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.

Differential expression of the keratinocyte growth factor (KGF) and KGF receptor genes in human vascular smooth muscle cells and arteries.

Keratinocyte growth factor (KGF) is a secreted member of the fibroblast growth factor (FGF) family of heparin-binding proteins. Studies reported to date indicate that it functions primarily as an important paracrine mediator of epithelial cell growth and differentiation. KGF appears to act via binding to a specific FGF receptor-2 isoform generated by an alternative splicing mechanism. To determine whether KGF may play a role in vascular smooth muscle cell (SMC) biology, we investigated KGF and KGF receptor gene expression in human SMC cultured in vitro as well as in several human nonatherosclerotic artery and atheroma specimens. KGF mRNA but not KGF receptor mRNA was expressed by SMCs, as determined by Northern blot hybridization analysis or reverse transcription-polymerase chain reaction assays, respectively. Additional experiments demonstrated that 1) human SMCs produce and secrete mitogenically active KGF and that 2) the cytokine interleukin-1 increases KGF mRNA and protein levels in human SMCs. We also found that KGF transcripts but not KGF receptor transcripts were expressed in control and atherosclerotic human arteries. Taken together, these results indicate that KGF is unlikely to be involved in SMC growth regulation unless it can function intracellularly or interact with a presently unidentified KGF receptor.

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.

Identification by targeted differential display of an immediate early gene encoding a putative serine/threonine kinase.

Fibroblast growth factor (FGF)-1 mitogenic signal transduction is mediated in part by gene products that are specifically expressed in response to cell surface receptor binding and activation. We have used a targeted differential display method to identify FGF-1-inducible genes in murine NIH 3T3 fibroblasts. Here we report that one of these genes is predicted to encode a novel serine/threonine-specific protein kinase. This putative kinase has been named Fnk, for FGF-inducible kinase. The deduced Fnk amino acid sequence has 49, 36, 33, 32, and 22% overall identity to mouse serum-inducible kinase (Snk), mouse polo-like kinase (Plk), Drosophila polo, Saccharomyces Cdc5, and mouse Snk/Plk-akin kinase (Sak), respectively. These proteins are all members of the polo subfamily of structurally related serine/threonine kinases. The Plk, polo, Cdc5, and Sak kinases are required for cell division. FGF-1 induction of Fnk mRNA expression is first detected at 30 min after mitogen addition, reflects transcriptional activation, and does not require de novo protein synthesis. FGF-2, platelet-derived growth factor-BB, calf serum, or phorbol myristate acetate treatment of quiescent cells also induces fnk gene expression. Fnk mRNA is expressed in vivo in a tissue-specific manner, with relatively high levels detected in newborn and adult mouse skin. These results indicate that Fnk may be a transiently expressed protein kinase involved in the early signaling events required for growth factor-stimulated cell cycle progression.

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.

A delayed-early gene activated by fibroblast growth factor-1 encodes a protein related to aldose reductase.

The addition of polypeptide mitogens to quiescent cell lines induces the expression of various gene products, some of which are likely to perform functions critical for cell cycle progression, DNA synthesis, and mitosis. We have used a differential display approach to identify fibroblast growth factor (FGF)-1-inducible genes in NIH-3T3 cells. One of these genes, termed FGF-regulated (FR)-1, encodes a 316-amino acid protein with approximately 82% amino acid sequence identity to an abundant protein expressed in mouse vas deferens and approximately 70% identity to human aldose reductase. The function of the vas deferens protein is unknown; however, aldose reductase is an NADPH-dependent monomeric oxidoreductase implicated in the pathogenesis of diabetic complications. FGF-1 induction of FR-1 mRNA expression is first detectable at 4 h after mitogen addition and is dependent on de novo RNA and protein synthesis. FGF-2 or phorbol ester treatment can also increase FR-1 mRNA levels; in contrast, whole blood serum or individual growth factors present in serum have only minimal effects on FR-1 mRNA expression. FR-1 mRNA is detectable in a number of mouse tissues but is most abundant in newborn liver and in adult intestine, ovary, and testis. These results raise the possibility that aldose reductase-related proteins may play a role in FGF-1- and FGF-2-stimulated mitogenesis.

Fibroblast growth factor-1 induces phosphofructokinase, fatty acid synthase and Ca(2+)-ATPase mRNA expression in NIH 3T3 cells.

Polypeptide growth factors act in part by inducing the expression of specific proteins that perform functions critical to cell cycle progression. We have used a differential display technique to identify genes that are expressed at higher levels following fibroblast growth factor (FGF)-1 (acidic FGF) stimulation of quiescent murine NIH 3T3 fibroblasts. Three such genes--liver (B-type) phosphofructokinase (PFK), fatty acid synthase (FAS) and sarco(endo)plasmic reticulum Ca(2+)-ATPase type 2 (SERCA2)--are described in this report. The level of FAS and SERCA2 mRNA expression is increased rapidly after FGF-1 addition; in contrast, PFK mRNA is induced with kinetics more typical of delayed-early genes. These results indicate that enhanced expression of the PFK, FAS and SERCA2 proteins may be important for FGF-1-stimulated cell proliferation.

Distinct patterns of expression of fibroblast growth factors and their receptors in human atheroma and nonatherosclerotic arteries. Association of acidic FGF with plaque microvessels and macrophages.

Because fibroblast growth factors (FGFs) modulate important functions of endothelial cells (EC) and smooth muscle cells (SMC), we studied FGF expression in human vascular cells and control or atherosclerotic arteries. All cells and arteries contained acidic (a) FGF and basic (b) FGF mRNA. Northern analysis detected aFGF mRNA only in one of five control arteries but in all five atheroma tested, while levels of bFGF mRNA did not differ among control (n = 3) vs. plaque specimens (n = 6). Immunolocalization revealed abundant bFGF protein in control vessels (n = 10), but little in plaques (n = 14). In contrast, atheroma (n = 14), but not control arteries (n = 10), consistently exhibited immunoreactive aFGF, notably in neovascularized and macrophage-rich regions of plaque. Because macrophages colocalized with aFGF, we tested human monocytoid THP-1 cells and demonstrated accumulation of aFGF mRNA during PMA-induced differentiation. We also examined the expression of mRNA encoding FGF receptors (FGFRs). All cells and arteries contained FGFR-1 mRNA. Only SMC and control vessels had FGFR-2 mRNA, while EC and some arteries contained FGFR-4 mRNA. The relative lack of bFGF in plaques vs. normal arteries suggests that this growth factor may not contribute to cell proliferation in advanced atherosclerosis. However, aFGF produced by plaque macrophages may stimulate the growth of microvessels during human atherogenesis.

Elevated expression of basic fibroblast growth factor in an immortalized rabbit smooth muscle cell line.

Intimal smooth muscle cell accumulation is regarded as an important component of atherosclerotic plaque formation, angioplasty-induced restenosis, and vascular graft occlusion. Vascular smooth muscle cells can both express and respond to acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF); therefore, under certain conditions these polypeptides may regulate smooth muscle cell growth in an autocrine manner. Previous studies using smooth muscle cells cultured in vitro have identified factors that can enhance aFGF and bFGF gene expression. In this study, we assayed fibroblast growth factor gene expression in a spontaneously immortalized rabbit smooth muscle cell line. In contrast to "normal" rabbit smooth muscle cells, these immortalized cells acquire an altered morphology and enhanced proliferative rate during; cell passaging in vitro. Both "normal" and immortalized rabbit smooth muscle cells express bFGF but not aFGF transcripts. RNA gel blot hybridization, reverse transcription/polymerase chain reaction amplification, and Western blotting techniques demonstrate that bFGF expression in the immortalized smooth muscle cell line increases as a function of passage level. This continuous cell line should prove valuable for studying both the regulation of bFGF synthesis and the control of vascular smooth muscle cell proliferation.

Endothelin-1 and endothelin receptor mRNA expression in normal and atherosclerotic human arteries.

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide implicated in a number of human diseases including atherosclerosis. ET-1 binds to two distinct G protein-coupled receptors, known as the ETA and ETB receptor subtypes. In this study, we have examined ET-1, ETA and ETB mRNA expression levels in human vascular cells cultured in vitro and in normal and atherosclerotic human arteries. The results indicate that (a) ET-1 mRNA is constitutively expressed by endothelial cells but not by smooth muscle cells, (b) endothelial cells express only ETB mRNA but smooth muscle cells co-express ETA and ETB mRNA, and (c) in comparison to normal aorta, ET-1 mRNA expression is elevated and endothelin receptor mRNA expression is repressed in atherosclerotic lesions.