive to subsequent rechallenge with the tumor promoter. The results demonstrate that there are at least two components to the action of media from v-sis transformed cells on EGF binding: a labile factor that confers protein kinase C independence and a stable factor that appears to be dependent on protein kinase C. The action of the first factor cannot be mimicked by transforming growth factor-beta or EGF in either the presence or absence of PDGF. The action of the second factor is similar to that of PDGF. These findings indicate that heterologous regulation of the EGF receptor can occur through both protein kinase C-dependent and -independent pathways.     

dsRNA-mediated innate immunity of epidermal keratinocytes

MIP-1alpha, a CC chemokine, recruits monocytes, natural killer cells, lymphocytes, and neutrophils, and plays a critical role in viral infection. Since, the lesional epidermis of herpes zoster expressed MIP-1alpha, we hypothesized that keratinocytes produce MIP-1alpha in response to virus-associated dsRNA via TLR3. To investigate this, we examined cultured human keratinocytes for MIP-1alpha production induced by poly(I:C), a TLR3 ligand. Poly(I:C) treatment induced MIP-1alpha production, interestingly, poly(I:C)-induced IFN-alpha and -beta production preceded MIP-1alpha production. A neutralizing antibody for IFN-beta significantly inhibited the poly(I:C)-induced MIP-1alpha production indicating that MIP-1alpha production is via IFN-beta. IFN-alpha priming enhanced TLR3 expression and MIP-1alpha production in poly(I:C)-treated keratinocytes. This suggests that IFN-alpha enhanced the TLR3 expression and reinforced the response of keratinocytes to poly(I:C), which resulted in an increase in MIP-1alpha production. In conclusion, normal human keratinocytes produce MIP-1alpha in response to dsRNA via TLR3, and this production is regulated by IFN-alpha/beta.     

Calcitonin gene-related peptide increases proliferation of human HaCaT keratinocytes by activation of MAP kinases

Psoriasis is a chronic disease characterized by keratinocyte hyperproliferation and inflammation. It has been demonstrated that the expression of calcitonin gene-related peptide (CGRP) is elevated in psoriasis lesions and CGRP-containing neuropeptide nerve fibers are denser in the psoriatic epidermis. CGRP has been previously described to influence proliferation of several cell types, such as Schwann cell, tracheal epithelial cells, and human gingival fibroblasts. In the present study, we determined the effect of CGRP on HaCaT keratinocyte proliferation and the role of mitogen-activated protein kinases (MAPKs) in CGRP induced keratinocyte proliferation. Our data indicate CGRP increased [³H]-thymidine incorporation and MTT activity of HaCaT in a concentration-dependent manner. CGRP also enhanced serum-induced HaCaT cell proliferation. HaCaT cells cultured with CGRP had a significant increase in phosphorylated ERK1/2, p38 and JNK, and CGRP induced DNA synthesis was inhibited by PD 98059 or SB 203580, selective inhibitors of MAP kinase kinase (MEK, which is upstream from ERK) and p38, respectively. These findings suggest that HaCaT cell proliferate in response to CGRP, which is mediated by phosphorylation of ERK1/2 and p38 MAPK.     

Overexpression of JKTBP1 induces androgen-independent LNCaP cell proliferation through activation of epidermal growth factor-receptor (EGF-R)

Heterogenous nuclear ribonucleoprotein D-like protein (JKTBP) belongs to a new member of hnRNPs. Previous studies implied that JKTBP1 may be associated with the progression of androgen-independent (AI) prostate cancer. In this study, we generated three stable LNCaP cell lines which expressed exogenous JKTBP1. Furthermore, the effect of ectopic JKTBP1 on the proliferation of LNCaP cells and its mechanism was investigated. We originally found that the ectopic JKTBP1 expression resulted in the proliferation of LNCaP cells in an AI way, as well as inducing the upregulated expression of EGF-R and prostate-specific antigen (PSA), but did not influence the expression level of AR. Moreover, AG1478 suppressed the effect of proliferation induced by JKTBP1. In addition, immunohistochemistry showed that JKTBP1 expression was significantly elevated in AI prostate cancer tissues when compared with the androgen-dependent (AD) prostate cancer and benign prostatic hyperplasia. Our data indicated that overexpression of JKTBP1 in LNCaP cells leads to abnormal cell proliferation and may be involved in the process of AD to AI through induction of EGF-R expression.     

The epidermal growth factor.

Epidermal growth factor (EGF) is a single polypeptide of 53 amino acid residues which is involved in the regulation of cell proliferation. Egf exerts its effects in the target cells by binding to the plasma membrane located EGF receptor. The EGF receptor is a transmembrane protein tyrosine kinase. Binding of EGF to the receptor causes activation of the kinase and subsequently receptor autophosphorylation. The autophosphorylation is essential for the interaction of the receptor with its substrates. These bind to the receptor by the so-called SH2 domains. The signal transduction pathways activated by EGF include the phosphatidylinositol pathway, leading to activation of protein kinase C and to increase in the intracellular Ca2+ concentration, and to the ras pathway leading to MAP kinase activation. Recently the cytoplasm has been implicated as playing an important role in EGF induced signal transduction. The EGF receptor has been demonstrated to be an actin-binding protein. In addition EGF causes a rapid actin depolymerisation and the formation of membrane ruffles. In particular these membrane ruffles have been shown to act as the first site of signal transduction after EGF binding, and thus may be considered as signal transduction structures. Finally evidence has been presented suggesting a positive role for EGF and/or the receptor in the nucleus.     

Barium promotes anchorage-independent growth and invasion of human HaCaT keratinocytes via activation of c-SRC kinase

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl(2)) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5-50 μM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 μM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5-5 μM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro.     

EphA3 is up-regulated by epidermal growth factor and promotes formation of glioblastoma cell aggregates

EphA3, a member of the Eph family of receptor tyrosine kinases, has been reported to be overexpressed in some human cancers including glioblastoma. Here, we found that expression of EphA3 is up-regulated in response to epidermal growth factor (EGF) stimulation and promotes formation of cell aggregates in suspension culture of glioblastoma cells. Suppression of EphA3 expression by short hairpin RNA-mediated knockdown or CRISPR/Cas9-mediated gene deletion inhibited EGF-induced promotion of cell aggregate formation, whereas overexpression of EphA3 promoted formation of cell aggregates in suspension culture. EGF-induced EphA3 expression and promotion of cell aggregate formation required Akt activity. Furthermore, N-cadherin, whose expression was regulated by EGF and EphA3, contributed to the formation of cell aggregates in suspension culture. These results suggest that the regulation of EphA3 expression plays a critical role in glioblastoma cell growth in non-adherent conditions.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

Plasma membrane phospholipid scramblase 1 promotes EGF-dependent activation of c-Src through the epidermal growth factor receptor

Phospholipid scramblase (PLSCR1) is a multiply palmitoylated, calcium-binding endofacial membrane protein proposed to mediate transbilayer movement of plasma membrane phospholipids. PLSCR1 is a component of membrane lipid rafts and has been shown to both physically and functionally interact with activated epidermal growth factor (EGF) receptors and other raft-associated cell surface receptors. Cell stimulation by EGF results in Tyr phosphorylation of PLSCR1, its association with both Shc and EGF receptors, and rapid cycling of PLSCR1 between plasma membrane and endosomal compartments. We now report evidence that upon EGF stimulation, PLSCR1 is phosphorylated by c-Src, within the tandem repeat sequence 68VYNQPVYNQP77. The in vivo interaction between PLSCR1 and Shc requires the Src-mediated phosphorylation on tyrosines 69 and 74. In in vitro pull down studies, phosphorylated PLSCR1 was found to bind directly to Shc through the phosphotyrosine binding domain. Consistent with the potential role of PLSCR1 in growth factor signaling pathways, granulocyte precursors derived from mice deficient in PLSCR1 show impaired proliferation and maturation under cytokine stimulation. Using PLSCR1-/- embryonic fibroblasts and kidney epithelial cells, we now demonstrate that deletion of PLSCR1 from the plasma membrane reduces the activation of c-Src by EGF, implying that PLSCR1 normally facilitates receptor-dependent activation of this kinase. We propose that PLSCR1, through its interaction with Shc, promotes Src kinase activation through the EGF receptor.     

Angiotensin II promotes smooth muscle cell proliferation and migration through release of heparin-binding epidermal growth factor and activation of EGF-receptor pathway

Transactivation of EGF-receptor (EGFR) by G-protein coupled receptors (GPCRs) is emerging as an important pathway in cell proliferation, which plays a crucial role in the development of atherosclerotic lesion. Angiotensin II (Ang II) has been identified to have a major role in the formation of atherosclerotic lesions, although the underlying mechanisms remain largely unclear. We hypothesize that Ang II promotes the proliferation and migration of smooth muscle cells through the release of heparin-binding epidermal growth factor like growth factor (HB-EGF), transactivation of EGFR and activation of Akt and Erk 1/2, with matrix metalloproteases (MMPs) playing a dispensable role. Primary rat aortic smooth muscle cells were used in this study. Smooth muscle cells rendered quiescent by serum deprivation for 12 h were treated with Ang II (100 nM) in the presence of either GM6001 (20 microM), a specific inhibitor of MMPs or AG1478 (10 microM), an inhibitor of EGFR. The levels of phosphorylation of EGFR, Akt and Erk 1/2 were assessed in the cell lysates. Inhibition of MMPs by GM6001 significantly attenuated Ang II-stimulated phosphorylation of EGFR, suggesting that MMPs may be involved in the transactivation of EGFR by Ang II receptor. Furthermore Ang II-stimulated proliferation and migration of smooth muscle cells were significantly blunted by inhibiting MMPs and EGFR and applying HB-EGF neutralization antibody, indicating that MMPs, HB-EGF and EGFR activation is necessary for Ang-II stimulated migration and proliferation of smooth muscle cells. Our results suggest that inhibition of MMPs may represent one of the strategies to counter the mitogenic and motogenic effects of Ang II on smooth muscle cells and thereby prevent the formation and development of atherosclerotic lesions.