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• W2029351044 abstract "Epidermal growth factor receptor (EGFR) is a critical mediator of several types of epithelial cancers. Skin cancer arising from exposure to ultraviolet B irradiation (UVB) from the sun is a prominent form of human cancer. Recent data indicate that in addition to cognate ligands, EGFR is activated by UVB irradiation. We used pharmacological and genetic approaches to investigate the function of EGFR in mediating UVB-induced signal transduction in human skin keratinocyte HaCaT cells. Pharmacological inhibition of EGFR tyrosine kinase significantly inhibited UVB-mediated induction of ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of UVB activation of EGFR also suppressed activation of AKT-, PKC-, and PKA-dependent signal transduction pathways. B82 mouse L cells devoid of EGFR were used to further investigate EGFR dependence of UVB-induced signal transduction. UVB failed to induce ERK, and JNK activation was reduced 60% in B82 cells compared to B82K+ cells, which express EGFR. In addition, UVB induced both c-Fos and c-Jun proteins in B82K+ cells, whereas neither were induced in B82 cells. Taken together, these data demonstrate that EGFR is required for UVB-mediated induction of multiple signaling pathways that are known to mediate tumor formation in skin. Epidermal growth factor receptor (EGFR) is a critical mediator of several types of epithelial cancers. Skin cancer arising from exposure to ultraviolet B irradiation (UVB) from the sun is a prominent form of human cancer. Recent data indicate that in addition to cognate ligands, EGFR is activated by UVB irradiation. We used pharmacological and genetic approaches to investigate the function of EGFR in mediating UVB-induced signal transduction in human skin keratinocyte HaCaT cells. Pharmacological inhibition of EGFR tyrosine kinase significantly inhibited UVB-mediated induction of ERK, p38, and JNK MAP kinases, and their effectors, transcription factors c-Fos and c-Jun. Inhibition of UVB activation of EGFR also suppressed activation of AKT-, PKC-, and PKA-dependent signal transduction pathways. B82 mouse L cells devoid of EGFR were used to further investigate EGFR dependence of UVB-induced signal transduction. UVB failed to induce ERK, and JNK activation was reduced 60% in B82 cells compared to B82K+ cells, which express EGFR. In addition, UVB induced both c-Fos and c-Jun proteins in B82K+ cells, whereas neither were induced in B82 cells. Taken together, these data demonstrate that EGFR is required for UVB-mediated induction of multiple signaling pathways that are known to mediate tumor formation in skin. Ultraviolet (UV) irradiation is a potent carcinogen, capable of causing cell transformation and promotion of tumor formation. The shorter wavelength UVB region (290 to 310 nm) of the UV spectrum (290 to 400 nm) contains the most highly energetic photons. UVB irradiation causes DNA damage that can result in mutations stemming from imperfect DNA repair. In addition, accumulating evidence indicates that UVB-induced cellular responses lead to skin damage, promoting an environment conducive to tumor formation.1Egeblad M Werb Z New functions for the matrix metalloproteinases in cancer progression.Nat Rev Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5133) Google Scholar, 2Itoh Y Nagase H Matrix metalloproteinases in cancer.Essays Biochem. 2002; 38: 21-36Crossref PubMed Scopus (231) Google Scholar, 3Pupa S Menard S Forti S Tagliabue E New insights into the role of extracellular matrix during tumor onset and progression.J Cell Physiol. 2002; 192: 259-267Crossref PubMed Scopus (285) Google Scholar The mammalian UV response comprises UV activation of cell surface growth factor and cytokine receptors and their attendant downstream signal transduction machinery. UVB activation of four major families of growth factor receptors has been demonstrated: epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor, fibroblast growth factor receptor, and insulin receptor.4Zheng Z Chen R Prystowsky J UVB radiation induces phosphorylation of the epidermal growth factor receptor decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV-40-transfomed human keratinocytes.Exp Dermatol. 1993; 2: 257-265Crossref PubMed Scopus (26) Google Scholar, 5Sachsenmaier C Radler-Pohl A Zinck R Nordheim A Herrlich P Rahmsdorf H Involvement of growth factor receptors in the mammalian UVC response.Cell. 1994; 78: 963-972Abstract Full Text PDF PubMed Scopus (406) Google Scholar, 6Peus D Vasa R Mebes A Pott M Beyerle A Squillace K Pittelkow M H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes.J Invest Dermatol. 1998; 110: 966-971Crossref PubMed Scopus (169) Google Scholar, 7Peus D Vasa RA Beyerle A Meves A Krautmacher C Pittelkow MR UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.J Invest Dermatol. 1999; 112: 751-756Crossref PubMed Scopus (214) Google Scholar, 8Huang RP Wu JX Fan Y Adamson ED UV activates growth factor receptors via reactive oxygen intermediates.J Cell Biol. 1996; 133: 211-220Crossref PubMed Scopus (253) Google Scholar In addition, UVB activates receptors for the primary cytokines interleukin-1 and tumor necrosis factor-α and the death receptor Fas.9Zhuang S Kochevar I Ultraviolet A radiation induces rapid apoptosis of human leukemia cells by Fas ligand-independent activation of the Fas death pathways.Photochem Photobiol. 2003; 78: 61-67PubMed Google Scholar, 10Rosette C Karin M Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factors and cytokine receptors.Science. 1996; 274: 1194-1197Crossref PubMed Scopus (943) Google Scholar, 11Bang B Gniadecki R Larsen J Baadsgaard O Skov L In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells.Exp Dermatol. 2003; 12: 791-798Crossref PubMed Scopus (30) Google Scholar UVB activation of these diverse cell surface receptors results in concomitant activation of multiple receptor-coupled signal transduction pathways, including the three MAP kinase signaling modules (ERK, JNK, and p38), Jak/STAT pathways, protein kinase-C pathways, integrin-coupled focal adhesion kinase pathways, and PI-3 kinase/AKT pathways.7Peus D Vasa RA Beyerle A Meves A Krautmacher C Pittelkow MR UVB activates ERK1/2 and p38 signaling pathways via reactive oxygen species in cultured keratinocytes.J Invest Dermatol. 1999; 112: 751-756Crossref PubMed Scopus (214) Google Scholar, 12Klotz L Briviba K Sies H Singlet oxygen mediates the activation of JNK by UVA radiation in human skin fibroblasts.FEBS Lett. 1997; 408: 289-291Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 13Huang C Ma WY Dong ZG Signal transduction through atypical PKCs, but not the EGF receptor, is necessary for UVC-induced AP-1 activation in immortal murine cells.Oncogene. 1997; 14: 1945-1954Crossref PubMed Scopus (36) Google Scholar, 14Aragane Y Kulms D Luger T Schwarz T Down-regulation of interferon gamma-activated STAT1 by UV light.Proc Natl Acad Sci USA. 1997; 94: 11490-11495Crossref PubMed Scopus (63) Google Scholar UVB stimulation of these signal transduction pathways directly stimulates activation of transcription factors, which in turn regulate target gene expression. UVB-inducible transcription factors include Ets family members, EGR-1, AP-1 components (c-Jun and c-Fos), and nuclear factor (NF)-κB.15Zhang W Chen S EGF-1, a UV-inductible gene in p53 (−/−) mouse cells.Exp Cell Res. 2001; 266: 21-30Crossref PubMed Scopus (18) Google Scholar, 16Karin M Takahashi T Kapahi P Delhase M Chen Y Makris C Rothwarf D Baud V Natoli G Guido F Li N Oxidative stress and gene expression: the AP-1 and NF-κB connections.Biofactors. 2001; 15: 87-89Crossref PubMed Scopus (136) Google Scholar, 17Legrand-Poels S Schoonbroodt S Matroule J Piette J NF-κB: an important transcription factor in photobiology.J Photochem Photobiol B. 1998; 45: 1-8Crossref PubMed Scopus (73) Google Scholar, 18Huang R Fan Y Boynton A UV irradiation upregulates Egr-1 expression at transcription level.J Cell Biochem. 1999; 73: 227-236Crossref PubMed Scopus (32) Google Scholar, 19Fisher G Wang Z Datta S Li X Kang S Voorhees J UV irradiation inhibits collagen synthesis via c-Jun mediated transcriptional repression that is reversed by all-trans retinoic acid in human skin in vivo.J Clin Invest. 2000; 106: 661-668Crossref Scopus (248) Google Scholar A prominent feature of the mammalian UV response is induction of AP-1 and NF-κB-regulated genes including numerous cytokines, adhesion molecules, cyclooxygenase-2 (cox-2), nitric-oxide synthase, and matrix metalloproteinases. In human skin, these UVB-induced gene products cause an inflammatory response characterized by vasodilation, recruitment of circulating immune cells into the skin, and breakdown of skin connective tissue.19Fisher G Wang Z Datta S Li X Kang S Voorhees J UV irradiation inhibits collagen synthesis via c-Jun mediated transcriptional repression that is reversed by all-trans retinoic acid in human skin in vivo.J Clin Invest. 2000; 106: 661-668Crossref Scopus (248) Google Scholar, 20Schwartz E Cruickshank F Christensen C Perlish J Lebwohl M Collagen alterations in chronically sun-damaged human skin.Photochem Photobiol. 1993; 58: 841-844Crossref PubMed Scopus (106) Google Scholar, 21Fisher G Datta S Talwar H Wang Z Varani J Kang S Voorhees J Molecular basis of sun-induced premature skin ageing and retinoid antagonism.Nature. 1996; 379: 335-339Crossref PubMed Scopus (1198) Google Scholar, 22Fisher G Wang Z Datta S Varani J Kang S Voorhees J Pathophysiology of premature skin aging induced by ultraviolet light.N Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1171) Google Scholar, 23Fisher G Esmann J Griffiths C Talwar H Duell E Hammerberg C Elder J Finkel L Karabin G Nickoloff B Voorhees J Cellular, immunologic and biochemical characterization of topical retinoic-acid treated human skin.J Invest Dermatol. 1991; 96: 699-707Abstract Full Text PDF PubMed Google Scholar, 24Gilchrest B Yaar M Ageing and photoageing of the skin: observations and the cellular and molecular level.Br J Dermatol. 1992; 127: 25-30Crossref PubMed Scopus (133) Google Scholar Recent evidence indicates that UVB-induced inflammation provides a microenvironment that promotes tumor formation by cells harboring permissive UVB-induced mutations.25Coussens L Tinkle C Hanahan D Werb Z MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.Cell. 2000; 103: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar The activation of these diverse cell surface receptors by UVB irradiation has been confirmed by several research groups.9Zhuang S Kochevar I Ultraviolet A radiation induces rapid apoptosis of human leukemia cells by Fas ligand-independent activation of the Fas death pathways.Photochem Photobiol. 2003; 78: 61-67PubMed Google Scholar, 10Rosette C Karin M Ultraviolet light and osmotic stress: activation of the JNK cascade through multiple growth factors and cytokine receptors.Science. 1996; 274: 1194-1197Crossref PubMed Scopus (943) Google Scholar, 11Bang B Gniadecki R Larsen J Baadsgaard O Skov L In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells.Exp Dermatol. 2003; 12: 791-798Crossref PubMed Scopus (30) Google Scholar What remains unclear is the relative contribution of each receptor type to specific downstream signaling pathways. Studies designed to address this question will be helpful to dissect the interconnections among UVB-induced signal pathways and to build a detailed map of the signal relay mechanisms. Binding of EGF family ligands to EGFR triggers a complex network of signaling pathways, culminating in responses ranging from cell division to death, and motility to adhesion proteolysis.26Hackel P Zwick E Prenzel N Ullrich A Epidermal growth factor receptors: critical mediators of multiple receptor pathways.Curr Opin Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar, 27Zwick E Hackel P Prenzel N Ullrich A The EGF receptor as central transducer of heterologous signalling systems.Trends Pharmacol Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 28Prenzel N Fischer O Streit S Hart S Ullrich A The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification.Endocr Relat Cancer. 2001; 8: 11-31Crossref PubMed Scopus (561) Google Scholar, 29Yarden Y Sliwkowski MX Untangling the ErbB signaling network.Mol Cell Biol. 2001; 2: 127-137Google Scholar Dysregulation of EGFR family protein tyrosine kinases (HER, erbB) has been reported in multiple epithelial human cancers.30Turkeri L Erton M Cevik I Akdas A Impact of the expression of epidermal growth factor, transforming growth factor alpha and epidermal growth factor receptor in the prognosis of superficial bladder cancer.Urology. 1998; 51: 645-649Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 31Scambia G Benedetti-Panici P Ferrandina G Distefano M Salerno G Romanini M Fagotti A Mancuso S Epidermal growth factor, oestrogen and progesterone receptor expression in primary ovarian cancer: correlation with clinical outcome and response to chemotherapy.Br J Cancer. 1995; 72: 361-366Crossref PubMed Scopus (97) Google Scholar, 32Mukaida H Toi M Hirai T Yamashita Y Toge T Clinical significance of the expression of epidermal growth factor and its receptor in esophageal cancer.Cancer. 1991; 68: 142-148Crossref PubMed Scopus (116) Google Scholar, 33Garcia I Vizoso F Martin A Sanz L Abdel-Lah O Raigoso P Garcia-Muniz J Clinical significance of the epidermal growth factor receptor and HER2 receptor in resectable gastric cancer.Ann Surg Oncol. 2003; 10: 237-241Google Scholar, 34Reinartz J George E Lindgren B Niehans G Expression of p53, transforming growth factor alpha, epidermal growth factor, and c-erbB-2 in endometrial carcinoma and correlation with survival and known predictors of survival.Hum Pathol. 1994; 25: 1075-1083Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 35Skirnisdottir I Sorbe B Seidal T The growth factor receptors HER-2/neu and EGFR, their relationship, and their effect on the prognosis in early stage (FIGO I-II) epithelial ovarian carcinoma.Int J Gynecol Cancer. 2001; 11: 119-129Crossref PubMed Scopus (89) Google Scholar, 36Lipponen P Eskelinen M Expression of epidermal growth factor receptor in bladder cancer as related to established prognostic factors, oncoprotein (c-erbB-2, p53) expression and long-term prognosis.Br J Cancer. 1994; 69: 1120-1125Crossref PubMed Scopus (236) Google Scholar Accumulating evidence has expanded the role of EGFR from solely mediating responses to EGF-like ligands to being a major transducer of diverse signaling systems and a switch point for cellular communication networks.26Hackel P Zwick E Prenzel N Ullrich A Epidermal growth factor receptors: critical mediators of multiple receptor pathways.Curr Opin Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar EGFR is an essential participant in signal transduction pathways engaged by G-protein-coupled receptors, cytokine receptors, integrins, ion channels, and other tyrosine kinase receptors.26Hackel P Zwick E Prenzel N Ullrich A Epidermal growth factor receptors: critical mediators of multiple receptor pathways.Curr Opin Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar, 27Zwick E Hackel P Prenzel N Ullrich A The EGF receptor as central transducer of heterologous signalling systems.Trends Pharmacol Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 37Carpenter G Zendegui J Epidermal growth factor, its receptor, and related proteins.Exp Cell Res. 1986; 164: 1-10Crossref PubMed Scopus (153) Google Scholar, 38Carpenter G Employment of the epidermal growth factor receptor in growth factor-independent signaling pathways.J Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar Various mechanisms have been proposed for this nonclassical transmodulation of the EGFR. These mechanisms include activation by intracellular, soluble tyrosine kinases such as those of the Src family and release of membrane-bound EGFR ligands by proteolysis.26Hackel P Zwick E Prenzel N Ullrich A Epidermal growth factor receptors: critical mediators of multiple receptor pathways.Curr Opin Cell Biol. 1999; 11: 184-189Crossref PubMed Scopus (546) Google Scholar, 27Zwick E Hackel P Prenzel N Ullrich A The EGF receptor as central transducer of heterologous signalling systems.Trends Pharmacol Sci. 1999; 20: 408-412Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 28Prenzel N Fischer O Streit S Hart S Ullrich A The epidermal growth factor receptor family as a central element for cellular signal transduction and diversification.Endocr Relat Cancer. 2001; 8: 11-31Crossref PubMed Scopus (561) Google Scholar Irrespective of the mechanisms of activation, it is becoming increasingly clear that EGFR functions not only to transduce signals in response to its cognate ligands but is also critical for activation of a diverse array of other cell surface receptors.38Carpenter G Employment of the epidermal growth factor receptor in growth factor-independent signaling pathways.J Cell Biol. 1999; 146: 697-702Crossref PubMed Scopus (247) Google Scholar UVB induction of EGFR tyrosine phosphorylation has been observed in several cell types. Although UVB-irradiation is known to activate EGFR, the role of this activation in UVB-induced signal transduction is not well understood. Given the central participation of EGFR in other diverse signaling pathways, we have used both pharmacological and genetic approaches to investigate the role of EGFR in UVB-induced signal transduction pathways. HaCaT cells were generously provided by Dr. N.E. Fusenig (German Cancer Research Center, Heidelberg, Germany). Dulbecco's modified Eagle's medium, heat-inactivated fetal bovine serum, Dulbecco's phosphate-buffered saline (PBS), and trypsin 2.5% were obtained from Life Technologies, Inc. (Grand Island, NY). The B82 cell line and its derived EGFR-expressing cell line B82K+ cells were generous gifts from Dr. Gordon Gill of University of California, San Diego, CA. EGFR, ERK, JNK, and p38 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphotyrosine antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Phospho-ERK, phospho-JNK, phospho-p38, phospho-AKT, PKC, and PKA substrate antibodies were purchased from Cell Signaling Technology (Beverly, MA). c-Fos and c-Jun antibodies were purchased from Oncogene Research Products (Cambridge, MA). Sodium orthovanadate was purchased from Sigma Chemical (St. Louis, MO). PD169540, a highly specific irreversible inhibitor of EGFR and ErbB2 tyrosine kinases, was provided by Dr. David Fry (Pfizer, Ann Arbor, MI). Cells were washed twice with ice-cold PBS, scraped from the dishes in WCE buffer (25 mmol/L HEPES, pH 7.2, 75 mmol/L NaCl, 2.5 mmol/L MgCl2, 0.2 mmol/L ethylenediaminetetraacetic acid, 0.1% Triton X-100, 0.5 mmol/L dithiothreitol, 20 mmol/L β-glycerophosphate), supplemented with 10 μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mmol/L phenylmethyl sulfonyl fluoride, and 1 mmol/L orthovanadate. The cell homogenates were centrifuged at 14,000 × g for 15 minutes, and supernatants were collected and used as whole cell lysate. Human keratinocyte HaCaT cells and mouse L cell lines B82 and B82K+ cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum under 5% CO2 at 37°C. An Ultralite Panelite lamp (Daavlin, Bryan, OH) containing six FS24T12 UVB-HO bulbs was used as the UVB irradiation source. A Kodacel filter (Kodak, Rochester, NY) was used to eliminate wavelengths below 290 nm (UVC). The spectral output was 70% UVB (290 to 320 nm) and 30% UVA2 (320 to 340 nm). Because the majority of the energy was in the UVB range, we refer to this UV irradiation as UVB. Irradiation intensity was monitored and calculated with an IL1443 phototherapy radiometer and a SED240/UVB/W photodetector (International Light, Newbury, MA), positioned at the same distance from the source as the cells. For UVB irradiation, human keratinocyte HaCaT cells and B82 cells were seeded in 10-cm dishes, grown to ∼80% confluence, and then exposed to UVB while covered with a thin film (6 ml) of Dulbecco's PBS. Media were then replaced and plates were returned to the incubator for the indicated times. Sham-irradiated cells were also kept in PBS for equal amount of time without UV irradiation. Human keratinocyte HaCaT cells were harvested, and total cellular RNA was purified using a Miniprep RNA isolation kit according to the manufacturer's instruction (Qiagen, Chatsworth, CA). Reverse transcription of total RNA was performed using a TaqMan reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time RT-PCR was performed using a TaqMan Universal PCR Master Mix kit (Applied Biosystems) and a 7700 Sequence Detector (Applied Biosystems) was used to run the PCR. Primer/probe combinations were produced by the Custom Oligonucleotide Synthesis Service (Applied Biosystems). Target gene mRNA levels (number of molecules/10 ng total RNA) were normalized to mRNA levels of 36B4 (internal control housekeeping gene). Antibodies that specifically recognize the phosphorylated forms of substrates for AKT, PKC, and PKA were used to determine the role of these protein kinase pathways. The phospho-AKT substrate antibody recognizes R/K-X-R/K-X-X-S/T motif when the serine (S) or threonine (T) residue is phosphorylated.39Obata T Yaffe M Leparc G Piro E Maegawa H Kashiwag A Kikkawa R Cantley L Peptide and protein library screening defines optimal substrate motifs for AKT/PKB.J Biol Chem. 2000; 275: 36108-36115Crossref PubMed Scopus (335) Google Scholar, 40Alessi D Cudwell B Andjelkovic M Hemmings B Cohen P Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase-1 and p70 S6 kinase.FEBS Lett. 1996; 399: 333-338Abstract Full Text PDF PubMed Scopus (550) Google Scholar The phospho-PKC substrate antibody recognizes the R/K-X-S-Hydrophobic-R/K motif when the serine (S) is phosphorylated.41Pearson R Kemp B Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations.Methods Enzymol. 1991; 200: 62-81Crossref PubMed Scopus (870) Google Scholar, 42Nishikawa K Toker A Johannes F-J Songyang Z Cantley L Determination of the specific substrate sequence motifs of protein kinase C isozymes.J Biol Chem. 1997; 272: 952-960Crossref PubMed Scopus (493) Google Scholar The phospho-PKA substrate antibody recognizes the R-X-X-T-X-X-X/R-R-X-S-X-X motif when the threonine (T) and serine(S) are phosphorylated.41Pearson R Kemp B Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations.Methods Enzymol. 1991; 200: 62-81Crossref PubMed Scopus (870) Google Scholar Equal amounts of whole cell lysate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P filter paper (Millipore, Bedford, MA). Immunoreactive proteins were visualized by enhanced chemifluorescence according to the manufacturer's protocol (Amersham Biosciences, Piscataway, NJ). Quantification was performed using a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA). EGF (10 ng/ml) and UVB irradiation (40 mJ/cm2) induced tyrosine phosphorylation of EGFR in intact human keratinocyte HaCaT cells (Figure 1). However, pretreatment of cells with 200 nmol/L PD169540, which specifically inhibits EGFR and related ErbB tyrosine kinase activities through covalent modification of a cysteine residue in the ATP binding site of EGFR,43Fry D Bridges A Denny W Doherty A Greis K Hicks J Hook K Keller P Leopold W Loo J McNamara D Nelson J Sherwood V Smaill J Trump-Kallmeyer S Dobrusin E Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor.Proc Natl Acad Sci USA. 1998; 95: 12022-12027Crossref PubMed Scopus (391) Google Scholar nearly completely blocked tyrosine phosphorylation of EGFR by both EGF and UVB irradiation (Figure 1). UVB irradiation induced a sevenfold increase in phosphorylation of p42 ERK and a fivefold increase in p44 ERK phosphorylation in HaCaT keratinocytes (Figure 2A). Pretreatment of HaCaT cells with PD169540 almost completely blocked UVB activation of both p42 and p44 isoforms of ERK. Treatment of cells with PD169540 alone, in the absence of UVB irradiation, did not alter basal ERK phosphorylation (Figure 2A). UVB irradiation induced a 13-fold increase in the level of p54 JNK phosphorylation and a 20-fold increase in the level of p46 JNK phosphorylation relative to levels in untreated, nonirradiated controls. Pretreatment of cells with PD169540 inhibited UVB-induced phosphorylation of p54 and p46 JNK by 50 and 30%, respectively (Figure 2B). Treatment of cells with PD169540 alone did not alter either p54 JNK or p46 JNK phosphorylation (Figure 2B). UVB irradiation induced a threefold increase in the level of p38 phosphorylation relative to untreated, nonirradiated HaCaT keratinocytes. Pretreatment of cells with PD169540 inhibited UVB-induced phosphorylation of p38 by 50% (Figure 2C). Pretreatment with PD169540 alone did not alter p38 phosphorylation (Figure 2C). Because UVB activation of the MAP kinase pathways results in induction of c-Fos and c-Jun expression, we investigated whether EGFR activation by UVB irradiation is required for stimulation of c-Fos and c-Jun gene expression. Human HaCaT keratinocytes were pretreated with vehicle or PD169540 (200 nmol/L) and irradiated with UVB (40 mJ/cm2). Cells were harvested for real-time RT-PCR analysis of c-Fos and c-Jun mRNA at 15 to 60 minutes after UVB irradiation. UVB irradiation induced a marked increase in the level of c-Fos mRNA as early as 15 minutes after UVB, peaking at 3.5-fold the level in untreated controls at 45 minutes after UVB and remaining elevated for at least 60 minutes (Figure 3A). Pretreatment with PD169540 before UVB irradiation inhibited c-Fos mRNA induction to levels in untreated, nonirradiated controls at all time points after exposure to UVB. UVB irradiation also induced a marked increase in the level of c-Jun mRNA as early as 15 minutes after UVB, peaking at fivefold at 45 minutes after UVB and remaining elevated for at least 60 minutes (Figure 3B). PD169540 pretreatment of human HaCaT keratinocytes before UVB irradiation also reduced UVB induction of c-Jun mRNA to levels observed in nonirradiated cells at all time points after exposure to UVB (Figure 3B). We used overnight treatment for logistical reasons. To address the specificity of overnight incubation of HaCaT cells with PD169540, we determined its effect on UV irradiation induction of TNF-α gene expression. Induction of TNF-α gene expression is a well-characterized EGFR-independent response to UV irradiation. We found similar levels of TNF-α mRNA induction (approximately eightfold over the mock-irradiated cells) in vehicle- and PD169540-treated HaCaT cells (data not shown). In addition to MAP kinases, EGFR effectors include PI3K/AKT, PLC/PKC, and PKA. To determine the role of EGFR in UVB activation of these effector pathways, we used antibodies that specifically recognize the phosphorylated forms of substrate proteins, which have been phosphorylated by AKT, PKC, or PKA protein serine/threonine kinases. Human HaCaT keratinocytes were pretreated with vehicle or PD169540 (200 nmol/L), and sham- or UVB-irradiated. Whole cell lysates were prepared 10 and 20 minutes after UVB irradiation and subjected to SDS-PAGE/Western blot using phospho-AKT substrate, phospho-PKC substrate, or phospho-PKA substrate antibody. UVB irradiation induced phosphorylation of at least four AKT substrate proteins. Treatment of keratinocytes with EGFR inhibitor PD169540 before UVB irradiation markedly decreased phosphorylation of these four AKT substrate proteins (Figure 4A) ranging in size from 32 to 120 kd. The identities of these phosphor-proteins are not known. UVB irradiation also induced phosphorylation of two major PKC protein substrates of molecular weights 34 kd and 200 kd (Figure 4B). Phosphorylation of both of these protein bands was substantially reduced by pretreatment of cells with EGFR kinase inhibitor PD169540 (Figure 4B). UVB irradiation stimulated phosphorylation of at least five protein substrates for PKA, ranging in molecular weight from 30 to 150 kd (Figure 4C). UVB-induced phosphorylation of these PKA substrates was markedly reduced by EGFR inhibition by PD169540. Although the identities of the phosphorylated substrate proteins are not known at this time, the above data demonstrate that EGFR is a critical mediator of MAP kinase-, AKT-, PKC-, and PKA-mediated UVB-induced signal transduction in cultured human HaCaT keratinocytes. To further investigate the importance of EGFR in the UVB-induced signaling, we used B82 cells, which do not express EGFR, and B82K+ cells, which are B82 cells made to express EGFR by stable transfection.44Bertics P Chen W Hubler L Lazar C Rosenfeld M Gill G Alteration of epidermal growth factor receptor activity by mutation of its primary carboxyl-terminal site of tyrosine self-phosphorylation.J Biol Chem. 1988; 263: 3610-3617Abstract Full Text PDF PubMed Google Scholar, 45Davies R Grosse V Kucherlapati R Bothwell M Genetic analysis of epidermal growth factor action: assignment of human epidermal growth factor receptor gene to chromosome.Proc Natl Acad Sci USA. 1980; 77: 4188-4192Crossref PubMed Scopus (94) Google Scholar B82 a" @default.
• W2029351044 created "2016-06-24" @default.
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• W2029351044 date "2006-09-01" @default.
• W2029351044 modified "2023-09-24" @default.
• W2029351044 title "Epidermal Growth Factor Receptor Is a Critical Mediator of Ultraviolet B Irradiation-Induced Signal Transduction in Immortalized Human Keratinocyte HaCaT Cells" @default.
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