The Fn14 immediate-early response gene is induced during liver regeneration and highly expressed in both human and murine hepatocellular carcinomas.

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 surface-localized 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 approximately 82% 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-alpha-driven and the hepatitis B virus X protein-driven transgenic mouse models of hepatocarcinogenesis. These results indicate that Fn14 may play a role in hepatocyte growth control and liver neoplasia.

The mitogen-inducible Fn14 gene encodes a type I transmembrane protein that modulates fibroblast adhesion and migration.

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 factor-inducible-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.

Angiotensin II and endothelin-1 increase fibroblast growth factor-2 mRNA expression in vascular smooth muscle cells.

The vasoactive hormone angiotensin II (Ang II) can stimulate vascular smooth muscle cell (SMC) hypertrophy and proliferation; thus, it may have an important role in the pathogenesis of hypertension, atherosclerosis and restenosis. Several studies have indicated that Ang II bioactivity on SMC may depend, at least in part, on its ability to induce the expression of polypeptide growth factors that can function in an autocrine manner. Here we report that Ang II treatment of rat aortic SMC increases fibroblast growth factor-2 (FGF-2) but not FGF-1 mRNA levels. Increased FGF-2 mRNA expression is first detectable at 30 min after Ang II addition and maximal levels are present at 8 hr. Ang II induction of FGF-2 mRNA levels is dependent on de novo RNA and protein synthesis. The Ang II effect can be blocked by treatment with either the Ang II type 1 receptor-selective antagonist CI-996 or the tyrosine kinase inhibitor genistein. The potent vasoconstrictor and SMC mitogen endothelin-1 can also induce FGF-2 mRNA levels in rat aortic SMC. These results indicate that FGF-2 gene expression is up-regulated by two distinct vasoactive peptides implicated in vascular SMC growth control in vivo.

Differential control of murine aldose reductase and fibroblast growth factor (FGF)-regulated-1 gene expression in NIH 3T3 cells by FGF-1 treatment and hyperosmotic stress.

Aldose reductase (AR) is an NADPH-dependent aldo-keto reductase implicated in cellular osmoregulation and detoxification. Two distinct murine genes have been identified that are predicted to encode proteins with significant amino acid sequence identity with mouse AR: mouse vas deferens protein and fibroblast growth factor (FGF)-regulated-1 protein (FR-1). Here we report that the AR and FR-1 genes are differentially regulated in NIH 3T3 fibroblasts. FGF-1 stimulation of quiescent cells induces both AR and FR-1 mRNA levels, but the effect on FR-1 mRNA expression is significantly greater. FGF-1 treatment also increases FR-1 protein expression, as determined by Western-blot analysis using FR-1-specific polyclonal antiserum. Calf serum stimulation of quiescent cells increases AR mRNA expression but not FR-1 mRNA expression. Finally, when NIH 3T3 cells are grown in hypertonic medium, AR mRNA levels are significantly increased whereas FR-1 mRNA levels are only slightly up-regulated. These results indicate that the AR and FR-1 genes are differentially regulated in murine fibroblasts by two different growth-promoting agents and by hyperosmotic stress. Therefore these structurally related enzymes may have at least some distinct cellular functions; for example, although both AR and FR-1 activity may be important for the metabolic changes associated with cellular proliferation, AR may be the primary aldo-keto reductase involved in cellular osmoregulation.

Postnatal regulation of fibroblast growth factor ligand and receptor gene expression in rat thoracic aorta.

Fibroblast growth factor (FGF)-1 and FGF-2 are potent angiogenic factors and vascular smooth muscle cell (SMC) mitogens in vivo. They function via binding to a family of structurally related cell surface receptors that possess intrinsic tyrosine kinase activity. Several studies have indicated that increased FGF and/or FGF receptor (FGFR) expression may correlate with adult SMC proliferation in vivo. In this study, we used Northern blot hybridization and reverse transcription-polymerase chain reaction assays to compare the FGF and FGFR mRNA levels in newborn rat aorta, where SMCs have a high replication index, to those in adult rat aorta, where SMCs are relatively quiescent. We found that FGF-2 and FGFR-2 mRNA expression was elevated 8.2- and 5.6-fold, respectively, in adult aorta. Increased FGF-2 protein expression in the adult aorta was confirmed by Western blot analysis. We also examined FGF and FGFR mRNA expression levels in SMC cultures derived from newborn or adult rat aorta. FGF-1 transcripts were more abundant in newborn SMCs whereas FGF-2 and FGFR-1 mRNA expression was higher in adult SMCs. Furthermore, FGF-1 and FGF-2 mRNA expression levels were altered by cell culture density and by serum treatment. We conclude that elevated FGF ligand and receptor expression does not always correlate with a high SMC proliferative index, that FGF-1 or FGF-2 may not be the primary mitogens responsible for newborn SMC growth in vivo, and that FGF-1 and FGF-2 may serve nonmitogenic functions within the mature, adult vessel wall.

Fibroblast growth factor-1 stimulation of quiescent NIH 3T3 cells increases G/T mismatch-binding protein expression.

Polypeptide growth factors promote cell-cycle progression in part by the transcriptional activation of a diverse group of specific genes. We have used an mRNA differential-display approach to identify several fibroblast growth factor (FGF)-1 (acidic FGF)-inducible genes in NIH 3T3 cells. Here we report that one of these genes, called FGF-regulated (FR)-3, is predicted to encode G/T mismatch-binding protein (GTBP), a component of the mammalian DNA mismatch correction system. The murine GTBP gene is transiently expressed after FGF-1 or calf serum treatment, with maximal mRNA levels detected at 12 and 18 h post-stimulation. FGF-1-stimulated NIH 3T3 cells also express an increased amount of GTBP as determined by immunoblot analysis. These results indicate that elevated levels of GTBP may be required during the DNA synthesis phase of the cell cycle for efficient G/T mismatch recognition and repair.

Heparin-binding epidermal growth factor-like growth factor regulates fibroblast growth factor-2 expression in aortic smooth muscle cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a vascular smooth muscle cell (SMC) mitogen and chemotactic factor that is expressed by endothelial cells, SMCs, monocytes/macrophages, and T lymphocytes. Both the membrane-anchored HB-EGF precursor and the secreted mature HB-EGF protein are biologically active; thus, HB-EGF may stimulate SMC growth via autocrine, paracrine, and juxtacrine mechanisms. In the present study, we report that HB-EGF treatment of serum-starved at aortic SMCs can induce fibroblast growth factor (FGF)-2 (basic FGF) gene expression but not FGF-1 (acidic FGF) gene expression. Increased FGF-2 mRNA expression is first detectable at 1 hour after HB-EGF addition, and maximal FGF-2 mRNA levels, corresponding to an approximately 46-fold level of induction, are present at 4 hours. The effect of HB-EGF on FGF-2 mRNA levels appears to be mediated primarily by a transcriptional mechanism and requires de novo synthesized proteins. HB-EGF induction of FGF-2 mRNA levels can be inhibited by treating cells with the anti-inflammatory glucocorticoid dexamethasone or the glycosaminoglycan heparin. Finally, Western blot analyses indicate that HB-EGF-treated SMCs also produce an increased amount of FGF-2 protein. These results indicate that HB-EGF expressed at sites of vascular injury or inflammation in vivo may upregulate FGF-2 production by SMCs.

Identification of a murine TEF-1-related gene expressed after mitogenic stimulation of quiescent fibroblasts and during myogenic differentiation.

Fibroblast growth factor (FGF)-1 binding to cell surface receptors stimulates an intracellular signaling pathway that ultimately promotes the transcriptional activation of specific genes. We have used a mRNA differential display method to identify FGF-1-inducible genes in mouse NIH 3T3 fibroblasts. Here, we report that one of these genes, FGF-regulated (FR)-19, is predicted to encode a member of the transcriptional enhancer factor (TEF)-1 family of structurally related DNA-binding proteins. Specifically, the deduced FR-19 amino acid sequence has approximately89, 77, and 68% overall identity to chicken TEF-1A, mouse TEF-1, and mouse embryonic TEA domain-containing factor, respectively. Gel mobility shift experiments indicate that FR-19, like TEF-1, can bind the GT-IIC motif found in the SV40 enhancer. The FR-19 gene maps in the distal region of mouse chromosome 6, and analysis of several FR-19 cDNA clones indicates that at least two FR-19 isoforms may be expressed from this locus. FGF-1 induction of FR-19 mRNA expression in mouse fibroblasts is first detectable at 4 h after FGF-1 addition and is dependent on de novo RNA and protein synthesis. FGF-2, calf serum, platelet-derived growth factor-BB, and phorbol 12-myristate 13-acetate can also induce FR-19 mRNA levels. We have also found that FR-19 mRNA expression increases during mouse C2C12 myoblast differentiation in vitro. The FR-19 gene is expressed in vivo in a tissue-specific manner, with a relatively high level detected in lung. These results indicate that increased expression of a TEF-1-related protein may be important for both mitogen-stimulated fibroblast proliferation and skeletal muscle cell differentiation.

Fibroblast growth factor-1-inducible gene FR-17 encodes a nonmuscle alpha-actinin isoform.

Polypeptide growth factor binding to cell surface receptors activates a cytoplasmic signaling cascade that ultimately promotes the expression of specific nuclear genes. As an approach to investigate the molecular mechanism of fibroblast growth factor (FGF)-1 mitogenic signaling, we have begun to identify and characterize FGF-1-inducible genes in murine NIH 3T3 cells. Here we report that one of these genes, termed FGF-regulated (FR)-17, is predicted to encode a nonmuscle isoform of alpha-actinin, an actin cross-linking protein found along microfilaments and in focal adhesion plaques. FGF-1 induction of alpha-actinin mRNA expression is first detectable at 2 h after mitogen addition and is dependent on the novo RNA and protein synthesis. Maximal alpha-actinin mRNA expression, corresponding to an approximately nineteenfold level of induction, is present after 12 h of FGF-1 stimulation. Western blot analysis indicated that FGF-1-stimulated cells also produce an increased amount of alpha-actinin protein. The FGF-1-related mitogen FGF-2, calf serum, several of the polypeptide growth factors present in serum, and the tumor promoter phorbol myristate acetate can also induce alpha-actinin mRNA expression. Finally, nonmuscle alpha-actinin mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in adult mouse intestine and kidney. These results indicate that nonmuscle alpha-actinin is a serum-, polypeptide growth factor-, and tumor promoter-inducible gene in mouse fibroblasts.

Differential regulation of acidic and basic fibroblast growth factor gene expression in fibroblast growth factor-treated rat aortic smooth muscle cells.

The acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) proteins are potent vascular smooth muscle cell (SMC) mitogens that are expressed by endothelial cells and SMCs in vivo. Overexpression of these proteins in transfected cell lines can result in autocrine transformation; therefore, the precise control of fibroblast growth factor gene expression in the vessel wall may be an important mechanism regulating vascular cell growth. In the present study, we demonstrate that bFGF can induce bFGF mRNA expression, but not aFGF mRNA expression, in serum-starved rat aortic SMCs. bFGF autoinduction is maximal at 4 hours, requires de novo RNA and protein synthesis, and is mediated predominantly by a protein kinase C-dependent signaling pathway. Furthermore, aFGF treatment of rat SMCs also increases bFGF mRNA and protein expression; however, aFGF mRNA levels are only slightly modulated. These results suggest that the local release of aFGF or bFGF within the vessel wall could promote a prolonged period of elevated bFGF synthesis. This, in turn, could be of importance in the SMC hyperplasia that occurs in response to vascular injury and during atherosclerotic plaque formation.

Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide originally purified from endothelial cell-conditioned medium. It has multiple biological activities and has been implicated in a number of human diseases, including hypertension and atherosclerosis. Contradictory reports have been published regarding whether ET-1 is a mitogen for vascular smooth muscle cells (SMC); thus, this issue is presently unresolved. In this study, we demonstrate that rat aortic SMC express functional endothelin cell surface receptors but do not proliferate when ET-1 is added to serum-free culture medium on every other day for a period of 1 week. To determine whether ET-1 could function in an autocrine manner to promote SMC growth, we transfected this same cell line with an ET-1 expression plasmid. Several independent lines expressing variable levels of ET-1 mRNA and biologically active ET-1 were obtained. Cell proliferation assays indicated that the transfected SMC line secreting the highest level of ET-1 had an enhanced growth rate when compared with untransfected or vector-alone transfected cells. The growth rate of this SMC line, but not of untransfected cells, was significantly reduced when the ETA receptor subtype-selective antagonist BQ-123 was included in the culture medium. These results indicate that constitutive ET-1 overexpression can promote SMC proliferation. Therefore, it is possible that under certain conditions ET-1 could be an important factor controlling SMC replication in vivo.

Tumor promoters induce basic fibroblast growth factor gene expression in human dermal fibroblasts.

Tumor-promoting phorbol esters have been shown previously to either induce or repress the expression of numerous cellular genes, and this property is likely to be important for the in vitro and in vivo biological effects of these compounds. In this report, we demonstrate that phorbol 12-myristate 13-acetate induces the accumulation of basic fibroblast growth factor mRNA and protein in human dermal fibroblasts. In contrast, acidic fibroblast growth factor expression was unaffected by this compound. The enhancement of basic fibroblast growth factor gene expression by phorbol 12-myristate 13-acetate was blocked by the isoquinolinesulfonamide derivative H7, a potent inhibitor of protein kinase C. Two additional tumor promoters that bind to and activate protein kinase C, phorbol 12,13-didecanoate and mezerein, also increased basic fibroblast growth factor mRNA levels. Basic fibroblast growth factor is a mitogen for many cell types and can stimulate angiogenesis; thus, some tumor promoter-induced cellular responses may be mediated by this polypeptide.