110 Europeon Journol of Cell Biology 76, 110-118 (1998, June) . © Gustov Fischer Verlog . Jeno ... : Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D

Colin Farquharson1a, Jacqueline L. Berryb, E. Barbara Mawerb, Elaine Seawrighta, Colin C. Whiteheada

a Roslin Institute (Edinburgh), Roslin, Midlothian, Scotland/UK b Bone Disease Research Centre, University Department of Medicine, Manchester Royal Infirmary,

Manchester, England/UK

Received July 22, 1997 Accepted January 9, 1998

Chondrocyte - differentiation - ascorbic acid - vitamin D - extracellular matrix

Chondrocyte terminal differentiation is associated with cellular hyper­trophy, increased activity of plasma membrane alkaline phosphatase and the synthesis of collagen type X. The hypertrophic phenotype of cultured chondrocytes can be stimulated by ascorbic acid but the underlying mechanisms for this phenotypic change are unclear. As ascorbic acid is central to many hydroxylation reactions, the possibility was examined that its pro-differentiating effects are mediated by its effects on collagen and vitamin D metabolite formation. In vitro stud­ies indicated that ascorbic acid-induced chondrocyte alkaline phospha­tase activity was inhibited by the addition of both collagen and proteo­glycan synthesis inhibitors. The addition of arginine-glycine-aspartic acid (RGD)-containing peptides also resulted in lower alkaline phos­phatase activity. Chicks supplemented with dietary ascorbic acid had higher concentrations of both collagen and proteoglycans within their growth plates but the chondrocyte maturation rate was unaltered. No evidence was obtained to suggest that ascorbic acid-induced collagen production was mediated by lipid peroxidation. In addition, supple­mentation with dietary ascorbic acid resulted in higher serum 1,25-dihydroxyvitamin D3 concentrations and increased chondrocyte vita­min D receptor number. Ascorbic acid-treated chondrocytes main­tained in vitro also had increased vitamin D receptor numbers but chondrocyte receptor affinity for 1,25-dihydroxyvitamin D3 was unal­tered. These results indicate that ascorbic acid promotes both chon­drocyte matrix production and 1,25-dihydroxyvitamin D3 synthesis, accompanied by upregulation of the vitamin D receptor. Thus, ascorbic acid may be causing amplification of the vitamin D receptor­dependent genomic response to 1,25-dihydroxyvitamin D, resulting in promotion of terminal differentiation. Strong evidence is provided to support the hypothesis that ascorbic acid-induced chondrocyte termi­nal differentiation is mediated by interactions between integrins and RGD-containing cartilage matrix proteins.

Abbreviations: ALP Alkoline phosphatase. - 1,25-D 1,25-Dihydroxyvitamin D3. - VDR Vitamin D receptor.

1) Dr. Colin Farquharson, Roslin Institute (Edinburgh), Roslin, Mid­lothian, EH25 9PS, Scotland/UK.

Introduction

Chondrocytes of the epiphyseal growth plate undergo an orderly maturation process resulting in longitudinal bone growth. The control of this process is complex and involves a variety of local and systemic factors which stimulate both pro­liferation and terminal differentiation leading to the attain­ment of a hypertrophic phenotype and the production of a mineralized matrix [29]. A number of cellular changes are associated with the development of the terminally differenti­ated phenotype including increased activity of plasma mem­brane alkaline phosphatase (ALP) and increased expression of the vitamin D receptor (VDR) [1, 4]. Concomitant changes occur within the extracellular matrix such that each distinct maturational zone of the growth plate contains a unique mix­ture of various matrix proteins [2, 38]. Interactions between collagen and proteoglycans, the two principal components of the cartilage matrix, are central to the formation of the growth plate extracellular matrix. Furthermore, establishment of cell­matrix interactions are an important part of the control mechanisms associated with tissue development and chondro­cyte hypertrophy [8, 41]. Collagen type II, the principal struc­tural protein and predominant collagen of the growth plate, interacts with collagens type IX and XI to form heterotypic collagen fibrils [44]. Although these collagen types are distrib­uted throughout the cartilage matrix, their expression de­creases with the onset of chondrocyte hypertrophy and the increase in expression of collagen type X and increased ALP activity [1, 39]. Proteoglycans interact with collagen, cell membranes and tissue specific proteins within the growth plate [9] and consist of a core protein to which glycosamino­glycans are linked covalently. Recent evidence suggests that during hypertrophy chondrocytes switch their synthesis from aggrecan to include a significant amount of decorin and bigly­can [5].

Considerable attention has been directed to an understand­ing of the role of growth factors, hormones and vitamins in the regulation of chondrocyte development and matrix formation

and although a role for ascorbic acid in endochondral bone growth has been suggested [6] the underlying mechanism remains unclear. In studies using ascorbic acid-deficient gui­nea pigs, chondrocytes became degenerate with widening of the calcified cartilage region and their replacement with endo­chondral bone was impaired [6].

In vitro studies have indicated consistently that ascorbic acid causes an elevation in ALP activity and collagen type X mRNAexpression within chondrocytes [17, 26, 31, 42]. Stimu­lation of the differentiated phenotype by ascorbic acid has also been noted in a number of other mesenchymal derived cells such as adipocytes [43], osteoblasts [21] and myoblasts [34]. Franceschi [20] proposed that the pro-differentiating capabil­ities of ascorbic acid may be related to its requirement as a cofactor for prolyl and Iysyl hydroxylase in the synthesis and secretion of stable triple-helical collagen [37]. The formation of a collagen-rich matrix would promote cell-matrix interac­tions (via integrins) and provide a permissive environment for subsequent tissue specific gene expression . The results of stud­ies using cultured osteoblasts [21, 27] and myoblasts [34] are in accord with this proposal but for chondrocytes maintained in vitro, ascorbic acid-induced collagen synthesis was found not to influence ALP activity [42].

In addition to its role in collagen synthesis, ascorbic acid is also central to other hydroxylation reactions in a number of biosynthetic pathways, such as carnitine and norepinephrine biosynthesis (for review see [32]). There is limited evidence that ascorbic acid may enhance hydroxylation of 25-hydroxyvitamin D3 into its active hormonal metabolite , 1,25-dihydroxyvitamin D3 (1,25-0), [40, 45]. The activity of lu­hydroxylase is principally regulated by the systemic calcium and phosphorus status [7]; however, if ascorbic acid also enhances 1,25-0 synthesis it is possible that some of the skele­tal effects attributed to ascorbic acid are, in fact , mediated via 1,25-0 acting through its intracellular receptor (VOR) . 1,25-0 and VOR play profound roles in the regulation of chondrocyte metabolism and terminal differentiation [4, 10, 16, 36] and appear to be essential for endochondral ossification [15].

In an attempt to clarify the mode of action of ascorbic acid­induced chondrocyte terminal differentiation we have ex­amined both in vivo and in vitro the role of the principal carti­lage matrix proteins and their interaction with their cell sur­face adhesions receptors in this process. In addition, the effect of ascorbic acid on 1,25-0 synthesis and chondrocyte VDR number was studied in order to establish whether interactions existed between ascorbic acid and vitamin 0 metabolism .

Materials and methods

Materials Dulbecco's modified Eagles medium (DMEM), fetal bovine serum (FBS), gentamycin, pyruvate and dispase were obtained from GIBCO (Paisley, Scotland) and collagenase type A, porcine pepsin and colla­gen type I from Boehringer-Mannheim (Lewes , Sussex, UK) . L-[2 ,3-3H1proline (44.0 Ci/mmol) and [35S1Na2S04 (carrier free , lOoo Cit mmol) were purchased from Amersham International (Little Chalfont, Buckinghamshire, UK). Culture plastic was from Costar (High Wycombe, Buckinghamshire, UK) and RGD-containing peptides (GRGDSP and GRGESP) from Bachem (Saffron Walden , Essex, UK). I.-ascorbic acid phosphate and alkaline phosphatase assay kit were obtained from Alpha Laboratories (Eastleigh, Hampshire, UK). Biomatrix was obtained from TCS Biologicals Ltd. (Botolph Claydon,

Ascorbic acid-induced chondrocyte terminal differentiation 111

Buckinghamshire, UK). Lipid peroxidation kit was provided by Calbiochem-Novabiochem (Nottinghamshire, UK) and 1,9-dime­thylmethylene blue was obtained from Aldrich Chemical Co. (Poole, Dorset, UK). 125I-l,25-dihydroxyvitamin D3 assay kit was obtained from IDS Ltd. (Boldon , Tyne & Wear, UK). Collagen type II anti­bodies were supplied by Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, USA) . All other chemicals were pur­chased from Sigma (Poole, Dorset , UK).

In Vitro Studies Chondrocyte culture. Growth plates from proximal tibiotarsi of 3-week-old chicks were dissected and diced into 1-2 mm cubes. For the analysis ofYDR number and affinity the growth plate was divided into the upper proliferating and hypertrophic zones [17]. Chondrocytes were isolated from their surrounding matrix by digestion in DMEM containing 10 % FBS, gentamycin and 0.1 % collagenase for 18 h at 3rC [17]. Chondrocytes were cultured in DMEM containing 10 % FBS, pyruvate and gentamycin (complete medium) in multi-well plates at a density of 1000OO/cm2 and maintained at 37 °C under an atmosphere of 9S % airlS % CO2. Unless otherwise stated the cells were grown to confluency (day 6) at which time they were induced to differentiate by the addition of ascorbic acid (100 [tM). At the same time RGD-containing peptides (GRGDSP and GRGESP, 2S0 [tg/ml) or inhibitors of collagen (3 ,4-dehydroproline , O.S mM) and proteogly­can (4-methylumbelliferyl-(:\-D-xyloside, 1 mM) synthesis were added to appropriate cultures for a further 7 days with 3 complete medium changes. Chondrocytes were also cultured for a total of 7 days in 24-well plates that had previously been coated with either collagen type I (S [tg/c(2) or Biomatrix (basement membrane extract: 10 [tg/cm2) in the presence (added from day 1) or absence of ascorbic acid (l00 [tM). Cartilage pellet cultures were also set up as described previously [18] . In the presence of ascorbic acid (100J.lM) the cells were grown for a total of 7 days in the presence or absence of D-xyloside (1 mM). Assays. Alkaline phosphatase and protein content: Cell layers were washed twice with phosphate-buffered saline (PBS) and detached from the plastic by the use of a cell scraper. The cell suspension or the homog­enized cartilage pellets were resuspended in 0.9 % NaCI , 0.2 % Triton X-100 and centrifuged at 12000g for IS min at 4 °C. The supernatant was assayed for ALP activity and for cellular protein [17, 18]. Total ALP activity was calculated and expressed as nmoles p-nitrophenyl phosphate (pNPP) hydrolyzed/min/mg protein. Collagen analysis: Total hydroxyproline concentrations were deter­mined in the cell-matrix layers and pellet cultures [19] from which the collagen content of each sample was determined.

The rate of collagen accumulation into the cell-matrix layer was measured as previously described [46] . Essentially, cells were incu­bated for the last 24 h of culture with L-[2,3-3H]proline (S [tCilml) con­taining 100 [tg/ml ~-aminoproprionitrile. The cellular layer was agi­tated in the presence of lS0mM potassium phosphate buffer (pH 7.6) containing protease inhibitors at 4 °C for 3 h. The collagen was precip­itated overnight by the addition of ammonium sulfate to 30 % satura­tion in the presence of protease inhibitors and recovered by centrifuga­tion. The collagen was dissolved in O.S M acetic acid containing pepsin (0.01 %) for 18 h at 4 °C to remove non-collagenous proteins. Proteins were finally precipitated with 10 % trichloroacetic acid, washed with acetone and resuspended in Laemmli buffer (O.OS M Tris-HCI, pH 6.8, containing 1 % SDS and 10 % glycerol) and counted. Proleoglycan analysis: Total proteoglycan concentration in the cell­matrix layer and pellct cultures was determined using 1,9-dimethyl methylene blue [18].

The rate of proteoglycan incorporation into the cell-matrix layer was measured as previously described [461. In brief, cells were incubated for the last 24 h of culture with eSS]Na2S04 (S ftCi/ml) and the cell­matrix layer was solubilized overnight in 0.07S M sodium acetate buffer (pH 7.8) containing 4 M guanidine hydrochloride and protease inhibitors . An aliquot (10 % v/v) was added to ethanol and left over­night at 4°C in order to precipitate the proteoglycans. After washing in ethanol , the pellet was resuspended in Laemmli buffer and counted. Biochemical analysis of VDR: Previous in vivo studies have indicated

112 C. Farquharson, J. L. Berry, E. B. Mawer et al.

that hypertrophic chondrocytes have significantly more VDR than the less mature proliferating chondrocytes [4]. We therefore examined chondrocytes isolated from both growth plate zones in order to deter­mine whether ascorbic acid differentially regulated their VDR number. Further, as VDR expression is likely to change with time in culture we added ascorbic acid from day 1 in order to try and maintain the differ­ent VDR expression noted in vivo.

Chondrocytes were detached from the growing surface using both collagenase (0.1 %) and dispase (0.25 %) for 10 min, counted and cen­trifuged. Receptor preparations were made as previously described [3]. Briefly, chondrocytes were sonicated in Tris/KCl buffer, pH 7.5, containing 0.1 % gelatin. After centrifugation each preparation was made up with Tris/KCl buffer to contain 2-3 X 106 cells per ml. Dupli­cate 0.5 ml aliquots were incubated with 6000 dpm eH]1,25-D (6.48 TBq/mmol) and increasing concentrations of unlabeled 1,25-D for 1 h, as described previously [28]. Immunocytochemistry: The collagen type II antibody (II-II6B3) was diluted to 6 [tg IgG/ml and used in conjunction with an indirect peroxi­dase method [18] to localize collagen type II in chondrocyte monolayer cultures. Lipid peroxidation: Cells were detached from the culture surface by treatment with collagenase and dispase. The cells were lysed with re­peated freeze-thawing and the products of lipid peroxidation, malonal­dehyde and 4-hydroxyalkenals were measured by a commercial kit according to manufacturer's instructions.

In Vivo Studies Animals and tissue preparation. Chicks were fed from hatching to 3 weeks of age on a standard broiler diet or one supplemented with 1 gI kg ascorbic acid. The chicks were injected intra-peritoneally with 25 mg bromodeoxyuridine (BrdU)/kg body weight 21 h before being killed. Samples of the proximal tibiotarsi were immersed in 5 % polyvi­nyl alcohol and chilled immediately at -70°C. Serial sections (10[tm thick) were cut in the direction of growth on a cryostat and were pro­cessed for immunocytochemistry. Immunocytochemistry. Nuclei with incorporated BrdU were detected using indirect immunofluorescence. The distance from the top of the growth plate to the furthest BrdU-positive chondrocyte down the growth plate in a longitudinal direction was measured [16]. Five mea­surements of each parameter were taken along the breadth of the growth plate from two serial sections of each bone from each of six chicks in each experimental group. Collagen, proteoglycan and VDR analysis. The proximal tibiotarsi growth plates were dissected out, weighed and analyzed for hydroxy­proline and proteoglycan content as described above. For VD R analy­sis, chondrocytes were isolated from the growth plate of the proximal tibiotarsi, by collagenase digestion, and pooled with respect to their dietary group. Biochemical analysis was as described for the in vitro experiments. Plasma 1,25-dihydroxyvitamin D3 analysis. Blood samples were taken immediately before death and plasma was collected and frozen at -20°C until assay. Plasma samples were assayed using the IDS 1251_1,25_ D radioimmunoassay kit following the manufacturer's instructions [22]. Briefly, 0.5 ml of plasma was delipidated with 50 [tl of a solution of dextran sulfate and magnesium chloride. 1,25-D was extracted from duplicate 100 [tl aliquots of the supernatant by incubation for 3 h with a highly specific solid phase monoclonal antibody to 1,25-D. Following immunoextraction, purified 1,25-D was eluted directly into glass tubes and evaporated to dryness under nitrogen. The dried extracts were reconstituted with assay buffer. Extracts and calibrators were incu­bated overnight at 4°C with sheep-anti 1,25-D polyclonal antibody. 125I_1,25_D was added and the incubation continued for 2h at room temperature. Separation of bound from free label was performed using Sac-Cel® solid phase and the precipitates were counted for 2 min in a gamma counter. Bound radioactivity was inversely proportional to the concentration of 1,25-D.

Statistical analyses All in vitro experiments were performed a minimum of 3 times and although slight variations in absolute values between individual experi­ments existed the trends shown in the representative experiments reported were noted in all experimental repeats. All data are expressed as the mean ± SEM and statistical analyses were performed using Student's t-test or one-way analysis of variance (ANOVA).

Results

Effect of ascorbic acid on chondrocyte alkaline phosphatase activity and collagen synthesis Addition of ascorbic acid for 7 days to confluent chondrocytes significantly increased ALP activity (9.36 ± 0.52 vs. 20.8±2.83 nmoles pNPP hydrol/minlmg protein, P<O.01) in comparison to cultures without ascorbic acid. Immunolocal­ization of collagen type II clearly indicated that in the pres­ence of ascorbic acid an extensive extracellular collagen net­work was formed, whereas in unsupplemented cultures the collagen is retained within the cell with little, if any, extracellu­lar staining (result not shown). Although collagen type X syn­thesis is well accepted as a marker of chondrocyte terminal dif­ferentiation it could not be used in this present study. Its syn­thesis, together with other collagen types, would be affected directly by the ascorbic acid status of the cultures and its rate of induction would not reflect the true differentiation state of the chondrocytes. We therefore limited the assessment of chondrocyte differentiation to ALP activity.

Effect of collagen and proteoglycan inhibitors on matrix production and chondrocyte alkaline phosphatase activity In cell culture, matrix components are either soluble and se­creted into the culture medium or retained in the cell-matrix layer. As it is the matrix components of the latter that influ­ence chondrocyte metabolism we restricted our analysis to matrix accumulated in this fraction.

Chondrocyte ALP activity and total collagen and proteogly­can concentrations within the cell-matrix layer were all signifi­cantly higher in cultures supplemented with ascorbic acid than in the unsupplemented controls (Fig. 1). Addition of inhibitors of collagen (3,4-dehydroproline) or proteoglycan (xyloside) synthesis to the ascorbic acid-supplemented cultures resulted in a reduction in ALP activity and in the concentration of ma­trix components to concentrations close to those of unsupple­mented cultures. An indication of the collagen and proteogly­can interactions that exist within the cell-matrix layer is also shown in Fig. 1. Collagen synthesis inhibitors lowered ALP activity and decreased the concentration of both collagen and proteoglycans. A similar inhibition of both matrix components and ALP activity was observed in the presence of xyloside. These results were corroborated by the measurement of the uptake rate of FH]proline and [35S]S04 into the cell-matrix layer. Inhibition of matrix accumulation within the cell-matrix layer was again associated with decreased chondrocyte ALP activity (Table I).

Chondrocytes cultured in a pellet reorganize into a mineral­ized cartilage-like mass. There is an extensive extracellular matrix, containing collagen types II and X and ALP activity similar to that of growth plates in vivo [18]. Using this physio­logical system we noted that xyloside treatment inhibited ALP activity and decreased both proteoglycan and collagen con-

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centrations by 48.5% (P<O.Ol), 70.3% (P<O.OOl) and 28.3 % (P <0.001), respectively (Fig.2a). Cartilage-like pel­lets were also visibly smaller in the presence of xyloside but failed to form when the cells were cultured in the absence of ascorbic acid or in the presence of collagen synthesis inhibi­tors.

Effect of preformed matrix on chondrocyte alkaline phosphatase activity Chondrocytes grown for 7 days on plastic, collagen type I or Biomatrix in the presence of ascorbic acid from day 1 of cul­ture all had similar ALP activity (45.8-54.5 nmoles of activity/ min/mg protein) which was significantly higher than their respective unsupplemented controls (Fig. 2b). These results also demonstrate that the extent of the chondrocyte ALP response to ascorbic acid is dependent upon the time of its addition to the cultures. The addition of ascorbic acid at the time of plating results in much higher ALP activity (Fig. 2b) when compared to cultures supplemented at confluency (Fig. 1).

Tab. I. In vitro effect of ascorbic acid (AA) and matrix synthesis inhibitors on chondrocyte alkaline phosphatase activity and the rate of collagen and prot eo glycan incorporation into the cell-matrix layer.

Treatment

+AA

-AA

+AA +dehydro­proline

+AA +xyloside

Alkaline [3H]proline [355]50,

Phosphatase Activity (nmoles pNPP (dpm/culture (dpm/culture hydrol/min/mg well) well) protein)

17.14±0.55 6342.0 ± 1027.0 4521.6 ± 225.8

9.85 ± 0.54*** 380.8± 54.2*** 1744.7 ± 240.7***

5.49 ± 0.24*** 495.8± 16.2*** 1104.6 ± 101.0***

6.54 ± 0.57*** 3239.9 ± 278.9** 1637.2 ± 263.5***

Values expressed as mean ± SEM from four replicates within each group. Significantly different from their respective +AA treatment, **P<0.01, *** P<0.001.

Ascorbic ocid-induced chondrocyte terminol differentiotion 113

Proteoglycan Collagen

four replicates in each group. Where error bars are missing they are too small to be shown. Significantly different from their respective +AA treatment, * P<O.05, ** P<O.01, *** P<O.OOl.

No evidence was obtained to suggest that the matrix synthe­sis inhibitors were toxic to the chondrocytes. Chondrocyte morphology was unaltered and no significant effect on chon­drocyte number (measured as cellular protein) was observed after 7 days in the presence of either inhibitor. After removal of the inhibitor the cells, in the presence of ascorbic acid, con­tinued to grow at the same rate as the ascorbic acid supple­mented cells, providing further evidence of the non toxic nature of the matrix inhibitors. The chondrocyte antiprolifera­tive effects of ascorbic acid are clearly seen at day 14 (Fig. 3).

The effect of exogenous RGD-containing peptides on chondrocyte ALP activity In the presence of ascorbic acid and RGD-containing peptides, chondrocyte ALP activity was significantly lower (P<O.Ol) than in chondrocytes cultured in the absence of RGD-containing peptides and similar to those without ascorbic acid supplementation. The presence of the control peptide GRGESP had no effect on ALP activity (Fig. 4a).

The effect of ascorbic acid on the vitamin D receptor Chondrocytes isolated from the proliferating and hypertrophic zone of the growth plate had similar VDR numbers after 6 days in culture. In the presence of ascorbic acid, the VDR number of both chondrocyte populations increased but this only reached significance in the proliferating chondrocytes (54.6%; P<0.05). There was no effect of ascorbic acid on chondrocyte VDR affinity for 1,25-D (Table II).

Lipid peroxidation and collagen synthesis: the effect of ascorbic acid Only small amounts of malonaldehyde and 4-hydroxyenals were produced by chondrocytes in vitro and no evidence was obtained to indicate that ascorbic acid induced lipid peroxida­tion (0.97 ± 0.04 vs. 0.94 ± 0.05 [.lmoles/106 cells). Incubation of chondrocytes in the presence of ascorbic acid and free rad­ical scavengers (vitamin E) and cell-impermeable iron chela­tors (desferrioxamine and EDTA) had no effect on collagen incorporation into the cell matrix layer (Fig. 4b).

114 C. Farquharson, J. L. Berry, E. B. Mawer et al.

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Tab. II. In vitro effect of ascorbic acid (AA) on chondrocyte vitamin D receptor number and affinity.

Proliferating chondrocytes (n = 5) Hypertrophic chondrocytes (n = 3)

Rec/cell Kd (pM)

Rec/cell Kd (pM)

-AA +AA

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1885 ± 261 2898 ± 260 35.3± 1.4 77.6± 25.9

Values expressed as mean ± SEM. Significantly different from their respective +AA treatment, * P < 0.05.

....... 1500

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Fig. 3. Effect of dehydroproline and xyloside on protein content of cultures after 7 days incubation and also after a further 7 days (14 days total) in which the inhibitors were removed. Results are from repre­sentative experiments and are the means ± SEM from four replicates in each group. Where error bars are missing they are too small to be shown. Significantly different from their respective + AA treatment, **P<O.Ol.

In vivo studies: The eHect of ascorbic acid on growth plate matrix composition, chondrocyte terminal diHerentiation and vitamin D receptor number Growth plates of chicks fed a diet supplemented with ascorbic acid had significantly higher concentrations of collagen (26.2%, P<O.01) and proteoglycans (9.8%, P<O.01) in comparison to tissue from unsupplemented birds. The dis­tance moved down the growth plate by the BrdU-labeled chondrocytes was similar in the ascorbic acid-supplemented and control birds (Table III). In ascorbic acid-supplemented birds there were significant increases in plasma concentrations of ascorbic acid (121.4 %; P <0.01) and chondrocyte VDR number (31.7 %; P <0.05). Chondrocyte affinity for 1,25-D was similar in both groups of birds (Table IV). Serum 1,25-D concentrations were higher in the supplemented birds although this difference did not quite reach statistical signifi­cance (P = 0.054).

Discussion

Promotion by ascorbic acid of the differentiated hypertrophic phenotype has been reported for a variety of mesenchymal cells maintained in culture and this effect has generally been considered to be a consequence of matrix secretion and inter­action with cell surface receptors. Establishment of cell-matrix interactions may generate signals that stimulate subsequent marker gene expression. Although it is well accepted that ascorbic acid elevates chondrocyte ALP activity, conflicting reports exist as to the importance of the matrix in this process. In agreement with the present results, Habuchi et al. [26] reported that ascorbic acid-induced chondrocyte terminal dif-

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Fig. 4. (a) In vitro effect of RGD-containing peptides or control (RGE) peptides on chondrocyte ALP activity. (b) The effect of ascorbic acid (AA) and vitamin E and cell-impermeable iron chelators on collagen incorporation into the cell-matrix layer. Results are from representative experiments and are the means ± SEM from four repli­cates in each group. Where error bars are missing they are too small to be shown. Significantly different from the + AA treatment. ** P < 0.01, *** P < 0.001.

Ascorbic acid-induced chondrocyte terminal differentiation 115

ferentiation was dependent upon collagen secretion, whereas a more recent study indicated that elevated transcription rates for ALP and collagen type X were not dependent upon the presence of a collagen-rich matrix [42].

Previous studies [35] have indicated that collagens and pro­teoglycans are secreted independently by chondrocytes in response to ascorbic acid and that proteoglycan secretion is unaffected by ascorbic acid treatment. Our results clearly show, however, that the accumulation of both matrix compo­nents in the cell-matrix layer is influenced by the ascorbic acid status of the cultures. This is probably a result of the chondro­cyte responding to the properties of the surrounding matrix and regulating its own composition accordingly. The results of the experiments using matrix synthesis inhibitors reported in this study further indicate that the accumulation of collagen and proteoglycans within the cell-matrix is interdependent. Such interdependency has also been noted in other studies [33]. From this observation it is not possible to separate the relative importance of these individual matrix proteins in the regulation of chondrocyte ALP activity.

Addition of xyloside to chondrocytes grown in pellet cul­tures resulted in decreased matrix accumulation and chon­drocyte ALP activity. The cartilage pellets formed were also smaller, probably reflecting the low amounts of glycosamino­glycans and their associated water molecules. Similarly, chick limb bud mesenchymal cells treated with xyloside formed into nodules with lower amounts of chondroitin sulfate proteogly­can present. Chondrogenesis was not impaired in this model [33] but the effect of xyloside on chondrocyte differentiation was not studied.

The matrix synthesis inhibitors used in this present study have been widely used [26, 33, 42]. Problems with toxicity and chondrocyte phenotype stability have not been reported in these studies where the length of incubation in their presence was up to 8 days. We also noted no toxic effects and therefore it is unlikely that the results of this study are due to non­specific toxic effects of the matrix inhibitors.

In this study we found no evidence in the in vitro experi­ments to support the suggestion that the mechanisms underly­ing ascorbic acid-induced collagen synthesis in fibroblasts are related to membrane lipid peroxidation [12, 24]. Chondrocyte peroxidation was not higher in ascorbic acid-treated cells and cell-impermeable iron chelators (EDTA and desferrioxamine) or peroxidation inhibitors (a-tocopherol) did not effect colla­gen synthesis. A related study has recently reported that lipid peroxidation and collagen synthesis can be dissociated [14].

Tab. III. In vivo effects of dietary ascorbic acid (AA) supplementa­tion on collagen and proteoglycan concentration of the growth plate and the distance of chondrocyte movement down the growth plate.

Treatment

+AA

-AA

Collagen

(mg/g wet weight)

24.8±0.91

19.7 ± 0.91 **

Proteoglycan Movement down growth Plate

(mg/g wet (% of growth plate weight) width)

52.9 ± 0.97 68.8±0.98

48.2±0.61** 71.6±0.85

Values expressed as mean ± SEM from six chicks within each group. Significantly different from their respective +AA treatment, ** P < 0.01.

116 C. Farquharson, J. L. Berry, E. B. Mower et 01.

Tab. IV. In vivo effect of ascorbic acid (AA) on serum 1,25-dihydroxyvitamin D3 concentration and chondrocyte vitamin D recep­tor number and affinity.

n -AA +AA

Plasma 1,25-D (pmol/l) 34 181.3± 8.3 204.5± 9.5 Receptors per cell 9 1095.0 ± 113 1442.0±184* Kd (pM) 9 36.4± 6.8 36.3± 6.0 Plasma AA (!lmol/l) 6 47.0± 6.4 104.1 ± 9.0**

Values expressed as mean ± SEM. Significantly different from their respective +AA treatment, * P < 0.05, ** P < 0.01.

To determine whether the observations noted in culture could be made also in vivo, we analyzed the effects of dietary ascorbic acid supplementation on matrix synthesis and the speed of chondrocyte movement through the growth plate. The latter measurement, which is an indicator of the rate of movement towards terminal differentiation [16], was unal­tered by ascorbic acid but both collagen and proteoglycan con­centrations within the growth plate were significantly increased. In response to ascorbic acid the percentage increase in chondrocyte matrix accumulation was severalfold higher in vitro than in vivo. In the chick endogenous synthesis of ascorbic acid led to near maximal matrix synthesis, whereas in vitro matrix secretion was severely limited in the unsupple­mented cultures. It is possible, therefore, that the small increase in the matrix concentration noted in the ascorbic acid-supplemented chicks, which is in agreement with previ­ous studies [45], was not sufficient to influence the movement towards terminal differentiation in vivo.

The addition of exogenous peptides containing the RGD tripeptide, arginine-glycine-aspartic acid, was found to inhibit ascorbic acid-induced chondrocyte ALP activity. These pep­tides compete with the synthesized matrix, effectively block­ing the cell surface receptor and leading to impairment of nor­mal cell-matrix interactions. This observation provides further evidence for the importance of such interactions in the organi­zation of cartilage tissue and the regulation of chondrocyte ter­minal differentiation. Chondrocytes have been shown to con­tain a number of cell surface adhesion receptors such as chondronectin and anchorin and RGD recognition sites have been located within collagen and other growth cartilage matrix proteins such as fibronectin, osteopontin and bone sialopro­tein [47]. Ligand-receptor interactions would give the chondrocytes the potential to sense the composition and prop­erties of the surrounding matrix and also provide a direct means for signals from the matrix to influence gene expression and terminal differentiation [13].

From the above, it would seem probable that a collagen-rich matrix could replace the requirement for ascorbic acid for chondrocyte terminal differentiation to take place. As this was not the case in this and other studies [42] it must be concluded that plastic coated with a preformed matrix cannot mimic the intimate cell-matrix interactions that occur between a cell and its endogenously produced matrix.

In addition to the known requirement for ascorbic acid in the hydroxylation events associated with collagen synthesis, some evidence exists for a possible role for this vitamin in ste­roid hydroxylation [32] and the synthesis of vitamin D metab­olites [40]. Our study indicated the existence of a positive rela­tionship between VDR number and plasma 1,25-D concentra-

tion in chicks supplemented with ascorbic acid. As 1,25-D is known to increase the number ofVDR binding sites [25], it is likely that the effect of ascorbic acid on VDR number is medi­ated by the higher circulating concentrations of 1,25-D. An increase in VDR number in response to ascorbic acid was also noted in vitro but the extrarenal conversion of 25-D to 24,25-dihydroxyvitamin D3 and not 1,25-D in cultured chick chondrocytes [3, 23] suggests that this upregulation of VDR number by ascorbic acid in culture may not be related to 1,25-D production but secondary to the promotion of the termi­nally differentiated phenotype in these cells. We and others have demonstrated by immunocytochemistry and Scatchard analysis that hypertrophic chondrocytes have more VDR than the less mature, proliferating chondrocytes [4, 30].

Our in vivo results are in agreement with others, where die­tary supplementation with ascorbic acid resulted in elevated serum 1,25-D concentrations in chicks [45] and humans [11] and ascorbic acid deprivation led to lower circulating 1,25-D levels and lower numbers of intestinal VDR [40]. In vivo stud­ies have indicated that 1,25-D influences chondrocyte arrange­ment [36] and cartilage growth [10] and we have further shown that dietary supplementation with 1,25-D accelerates the onset of chondrocyte maturation in chicks [16]. It is recognized that circulating concentrations of 1,25-D are carefully regulated and it is therefore possible that the small rise in plasma 1,25-D noted in the ascorbic acid-supplemented chicks was insuffi­cient to promote terminal differentiation.

In conclusion, the results of the chondrocyte culture studies indicate that promotion of the terminally differentiated phe­notype by ascorbic acid is mediated by the cells own extracel­lular matrix and involves interactions between the cell surface receptor and recognition peptides (RGD) within the matrix proteins. Dietary supplementation with ascorbic acid led to increased matrix production by the growth plate chondrocytes but increased terminal differentiation was not noted. Evi­dence is provided for a role of ascorbic acid in the metabolism of vitamin D and upregulation of its intracellular receptor. This observation requires further experimentation but is important because amplification of the VDR dependent genomic response to 1,25-D would result in promotion of ter­minal differentiation.

Acknowledgements. This study was funded by the Ministry of Agricul­ture Fisheries and Food, the CEC Directorate-General for Agricul­ture, a linked research grant from the Biotechnology and Biological Sciences Research Council, and F. Hoffmann-La Roche Ltd, Basel, Switzerland. The authors are grateful to Heather McCormack for the provision and formulation of the diets and to Professor Mike Lean, Department of Human Nutrition, University of Glasgow, Scotland for the plasma ascorbic acid analysis.

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