Association of EGF and LDL receptors with the cytoskeleton of cultured keratinocytes

Cell Biology international

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ASSOCIATION OH' PCP AND CYTO~LETONOS'CULTURED~TI-S Marinus F.W. te Pasl' Bergen en Henegouwen", Havekes3), and Johannes 1)

2) 3)

LDL

989

1990

RECERTQRS

WITH

TEB

Maria Ponecl', Paul Paola Lombardi3', Boonstra"

M.P. Louis

van M.

Department of Dermatology, University Hospital, The Netherlands; Leiden, Department of Molecular Cell Biology, University of Utrecht, Utrecht, The Netherlands; Gaubius Institute, Leiden, The Netherlands.

ABSTRACT isolated In this paper we demonstrate that cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This the binding is 300% higher than approximately binding of intact cells. cell carcinoma In contrast, various squamous under lines or normal cultured keratinocytes EGF differentiation conditions exhibit retarding binding to isolated cytoskeletons which is around lo-20% of the binding to intact cells. Incubation of of arotinoid normal keratinocytes in the presence ethyl sulfone resulted in a marked decrease of the ability of differentiate, and a the cells to decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding. INTRODUCTION Proliferation and differentiation of keratinocytes in cell culture requires the presence of a variety of factors, such as epidermal growth factor insulin, transferrin and (EGF) , hydrocortisone (Hheinwald and Green 1977; Taylor et al., 1985). These factors exert their effects on the cells via interaction with specific plasma membrane 0309-l

651/90/l

10989-l

l/%03.00/0

@ 1990 Academic

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or cytoplasmic receptors (Boonstra et al., 1985). The expression of the receptors on the cell surface is an essential feature in the regulation of cell proliferation and differentiation. Therefore, we have studied recently the regulation of EGF and low density lipoprotein (LDL) receptors in normal and a variety of transformed keratinocytes in relation to their differentiation capacity (Te Pas et al., 1989; Ponec et al., 1985; 1989). These studies revealed that regulation of EGF and LDL receptor expression occurs at the DNA level (gene amplification), mBNA and protein level, depending upon the cell line. In addition to regulation of receptor biosynthesis and processing, the composition of the plasma membrane has been demonstrated to play an important role in receptor function in keratinocytes (Ponec et al., 1987a, 1987b). In normal keratinocytes the regulation of EGF receptor expression appears to occur post-translationally. In this respect it is of interest that it has been demonstrated recently that a wide variety of plasma membrane located receptors are associated to the cytoskeleton, the EGF receptor being no exception (Wiegant et al., 1986; Van Bergen en Henegouwen et al., EGF binding studies 1989). demonstrated that high-affinity EGF receptors in particular are associated to the cytoskeleton (Van Bergen en Henegouwen et al., 1989). This finding is of interest since it has been demonstrated recently that the high-affinity receptors play a major role in EGF-induced signal transduction (Defize et al., 1989: Bellot et al., 1990). interaction of the EGF We now report the normal receptor with the cytoskeleton in keratinocytes and various transformed keratinocyte cell lines. In LDL-receptor addition, the association investigated to the cytoskeleton was because we observed a great deal of have similarities between the regulation of EGF and LDL receptor expression in cultured keratinocytes (Te The external Ca*+ conditions (0.06 Pas et al., 1989). mM) exhibit EGF receptor-cytoskeleton other cell lines (Van interaction as observed in However, when Bergen en Henegouwen et al., 1989). normal keratinocytes were cultured under normal Ca*' conditions, the fraction of EGF receptors associated to the cytoskeleton was greatly increased compared were t0 lines. Similar observations other cell obtained for the LDL receptor.

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culture

Human normal keratinocytes were isolated from described in juvenile cultured as foreskin and detail previously (Boonstra et al., 1985). Culture carcinoma cell conditions and origin of squamous keratinocytes lines transformed and sv40 (SCC) (SVK,,) have been described previously (Boonstra et al., 1985). For experiments, differentiation-inhibition normal keratinocytes were grown for two weeks under low calcium deprived of conditions in medium refed under hydrocortisone. than The cells were yellow light 10, 7 and 4 days prior to the EGF binding experiment with culture medium of the same composition supplemented with 3 pM retinoid Ro 151570 (Hoffmann-La Roche, Retinoid stock Basel). solutions (2 mM) in ethanol were stored frozen in liquid nitrogen. Two days prior to the EGF binding experiment the culture medium was renewed either with medium containing normal calcium low or concentration with or without Ro 15-1570.

EGF and LDL binding

studies

Specific EGF binding was determined essentially as described previously (Boonstra et al., 1985) using 2 lz51-EGF. EGF (receptor grade; q/ml Collaborative Research, Waltham, Lexington, MA) was radiolabeled with lZ5 [I] (Amersham, the UK) by chloramine T method. LDL binding was determined described previously (Te Pas et al., 1989) usinga? pg/ml 1251LDL. Human LDL were isolated as described (Te Pas et al., 1989). Cytoskeletons were isolated either before or after the binding assays as indicated, using 0.5% Triton X-100 in 25 mM Hepes (pH 7.4) containing 1 mM PMSF as described (Van Bergen en Henegouwen et al., 1989).

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RESULTS EGB-receptor

cytoskeleton

association

normal binding determined in EGF was transformed SCC-12F2 cells, keratinocytes and in grown either under low (0.06 mM) or normal (1.6 mt4) Under normal Ca2+ concentration, the Ca'+ conditions. high capacity to exhibit a relatively cells plasma number of have a low differentiate and while the sites, located EGF binding membrane low Ca'+ conditions under opposite is measured In order to compare the (Boonstra et al., 1985). effects of a Triton X-100 extraction before or after the binding assay in cells grown either under low or exhibiting a thus and normal Ca2+ conditions, different number of cell surface receptors (Boonstra normalized taking the data were et al., 1985), binding on intact cells as a reference. As shown in 1 a Triton X-100 extraction following "'1-EGF Fig. binding resulted in 60-70% decrease of EGF binding in SCC-12F2 cells grown under both Ca2+ conditions. scGl2F2

a

Fig.

1:

noml

123 123 123 123 and normal SCC-12F2 cells binding in EGF Cells were cultured under low or normal keratinocytes. under Materials and Ca'+ respectively, as described Results are normalized taking for each set of Methods. data results obtained without incubation with Triton 1, binding to intact cells; Numbers refer to: x-100. followed by Triton X-100 to intact cells 2, binding 3, binding to isolated cytoskeletons. extraction;

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1990

cell-surface, represent the These binding sites cytoskeleton-associated EGF receptors. Isolation of of SCC-12F2 cells followed by EGF cytoskeletons binding yielded a 70-80% decrease of EGF binding as compared to intact cells (Fig. 1). Similar results carcinoma cell were obtained using other squamous are in lines the data (not shown). As such, EGF association of agreement with data on the receptors with the cytoskeleton of A431 cells (Van Bergen en Henegouwen et al., 1989). normal Similar results were obtained in keratinocytes cultured low Ca2+ conditions under (Fig. 1). However, isolation of cytoskeletons from normal normal Ca2+ keratinocytes grown under conditions followed by "1-EGF binding revealed a in binding (Fig. l).These results strong increase indicate that these cells contain a high number of intracellular, cytoskeleton-associated receptors, in so-far (Van studied all cell contrast to lines Bergen en Henegouwen et al., 1989). The fraction of cell-surface, cytoskeleton-associated receptors in these cells, as determined by binding followed by Triton X-100 extraction, is comparable to SCC-12F2 and normal keratinocytes cultured under low Ca2+ conditions (Fig. 1). Effect

of

arotinoid

ethyl

aulfone

on PCB binding

In order to establish the relationship between differentiation capacity and EGF binding to isolated cytoskeletons, we studied the effects of arotinoid ethyl sulfone 15-1570). Recently we have (Ro demonstrated that Ro 15-1570 causes increased EGF binding to normal keratinocytes and a decreased ability of the cells to differentiate (Ponec et al., 1987b). Therefore, cells were grown under low Ca" conditions in the absence of hydrocortisone and incubated for 10 days with or without Ro 15-1570. Subsequently, part of the cultures were exposed to normal Ca2+ conditions for two days as indicated. As shown in Fig. 2, the Ro 15-1570 had no significant effect on EGF binding in low Ca'+-cells, although the binding of EGF to isolated cytoskeletons was lower in the presence of the retinoid. Keratinocytes exposed for two days to normal Ca2+ conditions exhibited an increased EGF binding to the cytoskeleton. However, in the

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r 0 123

Fig.

2:

123

123123

Effect of Ro 15-1570 on EGF binding. Cells were cultured for 10 days under low Ca'+ conditions in the ,absence of hydrocortison with or without 2 pM Ro 151570, as indicated. Part of the cell cultures were subsequently exposed for 2 days to normal Ca" concentrations as indicated. The relative EGF binding was calculated as in Fig. 1.

presence of decreased to Thus Ro 15-1570 intact cells of the relative These results differentiation binding sites

LDL receptor

Ro binding 15-1570 this level of values comparable to low Ca'+-cells. caused an increase of EGF binding to (Ponec et al., 1987b), but a decrease EGF binding to the cytoskeleton. keratinocyte suggest that capacity and the distribution of EGF are closely related.

cytoakaleton

interaction

Recently, we have demonstrated a great number of similarities in the regulation of EGF-receptor and LDL-receptor expression in normal keratinocytes and various transformed keratinocyte cell lines (Te Pas et al., 1989; Ponec et al., 1985; 1989). No evidence is presently available for a possible interaction between the LDL receptor and the cytoskeleton. Since LDL regulates LDL receptor expression (Goldstein and Brown, 1984), the cells were cultured in medium supplemented with lipoprotein deficient serum (LPDS) for 2 days prior to the binding assays.

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1990

995

SCG12F2

123 Fig.

3:

123

123

123

LDL binding in SCC-12F2 and normal cells Cell culture and normalization of the keratinocytes. binding data as described in Legend Fig. 1. Numbers 1 to 3 as in Legend Fig. 1.

As shown in Fig. 3, LDL binding followed by a Triton X-100 extraction in SCC-12F2 cells resulted in a strong decrease of the binding regardless of the extracellular Ca2+ concentration, comparable to that found for EGF binding (Fig. 1). However, binding of LDL to isolated cytoskeletons of SCC-12F2 cells revealed a significant higher level of binding, indicating the presence of intracellular, cytoskeleton associated LDL receptors. Furthermore, under normal Ca2+ conditions this relative amount of intracellular cytoskeleton associated-receptors was significantly higher than in cells cultured under low Ca2+ conditions. As LDL such the binding properties of SCC-12F2 cells differ from the EGF binding properties. Similar results were observed in normal keratinocytes cultured under low Ca*+ conditions while culturing normal keratinocytes under (Fig. 3), normal Ca2+ conditions resulted in a relative cytoskeleton LDL binding comparable to the EGF binding. Thus in latter these cells, the intracellular cytoskeleton-associated fraction of

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LDL binding sites exceeded the total surface binding sites by a factor of 3) -

number of cell 2.5 fold (Fig.

DISCUSSION In this paper we demonstrated that normal keratinocytes grown under normal Ca'+ exhibit a relatively high number of intracellular EGF and LDL binding sites associated with the cytoskeleton. These findings are novel; these high levels of intracellular, cytoskeleton-associated EGF binding sites have never been observed in other cell lines (Wiegant et al., 1986; Van Bergen en Henegouwen et al. 1989), nor in various squamous carcinoma cell lines (unpublished results). Further we now show that, in addition to a large number of plasma membrane located receptors, LDL receptors are also associated with the cytoskeleton. The relatively high levels of intracellular, cytoskeletonassociated LDL receptors in normal keratinocytes grown under normal Cazt conditions is in a good agreement with studies on the localization of LDL receptors in epidermis (Mommaas et al., 1990). In basal human LDL cells of normal epidermis the receptors were distributed evenly between the cell surface and the cytoplasm, while in suprabasal cells most of the LDL receptors were located inside the cells. findings These strongly suggested a relationship between the proliferative state of the cells and the presence of LDL receptors on the cell surface similar (Mommaas et al., 1990). A relationship appears to be present for the EGF receptor. At present little is known about the function of cytoskeleton receptors. It is tempting to speculate that changes in cytoskeletal organization, i.e. synthesis of various keratins (Fuchs and Green, 1980; 1981; van Muyen et al., 1987), are responsible for the observed changes. Since a great deal of receptors are localized inside the cells, it seems possible that the association to the cytoskeleton is functioning as a regulatory because the step, receptors become inaccessible to the ligand. Consequently the ligands are not able to trigger signal transduction anymore, causing the cells to differentiate. It will be future clear that

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experiments are needed to elucidate the nature associated to the receptor subpopulation cytoskeleton as well as the nature of cytoskeletal domain involved in this association.

of the the

Bellot, F., Moolenaar, W., Kris, K., Mirakhur, B., Verlaan, I., Schlessinger, J., and Ullrich, A., Felder, S. (1990) High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses. J. Cell Biol. 110, 491-502. Boonstra, J., De Laat, Epidermal growth factor to differentiation transformed keratinocytes. 433.

S.W., and Ponec, M. (1985) receptor expression related normal capacity in and Exp. Cell Res. 161, 421-

Boonstra, J., (1989)Proliferating, and Ponec, M. transformed keratinocytes cultured under low Ca2' conditions exhibit high-affinity epidermal growth factor receptors. Cell Biol. Int. Rep. 13, 609-617. Defize, L.H.K., Meisenhelder, J., Boonstra, J., Kruyer, W., Tertoolen, L.G.J., Tilly, B.C., Hunter, T Van Bergen en Henegouwen, P.M.P., Moolenaar, wzi., and De Laat, S.W. (1989) Signal transduction epidermal factor occurs growth through the by subclass of high affinity receptors. J. Cell Biol. 109, 2495-2507. Fuchs, E., and gene expression the keratinocyte.

Green, during Cell

Fuchs, E., and Green, terminal differentiation keratinocytes by vitamin

H. (1980) Changes in terminal differentiation 19, 1033-1041. H.

(1981) Regulation of cultured A. Cell 25, 617-626.

keratin

of

of human

Goldstein, J.L., and Brown, M.S. (1984) Progress in understanding the LDL receptor HMG-CoA and reductase, two membrane proteins that regulate the plasma cholesterol. J. Lipid Res. 25, 1450-1461.

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Mommaas, M., Tada, J., Wijsman, M.C., Onderwater, J.J.M., and Vermeer, B.J. (1990) Differences in low density lipoprotein receptor the expression in suprabasal layer of normal and psoriatic epidermis. J. Derm. Sci. 1, 15-22. Ponec, M., Lavrijsen, S., Havekes, Kempenaar, J., L (1985) SV,,-transformed (SVK,,) and Boonstra, J. and normal keratinocytes: the similarity in expression of EGF, LDL and glucocorticoid receptors and the regulation of lipid metabolism. J. Invest. Dermatol. 85, 476-482. Ponec, M., and Kempenaar, J., Weerheim, A., human Boonstra, J. (1987a) Differentiation of keratinocytes: changes in lipid synthesis, plasma membrane lipid composition and 1251-EGF binding upon and administration of 25-hydroxycholesterol mevinolin. J. Cell Physiol. 133, 358-364. Ponec, M., Weerheim, A., Havekes, L., and Boonstra, Effects of retinoids on differentiation, J. (1987b) lipid metabolism, epidermal growth factor and low density lipoprotein binding in squamous carcinoma cells. Exp. Cell. Res. 171, 426-435. Ponec, M., te Pas, M.F.W., and Boonstra, J. (1989). modulation of EGF receptor Growth factor receptors: during keratinocyte differentiation. In: The Dermis, Pierard and C. from biology to diseases (G.E. Mon. Derm. Pierard-Franchimont, eds.1, PP. 62-71. Liegeoises, Liege, Belgium. Epidermal (1977) and Green, H. Rheinwald, J.G., growth factor and the multiplication of cultured human epidermal keratinocytes. Nature 265, 421-424. (1985) B.L.M., and Watt, F.M. Taylor, A., Hogan, Biosynthesis of EGF receptor, transferrin receptor, and colligin by cultured human keratinocytes and the effect of retinoic acid. Exp. Cell Res. 159, 47-54. Havekes, L., M.F.W., Boonstra, J., Te Pas, Hesseling, S.C., and Ponec, M. (1989) The competence to differentiate is of transformed keratinocytes accompanied by amplification of the LDL and EGF receptor genes but not of the insulin receptor gene. Cell Biol. Int. Rep. 13, 237-249.

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1990

Van Bergen en Henegouwen, P.M.P., Defize, L.H.K., De and Kroon, J., Van Verkleij, A.J., Damme, H., Boonstra, J. (1989) Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells. J. Cell. Biochem. 39, 455-465. Van Muyen, and Ponec, M. G.M.P., Warnaar, S.O., (1987) Differentiation-related changes of cytokeratin expression in cultured keratinocytes and in fetal, new born, and adult epidermis. Exp. Cell Res. 171, 331-345. Wiegant, F.A.C., Blok, F.J., Defize, L.H.K., Linnemans, W.A.M., Verkleij, A.J., and Boonstra, J. (1986) Epidermal growth factor receptors associated to cytoskeletal elements of epidermoid carcinoma (A431) cells. J. Cell Biol. 103, 87-94. Paper received

11.07.90.

Paper accepted

20.08.90.