Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-laminin receptor

Mechanisms of Ageing and Development 106 (1999) 241 – 260

Effect of in vitro aging on the biosynthesis of glycosaminoglycans by human skin fibroblasts. Modulation by the elastin-laminin receptor I. Fodil-Bourahla a, I. Drubaix b, L. Robert a,* a Laboratoire Uni6ersitaire de Recherche sur les The´rapeutiques Substituti6es en Ophtalmologie, Hoˆtel-Dieu, escalier B3, 6e`me e´tage, 1, place du par6is Notre-Dame, 75181 Paris Cedex 04, France b Centre de Recherches Biocliniques sur le Vieillissement, Groupe Hospitalier Charles Foix-Jean Rostand, 7 a6enue de la re´publique, 94205 I6ry sur Seine Cedex, France

Received 6 May 1998; received in revised form 5 August 1998; accepted 14 August 1998

Abstract The incorporation of a radioactive precursor 3H-glucosamine in glycoconjugates, essentially glycosaminoglycans (GAG) was evaluated in the culture medium and cell fraction of human skin fibroblasts. Using increasing passage numbers, we could estimate the effect of in vitro aging on these biosynthetic activities. The incorporation in different free (hyaluronan) and protein bound (proteoglycans) GAGs was evaluated after specific enzymatic digestion. Most newly synthesized GAGs were excreted in the extracellular medium. Incorporation of the tracer in hyaluronan, the major biosynthetic product, increased with passage number but its titratable concentration decreased with in vitro aging, suggesting a rapid post-synthetic degradation. The proportion of chondroitin sulfates 4 (A) and 6 (C) and heparan sulfate decreased and that of dermatan sulfate increased with increasing passage number. We explored the modulation of these biosynthetic activities by the elastin laminin receptor. Using agonists (elastin peptides) and an antagonist (melibiose) of the receptor, their action on GAG biosynthesis was evaluated. Both elastin peptides and melibiose increased incorporation of the tracer in GAGs, but only melibiose inhibited post-synthetic degradation of hyaluronan, therefore increasing its concentration. The effect of passage number on the

* Corresponding author. Fax: + 33 1 56244045. 0047-6374/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0047-6374(98)00108-0

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receptor mediated modulations was also investigated. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Glycosaminoglycans; Hyaluronan; Proteoglycans; Elastin-laminin receptor; Aging; Fibroblasts

1. Introduction The presence of the elastin-laminin receptor (ELR) was demonstrated on a variety of cell types: mesenchymal cells, white blood cells and others (Wrenn et al., 1988; Robert et al., 1989; Pe´terszegi et al., 1996). It is composed of three subunits: two transmembrane proteins of 55 and 61 kDa, and a 67 kDa membrane-localized subunit, also called the elastin binding protein, responsible for the interaction with tropoelastin, laminin and elastin peptides. This subunit is a lectin with a b-galactoside fixation site, a galectin (Hinek et al., 1988; Mecham et al., 1989). The activation of the ELR by elastin peptides modulates several biological reactions such as chemotactic migration of cells, adhesion of fibroblasts, smooth muscle cells and malignant cells to elastic fibers (Hornebeck et al., 1986; Timar et al., 1991; Senior et al., 1980), modifications of ion fluxes (Fu¨lo¨p et al., 1986; Jacob et al., 1987), increase of the biosynthesis and release of elastase type proteases (Fu¨lo¨p et al., 1986; Archilla-Marcos et al., 1993) and increase of free radical ’ production (superoxide) (Fu¨lo¨p et al., 1986), NO and endothelium-dependent vasodilation (Faury et al., 1995). Preliminary evidence indicated that this same receptor can modulate the relative rates of biosynthesis of some of the extracellular matrix macromolecules (Ghyusen-Itard, 1993). We investigated the action of agonists of this receptor (elastin peptides) (Fu¨lo¨p et al., 1986; Jacob et al., 1987) and one of its antagonists (melibiose) on the biosynthesis of proteoglycans and glycosaminoglycans (GAGs), actively synthesized by human skin fibroblasts in culture in order to explore the modifications of their synthesis as a function of population doublings (PD), considered as a model of in vitro aging (Hayflick, 1977). Variations in the relative rates of biosynthesis of extracellular matrix components by mesenchymal cells were demonstrated in physiological as well as in pathological conditions such as aging, UV radiations, atherogenesis and a variety of skin diseases (Hayflick, 1977; Labat-Robert et al., 1981; Furth, 1991; Rasoamanantena et al., 1994).

2. Materials and methods

2.1. Cell cultures Primary cultures of human skin fibroblasts were obtained during othoplasty of a 13-year-old Caucasian girl (PD level max= 21 PD).

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Cells were cultured in Dulbecco Modified Eagle’s medium (DMEM GLUTAMAX, Gibco) with 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml fungizone and supplemented with 10% foetal bovine serum (FBS) at 37°C in humid atmosphere with 5% CO2/95% air. They were seeded in 6 mm diameter (NUNC) dishes at a density of 3×105 cells per dish with 5 ml of culture medium. Successive population doublings were obtained with a 1:2 split ratio after trypsinisation. The population doubling time was 5 days for a saturation density of 3×105 cells/cm2. We used early passage cells at the fifth and tenth PD levels and ‘‘older’’ cells at the 15th PD level. All tested cells were in phase II of the Hayflick model (Hayflick, 1977). Experiments for the fifth passage were carried out with cells frozen at the second passage and subcultivated until the fifth PD level (split ratio 1:2). For the two other experiments, cells were frozen at the sixth passage and subcultivated until the 10th and the 15th population doubling levels. All the experiments were carried out consecutively.

2.2. Agonists and antagonists of the ELR The agonists used were elastin peptides prepared as described (K-elastin; Jacob et al., 1985) with an average molecular weight of 30 kDa. The antagonist used was melibiose (Sigma) at the concentrations indicated in the legends of the figures and tables.

2.3. Proteoglycan and GAG biosynthesis The cell culture medium was removed at the preconfluent state (5 days after seeding) and renewed for a fresh one containing kappa elastin (1 mg/ml) and/or melibiose at concentrations of: 0.1 mg/ml (0.3 mM); 1.0 mg/ml (2.9 mM) and 5.0 mg/ml (14.6 mM). Fresh medium (3 ml) containing bactericides (100 U or mg/ml), fungicides (0.25 mg/ml) and 10% FBS was added at the same time as kappa elastin or melibiose with 1 mCi/ml of D-[6-3H] glucosamine hydrochloride (Amersham; 4.41 Gbq/mmol) for 24 h at a cell density of 2×104 cells/cm2. (Preliminary studies for time dependent incorporation of radiolabelled tracer were carried out and showed a 24 h incubation time to be the most appropriate. At later times, incorporation declined.) The medium was then removed, the cells rinsed with PBS and disrupted in 1 M urea containing 2 mM PMSF – EDTA as proteinase inhibitors and scraped with a rubber policeman. Both culture supernatants and cell lysates were dialysed overnight against PBS (pH 7.4) containing 0.01% NaN3 and 2 mM PMSF and EDTA (protease inhibitors). The unbound radioactive precursor was eliminated by gel filtration. All experiments were performed with six parallel culture dishes; one for cell counting and the others for experimentation. The precipitations were carried out in duplicate and the results compared with controls without agonist or antagonist cultured in the same medium as described above.

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2.4. Glycoconjugate biosynthesis Total glycoconjugate biosynthesis was determined by incorporation of [3H]-glucosamine into macromolecular glycoconjugates separately in cells and in the culture media. The [3H]-glucosamine incorporation in the glycoproteins was calculated as the difference between the total [3H]-glucosamine incorporation and the GAG fractions determined after selective cleavage by specific enzymes as described later (see Table 1).

2.5. Biosynthesis of hyaluronan Hyaluronan biosynthesis was evaluated after enzymatic hydrolysis by hyaluronidase from Streptomyces hyaluroniticus (E.C. 4.2.99.1 Calbiochem). It is a hyaluronan specific enzyme (Ohya et al., 1970) which hydrolyses b-Glc, Nac-[1“ 4] glycosidic bounds leading to D4,5-tetra or hexa unsaturated saccharides. One turbidity reducing unit of hyaluronidase was added per ml of sample and incubated overnight at 60°C. Then the enzyme is denatured for 5 min at 100°C and the macromolecules are precipitated as described by Yamagata et al. (1968), using 1% potassium acetate in ethanol. One hundred microliters of a carrier solution of chondroitine sulfate A and C (Sigma; 12 mg/ml) were added to the mixture at −20°C for 30 min, and then the macromolecules were sedimented by centrifugation. The radioactivity of the supernatants represents the hyaluronidase hydrolysed fraction.

2.6. Hyaluronan determination by enzyme linked sorbent assay We used hyaluronectin (HN), a protein with high affinity to hyaluronan (HA), with an indirect detection method described by Delpech (Delpech et al., 1981, 1991, 1995), which is an enzyme linked sorbent assay (ELSA) where HN-alcaline phosphatase is incubated with samples for 1 h at 37°C then added to a 96-well plate coated with known quantities of hyaluronan. The inhibition of alcaline phosphatase activity determined by reading the optical density at 405 nm (in an EPSON LX 800 ELISA reader) is proportional to the sample concentration of hyaluronan. This method is highly specific with a detection limit of 1 mg/l.

2.7. Biosynthesis of sulfated GAGs Chondroitin sulfates 4 (A) and 6 (C) are polymers of b-glucuronic acid [1“ 3]Nacetyl-b-galactosamin units with one sulfated carbon per unit (C4 for chondroitin sulfate A and C6 for Chondroitin sulfate C). These glycosaminoglycans were hydrolysed by chondroitinase ABC (chondroitin ABC Lyase, EC 4.2.2.4, from Proteus Vulgaris, Sigma) which hydrolyses chondroitin sulfates A and C more efficiently and chondroitin sulfate B or dermatan sulfate less efficiently (Yamagata et al., 1968).

Extracellular Intracellular Total Extracellular Intracellular Total

PD 5

Hyaluronan

24078 99554 2110 9349 26188 9 9903 35602 9 4465 4761 9 619 40363 95084

Total 3H-glucosamine incorporation per 106 cells 76892 99925 14386 93198 91278 913123 111292 94668 21579 92283 132871 96951

14751 9 2279 4417 9 504 19168 9 2783 27959 9 4761 4692 9 765 32651 9 5526

Chondroitin sulfates A, B, C

1439292720 60809916 204729 3636 3152193078 97009 1519 412219 4579

23671 1779 25450 16210 2426 18636

Heparan sulfates Non-GAG glycoconjugates

The radioactivity incorporated in the different GAG fractions was determined by their selective enzymatic release as described in Section 2.

PD 15

Pool

Population doubling level

Table 1 [3H]-glucosamine incorporation in total glycoconjugates and in the different glycosaminoglycan fractions expressed as cpm per 106 cells at the fifth (PD5) and the 15th (PD15) population doubling level of fibroblasts

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Samples were diluted 1:2 in a 100 mM Tris–HCl buffer, containing 120 mM sodium acetate, 0.02% bovine serum albumin, pH 8. The chondroitinase ABC was added at 0.001 U/ml to the samples. The mixture was incubated overnight at 37°C. After inactivation of the enzyme (100°C for 1 min), the macromolecules were precipitated as described previously and the [3H]-glucosamine incorporation in hydrolysed fractions evaluated on a Rackbeta counter in both cell extracts and in culture media.

2.8. Heparan sulfate biosynthesis Heparan sulfate was determined using a method described by Shievley et al. (1976). At very low pH, the 2-amino-2-deoxy-D-glycosidic bonds in heparin and heparan sulfates can be hydrolysed to disassacharides which can be cleaved to the monomeric state. This was realised by adding nitrous acid prepared extemporarily by mixing 1 ml of 0.5 mmol NH2SO4 with 0.5 mmol Ba(NO3)2 at 4°C. The supernatant (pH 1.5) was added to the sample for 30 min. The mixture was neutralized with 2 N Na2CO3 and macromolecules precipitated as already described.

2.9. Biosynthesis of dermatan sulfates For this determination, we used a dermatan sulfate specific hydrolytic enzyme: chondroitinase B (chondroitin sulfate B Lyase from Flavobacterium Heparinum, Sigma). The method was as described for hyaluronan biosynthesis.

2.10. Statistics The non-parametric Mann – Whitney U-test was used.

3. Results

3.1. Distribution of 3H-glucosamine incorporation in glycoconjugates Table 1 shows the results of the determination of [3H]-glucosamine incorporation in total glycoconjugates (GAGs and glycoproteins) as well as the incorporation in the different GAGs, free (hyaluronan) or protein bound (proteolgycans), at the fifth and the 15th passages of fibroblasts. Total incorporation increased by about 45% (PB 0.001) between the fifth and 15th passages. This increase is due to the increase of radioactive glycoconjugates in the extracellular fraction (+44%; PB 0.001). Incorporation of [3H]-glucosamine in hyaluronan represents about 28% (Table 1) of total [3H]-glucosamine incorporation and about 40% (Table 2) of its incorporation in all GAG fractions. The chondroitin sulfates and heparan sulfates incorporated nearly the same amount of [3H]-glucosamine (about 22% of the total for

65828 916322 49145 910103 114235 915207

PD 5 PD 10 PD 15

40 34 35

29 26 28

(%)

(%)

16 17 22

Dermatan sulfates* (%)

Chondroitin sulfates A, B, C

Hyaluronan

31 40 36

(%)

Heparan sulfates

The dermatan sulfate fraction is comprised in the column of the chondroitin sulfates A, B and C and was also determined seperately (second part of the third column*). The difference between the two parts of the third column gives the chondroitin sulfate A–C fraction.

Total incorporation in GAGs cpm/106 cells

Population doubling level

Table 2 Distribution of [3H]-glucosamine incorporated in the different GAG fractions, expressed as a percentage of the total [3H]-glucosamine incorporation

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each). Most of this incorporation was found in the excreted GAG fraction, 76% of total chondroitine – dermatane sulfates at the fifth PD level and 85% at the 15th PD level. It has to be remembered, however, that chondroitinase ABC might not digest completely the dermatane sulfate GAGs. We therefore carried out separately a digestion with a dermatanase enzyme (see Section 2). When the results of this digestion are subtracted from the radioactivity in the total chondroitinase ABC released fraction, it appears that the dermatan sulfate fraction of total GAGs increased with PD levels and the chondroitin 4–6 sulfate fraction decreased (Table 2 and Fig. 1). The radioactivity incorporated in the heparan sulfate fraction increased significantly with PD levels (Table 1). This fraction represented about 22% of total [3H]-glucosamine incorporated at the fifth passage and 30% at the 15th passage. Most of these GAGs (70%) were excreted at the fifth passage and about 76% at the 15th passage. It appears, therefore, that the biosynthesis of heparan sulfate proteoglycans increased rapidly with the PD level. The fraction of total [3H]-glucosamine incorporated in glycoconjugates other than GAGs, mainly glycoproteins and also some glycolipids, represented about 27% of total incorporation at the fifth PD level and 14% at the 15th PD level (Table 1). The absolute value of incorporation also decreased slightly in this fraction from about 25 ×103 cpm/106 cells at the fifth PD level to 18 × 103 cpm/106 at the 15th PD level. It appears that the biosynthesis of non-GAG glycoconjugates decreased

Fig. 1. 3H-glucosamine incorporation in chondroitin sulfates A and C, in dermatan sulfate and in heparan sulfates. The values are expressed in cpm per 106 cells.

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Fig. 2. Hyaluronan concentrations determined in human skin fibroblast cultures by ELSA expressed as ng per million cells. Determinations were made separately in culture medium and in the cellular fraction. Only total concentrations are shown ( ).

with PD levels. The precise nature of these glycoconjugates remains to be determined. This was certainly not the case of fibronectin, which was shown previously to increase with PD levels (Rasoamanantena et al., 1994; Fodil-Bourahla et al., 1998).

3.2. Biosynthesis of hyaluronan Hyaluronan biosynthesis was studied in more detail for several reasons. This is the principal GAG synthesised by fibroblasts, strongly involved in a number of biological phenomena like wound healing, angiogenesis or cell migration (Ellis et al., 1996; Montesano et al., 1996; Turley, 1994). As can be seen in Table 1, the radioactivity of [3H]-glucosamine incorporated in hyaluornan increased significantly with PD levels: from about 26 ×103 cpm per 106 cells at passage 5 to above 40 × 103 cpm/106 cells at the 15th PD level (Table 1). This increase represented about 44% of the increase of total incorporation between the fifth and 15th PD levels. Most of this freshly incorporated glucosamine was in the excreted hyaluronan fraction: 92% at the fifth PD level and 88% at the 15th PD level. Curiously, however, when the actual hyaluronan concentration of the culture media and of the cellular fractions was determined by the ELSA procedure, a strong decrease of the

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titratable hyaluronan content was observed (Fig. 2). This could only be attributed to a rapid post-synthetic degradation of hyaluronan as observed also by Tammi et al. (1991) and Agren et al. (1997). The distribution of hyaluronan between the extracellular and cellular fractions was progressively inverted with increasing passages. Hyaluronan determined by ELSA was mainly extracellular at the fifth PD level (87% of total) and became mainly intracellular at the 15th PD level. Only 17% of total hyaluronan were recovered in the culture medium at this PD level (Table 3).

3.3. Effect of elastin peptides When 1 mg/ml elastin peptides were added to the culture medium of human skin fibroblasts, the incorporation of [3H]-glucosamine in chondroitin 4 and 6 sulfates and dermatan sulfate was significantly increased at the 15th PD level (Fig. 3) with no effect at PD level 10. Incorporation both in dermatan sulfate (+ 30%; PB 0.01) and heparan sulfate (+90%; P B0.001) was increased between the fifth and the 15th PD levels in the presence of elastin peptides. Incorporation of [3H]-glucosamine in hyaluronan presented a significant increase by about 50% (PB 0.001) at the 15th PD level in the presence of elastin peptides as compared to the fifth PD level (Fig. 4a). As shown in Fig. 4a, elastin peptides did not modify the incorporation of [3H]-glucosamine at the fifth and the 10th PD levels in hyaluronan, but did produce a significant increase at the 15th PD level. The titratable hyaluronan concentration (by ELSA) increased only slightly at the fifth PD level but significantly at the 10th and the 15th PD levels (Fig. 4b). This increase concerned only the intracellular fraction (Table 4). Hyaluronan in the extracellular fraction did not change or even decrease (not significant, NS) in the presence of elastin peptides. Incorporation of the [3H]-glucosamine in the other GAG fractions increased at the 15th PD level in the presence of kappa-elastin (Table 4). It appears that elastin peptides did produce a moderate increase of incorporation of the tracer in all GAG fractions but they did not modify, apparently, the post-synthetic degradation of hyaluronan, and as a consequence did not modify its concentration. Table 3 Hyaluronan concentration expressed as ng/106 cells, determined by ELSA in human skin fibroblast cultures at different population doubling levels Population doubling level

Total ng/106 cells

Extracellular fraction (%)

Intracellular fraction (%)

PD 5 PD 10 PD 15

5195429 115232 2686439 98965 218839 5984

87 31 17

13 69 83

Distribution as a percentage of the total concentration between the media and the cellular fraction is also shown.

Extracellular Intracellular Extracellular Intracellular Extracellular Intracellular

PD 5 +28 +18 +6 +143 −30 +252

% Variation

Hyaluronan concentrationa

NS NS NS *** NS ***

P −18 +35 +3 +16 +48 +54

% Variation

Hyaluronan

NS * NS NS *** ***

P −2 +9 +13 +7 +49 +28

% Variation

NS NS * NS *** ***

P

Chondroitin sulfates ABC

[3H]-glucosamine incorporation in

+11 +32 +6 +12 +32 +75

% Variation

NS * NS NS ** ***

P

Dermatan sulfates

+8 +17 +9 −6 +90 +70

% Variation

Heparan sulfates

a

Results are expressed as the percentage variation compared to the control cultures without kappa-elastin, the significance of the difference (P) is also indicated. NS, non significant; * PB0.05; ** PB0.01; *** PB0.001. Determined by ELSA.

PD 15

PD 10

Pool

Population doubling level

Table 4 Effect of 1 mg/ml kappa-elastin added to the culture medium on the biosynthesis and concentration of different GAGs at increasing population doubling levels

NS NS NS NS *** ***

P

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Fig. 3. Incorporation of [3H]-glucosamine in chondroitin 4 and 6 sulfates and dermatan sulfate by human skin fibroblasts cultured in the presence of 1 mg/ml elastin peptides (a) as a function of population doubling level. The incorporation was compared to controls without kappa-elastin ( ); *** PB 0.001.

3.4. Effect of melibiose The effect of melibiose, an antagonist of the ELR was tested at different concentrations: 0.1, 1.0 and 5 mg/ml. Melibiose increased the rate of incorporation of labelled glucosamine in hyaluronan and also its titratable concentration. As shown in Table 5, at the fifth PD level, an important increase of hyaluronan concentration was observed in the presence of 1 and 5 mg/ml melibiose, using ELSA determination. The concentrations of 1.0 and 5.0 mg/ml melibiose produced an increase of incorporation of [3H]-glucosamine in hyaluronan from 30 to 130% as compared to the control without melibiose at the 10th PD level (PB0.001). At the 10th and the 15th PD levels, the presence of melibiose not only increased [3H]-glucosamine incorporation in hyaluronan (Table 5) but also the titratable concentration of hyaluronan (determined by ELSA). At the 10th PD level, melibiose, at all three concentrations tested produced an increase in titratable hyaluronan concentration in the extracellular fraction (Table 5). At the 15th PD level, 0.1 mg/ml melibiose increased significantly the titratable hyaluronan concentration in the cellular fraction, while 5 mg/ml were necessary to

Extracellular Intracellular Extracellular Intracellular Extracellular Intracellular

0.1

NS *** *** *** *** * +180 +29 +302 −40 +116 −46

*** NS *** * *** NS

P

% Variation −39% −6% −30% +13% −23% +8%

P *** NS * NS NS NS

% Variation +20% +33% +39% +31% +117% +130%

P NS ** *** ** *** ***

Hyaluronan biosynthesis ([3H]-glucosamine incorporation)

+10 +117 +30 +150 +160 +120

% Variation

% Variation P

PD 10

PD 5

Hyaluronan concentration at increasing PD level

% Variation +13 +16% +52% +70% +32% +16%

+13 +186 +127 +10 +560 −60

% Variation

PD 15

P NS NS *** *** *** NS

NS ** * NS *** NS

P

Melibiose was added to human skin fibroblast cultures at the fifth, 10th and 15th population doubling level at 0.1, 1.0 or 5.0 mg/ml and hyaluronan concentration was determined in extracellular and intracellular fractions by ELSA as described in Section 2. Hyaluronan biosynthesis was estimated by [3H]-glucosamine incorporation in the fraction hydrolysed by hyaluronidase as described in Section 2. The results are expressed as the percentage variation of concentration of hyaluronan or [3H]-glucosamine incorporation in hyaluronan as compared to the control samples. P values are also indicated. * PB0.05; ** PB0.01; *** PB0.001.

5

1

5

1

Extracellular Intracellular Extracellular Intracellular Extracellular Intracellular

Pool

0.1

(mg/ml)

Concentration of melibiose added

Table 5 Effect of melibiose on hyaluronan concentration and biosynthesis

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Fig. 4.

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255

also increase its concentration in the extracellular fraction (Table 5). It appears, therefore, that melibiose increased the incorporation of the tracer in hyaluronan and decreased its post-synthetic degradation. At the lowest concentration (0.1 mg/ml) in the fibroblasts culture medium, melibiose induced a decrease of incorporation of [3H]-glucosamine in the chondroitin sulfates by 33% (P B 0.001) at the fifth PD level (not shown). At higher concentrations, this effect was inverted. An increase of 40% at 1 mg/ml for the 10th and the 15th PD levels, as compared to the fifth PD level, was observed (PB 0.01 for the 10th and P B0.001 for the 15th PD levels) (Table 6). At the fifth PD level, incorporation in dermatan sulfate was increased by 26% (PB0.001) in the presence of 5 mg/ml melibiose. At the 10th PD level, 1 mg/ml melibiose produced an increase of about 50% (PB 0.01). At the concentration of 5 mg/ml, the increase was over 100% (PB0.001; Table 6). At the 15th PD level, the increase produced by 1 mg/ml also reached 90% (PB 0.001) in the cellular fraction. Concerning the incorporation of [3H]-glucosamine in heparan sulfates, the same variations as for dermatan sulfates were observed in the presence of melibiose. At the fifth PD level, the increase in presence of melibiose was of about 27% (PB 0.001). At the 10th PD level, 1 mg/ml produced an increase of about 46% and 5 mg/ml an increase of more than 100% (PB 0.001). At the 15th PD level, all three concentrations of melibiose (0.1, 1.0 and 5.0 mg/ml) increased incorporation in heparan sulfate by more than 40% (PB 0.001).

4. Discussion Fibroblasts are mesenchymal cells specialised in extracellular matrix (ECM) biosynthesis. Their differentiation, acquired during embryonic development, results in the precise adjustment of the relative rates of biosynthesis of a selection of ECM components which vary with their tissue specificity and with age (Labat-Robert et al., 1988; Robert, 1992). Their differentiation involves the selection of genes coding specific ECM macromolecules and the regulation of the relative rates of their synthesis and degradation. The ECM macromolecules synthesised by human skin fibroblasts comprise members of the collagen family, proteoglycans and hyaluronan, elastin and several matrix glycoproteins as fibronectin (Robert, 1992). The relative rates of synthesis and renewal of these macromolecules changes with age. The incorporation in non-GAG glycoconjugates decreased with population doublings from about 25× 103 cpm/106 cells (28% of total incorporation) at the fifth PD level to about 18×103 cpm/106 cells (14% of total incorporation) at the 15th Fig. 4. (see left) (a) Effect of kappa-elastin on hyaluronan biosynthesis by human skin fibroblasts in culture as a function of population doubling level (a) as compared to control cultures ( ). Values are average9 S.D. for five parallel cultures. (b) Effect of kappa-elastin on hyaluronan concentration in human skin fibroblasts in culture as a function of population doubling level (a) as compared to control cultures without kappa-elastin ( ). Significances are calculated between the control and the cells in the presence of kappa-elastin at the same population doubling level (*** PB0.001).

+1%NS 0%NS +57%** +37%*** +36%* +93%***

0%NS +2%NS +46%*** +22%NS +50%*** +73%***

Heparan sulfates

+25%** +14%NS +129*** +80%** +14%NS +11%NS

Chondroitin sulfates A, B and C

+26%*** +12%NS +135%*** +130%*** +4%NS +37%*

Dermatan sulfates

Melibiose concentration = 5 mg/ml

+27%*** +4%NS +140%*** +68%*** +44%*** +26%**

Heparan sulfates

Melibiose was added to human skin fibroblast cultures at the fifth, 10th and 15th population doubling level at 1.0 or 5.0 mg/ml and [3H]-glucosamine incorporation was determined in extracellular and intracellular fractions. The variations are expressed in percent as compared to control samples, and P values are also indicated.

PD 15

PD 10

+2%NS +3%NS +40%** +3%NS +40%*** +10%NS

Extracellular Intracellular Extracellular Intracellular Extracellular Intracellular

PD 5

Dermatan sulfates

Melibiose concentration = 1 mg/ml

Chondroitin sulfates A, B and C

Pool

level

Population doubling

Table 6 Effect of melibiose on [3H]-glucosamine incorporation in different sulfated GAGs

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PD level. In the present experiments, we concentrated on GAGs, free (hyaluronan) and protein bound (proteoglycans). Their biosynthesis was estimated by the incorporation of [3H]-glucosamine, followed by the selective hydrolysis of glycosaminoglycans using specific enzymes as described in Section 2. We compared a culture at an early PD level (fifth PD) and a relatively late PD level (15th PD) as far as GAG biosynthesis was concerned. We did not want to study later PD levels, near the exhaustion of division potential (the so-called Hayflick limit), because of the doubtful in vivo significance of such cells. For some experiments, an intermediary PD level, 10th PD level cells, were also studied. It appeared that total [3H]-glucosamine incorporation, expressed as cpm/106 cells, increased with PD levels by about 45% between the fifth and 15th passages. Most of the newly synthesised glycoconjugates were excreted in the culture medium: 84% at the fifth PD level and 83% at the 15th PD level. Less than one-third of the total incorporation occurred in non-GAG glycoconjugates, glycoproteins and, or glycolipids: 28% at the fifth PD level and 14% at the 15th PD level. All the rest of the incorporated [3H]-glucosamine appeared in GAGs. Hyaluronan represented a major product of the biosynthetic activity of fibroblasts: 28% of total incorporation and 40% of incorporation in glycosaminoglycans was found in hyaluronan at the fifth PD level. This decreased slightly to 30% of total incorporation at the 15th PD level. About 90% of this newly synthesised hyaluronan was in the extracellular fraction at this early PD level. When, however, hyaluronan concentration was determined by the ELSA procedure, no accumulation of hyaluronan could be demonstrated. On the contrary, the titratable hyaluronan concentration decreased with PD levels (Fig. 2). This can only be explained by a rapid post-synthetic degradation of newly synthesised hyaluronan. According to Agren et al. (1997) and other authors (Deguine et al., 1997, 1998), reactive oxygen species may be involved in hyaluronan catabolism besides receptor mediated intracellular degradation (Fraser et al., 1989). The chondroitin and dermatan sulfates represented about 21–24.5% of total incorporation with no significant change with PD levels. There was, however, a modification in the relative amounts of the different GAG chains synthesised: the proportion of chondroitin 4 and 6 sulfates decreased and that of dermatan sulfate increased with PD levels. Heparan sulfates represented about 22% of total incorporation at the fifth PD and about 30% at the 15th PD level. As human skin fibroblasts express the ELR as shown previously (Wrenn et al., 1988; Robert et al., 1989), it was interesting to study the effect of agonists (elastin peptides) and of antagonists (melibiose) of the receptor on GAG biosynthesis. It occurred that elastin peptides increased incorporation in some fractions, but did not produce an accumulation of hyaluronan in the fibroblast cultures. There was apparently no effect of elastin peptides on post-synthetic degradation of hyaluronan. Melibiose, the antagonist of ELR did increase incorporation of [3H]-glucosamine in all GAG fractions and did increase also the titratable concentration of hyaluronan in the cultures. This result can be taken as an indication of the inhibition of

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post-synthetic degradation of hyaluronan by melibiose. The most effective concentration of melibiose and the dose dependence of its action varied with PD level. The effect on incorporation of the tracer in hyaluronan was modest at the fifth PD level, dose dependent at the 10th PD level and showed a maximum at the 15th PD level at 1 mg/ml concentration of melibiose. Titratable hyaluronan concentration increased in a dose dependent fashion at the fifth and the 15th PD levels in the extracellular fraction, suggesting that in presence of melibiose, the increased hyaluronan biosynthesis appeared in the excreted fraction. These modifications of effective optimal concentration of melibiose might be due to PD level dependent variations of the expression and/or coupling of the receptor. Variation in receptor expression and intracellular coupling mechanisms were found during our previous studies of reactions mediated by the ELR on different cell types (Jacob et al., 1987; Varga et al., 1989; Archilla-Marcos et al., 1993; Pe´terszegi et al., 1996). It cannot be excluded, however, that melibiose might also act as a free radical scavenger, as shown by the inhibition of the decrease of viscosity of hyaluronan produced by free radicals (Deguine et al., 1997) in the presence of melibiose (unpublished results). This effect needed, however, higher concentrations of melibiose than those used in these experiments. It appears from these results that agonists and antagonists of the ELR may well play a role in the regulation of the population doubling dependent variations of glycoconjugate biosynthesis by human skin fibroblasts.

Acknowledgements We thankfully acknowledge the supply of human skin samples by Dr Dominique Rheims (Robert Debre´ Hospital, Paris). This work was supported by RoC-Johnson and Johnson and Institut Derm with a fellowship to I.F.-B.

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