The binding of polypeptide growth factors to their appropriate cell surface transmembrane receptors triggers numerous biochemical responses, including the transcriptional activation of specific genes. We have used a differential display approach to identify fibroblast growth factor-1-inducible genes in murine NIH 3T3 cells. Here, we report that the fibroblast growth factorinducible-14 (Fn14) gene is a growth factor-regulated, immediate-early response gene expressed in a developmental stage-and adult tissue-specific manner in vivo. This gene, located on mouse chromosome 17, is predicted to encode an 129-amino acid type Ia membrane protein with no significant sequence similarity to any known protein. We have used two experimental approaches, direct fluorescence microscopy and immunoprecipitation analysis of biotinylated cell surface proteins, to demonstrate that Fn14 is located on the plasma membrane. To examine the biological consequences of constitutive Fn14 expression, we isolated NIH 3T3 cell lines expressing variable levels of epitope-tagged Fn14 and analyzed their phenotypic properties in vitro. These experiments revealed that Fn14 expression decreased cellular adhesion to the extracellular matrix proteins fibronectin and vitronectin and also reduced serum-stimulated cell growth and migration. These results indicate that Fn14 is a novel plasma membrane-spanning molecule that may play a role in cell-matrix interactions.Complex cellular processes such as proliferation, migration, differentiation, and apoptosis are regulated in part by a diverse group of molecules known as polypeptide growth factors. These factors act by binding and thereby activating specific transmembrane receptor tyrosine kinases. The activation of cell surface receptors by polypeptide ligands triggers downstream intracellular events, including the stimulation of protein phosphorylation cascades and the transcriptional activation of numerous genes (1, 2). Many mitogen-inducible genes have been identified, and they encode a diverse group of proteins including transcription factors, protein kinases and phosphatases, cell cycle regulators, and cytoskeletal and extracellular matrix proteins (2, 3). A recent study using cDNA microarray technology has demonstrated that Ͼ500 genes are transcriptionally activated after serum stimulation of quiescent human fibroblasts and that a subset of these genes encode proteins implicated in the wound healing process in vivo (3).Our laboratory has been studying fibroblast growth factor-1 (FGF-1) 1 -regulated gene expression in murine NIH 3T3 cells. FGF-1 (also referred to as acidic FGF) is one of the most extensively characterized members of the FGF family of heparin-binding proteins (4 -6). It is a potent mitogenic, chemotactic, angiogenic, and neurotrophic factor both in vitro and in vivo. These cellular responses are mediated via high affinity binding to a family of related membrane-spanning tyrosine kinase receptors (4 -6). We have shown by Northern blot hybridization analysis that FGF-1 stimulation of quies...

Polypeptide growth factors stimulate mammalian cell proliferation by binding to specific cell surface receptors. This interaction triggers numerous biochemical responses including the activation of protein phosphorylation cascades and the enhanced expression of specific genes. We have identified several fibroblast growth factor (FGF)-inducible genes in murine NIH 3T3 cells and recently reported that one of them, the FGF-inducible 14 (Fn14) immediate-early response gene, is predicted to encode a novel, cell surfacelocalized type Ia transmembrane protein. Here, we report that the human Fn14 homolog is located on chromosome 16p13.3 and encodes a 129-amino acid protein with ϳ82% sequence identity to the murine protein. The human Fn14 gene, like the murine Fn14 gene, is expressed at elevated levels after FGF, calf serum or phorbol ester treatment of fibroblasts in vitro and is expressed at relatively high levels in heart and kidney in vivo. We also report that the human Fn14 gene is expressed at relatively low levels in normal liver tissue but at high levels in liver cancer cell lines and in hepatocellular carcinoma specimens. Furthermore, the murine Fn14 gene is rapidly induced during liver regeneration in vivo and is expressed at high levels in the hepatocellular carcinoma nodules that develop in the c-myc/transforming growth factor-␣-driven and the hepatitis B virus X protein-driven transgenic mouse models of hepatocarcinogenesis. These results indicate that Polypeptide mitogens such as fibroblast growth factor (FGF)-1 and platelet-derived growth factor-BB stimulate cell cycle progression by binding to specific receptor tyrosine kinases and thereby activating intracellular signal transduction pathways.1 The activation of cytoplasmic signaling molecules promotes changes in gene expression that are critical for the cellular growth response. Numerous growth factor-and/or serum-inducible genes have been identified and classified into one of three groups: immediate-early, delayed-early, or late response genes.2 Immediate-early response genes are rapidly and transiently expressed following mitogenic stimulation of quiescent cells and their transcriptional activation does not require de novo protein synthesis. Delayed-early response genes are first expressed a few hours later, in the early to middle portions of the G1 phase, and transcript levels often remain elevated for the remainder of the cell cycle. Late response genes are generally expressed only during the S phase of the cell cycle. Both delayed-early and late response genes require de novo protein synthesis for their transcriptional activation. Growth factor-inducible genes encode many types of proteins, including transcription factors, cell cycle regulators, extracellular matrix proteins and metabolic enzymes. [2][3][4]

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C60OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

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Chronic incubation of 3T3-L1 adipocytes with tumor necrosis factor (TNF) induces a state of insulin resistance characterized by a diminished ability of insulin to induce phosphorylation of the beta subunit of its own receptor and insulin receptor substrate 1 (IRS-1). When adipocytes are briefly pretreated with TNF and then stimulated with insulin, tyrosine phosphorylation of IRS-1 increases above the level induced by insulin alone. By itself, TNF induces the time-dependent tyrosine phosphorylation of proteins in 3T3-L1 adipocytes. Among these is IRS-1, a docking protein with tyrosine phosphorylation sites that bind cytoplasmic signaling molecules that contain Src homology 2 (SH2) domains. TNF stimulation of 3T3-L1 adipocytes also promotes the association of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) with IRS-1 and also its tyrosine phosphorylation. In murine 3T3-L1 adipocytes, IRS-1 and PI 3-kinase phosphorylation and the association of these proteins are promoted by murine TNF, which interacts with the type 1 and type 2 TNF receptors. Human TNF, which binds to the murine type 1 TNF receptor selectively, also promotes IRS-1 phosphorylation and binding of IRS-1 to PI 3-kinase. This is the first demonstration that a member of the TNF/nerve growth factor receptor superfamily can use an IRS-1 signaling system as a component of its cellular response and provides a mechanism through which TNF receptors may engage downstream elements in signaling pathways.