Inhibition of bovine gingival laminin receptor by bacterial lipopolysaccharide

1991 ArchsoralBid.Vol. 36,No. 5,pp.341-346, Printed in Great Britain. All rights reserved

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INHIBITION OF BOVINE GINGIVAL LAMININ RECEPTOR BY BACTERIAL LIPOPOLYSACCHARIDE B. L. SLOMIANY,S. SENGUPTA,J. PIOTROWSKI,F. S. SHOVLINand A. SLOMIANY Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. (Accepted 19 December

1990)

laminin receptor was isolated from bovine gingival epithelial-cell membrane. After solubilization with octylglucoside, the receptor was subjected to affinity chromatography on laminincoupled Sepharose and eluted with cation-free EDTA buffer yielding on SDS-PAGE a 67 kDa protein Summary-A

band. After radioiodination, the protein was incorporated into liposomes which displayed specificaffinity towards laminin-coated surfaces, as well as to tooth cementum. The binding of receptor protein to cementum was inhibited by lipopolysaccharide from Bacteroides gingiualis. Preincubation of cementum with the lipopolysaccharide decreased the binding of the liposomal laminin-receptor preparation by 35.8%, while a 59.2% decrease in binding occurred when the lipopolysaccharide was preincubated with the receptor, suggesting that the lipopolysaccharide interfered with the laminin binding site on the receptor. The results demonstrate the existence of a specific gingival cell-surface laminin receptor, show that it is capable of binding to cementum, and provide evidence for the disruption of this process by bacterial lipopolysaccharide. This mechanism may account for the loss of gingival attachment in the pathogenesis of periodontal disease. Key words: laminin receptor, gingival epithelium, bacterial lipopolysaccharide,

INTRODUCIION The attachment of cells to a substratum requires the specific interaction of cell-surface receptors, termed integrins, with distinct extracellular-matrix adhesive proteins, one of which is laminin (Kleinman, Klebe and Martin, 1981; Kleinman et al., 1988; Rouslahti

and Pierschbacher, 1987; Beck, Hunter and Engel, 1990). This large, multidomain, 900 kDa glycoprotein is located in the lamina lucida of basement membrane, interposed between the cell’s basal surface and the supporting matrix of typ IV collagen in the basement membrane. Globular and rod-like domains of laminin. arranged in an extended, cruciform shape, make this protein particularly well suited for mediating various types of interactions between distant sites on cells and other components of the extracellular matrix (Beck et al., 1990). Indeed, in addition to a domain binding to collagen, a cell signalling site with mitogenic action, and a region involved in calcium-dependent aggregation, the protein also has receptor-mediated, cellattachment sites (Graf et al., 1987a, b; Gehlsen et al., 1988; Beck et al., 1990). To unravel the molecular basis of attachment of gingival tissue to tooth, we have sought to isolate the laminin receptor from the gingival junctional epithelium and study its adhesion to tooth cementum in the absence and the presence of Bacteroides gingivalis lipopolysaccharide. The nature of this mechanism is of vital importance to gingival health as the involveAbbreviations: GRGDSP, glycyl-L-arginyl-glycyl-L-aspartyl-

L-seryl+proline; PBS, phosphate-buffered saline; PMSF, phenylmethylsulonyl fluoride; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

interaction.

ment of subgingival microbial flora is a major factor in periodontal disease, and the pathogenic action of Gram-negative bacteria is thought to be mediated by their membrane lipopolysaccharide component (Rosen et al., 1989; Parent, 1990). EXPERIMENTAL PROCEDURES Materials

Bovine gingival tissue was obtained from Rockland Company, Gilbertsville, PA, and bovine teeth were from Max Insel Cohen Inc., Livingston, NJ. Laminin, GRGDSP, CNBr-activated Sepharose and egg-yolk phosphatidylcholine were purchased from Sigma Chemical Co., St Louis, MO. Enzymobead-lactoperoxidase-glucose oxidase system and Bio-Gel PdDG, and the reagents for SDS-PAGE gels were from BioRad, Rockville Center, NY, octyl-P-D-thioglucoside (octylglucoside) from Boehringer Mannheim, Indianapolis, IN and Dulbecco’s PBS from Gibco Laboratories, Grand Island, NY. B. gingivalis, used for lipopolysaccharide isolation, was kindly donated by Dr P. Murray, NJDS. Receptor

isolation

Gingival epithelium-cell membranes were prepared by a modification of the method of Hock and Hollenberg (1980). Fresh tissue, cut near the junctional epithelium, was suspended in ice-cold buffer consisting of 0.25 M sucrose with 25 mM tris-HCl buffer, pH 7.4 containing 1 mM PMSF, 100 TIU/ml aprotinin and 1 fig/ml leupeptin, minced, and homogenized with a Tekmar Tissumizer for 40 s. The homogenate was centrifuged at 600 x g for 15 min, the supernatant adjusted with 0.1 M NaCl and 0.2 mM MgSO,, and 341

B. L. SLQMIANY et al.

342

centrifuged at 40,000 x g for 40 min. The pellet was washed with 0.1 M tris-HCl, pH 7.2 containing 1 mM PMSF, 100 TIU/ml aprotinin and 1 pg/ml leupeptin. The final pellet was resuspended in the same buffer, treated with 25 mM octylglucoside for 30 min at 4°C and centrifuged at 105,000 x g for 90 min. The resulting supernatant was applied to the affinity matrix consisting of laminin coupled to CNBr-activated Sepharose 4B (Gehlsen et al., 1988). The column was washed with 3 ml of 0.1 tris-HCl buffer, pH 7.2, and the fractions were eluted with 2 ml buffer containing GRGDSP at 1 mg/ml followed by 2 ml of cation-free 20mM EDTA. Protein concentration of the eluted material was measured by bicinchonic acid protein assay kit, (Pierce, Rockford, IL), and the fractions were analysed by electrophoresis on SDS-PAGE using 7.5% gels (Laemmli, 1970). The gels were silver-stained according to the method of Oakley, Kirsch and Morris (1980).

were aspirated and wells were washed three times with PBS. Bound liposomes were dissolved in 1% SDS (100 PI/well) and quantitated in Packard gamma counter. Lipopolysaccharide preparation

A 500 ng sample of isolated receptor protein was concentrated to a 10 ~1 volume in tris-HCl, pH 7.2, and then mixed with 50 ~1 of an immobilized preparation of lactoperoxidase and glucose oxidase, 1.OmCi Na[‘251] and 1% fi-n-glucose for 25 min at room temperature. After incubation, the mixture was subjected to gel filtration on a Bio-Gel PdDG column, and the first eluted peak containing the iodinated receptor protein was collected.

The harvested B. gingivalis were washed with distilled water, treated with ethanol and acetone, and dried in vacuum over CaCl, . The dried bacteria were suspended in an extraction mixture consisting of liquid phenol-chloroforn-petroleum ether (2 : 5 : 8, by Vol), and homogenized in a glass homogenizer for 2 min (Galanos, Luderitz and Westphal, 1969; Parent, 1990). The suspension was then centrifuged at 5,000 x g for 15 min, the resulting supernatant containing the lipopolysaccharide was filtered through filter paper, and the bacterial residue was extracted once more. Petroleum ether and chloroform were removed from the pooled supernatant solution on a rotary evaporator, and the lipopolysaccharides precipitated by the addition of water. The precipitate was centrifuged, washed with 80% phenol solution followed by ether, and dried. The dry residue was dissolved in a small volume of water at 45°C and centrifuged at 100,000 x g for 4 h. The resulting lipopolysaccharide sediment was then redissolved in water, and freeze-dried. This prepared lipopolysaccharide was essentially free of nucleic acids as determined by absorption at 260 nm, and its protein content, measured according to Lowry et al. (1951) was less than 0.2%.

Liposomai reconstitution of [‘z51]receptor protein

Preparation of tooth cementum

Liposomes were prepared by the method of Mimms et al. (1981). Egg-yolk phosphatidylcholine (63 pmol) was dried onto a glass tube and dissolved in PBS containing 50 nM octylglucoside with [‘251]-1abelled 67 kDa receptor protein. Detergent was removed by dialysis against PBS for 24 h at 4°C thus yielding liposomes. For further purification, a suspension of liposomes was made with 45% sucrose, overlaid with 2 ml of 30% sucrose and 1 ml of 10% sucrose, and then centrifuged at 4°C for 18 h at 45,000 x g in a Beckman SW50 rotor. The liposomes, recovered as a white band at the top of the 10% sucrose layer, were suspended in the PBS containing 1 mg/ml albumin and used for assays.

The teeth were removed, within 30 min of killing, from the anterior parts of bovine upper and lower jaws, and the crowns were separated from the roots at the cementurn-enamel junction. The pulp canals were debrided and the roots sectioned vertically to produce relatively flat areas of cementum at the cervical one-third of the root. Test samples were prepared with ‘windows’ of equal area (2 mm in diameter) overlying the cervical one-third of the cementum. A uniform size of window was assured by first cutting a standardized hole in a small strip of plastic adhesive tape. The tape was then attached to the cementum and the cementum surfaces surrounding the taped window were covered with nail varnish. Windows prepared in this way held 5 ~1 of solution in contact with the exposed cementurn.

Receptor protein [‘251]radiolabelling

Liposome attachment assay

This was done by the method of Pytela, Pierschbather and Rouslahti (1985). The wells of a polystyrene microtitre plate (Nunc-Immuno Plate, 300 p 1 size) were coated with laminin solution (in an increasing-concentration manner) in PBS and incubated overnight at 4°C. Unoccupied binding sites were then saturated by incubation with 2mg/ml bovine serum albumin for 2 h at 37°C. Liposomes suspended in PBS containing 1 mg/ml albumin were added to wells and incubated overnight at 4°C. The supernatants

Binding of [‘251]receptor to cementum

Before the experiment, the adhesive tapes were peeled off to expose the cementum surface, which was washed thoroughly with Dulbecco’s PBS. Two types of binding assay were used. In one, the cementum with the exposed areas was incubated in 0.1 M PBS, pH 7.0, in a final volume of 5 ~1 for 90 min at room temperature with [‘2SI]-labelled 67 kDa receptor protein directly; in the second group, the [‘251]-1abe11ed

Plate I Fig. 1. SDS-PAGE analysis of laminin receptor protein isolated from octylglucoside extracts of gingival membrane by affinity chromatography on laminin. Line 1, molecular-weight markers; line 2, 67 kDa receptor protein before incorporation into liposomes; line 3, receptor protein from the sucrose gradientpurified liposomes. The bands were visualized by silver stain.

Gingival laminin receptor

343

,

92K

43K

*

31 K .

Plate 1

B. L. SLOMIANYet al.

344

receptor protein incorporated into liposomes was used. The supernatants were aspirated, the windows were washed three times with PBS, and the bound [‘251]receptor protein was desorbed with 1% SDS (Pytela et al., 1985), and quantitated in a Packard gamma counter. Non-specific binding was determined in the presence of lOOO-fold excess of unlabelled receptor protein. Binding of [‘251]receptor to cementum in the presence of exogeneous laminin

In these experiments, cementum was preincubated with laminin (lOOO-foldconcentration) for 1 h at room temperature and then incubated for 90 min with [1251]labelled receptor protein directly or with liposomebound, [‘251]-labelled receptor protein. The reaction was ended by adding 1.0 ml of ice-cold 10 mM trisHCl, pH 7.0 containing 0.5% bovine serum albumin. The cementum-bound, [‘251]-labelled receptor protein was separated by centrifugation, followed by aspiration of the supernatant and washing with PBS. The bound [1251]receptorwas then desorbed with SDS and subjected to gamma counting. Binding of [‘251]receptor to cementum in the presence of lipopolysaccharide

with izsI resulted in an [‘251]-labelled laminin receptor protein that exhibited significant binding to cementurn (Table 1). Furthermore, the protein also showed affinity towards other laminin-coated surfaces, such as polystyrene microtitre wells. Hence, in the further studies, we investigated if the protein could be incorporated into the liposome model membrane, which conforms to the structure of a membrane-bound protein. Liposomal incorporation of receptor protein

The phosphatidylcholine liposomes containing the 67 kDa receptor protein were prepared by sucrosegradient centrifugation, loading the sample at the bottom of the gradient and recovering the purified, [‘251]-labelled vesicles from the top of the 10% sucrose layer. The latter, when subjected to SDS-PAGE, showed the presence of 67 kDa protein identical to that of the intact preparation (Fig. 1). The liposomeincorporated 67 kDa protein, furthermore, behaved as a membrane-bound laminin receptor, showing concentration-dependent attachment to polystyrene surfaces coated with laminin. The optimum range for binding was found to be between 0.25-0.8 pg laminin/ml, giving a correlation coefficient of 0.96.

The laminin receptor protein, either directly or after incorporation into liposomes, was incubated for 30 min at room temperature with B. gingivalis hpopolysaccharide (O-125 pg) and then subjected to the binding assay with cementum. Binding assays were also conducted with cementum preincubated at room temperature for 30 min with the lipopolysaccharide (O125 pg). The cementum-bound, [‘251]-labelledreceptor protein was separated by centrifugation, aspiration and washing, followed by SDS solubilization and gamma counting. Data analysis

All experiments were carried out in triplicate, and the results are expressed as means f SD. Student’s t-test was used to determine significance, and p values of 0.05 or less were considered as significant. RESULTS

Laminin receptor

The laminin receptor was purified from the octylglucoside extracts of gingival epithelial-cell membranes on a column of Sepharose-bound laminin. The receptor was eluted from the affinity matrix with cation-free buffer containing EDTA. This fraction, when subjected to SDS-PAGE, gave, on silver staining, a protein band of 67 kDa (Fig. 1). Iodination Table

1. Binding

Type of receptor preparation Intact receptor

protein

Liposome-incorporated receptor protein

Receptor binding to cementum

Cementum sections were incubated with [‘*‘I]labelled 67 kDa receptor protein, directly or after incorporation into liposomes, in the absence (total binding) or presence (non-specific binding) of unlabelled receptor at lOOO-foldconcentration. Although the total binding values were different in the two cases, the patterns clearly showed that in the presence of unlabelled receptor, due to the competition between the two receptors for laminin, the non-specific binding decreased significantly (Table 1). These results indicated that liposomes containing [‘251]-labelled 67 kDa protein truly behaved as membrane-bound laminin receptors and that the cementum contains laminin that binds the laminin receptor of gingival membrane. Binding of receptor to cementum in the presence and absence of Iaminin

Incubation of cementum in the absence (total binding) and presence of laminin showed that total binding was dramatically decreased in the presence of soluble laminin (preincubated for 1 h), indicating competition between the soluble laminin and that of cementum-bound for the receptor. The soluble laminin bound to the receptor was thereby able to significantly decrease the laminin binding of the receptor protein (Table 2). This finding lends further support to the notion that the isolated 67 kDa protein is, indeed, a

of [‘Z51]-labelled laminin

receptor

protein

to cementum

Binding (cpm x IO’/cm’ of tooth cementum ._ Total

Non-specific

surface)

Specific

84,738 + 2967

3337 + 356 p < 0.001

81,401 + 2863

355 + 29

110 * 10 p i 0.001

245 k 26

Values are the means k SD of four separate

experiments

performed

in triplicate.

Gingival laminin receptor

345

Table 2. Binding of [‘2SI]-labelled laminin receptor protein to cementurn in the absence

and the presence of laminin Binding (cpm x 103/cm2of tooth cementurn surface) Type of

receptor preparation Intact receptor protein

In the absence of exogeneous laminin

exogeneous laminin

In the presence of

84,738 k 2967

1476 k 62.1

p < 0.001 Liposome incorporated receptor protein

35.5k 29

68.7 + 0.4 p < 0.001

Values are the means + SD of three separate experiments performed in triplicate. Table 3. Binding of [‘2SI]-labelled laminin receptor protein to cementum preincubated with B. gingivalislipopolysaccharide Binding (cpm x 103/cm2of tooth cementurn surface) Type of receptor preparation Intact receptor protein

Absence of lipopolysaccharide

Presence of lipopolysaccharide*

84,738 + 2967

46,912 & 8654 p < 0.03

Liposome incorporated receptor protein

355 f 29

228 + 15 p < 0.005

‘30 min preincubation with 100 pg of lipopolysaccharide. Values are the means k SD of four separate experiments performed in triplicate. Table 4. Binding of [‘2SI]-labelledlaminin receptor protein preincubated with B. gingivalis lipopolysaccharide (100 pg) to cementum Binding (cpm x 103/cm2of tooth cementum surface) Type of receptor preparation Intact receptor protein

Absence of lipopolysaccharide

Presence of lipopolysaccharide

84,738 f 2967

26,836 f 874 p < 0.001

Liposome incorporated receptor protein

355 f 29

145 & 17 p < 0.001

Values are the means f SD of three separate experiments performed in triplicate.

laminin receptor, because exogenous laminin competed with the laminin present within the cementum. Effect of Iipopolysaccharide

The data on the effect of B. gingivalis lipopolysaccharide on the laminin receptor binding to cementurn are presented in Tables 3 and 4. Preincubation of lipopolysaccharide with cementurn or the receptor before assay caused, in both cases, a decrease in binding. This inhibitory effect was proportional to the concentration of lipopolysaccharide and reached a maximum at IOO~g in both. However, the extent of inhibition differed greatly. Preincubation of cementurn with lipopolysaccharide decreased the receptor protein binding by 35.8% for the liposomal preparation and by 44.6% for the intact receptor protein (Table 3), whereas with preincubation of lipopolysaccharide with the receptor, the binding of its liposomal preparation decreased by 59.2% and that of intact protein by 68.3% (Table 4). DISCUSSION

Although the exact sequence of events involved in the pathogenesis of periodontal disease remains

elusive, the concensus is that it results from interaction between the subgingival microbial flora and the host immune and inflammatory systems (Page and Schroeder, 1976; Sengupta et al., 1990). Understanding the nature of this mechanism requires knowledge of the molecular interactions implicated in the attachment of gingival tissue to tooth cementum. We here provide evidence for the involvement of interaction between the cementum laminin and its gingival receptor in the maintenance of the cementumepithelial junction and show that this interaction is greatly hampered by the cell-wall lipopolysaccharide of B. gingivalis. Our findings that the isolated protein serves as receptor for laminin is supported by the fact that the protein bound to laminin-affinity matrix and was displaced by EDTA after removal of fibronectin by GRGDSP. On SDS-PAGE, the protein gave a single 67 kDa band that displayed laminin receptor activity in the in vitro model system and, when incorporated into the liposomes, exhibited properties of a membrane-embedded receptor. Thus, the isolated gingival laminin receptor is similar to the laminin receptors described in other tissues (Rao et al., 1982; Malinoff and Wicka, 1983; Lesot, Kuhl and Vonder Mark,

346

B. L.

SLOMIANY et al.

1983). The gingival laminin receptor appears to function as an integral membrane protein exposed at the gingival epithelial-cell surface, as it is easily solubilized by detergent, and readily incorporated into the lipid vesicles, properties common to many membrane proteins (Mimms et al., 1981). The liposome-incorporated 67 kDa protein, furthermore, exhibited functional activity compatible with that of a laminin receptor, as the vesicles bound selectively to laminin-coated surfaces. Moreover, the receptor protein showed a strong and specific affinity for the cementum surface. This finding is of particular importance, as it demonstrates for the first time the involvement of laminin in the interaction between cementum and its gingival receptor. As the subgingival microflora and, in particular, Gram-negative bacteria pose a major threat to gingival health and the detrimental effects are exerted through their surface-membrane lipopolysaccharide component, we examined the influence of B. gingiualis lipopolysaccharide on the laminin receptor binding to cementum. A significant decrease in receptor binding occurred in the presence of lipopolysaccharide, and this effect was more pronounced when the lipopolysaccharide was preincubated with the receptor protein than with the cementum, thus indicating interference by lipopolysaccharide with the receptor binding site for laminin. It is conceivable, therefore, that bacterial cell-membrane lipopolysaccharide is also capable of disrupting cementum laminin-receptor interactions in vivo, leading to the loss of gingival attachment and subsequently periodontal disease. Acknowledgements-Supported by USPHS Grant No. DE05666-12from the National Institute of Dental Research and Grant No. AA05858-09 from the National Institute of Alcoholism and Alcohol Abuse, NIH.

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