0955-2235:9? $15.00 ,i:, 1993Pergamon PressLtd Pro,sye.\s in Growth Factor Rrsearch. Vol. 4. pp. 107-120, 1992 Printed in Great Britain. All rights reserved. INTERACTIONS OF FGFs WITH CELLS Dominique Ledoux, Leila Gannoun-Zaki TARGET and Denis Barritault* Laboratoire de Recherche sur la Croissance Cellulaire, La Reparation et la Regeneration Tissulaires, Jeune Formation INSERM n 9014 Universite Paris XII-Val de Marne Avenue du General de Gaulle, 94010 Creteil Cedex, France Growth factors play a key role in cellular communication. a necessary step for the development ofpluricellular organisms. Thejbroblast growth factors (FGF) are among these polypeptides andhave seven known members: FGF I to FGF 7 which are also known as acidic FGF, basic FGF, translation products of oncogenes hst, int 2, FGFS, FGF6 and FGF 7 or keratinoc.vte growth factor (KGF) respectivelyfl].? The best known and the most abundant in normal adult tissues are acidic and basic FGFs, or FGF I and 2 respectively, which have been subjected to extensive studies both in vitro and in vivo. These two factors have almost ubiquitous distribution and a wide spectrum of biological activity including action on cellular proltferation and differentiation. as well as neurotrophic and angiogenic properties[l]. These different activities are induced by triggering spec$c receptors present at the surface of the target cell. Following this interaction, the FGF-receptor complexes are internalized and activate intracellular pathways. An important effort of investigations has been produced to characterize these receptors and intracellular pathways. It is the purpose of this review to present this work which willfocus on FGFs I and 2. The existence of two classes of interactions has been reported as early as 1987 (52,53.54] suggesting the presence of high and low r@init~~ receptors for FGFs. Keywords:FGF, receptor, heparan sulfate, internalization. HIGH AFFINITY Biochemical RECEPTORS TO FGFs 1 AND 2 Characterization and Cellular Distribution The presence of high affinity receptors for FGFs 1 and 2 has been reported for many cell types as shown in Table 1 in agreement with previous observations describing a biological activity of FGF 1 on these cells [I]. These receptors are characterized *To whom correspondence should be addressed. tThe accepted nomenclature after the New York Academy of Sciences Meeting in La Jolla, CA. January 1618, 1991. 107 108 D. Ledoux et al. TABLE 1. Identification of cellular receptor to FGFs 1 and 2. References Endothelial cells Hepatoma cells Myoblasts Vascular smooth muscle Fibroblasts (3T3) Fibroblasts (BHK) Fibroblasts (CCL39) Chondrocytes Lens epithelial cells Fetal neurons External segment from retina Adrenocortical Bladder tumor Neuroblastoma MCF7 breast cell Pheochromocytome (PC12) FGFI,FGF:! FGF 1 FGF I FGF 1 FGF I FGF I FGF 1 FGF 2 FGF l,FGF2 FGF 2 FGF 2 FGF 2 FGF 2 FGF l,FGF2 FGF 2 FGF I FGF 2 FGF 2 FGF l,FGF2 24 108 IO,38 109 10.38 9,38,52 3.9.52.53 52 51 15 2 55 82 11 57 56 I IO by apparent dissociation constants ranging from 10 PM to 600 PM for both forms of FGFs. The number of sites per cell varies from 2000 to over 100 000 according to the cell type studied. Cross-linking experiments followed by polyacrylamide gel electrophoresis of the complexes formed between labelled FGF and the putative receptors allow the identification of several molecular entities which can bind specifically to FGFs 1 and 2. According to the cell types, the apparent molecular weight of these entities range from 105 to 165 kDa. The absence of modification of the electrophoretic migration patterns after treatment with reducing agents suggests that these high affinity binding sites have monomeric structures [2,3]. Further studies indicate that FGF 2 receptors on BHK cells are glycosylated on asparagine residues [4] and that the glycosylated moities (25-30 kDa) are important for proper FGF-receptor recognition. Different degrees of glycosylations are believed to explain some of the variations observed in the molecular weights of these receptors as mentioned above [5]. Despite this heterogeneity, the great majority of cellular receptors for FGF is common to FGF 1 and FGF 2 [&lo]. Only very few cases have been reported where a specific biological property was induced by receptors which were not recognized by both FGFs. As an example, we can mention a cell line derived from bladder carcinoma epithelium where FGF 1 was able to induce cellular changes in morphology and in cellular motility by interacting with high affinity receptors which could not be detected with FGF 2 [l 11. A particular high affinity receptor to FGF 1 has been identified on parathyroid cells [12]. This receptor contains several heparan sulfate chains which represent 10-l 5% of the molecular mass and are needed for the recognition and the proper binding of FGF 1. The variation in the number of FGF receptors appears as being an important event Interactions of FGFs with Target Cells IO9 in some biological processes such as cellular differentiation or embryonic induction. In the course of myogenic differentiation, receptors to FGFs are expressed during myoblast proliferation and gradually disappear when cells stop proliferating, align and fuse [13,14]. Terminal differentiation of chondrocytes is also associated with a reduction in the detectable receptors to FGF 2 [15]. In the developing Xenopus embryo, maximal expression of FGF 2 receptors occurs at stage 8 in association with mesodermal induction [16]. In the chicken, the number of receptors remains constant until day 7, then drastically drops [ 171. 112vitro studies indicate that the number of FGF receptors could be regulated by cellular density [3,18,19] or by other molecules such as phorbol esters [20]. A diminution in the number of receptors is also observed after FGF binding to its receptor and subsequent internalization [3,21,22]. FGF affinity for their receptors can also be modulated positively or negatively by a whole range of molecules. Heparin increases FGF I affinity to its receptors [23-251 while protamine, suramin and several basic proteins inhibit FGF 2 receptor binding [26-281. Purification and Primary Structure Like other membrane proteins, cell surface receptors are difficult to purify in a biological active form, i.e. to bind to FGF. Several forms of FGF receptors have been partially or totally purified from various sources: human hepatoma cells [29], epithelial lens cells [30], adult rat brain [31], bovine brain [32], 6 and 7 day old chick embryos [33,34]. All purification procedures include affinity chromatographies on WGA and on immobilized FGF. Purified receptors were obtained with molecular weights between 100 and 160 kDa. However, only protein sequence information was obtained from a purified form of the FGF receptor from more than 20,000 7 day old chick embryos [34]. This sequence information was identical to parts of primary sequences of the product of theflg gene (@s-like gene) [35]. A portion of this gene was previously isolated from a cDNA bank gene from human endothelial cells and it presented homology with the proto-oncogene ~fins. Based on this homology, a clone with the whole cDNA was isolated and the entire sequence of FGF 2 chicken receptor was deduced. Analysis of this primary sequence indicated that this trans-membrane protein possessed a tyrosine kinase domain and an organization analogous to other tyrosine kinase receptors [36]. Up to now. several other groups have identified multiple forms of FGF receptors presenting sequence homology withJEg. Four distinct families of receptors have been described, namely: FGFRl (f7g), FGFR2 (bek), FGFR3 and FGFR4 [37]. TISSUE DISTRIBUTION OF FGF RECEPTORS AND THEIR mRNAs Parallel studies were performed to characterize FGF receptors from cell cultures or from membrane preparations from chick or mouse embryos [33,34,38], mouse placenta [39], bovine brain [40] or various adult guinea pig tissues [41,42]. These receptors possess analogous characteristics to those identified from cell cultures. Kd values are from lo-30 pM for FGF 2 and loo-180 pM for FGF 1 as well as proteic entities with 110 D. Ledmu et ul. molecular weights ranging from 125 to 200 kDa. Messenger RNA transcripts coding for FGFRI, FGFR2 and FGFR3 are found in most adult tissues of adult guinea pig, rat and mice [42-451. FGFR4 mRNA presents a more restricted distribution and is mainly found in the lung [46,47]. However, in adult tissues, the proteins corresponding to these mRNA transcripts are difficult to detect. Indeed, it was not possible to combine cross-linking or immunoblots to detect high affinity receptors for FGFs in membranes of liver, kidney, stomach, lung, intestine or spleen although the mRNA was detected [41,42,48,49]. In contrast, high affinity receptors could be easily detected in adult brain membranes [4042]. In order to compare possible homologies between these high affinity brain derived FGF receptors and the other families of FGFR, covalent complexes between FGF and these membrane receptors were immuno-precipitated with anti-FGFRl and antiFGFR2 antibodies. The results obtained indicated that brain derived high affinity receptors are immunologically distinct from FGFRl and FGFR2 although these two forms of receptors are functional in the brain but present in minor quantities [48]. Measurement of the content of mRNA transcripts for FGFRl, FGFR2 and FGFR3 also indicates that these transcripts are no more abundant in adult brain than in other tissues [42,43,45]. Altogether, these results suggest the existence of another form of high affinity FGF receptor in adult brain different from those already known. These receptors could be similar to those lacking tyrosine kinase activity [33,50]. The presence of large amounts of high affinity receptors for FGFs only in adult brain compared to other adult organs suggests that these factors may not only play a role in homeostasis or tissue repair but may also fulfil other task functions in the nervous system e.g. a neurotrophic function, which could be mediated through specific neuronal receptors. LOW AFFINITY BINDING SITES TO FGFs 1 AND 2 A second family of interaction sites for FGF 1 and 2 has been described as low affinity binding sites on a large number of cell types including fibroblasts [51-531, endothelial cells [52,54], epithelial cells [52], neurons [55], tumoral cells such as bladder carcinoma cells [I 11, breast cancer cell line [56] and neuroblastoma cells [57]. Basement membranes were also shown to contain low affinity binding sites which were detected in membranes from embryonic [58], adult [59] or tumoral tissues [60]. These binding sites are characterized by an apparent Kd of between 2-20 nM and a number per cell ranging from 0.5 x 10h to several million. Incubation of the cells with pg/ml quantities of heparin or heparan sulfate inhibits the interaction of basic FGF with these low affinity sites while other glycosaminoglycans (GAG) such as dermatan, keratan or chondroitin sulfate have no effect [53]. Furthermore, interaction of FGF 2 with these low affinity binding sites is strongly reduced when the cells are pre-treated with heparinase [5153,588601. These results suggest that low affinity binding sites are related to proteoheparan sulfates. Proteoheparan sulfates are essentially localized in cellular membranes and in extracellular matrices and represent nearly as much as 70% of matritial glycosaminoglycans [61]. Proteoheparan sulfates bind specifically FGF 1 and FGF 2. They have been identified from murine sarcoma [60], endothelial cells and their conditioned medium [62,63] or their extracellular matrix [64]. Two major entities have been characterized: a proteoglycan of 250 kDa which appears to be a plasmic membrane component and an 800 kDa which belongs to the extracellular matrix. Interactions ?f‘FGFs with Target Cells 111 Recently, a 200 kDa proteoheparan sulfate which can specifically bind FGF 2 hasbeen described associatedto bone marrow cell membranesvia a phosphatidyl-inosito1[65]. Another step in the understanding of the structures of low affinity FGF binding sites was reached with the cloning of the proteic core of a membrane proteoheparan sulfate (FGF-HSPG) [66]. FGF-HSPG was isolated from a cDNA bank of hamster fibroblasts on the basisof the ability of its glycosaminoglycan (GAG) chains to bind FGF 2. The 33 kDa core protein has an extracellular domain containing 6 potential sites for GAG chains, a unique trans-membrane domain and a short cytoplasmic domain (34 residues) with no particular specific sequencesuch as a tyrosine kinase domain. The primary sequence of hamster FGF-HSPG presents 85% analogy with the sequenceof a previously cloned mouse membrane proteoglycan named Syndecan [67]. Syndecan is a polymorphic molecule with heparan and chondroi’tin sulfate chains associated to a 31 kDa trans-membrane polypeptide. This proteoglycan is mainly found at the surface of epithelial cells from mature tissues(skin, liver and mammary gland) and interacts with several other constituents of the matrix (fibronectin, collagen I, 111,IV, thrombospondin) and FGF 2 [68]. Expression of Syndecan during early mouse embryonic life indicates that this proteoglycan is present at all the stagesof development (4 cell stagesto 8.5 days of embryo life) and has different localization at different stages[69]. Moreover, although the size of the Syndecan core protein remains constant during the early mouse development, the size of the intact proteoglycan does change. thus indicating developmental alterations in its glycosaminoglycan composition. It is now clearly establishedthat both forms of FGFs I and 2 are found in significant amounts in extracellular matrices interacting with proteoheparan sulfates [70-741 although it is not excluded that other matrix components can also take part in the storage of these growth factors by interacting with FGF. Matrix bound FGFs are needed for endothelial cell growth as demonstrated by using anti-FGF 2 antibodies to block their growth [75]. Like in the FGF-heparin complex, the FGF bound to proteoheparan sulfate is protected against proteolysis [62] and its biological activity potentiated [64]. The major functions attributed to matrix associatedproteoheparan sulfates are a protecting role and a site for storage of FGFs. These ideas can be enforced by studies of tissue distribution of mRNAs coding for FGFs. Indeed, although these factors were isolated from a wide range of different tissuesin large amounts, their mRNAs are practically undetectable except in brain tissues [76,77]. This suggests that under normal conditions, there is a very weak rie ww synthesis of FGFs and that these factors bound to proteoheparan sulfates are protected from proteolysis and can become bioavailable to act locally after being releasedfrom their matritial storage places [78,79]. This releasecould be due to either partial or total proteolysis of the matrix by specific proteases secreted by cells. This hypothesis was studied with the model of the mammary tumor derived cell line MCF 7 which mainly secretedcathepsin D under estrogen stimulation. In this study, cathepsin D could liberate FGF 2 from its matrix storage places and this factor which was still biologically active could stimulate MCF 7 cells to proliferate [56]. Heparinase or heparitinase can also releasematrix bound FGF and other enzymes, such asurokinase or phospholipase C, can generate the formation of a soluble heparan sulfate-FGF 2 complex in the culture medium of endothelial cells or bone marrow cells [63,65]. More recent works have found that the role of either matrix or cellular bound 112 D. Ledoux et ul. proteoheparan sulfate can be extended from one natural storage function to a role in the FGF-receptor recognition. Using mutant cells which were deficient in heparan sulfates, Yayon et al. [80] have shown that FGF 2 could not bind to its high affinity receptor vg). Similarly, heparitinase treatment of skeletal muscle, adenocortical cells or of brain membranes induces the loss of the ability for FGFs to bind to their high affinity receptors, to simulate cell proliferation and to repress myoblast terminal differentiation [48,8 1,821. Hence proteoheparan sulfates would also act as accessory molecules which after complexing to FGFs would present the growth factor in a biologically active form to its high affinity receptor. Interestingly, this mechanism doesn’t seem to induce a conformation change of the protein as shown for FGF 1 by a variety of spectroscopic techniques [83]. Furthermore, the presence of heparan sulfate may not always be an absolute prerequisite to obtain proper binding for FGF to its high affinity receptors [33]. Using a ligand blotting technique, these authors have shown that a purified 150 kDa receptor containing no heparan sulfate chain could specifically retain FGF 2. INTERNALIZATION OF FGFs 1 AND 2 As for many other growth factors, interaction with their cellular receptors at 37” C yields to a rapid internalization of the receptor-factor complex and is followed by a decrease or down-regulation in the number of receptors at the cell surface. Similar observations were performed with FGFs added on fibroblast, astrocytes, neurons and human, bovine or mouse endothelial cells [3,22,5 I,84871. FATE OF FGF 1 AND FGF 2 AT 37” C Internalization of FGF 2 by Chinese hamster lung fibroblasts (CCL39 cells) after binding to its cell surface receptors has been originally shown to be remarkably resistant to degradation [52]. Indeed, after 2 h of incubation at 37” C on CCL39 cells, two fragments of 9 and 6 kDa were formed [86]. Further studies indicate that within 30 min after internalization, these fragments can be progressively detected and that they stay stable in the cell for more than 13 h [86]. Similar studies were performed with FGF 2 on endothelial cells [22,84] and on neurons and astrocytes [87]. According to the cell studied, different size of fragments were obtained. In bovine capillary endothelial cells, a 16 kDa fragment is found [22] while others report the formation of fragments of 15, 10 and 8 kDa in human microvascular endothelial cells [84]. Three fragments of 15.5,9 and 4 kDa are also described in astrocytes and neurons [87]. Kinetic studies indicate that the 15.5 kDa fragment is first detected after 4 h and is still present after 24 h while the 9 and the 4 kDa fragments appear after 4 h in astrocytes and 16 h in neurons but remain present for 24 and 48 h in astrocytes and neurons, respectively. Similar, although less extensive, studies were performed with FGF 1. In endothelial cells, FGF 1 is cleaved in two fragments of 15 and 10 kDa after 2 h at 37” C. The fragments remain stable in the cells after 12 h of incubation [85]. Interactions CELLULAR of‘FGFs 113 with Target Cells LOCALIZATION OF FGFs 1 AND 2 AND THEIR FRAGMENTS Lysosomotrophic agents such as chloroquine inhibit FGF degradation in endothelial cells [84,85] and fibroblasts [86]. These results suggest that the FGF cleavage takes place in lysosomal vesicles. According to the state of proliferation, exogenously added FGF 2 was localized in the cytoplasm as well as accumulated in nuclear and nucleolus. This nuclear uptake was correlated with overall stimulation of transcription and more specifically with increased transcription of genes coding for ribosomal RNA in the GO to Gl transition phase [88]. A long form of FGF 2 (18.4 kDa) is found in the nucleolus of exponentially endothelial cells but is not detected in the nucleus of confluent cells [89], while a 16.5 kDa form of FGF 2 is detected at all times in the cytoplasm. Similar results were obtained with Swiss 3T3 cells where 18.4 kDa and 16.5 kDa forms were detected in both the nucleus and cytoplasm but with a higher proportion of the 18.4 kDa in the nucleus than in the cytoplasm [90]. Stimulation of quiescent 3T3 cells by [“51] FGF 2 resulted after 8 h in a nuclear accumulation representing 8% of the total cellular incorporation [90]. The cellular compartmentalization of FGF 2 has also been further studied by indirect immuno-fluorescence techniques and confirmed that a fraction of FGF 2 was concentrated in the nucleus of growing 3T3 cells, showing bright staining of the nucleoli and diffuse staining of the nucleoplasmic network [90]. In neurons and astrocytes, internalized FGF 2 is mostly found associated with lysosomes and endosomes after 4 or 16 h. However, FGF 2 is also associated with the nuclear chromatin [87]. Endogenous FGF 2 also accumulates in the nuclei of various FGF 2 producing cells and of NIH 3T3 or BHK-21 cells transfected with cDNA expressing FGF 2 [91-961. Recently the interacting DNA sequences for FGF 2 were isolated from FGF 2 synchronously stimulated 3T3 cells using a DNA-protein cross-linking agent and a gel retardation technique from restriction enzymes (Dra-Hat III) DNA fragments. With this approach, the FGF 2 interacting nucleolus DNA sequences were shown to correspond to (GT) 28 putative ZDNA sequences located upstream of the spacer promoter [90]. Less is known about FGF 1. Indirect evidence of nuclear localization of FGF 1 has been provided using deletion mutants which were deprived of their putative nuclear translocation sequence NYKKPKL. These FGF 1 mutants bind to their high affinity receptors and enter the cells but fail to be mitogenic [97]. THE INTERNALIZATION OF FGF IS MEDIATED AFFINITY RECEPTORS BY HIGH AND LOW Evidence on the involvement of both high and low affinity receptors in the internalization pathway of FGF 2 comes from two different sets of experiments. At equilibrium and at concentrations close to the Kd value, FGF 2 is internalized via high affinity receptors (4700 molecules/cell) and via low affinity receptors (10” molecules/cell) [5 11.However, at higher concentration, internalization does not reach an equilibrium state and no apparent saturation was observed, suggesting other internalization pathways [51]. The second set of evidence comes from the need of long lasting interaction of FGF 2 with endothelial cells to induce proliferation [98]. Cells shifted from 4” C to 37“ C in the presence of FGF 2 rapidly internalize FGF 2 114 D. Ledou.u et (11. and high affinity receptors are shown to down-regulate in the first l-2 h [3,22]. However, a parallel and continuous internalization occurs after 68 h and addition of neutralizing antibodies more than 2 h after the treatment with FGF 2 also abolishes the mitogenic affect of FGF [98]. This late internalization pathway mediated by low affinity receptors was further supported by experiments where soluble heparin inhibited the prolonged internalization of FGF 2 and its binding to low affinity sites with the same potency as by heparinase treatment which also inhibited the prolonged internalization of FGF 2 [99]. The same authors also show that internalization of low affinity binding sites resulted in a partial protection of FGF from lysosomal degradation suggesting that bFGFcel1 surface-proteoheparan sulfate complexes are maintained during the internalization process. The involvement of both high and low binding sites in internalization has also been studied in neurons and astrocytes in the presence of heparin to inhibit low affinity binding and wheat germ agglutinin which inhibits high affinity binding [87]. The three degradation fragments of FGF 2 described for astrocytes and neurons were only obtained when cells were treated with heparin, suggesting that internalization in these cells was only mediated via the high affinity receptors. To visualize the intracellular complexes. photoactivatable cross-linking agents (HSAB) have been used [loo, 1011. FGF 2 can be covalently fixed to several sites on fibroblasts ranging from 80 to 320 kDa. These sites differ from the FGF high affinity receptors described in the first part of this review. The same molecular weight complexes are also formed once the FGFs have been internalized. After pre-treatment of the cells with heparitinase, the surface or intracellular complexes are no longer detected, suggesting that heparan sulfate proteoglycans are involved in these complexes. FURTHER DIRECTIONS Many questions concerning the FGF signal transduction pathways remain to be answered. FGF is internalized via its high and low affinity receptors into the lyososomal compartments but nothing is known on the routes to the nuclei. Can both internalization pathways also provide FGF for the nuclei? What are the mechanisms for FGF transportation to the nuclei? An intracellular 24 kDa protein putative FGF transporter has been recently described [ 1021. This protein binds covalently to FGF 1 after internalization and could transport FGF 1 to the nuclei. Another possibility where a soluble receptor could transport FGFs has also been proposed [ 1031. Why is only part of the internalized FGF found associated to the nuclei? Is it the same mechanism that regulates nuclear translocation of endogenously produced FGF 2, in this case, is FGF associated with chromatin or with the whole nucleus? For some authors, translocation towards the nucleus of higher molecular weight forms of endogenously produced FGF 2 (22.5 kDa and 24 kDa) could be due to extension of the NH2 terminal part of the factor [ 104,105]. This sequence has a particularly rich content in glycine and arginine similar to the nuclear targeting sequence (NTS) [ 1061. However, the demonstration of the presence of the three forms of endogenously produced FGF 2 in the nucleus indicates that the extended NH2 terminal sequence is not always needed for nuclear targeting [94]. Among the DNA sequences bound to FGF 2, the (AC 28: GT 28) rDNA region with a potential ZDNA formation is of particular interest. The location of the (GT 28) FGF Intrructions oj‘FGFs with Target ll_S Cells 2 binding site upstream from the spacer promoter suggests that this site might act as a regulatory element of rDNA transcription. Furthermore (GT) genomic repeated sequences are widely spread and may account for the pleiotrophic effect of FGF 2 in target cells [107]. However, other DNA binding sequences might also be involved and it would be of interest to see if other members of the FGF families interact with the same sequences. Other questions concern the possible different mechanisms of internalization mediated by the high and low affinity receptors, the role of the tyrosine kinase and the function, if any, of the long lasting FGF fragments. Is there also a transportation of the high or low affinity receptors to the nuclei? Do these receptors also interact with the DNA? Other questions concerning the function of the matrix in the control of cell growth and differentiation via the storage and the transportation of the FGFs to their cellular targets must also be addressed. 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