Density-induced down regulation of epidermal growth factor receptors

Summary Previous studies have shown that cell density can regulate the binding of several growth factors. To determine whether cell density exerts a uniform effect on the expression of epidermal growth factor (EGF) receptors, seven cell lines were examined in detail. For each cell line, EGF binding was found to decrease as cell density increases. Scatchard analysis of the binding data reveals that decreases in EGF binding are due to reductions in the number of cell surface EGF receptors. The only apparent exception is the effect of cell density on the binding of EGF to A-431 cells. For these cells, increases in cell density lead to two effects: decreases in the number of high affinity EGF receptors and increases in the total number of EGF receptors. In addition to the effects of cell density on EGF receptors, it was determined that increases in cell density can coordinately down-regulate receptors for as many as four different growth factors. Overall, the findings described in this report for EGF and those previously described for transforming growth factor type-β (TGF-β) and fibroblast growth factor (FGF) demonstrate the existence of a common mechanism for down-regulating growth factor receptors. This work was supported by grants from the Nebraska Department of Health (89-51), the National Cancer Institute (Laboratory Research Center Support Grant, CA36727), and the American Cancer Society (Core Grant ACS SIG-16). EDITOR'S STATEMENT The paper by Rizzino et al. demonstrates that receptor number decreases as a function of cell density. This may represent a mechanism by which cell proliferation is reduced as cell density increases.     

Effects of cell density and phorbol esters on the expression of epidermal growth factor receptors

Previously, it has been shown that the binding of epidermal growth factor (EGF) by a wide range of cells decreases as cell density increases. In this report, we demonstrate that KB cells treated chronically with phorbol esters continue to exhibit decreases in EGF receptor binding as cell density increases. This finding suggests that protein kinase-C may not be essential for density-induced down regulation of EGF receptors, since phorbol esters are known to down regulate protein kinase-C. We also report that short-term and long-term effects of phorbol esters on the binding of EGF are affected by density. As shown previously for several cell lines, the phorbol ester 12-0-tetradecanoylphorbol-13-acetate transiently reduces EGF binding. We now show that the magnitude of this reduction diminishes as cell density increases. In addition, we determined that long-term treatment of KB cells with phorbol ester increases EGF binding. Again, this effect is diminished at high cell densities. Finally, we report that the increases in EGF binding induced by long-term treatment with phorbol esters are due to increases in the number of EGF receptors.Abbreviations EGF epidermal growth factor - FGF fibroblast growth factor - PBS phosphate buffered saline - PDBu 4-phorbol-12,13-dibutyrate - PDGF platelet-derived growth factor - PK-C protein kinase-C - TGF- transforming growth factor- - TPA 12-0-tetradecanoylphorbol-13-acetate     

Inefficient internalization of receptor-bound low density lipoprotein in human carcinoma A-431 cells

Human epithelioid carcinoma A-431 cells are known to express unusually large numbers of receptors for the polypeptide hormone epidermal growth factor. The current studies demonstrate that this cell line also expresses 5- to 10-fold more low density lipoprotein (LDL) receptors per cell than either human fibroblasts or Chinese hamster ovary (CHO) cells. As visualized with an LDL-ferritin conjugate, the LDL receptors in A-431 cells appeared in clusters that were distributed uniformly over the cell surface, occurring over flat regions of the membrane as well as over the abundant surface extensions. Only 4% of the LDL receptors were located in coated pits. The LDL receptors in A-431 cells showed the same affinity and specificity as the LDL receptors in human fibroblasts and other cell types. In addition, they were subject to feedback regulation by sterols in the same manner as the LDL receptors in other cells. However, in contrast to other cell types in which the receptor-bound LDL is internalized with high efficiency, in the A-431 cells only a small fraction of the receptor-bound LDL entered the cell. In CHO cells approximately 66% of the LDL receptors were located over coated regions of membrane, and the efficiency of LDL internalization was correspondingly 10-fold higher than in A-431 cells. These findings support the concept that the rate of LDL internalization is proportional to the number of LDL receptors in coated pits and that the inefficiency of internalization in the A-431 cells is caused by a limitation in the ability of these cells to incorporate their LDL receptors into coated pits.     

Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.     

Reconstitution of epidermal growth factor receptor transmodulation by platelet-derived growth factor in Chinese hamster ovary cells

Platelet-derived growth factor (PDGF) causes an acute decrease in the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an increase in the phosphorylation state of the EGF receptor at threonine654. The hypothesis that PDGF action to regulate the EGF receptor is mediated by the activation of protein kinase C and the subsequent phosphorylation of EGF receptor threonine654 was tested. The human receptors for PDGF and EGF were expressed in Chinese hamster ovary cells that lack expression of endogenous receptors for these growth factors. The heterologous regulation of the EGF receptor by PDGF was reconstituted in cells expressing [Thr654]EGF receptors or [Ala654]EGF receptors. PDGF action was also observed in phorbol ester down-regulated cells that lack detectable protein kinase C activity. Together these data indicate that neither protein kinase C nor the phosphorylation of EGF receptor threonine654 is required for the regulation of the apparent affinity of the EGF receptor by PDGF.     

Production and characterization of antibody blocking epidermal growth factor:receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover

The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF-binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.     

Transfer of functional EGF receptors to an IL3-dependent cell line

Epidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175-kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175-kD protein and used this retrovirus to infect BAF3, a murine, bone marrow-derived cell line, which is dependent on the haematopoietic factor interleukin-3 (IL3) for its growth in culture. The EGF receptors expressed in the infected cells exhibit two affinity states, as well as EGF-stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short-term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175-kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

A delayed gonadotropin-dependent and growth factor-mediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates aromatase expression in granulosa cells

Human chorionic gonadotropin and human FSH (hFSH) elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors, human chorionic gonadotropin and human FSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6-9 h. Both the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of protein kinase A, the epidermal growth factor receptor kinase, metalloproteases, and MAPK kinase. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C. Because the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins, we tested the effects of these inhibitors on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors, but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and, when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSH receptor. A MAPK kinase inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by epidermal growth factor-like growth factors and that the delayed effect is partially mediated by protein kinase C and acts as a negative regulator of aromatase expression.     

Production and utilization of growth factors related to fibroblast growth factor by embryonal carcinoma cells and their differentiated cells

Previous studies have established that embryonal carcinoma (EC) cells produce several different growth factors, but express few, if any, receptors for epidermal growth factor, platelet-derived growth factor, or transforming growth factor type-beta. In this study, the production and utilization of fibroblast growth factor (FGF) by EC cells and their differentiated cells were investigated. We have determined that EC cells produce a heat-labile, heparin-binding factor that competes with FGF for binding to membrane receptors and appears to be immunologically related to FGF. The same or a similar factor is produced by three different EC cell lines, including a multipotent human EC cell line. However, production of this factor is apparently reduced when each EC cell line differentiates. Unlike the parental EC cells, the differentiated cells respond to FGF by growth stimulation and the growth responses to FGF correlate with increased binding of FGF. Although the binding data indicate that both the EC cells and their differentiated cells exhibit high affinity receptors for FGF, the differentiated cells express these receptors at levels approximately 10-fold higher. These findings suggest that the FGF-related growth factor could influence the growth of EC cells or their differentiated cells.     

Transforming growth factor beta-1 positively modulates the bioactivity of fibroblast growth factor on corneal endothelial cells

Transforming growth factor beta-1 (TGF beta-1), known as an inhibitor of vascular endothelial cell proliferation in vitro, stimulates bovine corneal endothelial cells (BCE) proliferation. It also positively modulates the response of BCE cells to fibroblast growth factor (FGF) and epidermal growth factor (EGF). This effect is concentration dependent within a physiological range of TGF beta-1, but it is blocked if cells are cultured on extracellular-matrix-coated dishes instead of plastic. TGF beta-1 does not modify the number or the affinity of bFGF receptors on BCE cell surface but increases the bFGF content of these cells. This suggests that TGF beta-1 might act through regulation of bFGF synthesis in BCE cells.     

Regulation of EGF signaling by cell polarity in MDCK kidney epithelial cells.

Although the presence of a dominant basolateral sorting signal ensures that the majority of newly synthesized epidermal growth factor (EGF) receptors are delivered directly to the basolateral surface in polarized epithelial cells, a fraction of the receptors are also delivered to the apical surface. Similar to most basolateral membrane proteins, the EGF receptor has an additional signal(s) that selectively targets molecules lacking a dominant basolateral signal to the apical surface. Although the physiological relevance of signal hierarchy is not known, alternative targeting may occur in different epithelial cell types or during development. The goal of this study, therefore, was to determine the effect of membrane domain location on EGF receptor function, focusing on EGF-induced MAP kinase signaling and DNA synthesis. Whereas ligand responsiveness was restricted to the basolateral domain in Madin-Darby canine kidney (MDCK) cells expressing a normal complement of receptors, apical ligand was effective if apical receptor density was increased by overexpression of an exogenous wild-type human gene. Unexpectedly, cells expressing apically localized, cytoplasmically truncated receptors, which behave as dominant negative mutations in other cell types, were also responsive to apical EGF. The cytoplasmically truncated molecules appear to have at least two effects: first, to increase the local concentration of ligand at the apical cell surface; and second, to facilitate activation of the relatively few native EGF receptors normally located at the apical surface. These results indicate that cell context is a critical determinant of receptor mutant protein phenotype.     

Cell density regulates the number of cell surface receptors for fibroblast growth factor

Previous studies have shown that the binding of fibroblast growth factor to several different non-transformed cell lines decreases as cell density increases. However, it was not determined whether this is due to a reduction in receptor number or to a decrease in receptor affinity. In this study, we demonstrate that the reduction in fibroblast growth factor binding is due to a reduction in receptor number. In addition, flow cytometric analysis indicated little or no difference in the cell cycle distribution of the cells at low and high cell densities, yet the binding of fibroblast growth factor was reduced substantially at high cell densities. Thus, the reduction in growth factor binding observed in this study does not appear to result from cell cycle-dependent regulation of growth factor receptors.     

Localization of neuropeptides in efferent terminals of the eye in the marine snail,Bulla gouldiana

Summary Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of ¹²⁵I-NGF to solubilized receptors. Analysis of ¹²⁵I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.Abbreviations DRG dorsal root ganglia - NGF nerve growth factor - RA retinoic acid     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth

Epidermal growth factor (EGF), a low-molecular-weight polypeptide (G. Carpenter and S. Cohen, 1979, Annu. Rev. Biochem. 48, 193-216), stimulates the proliferation and keratinisation of cultured embryonic epidermis (S. Cohen, 1965, Dev. Biol. 12, 394-407) and promotes epidermal growth, thickening, and keratinisation when injected into neonatal mice (S. Cohen and G.A. Elliott, 1963, J. Invest. Dermatol, 40, 1-5). We have determined the distribution of the available receptors for epidermal growth factor in rat skin using autoradiography following incubation of explants with 125I-labelled mouse EGF. EGF receptors are detected on the epithelial cells overlying the basement membranes of the epidermis, sebaceous gland, and regions of the hair follicle all of which have proliferative capacity. In marked contrast, tissues which have started to differentiate and lost their growth potential, carry either an undetectable or sharply reduced number of EGF receptors. The EGF receptor number and receptor affinity of epidermal basal cells freshly isolated from rats of increasing age has also been determined. We find that receptor affinity remains unchanged (3.3 nM) but that basal cell surface receptor number decreases markedly with age. This decrease in receptor number is similar in trend to the known drop in basal cell [3H]thymidine labelling index which occurs over the same time period. The data suggest that the distribution of EGF receptors and EGF cell surface receptor number in skin are important in the spatial and temporal control of epithelial proliferation.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Basic fibroblast growth factor-syndecan complex at cell surface or immobilized to matrix promotes cell growth.

Promotion of cell growth and differentiation by growth factors during early development and organ formation are both temporally and spatially very precise. Syndecan is a well characterized integral membrane proteoglycan that binds several extracellular matrix components via its heparan sulfate chains and is therefore suggested to participate in cell regulation. Syndecan-like molecules, as low affinity receptors for heparin-binding growth factors, have been recently suggested to also regulate growth factor activity. Heparin/heparan sulfate interaction is required before, e.g. basic fibroblast growth factor (bFGF) can associate with its high affinity cell surface receptors and trigger signal transduction. In this paper we show that syndecan, but not free heparan sulfate chains, can simultaneously bind both bFGF and extracellular matrix molecules. Moreover, increased DNA synthesis of 3T3 cells was observed when the 3T3 cells were exposed to beads coated with the fibronectin-syndecan-bFGF complex, indicating that bFGF remains biologically active even when immobilized to matrix via the heparan sulfate chains of syndecan. Finally, when bFGF was bound to the surface of another cell type (epithelial), co-culture with 3T3 cells stimulated 3T3 cell growth. Therefore, we suggest that syndecan-like molecules may determine sites of growth factor action at cell-matrix and cell-cell interfaces.