title: Src family kinase inhibitors block amphiregulin-mediated autocrine ErbB signaling in normal human keratinocytes.
abstract: c-Src potentiates proliferation, survival, and invasiveness in response to epidermal growth factor (EGF) in human mammary carcinoma cells. Tyrosine (Tyr) 845 of ErbB1 is phosphorylated by Src and has been implicated in control of malignant behavior. Although several lines of evidence also suggest important interactions of ErbB and Src family kinase signaling in normal epithelial cells, little is known about the mechanism of this interaction. Studying normal human keratinocytes (NHKs), here we demonstrate strong expression of the Src family kinases Src, Yes, and Fyn; Src family kinase-dependent stimulation of Tyr 845 by EGF; and potent inhibition of NHK proliferation and migration by two Src family kinase inhibitors PP1 and PD173952. EGF-stimulated extracellular signal-regulated kinase (ERK) phosphorylation occurred at much lower concentrations of EGF than required to phosphorylate Tyr 845. Moreover, the effect of Src family kinase inhibitors on EGF-stimulated ERK phosphorylation was transient, prompting a search for other targets of Src family kinase action. By enzyme-linked immunosorbent assay analysis, we found that three different Src family kinase inhibitors [6-(2,6-dichlorophenyl)-8-methyl-2-(4-morpholin-4-ylphenylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (PD173952), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1), and 2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (SU6656)] markedly inhibited elaboration of soluble amphiregulin by NHKs. The ErbB inhibitor PD158780 and the mitogen-activated protein kinase kinase inhibitor U0126 also markedly inhibited NHK proliferation, migration, and amphiregulin production. Together, these observations demonstrate that one or more Src family kinases act upstream as well as downstream of ErbB1 to promote amphiregulin-dependent autocrine stimulation of NHKs and suggest that autocrine NHK proliferation is more dependent upon ERK activation than upon Tyr 845 phosphorylation.
authors.affiliation: Department of Dermatology, University of Michigan Medical Center, 3312 CCGC, Box 0932, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0932, USA.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Kansra
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Stoll
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Johnson
authors.forename: James T
authors.initials: JT
authors.lastname: Elder
mesh_terms: D066258:Amphiregulin; D002478:Cells, Cultured; D066255:EGF Family of Proteins; D066246:ErbB Receptors / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006023:Glycoproteins / Q000037:antagonists & inhibitors*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D015603:Keratinocytes / Q000378:metabolism*; D010766:Phosphorylation; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D019061:src-Family Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology
chemical_list: C107875:6-(methylamino)pyrido(3,4-d)pyrimidine; C587848:AREG protein, human; D066258:Amphiregulin; D066255:EGF Family of Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011743:Pyrimidines; C015482:protein kinase modulator; D066246:ErbB Receptors; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases