Clinical and Laboratory Investigations Dermatologie;! 199(1; 180:60-65

Thrombospondin Binding by Kératinocytes: Modulation under Conditions which Alter Thrombospondin Biosynthesis Bmce /.. Riser. James Varani, Brian J. N ickoloffRaj S. Mitra, Vishva M. Dixit Department of Pathology. University of Michigan Medical School. Ann A rbor. Mich.. USA

Key Words. Keratinocyte ■Thrombospondin • Thrombospondin receptor • Adhesion • Interferon-'/ • Interferon-p • Tumor necrosis factor

Introduction Thrombospondin (TSP). a glycoprotein present in the basement membrane at the dermal-epidermal junction in normal skin |1|. is synthesized and secreted by'keratinocytes in culture [2], Our recent studies have shown that TSP is a potent adhesion factor for keratinocytes [3]. Our studies further suggest that TSP produced by the keratin­ ocytes themselves may play a critical role in mediating their adhesiveness. This is based on the findings that TSP is more effective than fibronectin or laminin in mediating keratinocyte cell-substrate attachment in vitro; that attachment of keratinocytes in the absence of exogenous adhesion factors can be partially inhibited with antibodies to TSP. and that conditions which inhibit keratinocyte production of TSP. including growth in the presence of

high Ca24 (1.4 mM) or treatment with interferon-'/ (IFN'/). concomitantly reduce adhesiveness |3 .4 j. Like other glycoprotein components of the extracellular matrix. TSP is thought to influence cell behavior by binding to specific cell surface receptors. In Chinese hamster ovary cells and endothelial cells, heparan sulfate proteoglycans are thought to be responsible for TSP binding [5.6|. In other cells (monocytes and certain human fibrosarcoma lines) TSP binding is to an 88-kDa membrane protein that also binds OKM5 antibody [7|. Receptor moieties with char­ acteristics of an integrin have been shown to mediate TSP binding by human umbilical vein endothelial cells [8] and sulfated glycolipids may serve as a TSP rceeptor in some cells [9], At present nothing is known about the moieties which arc responsible for TSP binding in normal human keratinocytes. However, we recently reported that squa-

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Abstract. Our recent studies have shown that thrombospondin (TSP) is a potent adhesion factor for normal human keratinocytes. Stimulation of adhesion is presumed to result from the binding of TSP to high-affinity receptors on the surface of responsive cells. The present study indicates that keratinocytes bind TSP in a receptor-like manner. Binding is time-and concentration-dependent, saturable, reversible and specific. Approximately 180 ngofTSPcanbe bound per 1 x 10s cells at saturation and half-maximal binding occurs at 22 nM. A series of monoclonal antibodies to various regions of the TSP molecule were examined for effects on TSP binding and TSP-induced adhesion. An antibody directed against the heparian-bindingdomain of the TSP molecule significantly inhibited TSP binding but had no effect on adhesion. In contrast, three antibodies which recognize epitopes in the 140-kDa fragment of the molecule inhibited both binding and adhesion. In a previous study we showed that treatment of keratinocytes with interferon-'/ inhibited TSP production and inhibited adhesion under unstimulated conditions as well as in response to TSP. The present study shows that interferon'/ also inhibits TSP binding by keratinocytes. When the data from the present study are taken together with our past data, they suggest that normal human keratinocytes have the capacity to bind TSP and use this capacity to bind endogenously synthesized TSP. This provides a mechanism for utilizing the endogenously produced TSP to stimulate adhesion.

TSP Binding by Kératinocytes

Materials and Methods Keratinocyte C’tiltures Primary keraiinocytc cultures were initiated from normal human skin obtained from adults after face lift surgery using the method of Liu and Karasek 1111. Keratinocytes( 1.7-2 .(lx 10" viable cells) were seeded onto 35-mm culture dishes (Lux. Flow Laboratories. McLean. Va.) as previously described |3 |. Culture medium was modified MCDB 153 medium (KG M: Clonetics: San Diego. Calif.). This is a serum-free cul­ ture medium which contains epidermal growth factor, insulin and pitui­ tary extract. Under these conditions, the keratinocytes remained in a rapidly proliferating condition for several passages [12]. Cells were grown in a humidified incubator at 37 C in 7% Co,/93% air. and were routinely used at passage No. 2-4 in these experiments. Cytokines Recombinant IFN-y was obtained from Dr. M. Shepard (Genenteclt. Inc.. San Francisco. Calif.) and had a specific activity of 3 x 10 U/ tng. The activity of the IFN-y was originally determined based on a viral plaque neutralization assay. We routinely monitored the stock using a radioimmunoassay procedure (Ccntocour. Melvern. Pa.). IFN'-|f was obtained from Dr. T. Basham (Stanford University. Calif.) and had a specific activity of 4.5 x 10 U/ntg. The activity of the 1FN-|S was deter­ mined. based on a viral plaque inhibition assay. Recombinant tumor necrosis factor-« (TNF) was obtained from Dr. M. Shepard (Genentech. Inc.) and had a specific activity of 5 x 10 U/mg. The activity of the TNF was monitored using the cvtopathic effect on a sensitive target cell line (1.929). / Imnnhospondm and Amiihromhospoinlin Monoclonal Antibodies TSP was purified from the supernatant fluid of thrombin-activated platelets as described previously ] 13]. The purified TSP migrated as a single protein band w ith an apparent molecular weight of 180 kDa when examined by sodium dodccvl sulfate-polyacrylamide gel electrophore­ sis (SDS-PAGE) under reducing conditions. The production and char­ acterization of monoclonal antibodies (MAB) A2.5. C6.7. A 6.I. and A 1.1 directed against TSP have been described previously |3. 14 16]. Antibody A2.5 recognizes an epitope in a 25-kDa fragment of the TSP molecule which contains the heparin-binding domain. Antibody C6.7 is

directed against the platelet-binding domain while antibodies A6.1 and A 4 .1 recognize epitopes within the 70-kDa trypsin-resistant core of the ISP molecule. The MABs were purified from ascites fluid bv ammo­ nium sulfate precipitation followed by affinity chromatography on pro­ tein A-Sepharose. Purity was determined by SDS-PAGE.

zl ddirional Reagents OKM5 MAB was obtained from Ortho Diagnostic Systems. Rar­ itan. N.J. The tetrapeptide Arg-Gly-Asp-Ser (RGDS) wax obtained from Penisula Laboratories. Inc.. Belmont. Calif. Heparin was obtained from Sigma Chemical Co. T SP Rinding

TSP purified as described above was iodinated using immobilized chloraminc-T on nonporous polystyrene beads (lodo-Bcads. Pierce Chemical Co.. Rockford. III.). Free iodide was removed by filtration on a SephadexG-25 colum n.1 4-TSPof high specific activity (2.2-3.2 x 10" cpm/ug) of protein was obtained. The I -IS P prepared in this way retained biological activity (i.e.. in the adhesion assay). This material was used to assess TSP binding. The binding studies were performed with cells in monolayer culture as described previously 11()|. Cells (5.0 x lit4) were seeded into wells of a 24-well culture dish using KGM as the culture medium. After incuba­ tion for 2-3 days at 37 C and 7% CO-. the cells were washed two times and reincubatcd in MEM supplemented with 200 pg of bovine serum albumin ( BSA)/ml. Two h later. 0.5 ml of fresh MEM containing 200 ug of BSA/ml and appropriate am ountsot1 T-TSP with or without 100-fold excess ot unlabeled TSP was added to the cells. After an appropriate incubation period (normally 30 min at 25 C), the binding medium was aspirated and the monolayers were washed four times with cold binding medium.The cells were then removed by trypsinization and the entire contents were transferred to a vial for counting. The wells were then washed three additional times with binding medium and the fluid was added to the corresponding vials. All assays were run in duplicate or trip­ licate with parallel cultures used to determine cell numbers. Specific binding was defined as the total radioactivity bound minus the amount bound in the presence of 100-fold excess unlabeled TSP and was adjus­ ted in each experiment to a standard cell number. In some experiments TSP binding to keratinocytes in suspension was also determined using a silicone centrifugation assay. For this, cells were harvested with trypsin, washed two times in MEM containing 10% fetal bovine serum and incubated at 37 C in polypropylene centrifuge tubes. One h later, cells were washed and 3.5 x It)' keratinocytes in 200 pi of binding medium were added to reaction tubes along with 200 pi of an appropriate dilution of the labeled ligand. In some cases, a 100fold excess of unlabeled TSP was also added to determine nonspecific binding. After incubation for 30 min at 25 C the mixture was removed and gently lave red onto 50(1 til ofSF 1250 silicone oil (density 1.05g/cm': (ieneral Electric. Silicone Products Department. W aterford. N Y.) contained in a 1.5-ml plastic microcentrifuge tube (W alter Sarstedt. Inc.. Princeton. N.J.). Controls received all of the reactants except cells. The tube was then centrifuged for 2 min in an Eppendorf Microcentrifuge (Brinkman Model S412). After aspiration of the buffer and oil. the bottom of the microfugc tube was cut off. and the radiation in the pellet counted in a gamma counter. Adhesion Assay Kcratinocyte attachment and spreading were measured as pre­ viously described |3], Briefly, various amounts o f TSP were incubated for2 It in 24-well culture plates (Costar. Cambridge. M ass.) using KGM

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mous carcinoma cells bound TSP in a reccptor-like fashion [10]. Indirect evidence suggested that there may be more than one cell surface moiety capable of binding TSP on these cells. Furthermore, we observed within a series of tumor cell lines, a direct relationship between TSP biosynthesis and TSP binding activity. This suggested that expression of the receptor for TSP and TSP production may be coordinatcly regulated. In the present report TSP binding by normal human kératinocytes is described. These cells bind TSP in a receptor-like fashion. There appears to be at least two regions of the TSP molecule capable of interacting with kératinocytes. Kératinocyte binding of TSP may be regu­ lated jointly with TSP production since incubation of the cells under conditions which inhibit TSP production simul­ taneously reduces TSP binding.




as buffer. Following this, the nonattached protein was removed and the wells washed two times. The wells were next incubated with (1.5 ml at KGM containing 1% BSA. Keratinocytes to be used in the adhesion assay were harvested by brief trypsinization (0.03% trypsin. (1.1% F.D T A -5 min) and added to untreated wells or toTSP-eoated wells in KGM containing 1% BSA. After 1 It. the nonattached keratinocytes were removed from the wells and counted with an electronic particle counter. The w'ells were then washed twice and fixed by adding 2% glutaraldehydc. The percentage of spread cells was determined using a phase contrast microscope with a calibrated grid.

Results TSP Binding by Human Keratinocytes Keratinocytes grown for 1-3 passages in KGM were examined for TSP binding. In the first set of experiments we examined the time course of l2:'I-TSP binding. Kera­ tinocytes bound the ligand in a time-dependent manner with maximum binding occurring by 30 min (fig. I). TSP binding to keratinocytes preceded TSP-induced adhesion (fig. 1). consistent with the suggestion that the adhesion response is a consequence of ligand binding. Experiments conducted at a single time point (30 min) showed that binding of n 'I-TSP was concentration-dependent over a range of 0.5-20 ug per reaction (fig. 2). The concentration

of TSP required for half-maximal binding was approxi­ mately 5 ¡.ig per reaction (22 n;W). This is in good agree­ ment with the amount needed to produce a half-maximal adhesion response (fig. 2). Additionally. TSP binding was saturable, specific (57-70% inhibition with 100-fold excess cold TSP) and reversible (75% after 15 min). Approximately 180 ngofTSP was specifically bound per 1x 105cells at saturation yielding2 x 106 receptors per cell. To eliminate the possibility that the majority of observed TSP binding was to the extracellular matrix as opposed to the cell surface, two types of experiments were conducted. In the first, keratinocytes in monolayer were exposed to l2'I-TSP, washed as previously described, then removed by gentle scraping and assayed for radioactivity. The remaining extracellular matrix was then removed fol­ lowing exposure to trypsin and also assayed. Approxi­ mately 24% of the total binding was to the extracellular matrix and the remaining 76% to the cell surface. In the second set of experiments. 1251-TSP binding to keratin­ ocytes in suspension was examined. Table 1 shows that total binding and specific binding to cells in suspension were similar to binding to cells in monolayer. Next, a standard amount of l25I-TSP (2 ug) was incu­ bated (1 It at 37 °C) with each of a series of anti-TSP MABs

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Fig. 1. A Time course of ' I-IS P binding.The experiment was carried out as described in Materials and Methods using a fixed amount o f 1 I-TSP (2(1 tig/ well) in the presence or absence of 50-fold excess unlabeled TSP. The values shown represent the average cpm of 1 'l-TSP bound at each time point based on duplicate samples in a single experiments where differences between averages and individual values were within 10%. ■ = Total bindingtO spe­ cific binding. The experiment was repeated four 20 30 times with similar results. B Time course of TSPng,25l-TSP mediated adhesion. The experiment was carried out as described in the Materials and Methods using 20 ttgof TSP per reaction. The values are averages ± differences between individual values and aver­ ages based on duplicate samples in a single exper­ iment. The experiment was repeated three times with similar results. Fig. 2. A Concentration-dependent binding of '■'l-TSP to keratinocytes. The assay was carried out < * as described under Materials and Methods using a single time point (30 min). Each concentration of la­ beled TSP was added to the cells in the presence or TIM E (MIN) M9 T S P absence of 100-fbld excess of unlabeled TSP. The values shown are averages based on duplicate samples in a single experiment in which the individual values were within 10% o f the averages. ■ = Total bindingtO = specific binding. The experiment was carried out three times with very similar results. B Concentration dependence of TSPmediated adhesion. The experiment was carried out as described in the Materials and Methods. The values are averages ± SD based on triplicate samples in a single experiment. The experiment was repeated three times with similar results.

TSP Binding bv Kératinocytes

Modulation o f 7SB Binding Three different cytokines (i.c.. IFN-y. IFN-fi and I NF) were examined for effects on TSP binding. We recently showed that IFN-y significantly inhibited TSP production by kératinocytes [41. This was associated with reduced I SP expression on the cell surface and reduced adhesiveness. IFN-|) and TNF did not alter TSP produc­ tion but I NF in combination with IFN-y produced greater inhibition than IFN-y alone. In the present study. 1- to 2day-old kératinocyte cultures were exposed for 2 days to ROM alone or to ROM containing 1.000 U/ml of 1FN-(3. 600 U/ml of IFN-y. 500 U/ml of TNF or 600 U/ml of IFN-y

Tabic 1. Comparison of '~T-TSP binding to kératinocytes in sus­ pension and kératinocytes in monolayer culture Condition

Nanograms of TSP Nanograms of TSP (total) bound specifically bound per2 x 10'cells per 2 x it)5cells

Percent specific binding

Monolayer Suspension

27.0 ± 5 23.8 ± 3

61 70

17.0 ± 3 16.7±2

Binding studies to cells in monolayer and to cells in suspension were carried out as described under Materials and Methods.Two micrograms of l:T-TSP were used per reaction in these studies in the presence or ab­ sence of 100-fold excess unlabeledTSP. Binding was for 30 min at 25 °C. Values represent averages ± SD based on four data points.

Table 2. Inhibition of 1251-TSP binding and TSP-induced attachment and spreading Agent

Percent inhibition of I-TSP binding Attachment and spreading

MABA2.5 1(H) ug M ABA2.5 25 ug MAB A4.1 100(tg MAB A4.1 25 ug MABA6.1 100 ug MABA6.1 25 ug MABC6.7 100 ug Heparin 100 pg Heparin 25 ug OKM5 100 ug OKM5 25 ug

90 ± 4 78 ± 3 45 ± 5 24 ± 2 58 ± 4 36 + 3 79 ± 1 64 ± 1 57 + 3 0 0

0 not done 86 ± 14 not done 88 ± 10 not done 52 ± 5 0 0 0 0

The T-TSP binding assay and the adhesion assay were carried out as described under Materials and Methods. The values represent the average percent inhibition ± the difference between the individual values and the averages based on duplicate sam­ ples in a single experiment. The experiment was repeated three times with similar results.

and 500 U/ml of TNF. Binding of TSP was then deter­ mined (fig. 3). Reratinocytes treated with IFN-y bound significantly less TSP than untreated control cells. Inhibi­ tion of binding waseven greater upon combined treatment with TNF and IFN-y. Interestingly, the reduction in bind­ ing was due almost entirely to inhibition of the specific component of binding. The nonspecific component of binding remained virtually unchanged in spite of the treatments. In contrast to the effects of IFN-y and IFN-y/ TNF. TNF by itself had no effect on binding and IFN-|3 actually potentiated the specific component of binding slightly (fig. 3). In additional studies it was shown that treatment of keratinocytes for 1or3days with IFN-ygave

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and then used in binding studies. Binding experiments were carried out in the normal manner (table 2). At the same time. anti-TSP MABs were tested for their ability to inhibit TSP-induccd attachment and spreading of kératin­ ocytes. For the adhesion experiments. TSP-coated wells were prepared as described in Materials and Methods and then incubated for I h at 37 °C with antibody. Following removal of unbound antibody, kératinocytes were added and attachment and spreading determined (table 2). While all of the M ABs were able to partially block ligand binding, antibody A2.3 (which recognizes the heparin-binding domain) was the most effective. This same MAB, how­ ever. was completely unable to inhibit attachment and spreading. Flic two MABs most effective at inhibiting attachment and spreading (A 4.1 and A 6 .1) were also moderately effective at blocking I'SP-binding (45 and 58% inhibition at 100 ug). Relatively high concentrations of antibodies were used in this study because the MABs have a relatively low affinity for the ligand [3. 14—1(5] and because the antibody preparations consisted of all of the protein-A-binding moieties in ammonium-sulfate-fractionated ascites fluid. In additional experiments, heparin was added to the cells in the binding assay and in the adhe­ sion assay. Exogenous heparin mimicked the effect of MAB A2.5. That is. it significantly inhibited TSP binding (64% inhibition at 100 ug per reaction) but did not inhibit attachment and spreading (table 2). OKM5 MAB and RODS tetrapeptide were also tested for their ability to inhibit TSP binding. OKM5 has been reported to block binding of TSP to monocytes as well as to the human fibro­ sarcoma cell line. HT 1080 |7|. RODS is the adhesion tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin [ 17], Neither were able to inhibit I SP binding when the monolayers were preincubated with the agents. Likewise, our previous studies showed that neither of these agents inhibited kératinocyte attachment and spreading on TSP (3).




Fig. 3. Modulation of I- I SP binding to kératinocytes by II N-y and IFN-y in combination with TNF. Subconfluent kératinocyte cul­ tures were exposed for 2 days to KG M alone or to KG M containing 600 U/ml of IFN-y or 500 U/ml oFI'NFor 600 l l/ml of IFN-y and 500 U/ml of I N For 1.000 U/ml of IFN-p. Binding was then determined as described under Materials and Methods using 2 [ig of TSP per well. Values are the averages from four experiments. The standard deviations were within 20% of the averages. White bars = specific component of binding: hatched bars nonspecific component of binding.

similar results to those shown in figure 3 1data not shown]. Dose-response studies indicated that treatment of cells with as little as 25 U/ml of IFN-y was sufficient to inhibit TSPbinding [data not shown). This value is in good accord with the amount needed to inhibit TSP biosynthesis and to inhibit attachment and spreading [4].

Discussion The results of this study indicate that human keratinocytes bind TSP in a receptor-like fashion. Binding was time- and concentration-dependent, saturable, inhibitable with excess unlabeled TSP. and reversible. At satura­ tion. approximately 1

Thrombospondin binding by keratinocytes: modulation under conditions which alter thrombospondin biosynthesis.

Our recent studies have shown that thrombospondin (TSP) is a potent adhesion factor for normal human keratinocytes. Stimulation of adhesion is presume...
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