The matrix fibronectin protein is a multifunctional adhesive molecule that promotes migration and invasiveness of many tumors including melanomas. Increased fibronectin synthesis has been associated with the metastatic potential of melanoma cells; however, the molecular mechanisms underlying fibronectin overexpression during melanoma development are poorly understood. We report that hepatocyte growth factor/scatter factor (HGF) induces fibronectin expression and its extracellular assembly on the surface of melanoma cells through activation of mitogen-activated protein (MAP) kinase pathway, and induction and transcriptional activation of Early growth response-1 (Egr-1). Inhibition of B-RAF/ MAP kinase pathway by dominant-negative mutants and by U0126-abrogated HGF-induced Egr-1, and chromatin immunoprecipitation showed that Egr-1 is bound to the fibronectin promoter in response to HGF. Exogenously expressed Egr-1 increased fibronectin levels, while blockage of Egr-1 activation by expression of the Egr-1 corepressor NAB2 interfered with the upregulation of fibronectin synthesis induced by HGF, indicating that Egr-1 exerts a significant role in fibronectin expression in response to HGF. Finally, analysis of the expression pattern of fibronectin in melanoma cells demonstrated that fibronectin levels are correlated with constitutive MAP kinase signaling. Our data define a novel mechanism that might have important implications in regulation of melanoma progression by autocrine HGF signaling or by constitutive activation of MAP kinase pathway.

Ikaros-a factor that positively or negatively controls gene transcription-is active in murine adult erythroid cells, and involved in fetal to adult globin switching. Mice with Ikaros mutations have defects in erythropoiesis and anemia. In this paper, we have studied the role of Ikaros in human erythroid development for the first time. Using a gene-transfer strategy, we expressed Ikaros 6 (Ik6)-a known dominant-negative protein that interferes with normal Ikaros activity-in cord blood or apheresis CD34 ؉ cells that were induced to differentiate along the erythroid pathway. Lentivirally induced Ik6-forced expression resulted in increased cell death, decreased cell proliferation, and decreased expression of erythroid-specific genes, including GATA1 and fetal and adult globins. In contrast, we observed the maintenance of a residual myeloid population that can be detected in this culture system, with a relative increase of myeloid gene expression, including PU1. IntroductionThe transcription factor Ikaros (also known as LyF-1) was first described to interact with Cd3␦ and TdT promoters in murine thymocyte cells. 1,2 Its sequence is highly conserved between mice and humans, [3][4][5] and its expression is high in the developing and adult hematopoietic systems. 1,6 Ikaros proteins are characterized by the presence of 2 Krüppel-like zinc finger domains. The N-terminal domain is involved in DNA binding, 2,7 while the C-terminal domain is required for homo-or heterodimerization with family members and is composed of 2 zinc fingers. 8 The Ikaros gene contains 7 translated exons and encodes 9 isoforms by means of alternative splicing that alters expression of exons 3 to 6. 2,7 In these different proteins, the number of N-terminal zinc fingers varies from 0 to 4; this combination determines DNA binding affinity. At least 3 zinc fingers in this domain are necessary to efficiently bind to DNA. Ik1, Ik2, Ik3, and IKX are considered as functional isoforms. However, Ik4 can only bind DNA on palindromic sequences. 7 Ik5, Ik6, Ik7, and Ik8 are considered dominantnegative (DN) isoforms because of their capacity to bind other isoforms and their inefficiency to bind DNA. 8 Gene-targeting studies evidenced the fundamental role of Ikaros in hematopoiesis, in particular in T and B lymphocytes, natural killer (NK) and dendritic cells, and stem cells. [9][10][11][12] In addition, analysis of Ikaros L/L mice (insertion of the ␤-Gal gene in exon-2 of the Ikaros gene) revealed that Ikaros is expressed at low levels in a majority of Ter-119 ϩ erythroid cells. 10 Other studies of Ikaros DN (deletion of exon 7) and null (deletion of dimerization domain) mice show that Ikaros is important for erythroid differentiation. 13 These mice display a decrease of erythroid precursor numbers, and a drop in hematocrit levels 2 to 3 weeks after birth. In MEL (murine erythroleukemia) cells, Ikaros is associated with the chromatin remodelling PYR complex which binds to an intergenic domain between the ␥-globin and ␤-globin genes, and facilitates the switch be...

Transmissible spongiform encephalopathies are accompanied by the recruitment of microglial cells in the vicinity of amyloid aggregates of the pathological prion protein (PrPres). We previously showed that PrPres itself triggered the recruitment of microglia by interacting with neurons leading to the up-regulation of the expression level of chemokines, mainly RANTES (regulated on activation normal T cell expressed and secreted). The intracellular mechanisms underlying the PrPres-inducible expression of chemokines in this setting are not clear. Here we demonstrate that the mitogen-activated protein kinase pathway is switched on shortly after PrPres exposure to neurons leading to the expression of early growth response factor-1 (Egr-1), a transcription factor initially linked to differentiation and growth and to up-regulation of RANTES mRNA expression. PD98059, a selective inhibitor of extracellular signal-regulated kinase1/2 activation, resulted in a decrease of RANTES mRNA expression and as a consequence to the lowering of microglial cell migration. Neuronal overexpression of Nab2, a corepressor of Egr-1, produced similar effects. PrPresinduced chemoattraction is independent of the presence of PrPc and the laminin receptor on the neuronal cell surface. Our report is the first demonstration that PrPres exposure on neurons results in the activation of the MAP kinase signaling pathway that acts as a master switch to trigger neuronal expression of regulators of chemoattraction. Transmissible spongiform encephalopathies (TSEs)1 are fatal neurodegenerative disorders that include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, scrapie, and chronic wasting disease in animals. Intraneuronal vacuolization, severe neuronal cell death, astrogliosis, and microglia activation are the main hallmarks of TSEs (1). At the molecular level, TSEs are characterized by progressive cerebral accumulation of a misfolded protease-resistant isoform (PrPres) of the host-encoded cellular prion protein (PrPc). No amino acid sequence or posttranslational differences have been detected between the normal PrPc and its pathological PrPres isoform (2). The molecular changes resulting in the formation of amyloid aggregates of PrPres have been extensively studied in vitro as well as in vivo. The PrPres fibril growth is thought to occur by binding of PrPc molecules followed by their conversion into a conformation physicochemically undistinguishable to PrPres (3). Conformational changes involved in the conversion step result in a lowering in ␣-helical content and an important increase of the ␤-sheet structures (4, 5).For decades, TSEs have been widely regarded as infectious diseases lacking inflammatory component (6). However, the accumulation of PrPres is correlated with the appearance of reactive microglia and astrocytes concomitantly with neuronal cell loss (7-9). Because they are frequently present in the vicinity of PrPres aggregates, activated microglial cells could play an important role in brain damages (10, 11). Growi...