Anne-Marie Freyria
email: am.freyria@ibcp.fr

Marie-Claire Ronzière

S Roche

Cécile F Rousseau

Daniel Herbage

Marie-Claire Ronzie

` Re

´phane Ste

Ce Roche

F Rousseau

Regulation of Growth, Protein Synthesis, and Maturation of Fetal Bovine Epiphyseal Chondrocytes Grown in High-Density Culture in the Presence of Ascorbic Acid, Retinoic Acid, and Dihydrocytochalasin B

Keywords: chondrocyte maturation, collagen types I, II, and X, ascorbic acid, retinoic acid, dihydrocytochalasin B

published or not. The documents may come

Regulation of growth, protein synthesis, and maturation

of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B.

During endochondral ossification, chondrocytes undergo maturation from resting and proliferating cells to hypertrophic cells [START_REF] Brighton | Structure and function of the growth plate[END_REF][START_REF] Hunzinker | Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes[END_REF]. This maturation process is controlled by programmed events, with changes in cell morphology and qualitative and quantitative variations in the synthesis of matrix macromolecules. Culture conditions and the presence of agents such as vitamins and growth or differentiation factors can modulate the phenotypic expression of the chondrocytes [START_REF] Solursh | Differentiation of cartilage and bone[END_REF][START_REF] Sandberg | Matrix in cartilage and bone development: current views on the function and regulation of major organic components[END_REF][START_REF] Adolphe | Different types of cultured chondrocytes. The in vitro approach to the study of biological regulation[END_REF][START_REF] Cancedda | Chondrocyte differentiation[END_REF][START_REF] Labourdette | Chondrocytes in culture: a useful tool for studying cartilage structure, development and repair[END_REF]Hickok et al., 1998]. After release from their cartilagenous matrix, rabbit differentiated articular chondrocytes grown in a monolayer on plastic surfaces rapidly lose their initial phenotype [START_REF] Benya | Independent regulation of collagen types of chondrocytes during the loss of differentiated function in culture[END_REF]. Under culture conditions that limit their attachment and proliferation, in liquid suspension [START_REF] Horwitz | The growth of cartilage cells in soft agar and liquid suspension[END_REF][START_REF] Pacifici | Stable phenotypic expression by chick chondroblasts in long-term suspension culture as determined by proteoglycan analysis[END_REF][START_REF] Tacchetti | In vitro morphogenesis of chick embryo hypertrophic cartilage[END_REF] or in a three-dimensional matrix such as agarose [START_REF] Benya | Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels[END_REF][START_REF] Aydelotte | Articular chondrocytes cultured in agarose gel for study of chondrocytic chondrolysis[END_REF], alginate beads [START_REF] Guo | Culture and growth characteristics of chondrocytes encapsulated in alginate beads[END_REF][START_REF] Ha ¨uselmann | Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads[END_REF], or collagen gels [START_REF] Gibson | Effects of matrix macromolecules on chondrocyte gene expression : synthesis of a low molecular weight collagen species by cells cultured within collagen gels[END_REF][START_REF] Kimura | Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures[END_REF], chondrocytes from various sources maintain their rounded configuration and a cartilage phenotype. Inhibition of chondrocyte spread by a high initial cell seeding density on plastic or by using the cell pellet technique has been shown by several authors to allow simple culture conditions for analysis of changes in chondrocyte gene expression during maturation in vitro. Under these conditions, fetal bovine epiphyseal or adolescent bovine articular cells [START_REF] Harmand | Proteoglycan synthesis in chondrocyte cultures from osteoarthritic and normal human articular cartilage[END_REF][START_REF] Kuettner | Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology[END_REF][START_REF] Daniel | Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III Effects of ascorbate[END_REF][START_REF] Ruggiero | Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density[END_REF][START_REF] Hering | Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension[END_REF]Freyria et al., 1995a,b;Ronziere et al., 1997], chicken growth plate cells [START_REF] Farquharson | Differentiation and mineralization in chick chondrocytes maintained in a high cell density culture: a model for endochondral ossification[END_REF] rabbit growth plate cells [START_REF] Kato | Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors[END_REF][START_REF] Iwamoto | Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor[END_REF], or rat epiphyseal cells [START_REF] Ballock | TGF-beta1 prevents hypertrophy of epiphyseal chondrocytes: regulation of gene expression for cartilage matrix proteins and metalloproteases[END_REF] rapidly synthesize their own extracellular matrix, form multicellular layers, and can be maintained in culture for longer than 1 month.

Ascorbic acid and retinoic acid, factors known to modulate the phenotype of the chondrocytes, were frequently added in the different culture systems described to study the chondrocyte maturation. Most of these studies were conducted in chicken embryo utilizing either vertebra [START_REF] Oettinger | Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures[END_REF][START_REF] Gerstenfeld | Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system[END_REF][START_REF] Adams | Rapid induction of type X collagen gene expression in cultured chick vertebral chondrocytes[END_REF] or sterna (caudal as immature chondrocyte zone and cephalic as mature chondrocyte region) [START_REF] Yasui | Coordinate regulation of type IX and type II collagen synthesis during growth of chick chondrocytes in retinoic acid or 5-bromo-2'-deoxyuridine[END_REF][START_REF] Horton | Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes[END_REF][START_REF] Bruckner | Induction and prevention of chondrocyte hypertrophy in culture[END_REF][START_REF] Leboy | Ascorbic acid induces alkaline phosphatase, type X collagen and calcium deposition in cultured chick chondrocytes[END_REF][START_REF] Dietz | Alterations of collagen mRNA expression during retinoic acid induced chondrocyte modulation: absence of untranslated alpha 1(I) mRNA in hyaline chondrocytes[END_REF]Iwamoto et al., 1993aIwamoto et al., ,b, 1994;;[START_REF] Sullivan | Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion[END_REF][START_REF] Chen | Osteogenic protein-1 promotes growth and maturation of chick sternal chondrocytes in serum-free cultures[END_REF][START_REF] Leboy | Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid[END_REF][START_REF] Venezian | Modulation of chondrocyte proliferation by ascorbic acid and BMP-2[END_REF]]. The chicken growth plate was also frequently used [START_REF] Castagnola | Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes[END_REF][START_REF] Takishita | Effect of retinoic acid on proliferation and differentiation of cultured chondrocytes in terminal differentiation[END_REF][START_REF] Wu | Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing plate chondrocytes[END_REF][START_REF] Nie | Retinoic acid treatment elevates matrix metalloproteinase-2 protein and mRNA levels in avian growth plate chondrocyte cultures[END_REF]. These studies and studies with chondrocytes from other species such as quail [START_REF] Sanchez | Alpha 2(I) collagen gene expression is up-regulated in quail chondrocytes pretreated with retinoic acid[END_REF], rat [START_REF] Ballock | TGF-beta1 prevents hypertrophy of epiphyseal chondrocytes: regulation of gene expression for cartilage matrix proteins and metalloproteases[END_REF], rabbit [START_REF] Kato | Terminal differentiation and calcification in rabbit chondrocyte cultures grown in centrifuge tubes: regulation by transforming growth factor beta and serum factors[END_REF][START_REF] Iwamoto | Hypertrophy and calcification of rabbit permanent chondrocytes in pelleted cultures: synthesis of alkaline phosphatase and 1,25-dihydroxycholecalciferol receptor[END_REF], bovine [START_REF] Hering | Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension[END_REF][START_REF] Koyano | Collagen and proteoglycan production by bovine fetal and adult chondrocytes under low levels of calcium and zinc ions[END_REF], and human [START_REF] Kirsch | Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and X collagen and matrix mineralisation[END_REF][START_REF] Stephens | Human articular surface chondrocytes initiate alkaline phosphatase and type X synthesis in suspension culture[END_REF] and from different tissues showed that all of the cell types analyzed could undergo a sequence of changes up to the final maturation with type X collagen synthesis and increase in alkaline phosphatase activity, depending on the culture conditions and on the presence of appropriate activating agents. Thus, cells from the reserve (resting) zone and the permanent articular region can undergo hypertrophy, although at a slower rate than cells from the growth plate.

In previous studies, we determined the composition and organization of the collagen network produced by fetal bovine epiphyseal chondrocytes in long-term culture at high density [START_REF] Ruggiero | Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density[END_REF] and the effect on these parameters of retinoic acid [Freyria et al., 1995a,b] and ascorbic acid [Ronziere et al., 1997]. In this paper, we report a study conducted to examine phenotypic modulation of the cells in the same culture model in the presence of ascorbic acid, retinoic acid (0.5 or 10 µM) and dihydrocytochalasin B (DHCB, 3, 10, and 20 µM), added separately or in combination (ascorbic acid ϩ retinoic acid or ascorbic acid ϩ DHCB). Cell morphology and proliferation, synthesis of total protein and collagen types I, II, and X in the cell layer and the medium and alkaline phosphatase activity were characterized under various culture conditions and various lengths of culture. The pattern of secreted proteins was determined by two-dimensional electrophoresis in order to elucidate the qualitative and quantitative modifications induced by these factors in fetal bovine epiphyseal chondrocytes.

MATERIALS AND METHODS

Cell Culture

Chondrocytes were isolated by enzymatic digestion from the reserve zone of the distal femoral epiphyses of 4-month-old bovine fetuses (five different animals were used in this study) after careful removal of the articular surface and the growth plate. They are noted here ''fetal bovine epiphyseal chondrocytes'' according to [START_REF] Koyano | Collagen and proteoglycan production by bovine fetal and adult chondrocytes under low levels of calcium and zinc ions[END_REF]. Chondrocytes were cultured at high density (0.8 ϫ 10 6 cells/cm 2 ) in RPMI/ NCTC medium containing 10% fetal calf serum and antibiotic supplements, in the absence or presence of freshly prepared ascorbic acid (25 µg/ml) throughout the culture. The medium was changed every three days, as previously described [START_REF] Ruggiero | Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density[END_REF]Freyria et al., 1995a;[START_REF] Ronzie | Analysis of types I, II, III, IX and XI collagens synthesized by fetal bovine chondrocytes in high-density culture[END_REF]. All-trans retinoic acid or DHCB was dissolved in 95% ethanol and added to the culture media at a volume of 0.2% after 2 days of culture and when the treated cultures were fed. Cells cultured with retinoic acid received 0.5 or 10 µM for 4, 11, 21, or 28 days, whereas cells cultured with DHCB received 3, 10, or 20 µM for 11 or 21 days; the control cultures received the same amounts of ethanol. The cultures were analyzed after 6, 13, 23, and 30 days of culture.

Electron Microscopy

For transmission electron microscopy, cultures were fixed in 2% glutaraldehyde in phosphate-buffered saline for 30 min at room temperature and post-fixed in 2% osmium tetroxide in phosphate-buffered saline for 1 h. After dehydration in successive dilutions of ethanol, the material was embedded in Epon. Thin sections were mounted on copper grids, stained with uranyl acetate, and lead citrate and observed on a Jeol 1200 EX electron microscope at a voltage of 80 kV.

For scanning electron microscopy, cultures were fixed in a 4% glutaraldehyde cacodylatebuffered solution for 1 h at room temperature then washed for 1 h with 0.4 M sodium cacodylate, pH 7.4, and 360 mOsmol/L. The samples were dehydrated by acetone diffusion, critical point-dried, coated with gold palladium, and viewed under a S800 Hitachi microscope at 15 kV. All of these experiments were performed at the Centre for Electron Microscopy Applied to Biology and Geology, Universite ´Claude Bernard, Lyon.

Analytical Procedures

Protein synthesis after 12 and 22 days of culture of DHCB-treated cells was measured by labelling duplicate cultures with 8 µCi/ml of 14 C-proline (290 mCi/mmol, NEN) for 24 h in fresh medium. For the retinoic acid-treated chondrocytes, labelling was performed after 5, 12, 22, or 29 days of culture with 20 µCi/ml of 35 S-methionine (1,000 Ci/mmol, SJ204 Amersham Pharmacia Biotech) for 14 h in fresh RPMI lacking methionine and in the absence of fetal calf serum, as previously reported [Freyria et al., 1995a]. After labelling, the media and cell layers were collected and treated separately, as previously described [Freyria et al., 1995a;[START_REF] Ronzie | Analysis of types I, II, III, IX and XI collagens synthesized by fetal bovine chondrocytes in high-density culture[END_REF].

The amounts and types of collagens were monitored in both the medium and the cell layer fractions after salt precipitation and pepsin digestion [Freyria et al., 1995a]. Pepsindigested samples were fractionated under reducing and non-reducing conditions on a 7% sodium dodecylsulfate-polyacrylamide gel (SDS) [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. Aliquots containing a known number of dpm were loaded onto the gels. The bands corresponding to the various types of collagens were visualized by fluorography, and the relative proportions of collagen types I and II were quantified by scanning densitometry with a Personal Densitometer (Molecular Dynamics). As the samples did not represent the same percentage of the corresponding matrix and culture medium and as they were not extracted from the same number of cells, these variations were integrated in the quantitative analysis.

The presence of type X collagen was detected by immunoblot analysis with a mouse anti-deer antibody (a generous gift from G. [START_REF] Gibson | Type X collagen is colocalized with a proteoglycan epitope to form distinct morphological structures in bovine growth cartilage[END_REF]). Pepsin-digested samples containing aliquots equivalent to 150 µl of the original cell layer extract were applied to each lane, reduced with dithiothreitol and electroblotted onto polyvinylidene difluoride membranes after electrophoresis on 10% polyacrylamide gels. After the membrane had been blocked with a 1% bovine serum albumin solution, the blotted proteins were immunostained with the antibody at a 1:200 dilution, a biotinylated second antibody, biotin-avidin-horseradish peroxidase complex and 4-chloro-1-naphtol as a color substrate.

The cellular DNA content was determined using bis-benzimidazole (Hoechst 33258) after extraction of the DNA according to [START_REF] Lipman | Fluorophotometric quantitation of DNA in articular cartilage utilizing Hoechst 33258[END_REF]. DNA calf thymus was used as standard. Cell number was calculated by using a conversion factor of 8 pg DNA per chondrocyte (personal data). Alkaline phosphatase activity was determined with para-nitrophenylphosphate as the substrate [START_REF] Freyria | Peritoneal macrophage response: an in vivo model for the study of synthetic materials[END_REF]. Aliquots (50 µl) of the cell layer lysate (0.1% Triton ϫ100 in 0.1 M Tris-HCl pH 8.1; 600 µl for a 25-cm 2 flask) were incubated after sonication with 250 µl of substrate. Hydrolysis of the substrate was followed for 5-10 mn at 405 nm and 37°C. Proteins in cell lysates were measured with the Pierce microbicinchoninic acid assay kit and bovine serum albumin as the standard. Student's t-test was performed and significant differences were taken as P Ͻ 0.02.

The secreted proteins were analyzed by twodimensional electrophoresis with Immobiline dry strips pH 3-10.5, pre-cast ExcelGel XL SDS, 12-14% acrylamide and ExcelGel SDS buffer strips (Amersham Pharmacia Biotech), as previously reported [Freyria et al., 1995a,b]. The culture media were labelled as for the collagen analyses with 50 µCi/ml of 35 S-methionine (1,000 Ci/mmol, SJ 204 Amersham). After dialysis and freeze-drying, they were solubilized in lysis solution (9.8 M urea, 2% Nonidet P-40, 2% carrier ampholytes [1.7% pH 5-7 (Serva) and 0.3% pH 3-10 (Bio Rad SA)] and 100 µM dithiothreitol. The same number of dpm were loaded for each sample at the cathodic side of the strip and focused at 15°C for 22 kVh. The Bio Rad two-dimensional polyacrylamide gel electrophoresis (PAGE) marker kit was used for determining molecular mass and pI. After SDS-PAGE, the gels were stained with Coomassie Blue, dried, and autoradiographed on reflection autoradiography film.

RESULTS

Cell Proliferation

When fetal bovine epiphyseal chondrocytes were grown for 30 days in high-density culture, the DNA content increased slightly throughout the culture in the absence of ascorbic acid, with 24 Ϯ 2.3 ϫ 10 6 cells per flask at day 6 and 40 Ϯ 4.6 ϫ 10 6 cells at day 30. The presence of ascorbic acid induced a three-fold increase in the DNA content at day 30 (70.2 Ϯ 1 ϫ 10 6 cells) as compared with the level in untreated cultures. The DNA content did not vary over time in the presence of 10 µM retinoic acid (21 Ϯ 3 ϫ 10 6 cells at day 30), whereas a small increase was measured at day 30 (39 Ϯ 1 ϫ 10 6 cells) in cultures treated with ascorbic acid ϩ retinoic acid as compared with the three-fold increase with ascorbic acid alone. The protein content of the cultures followed the changes in cell number.

When the cultures were treated with DHCB alone or with DHCB ϩ ascorbic acid, a rapid decrease in the DNA content was measured, as cells detached themselves from the culture flask. At day 13, a mean 30% decrease was seen with each dose, which reached 50% at day 23.

Cell Morphology

Within 4 weeks in culture under the various conditions, the cells changed in shape and size and in the abundance of extracellular matrix. In the absence of ascorbic acid, the cells had a characteristic round or polygonal morphology with a surrounding matrix after 23 days (Fig. 1a), but after 30 days they appeared to have lost their round shape (Fig. 1e). In the presence of ascorbic acid, the cells showed extensive accumulation of extracellular matrix (Fig. 1c,g). In the presence of 10 µM retinoic acid, the cells lost these characteristic traits and became longer and even smaller than in the control culture (Fig. 1b,f) as more and more were entrapped in the extracellular matrix. The ascorbic acid ϩ retinoic acid-treated cells were shorter and were surrounded by an abundant matrix (Fig. 1d,h). The average cell size did not increase with time in culture, regardless of whether ascorbic acid was present.

The addition of DHCB was followed by a change in cell shape: after 24 h of treatment with either dose of DHCB, the polygonal cells became rounded and kept this morphology with time of culture (Fig. 2). In contrast to the other culture conditions, in presence of DHCB was observed in only slight deposition of extracellular matrix (Fig. 2e).

Deposition and Composition of the Extracellular Matrix

As seen by transmission electronic microscopy, cells in the control cultures grew close together throughout the period of culture and were embedded in a loose matrix composed mainly of collagen fibrils of about 13-18 nm in width (Fig. 3a,e), which were either isolated, irregularly spaced, or organized in bundles. In the presence of ascorbic acid, the cells were widely spread at day 23 (Fig. 3c), and at this time many thin filaments were observed that interconnected the fibrils (Fig. 3g) and resembled a proteoglycan structure. During the 3 weeks of treatment with 10 µM retinoic acid, the cells appeared to grow tightly together, with many contacts (Fig. 3b). The extracellular matrix, deposited as a thin network at the boundaries of the cells, was composed mainly of collagen fibrils with a mean thickness of 15 nm (Fig. 3f). In the culture with the two vitamins (Fig. 3d), the organization and composition of the pericellular matrix (Fig. 3h) resembled that with retinoic acid alone. Chondrocytes grown with 10 µM DHCB were rounded and isolated (Fig. 2d), and there was a less abundant pericellular matrix containing fewer collagen fibrils (Fig. 2e); when they were present (Fig. 2f), they were of the same width as in the control culture (Fig. 2c).

Rates of Protein Synthesis and Collagen Typing

Changes in the total amount of protein, collagen synthesis, and typing in the presence of retinoic acid are presented on Figure 4. In control cultures with and without ascorbic acid, most total and pepsin-resistant protein synthesis occurred in the cell layer, with a net maximum at day 6. Addition of 0.5 µM retinoic acid suppressed this maximum, with an 80% decrease in type II collagen (Fig. 4c) and complete inhibition of the synthesis of type I collagen seen with ascorbic acid (Fig. 4d). On day 13, 10 µM retinoic acid were necessary to obtain simi- lar decreases in types I and II collagen. By day 30, retinoic acid was much less effective: the synthesis level was very low, and type I collagen synthesis was still occurring in cultures with 10 µM retinoic acid ϩ ascorbic acid, in both the medium and the cell layer (Fig. 4d).

In the study with DHCB, the rates of total and pepsin-resistant protein synthesis in the medium and the cell layer were comparable in control cultures on days 13 and 23, and a twoto three-fold increase in protein was seen with ascorbic acid in the cell layer (Fig. 5). With 3 µM DHCB, a mean 65% increase in total and pepsin-resistant proteins was seen, independently of the presence of ascorbic acid (Fig. 5a,b). A slight increase in protein synthesis was seen with 10 µM DHCB, whereas in the presence of 20 µM the level of total protein synthe- sis was close to or below that of the control culture (Fig. 5a). With all three doses, most of the total proteins (70%) and pepsin-resistant material (60%) was found in the cell layer. No change in type I or II collagen synthesis was observed in the cell layer at days 13 and 23 after treatment with 3 µM DHCB ϩ ascorbic acid (Fig. 5). With 10 and 20 µM DHCB ϩ ascorbic acid, a large decrease in type II collagen synthesis was observed on days 13 and 23, with complete inhibition of type I collagen syn-thesis; the contents of types I and II collagen in the medium were too low to be measured.

Chondrocyte Differentiation Markers

Synthesis of type X collagen was analyzed in both the culture medium and the cell layer after treatment with retinoic acid (Fig. 6). Type X collagen was absent from all culture media throughout culture and from the cell layer in the absence of ascorbic acid with or without retinoic acid. Type X collagen was observed in the cell layer of ascorbic acid-treated cultures at days 13 and 30 of culture and in ascorbic acid ϩ retinoic acid-treated cultures at day 30 (Fig. 6).

Table I shows the alkaline phosphatase activity in the cell layer during culture. A low mean level of 135.5 Ϯ 21 mU per 10 6 cells was measured on days 13, 23, and 30 in the control culture, and addition of ascorbic acid did not change this level at any time. In the presence of 0.5 µM retinoic acid, a two-to three-fold increase was observed only in the presence of ascorbic acid. With 10 µM retinoic acid and in the absence of ascorbic acid, the increase was only three-fold on day 30. The presence of both ascorbic acid and 10 µM retinoic acid strongly stimulated alkaline phosphatase activity in a time-dependent manner, increasing it statistically by as much as 12-fold by day 23 (P Ͻ 0.02) and 23-26-fold by day 30 (P Ͻ 0.02). No change in activity could be detected when the cells were grown in the presence of DHCB.

Analysis of Secreted Protein by Two-Dimensional Polyacrylamide Gel Electrophoresis

In order to examine the extent of the changes in protein synthesis induced by the various culture conditions, the media were subjected to two-dimensional electrophoresis. We selected three spots on the autoradiographs for comparison of the proteins expressed on the gels. The synthesis of proteins with a molecular mass of less than 29 kDa was down-regulated between days 6 and 13 in the absence of ascorbic acid (Fig. 7a vs. b), whereas acidic proteins with a mass of 45-95 kDa were expressed. The presence of ascorbic acid maintained a high level of expression of the low-molecular-mass proteins after 12 days (Fig. 7e,f). The subsets of proteins 1, 2, and 3 were also up-regulated in comparison with the untreated culture. Cells grown in the presence of 10 µM DHCB (Fig. 7c) ex-pressed nearly the same pattern of secreted proteins as untreated cells in the absence of ascorbic acid (Fig. 7b), but DHCB inhibited the large increase in protein synthesis due to ascorbic acid alone (Fig. 7g). When 10 µM retinoic acid were added to the culture, alone (Fig. 7d) or in combination with ascorbic acid (Fig. 7h), the cells had a different pattern of secreted protein from untreated cells (Fig. 7b,f): the lowmolecular-mass proteins were mainly downregulated as early as day 3 of treatment (not shown). Subsets 2 and 3 were no longer detected.

DISCUSSION

This study provides additional information [START_REF] Ruggiero | Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density[END_REF]Freyria et al., 1995a,b;[START_REF] Ronzie | Analysis of types I, II, III, IX and XI collagens synthesized by fetal bovine chondrocytes in high-density culture[END_REF] about modulation of the phenotype of fetal bovine epiphyseal chondrocytes cultured for up to 30 days in high density in RPMI/NCTC medium containing 10% fetal calf serum, with or without fresh ascorbic acid Fig. 7. Two-dimensional polyacrylamide gel electrophoresis autoradiographs of secreted proteins of chondrocytes collected on days 6 and 13 and labelled with 35 S-methionine for the last 14 h of culture. Samples containing the same number of counts per minute were separated in the first dimension on Immobiline dry strips pH 3-10.5 and in the second dimension on 12-14% acrylamide SDS ExcelGel XL. Control chondrocytes grown for 6 (a) or 12 days (b). Ascorbic acid-treated chondrocytes grown for 6 (e) or 13 days (f). Chondrocytes treated with 10µM dihydrocytochalasin B for the last 10 days in culture without (c) and with ascorbic acid (g); chondrocytes treated with 10 µM retinoic acid for the last 10 days in culture without (d) and with ascorbic acid (h). The apparent molecular masses of known proteins are indicated on the left side. Gels present the data from one representative single experiment. Comparable results were obtained with chondrocytes isolated from one other fetus.

and in the presence of various concentrations of retinoic acid or DHCB. In the absence of ascorbic acid, chondrocytes have a low proliferation rate (ϫ1.5 after 1 month), with a rapid decrease in total and pepsin-resistant protein synthesis after six days. The rate of type II collagen synthesis was very low at 13 and 30 days, and no type I collagen was synthesized. No deposition of type X collagen could be detected in the cell layer, and the alkaline phosphatase activity in the medium and cell layer remained very low. Thus, under these conditions, epiphyseal chondrocytes have a stable phenotype with low metabolic activity, as observed in vivo.

Addition of fresh ascorbic acid (25 µg/ml) induced extensive changes in chondrocyte growth and metabolism and in the formation of the extracellular matrix, leading to maturation of the cells: cell proliferation was induced (ϫ3 after 30 days), and type II collagen was still being synthesized after 13 days; type I collagen synthesis was seen after 6 days (in the cell layer), and type X collagen was present after 13 days. After 30 days, both types I and X collagen were present in the cell layer, whereas the rate of synthesis of type II collagen and the alkaline phosphatase activity were very low. The presence of type X collagen was not associated with cell hypertrophy, in accordance with results obtained in vivo in the chick growth plate [START_REF] Oshima | Developmental expression of genes in chick growth cartilage detected by in situ hybridization[END_REF]] and in vitro with immature sternal chondrocytes [Pacifici et al., 1991a,b] or with cells isolated from the mandibular ectomesenchyme [Ekayanake and Hall, 1994]. Similar data on proliferation, the expression of types I and II collagen genes and the synthesis of these types were observed [START_REF] Daniel | Synthesis of cartilage matrix by mammalian chondrocytes in vitro. III Effects of ascorbate[END_REF][START_REF] Hering | Modulation of extracellular matrix gene expression in bovine high-density chondrocyte cultures by ascorbic acid and enzymatic resuspension[END_REF] with adolescent bovine articular chondrocytes plated at high density and grown in the presence of ascorbic acid. Several studies with chicken chondrocytes (epiphyseal, growth plate, or sternal cells) have shown that ascorbic acid stimulates type X collagen synthesis and matrix calcification [START_REF] Tacchetti | In vitro morphogenesis of chick embryo hypertrophic cartilage[END_REF][START_REF] Leboy | Ascorbic acid induces alkaline phosphatase, type X collagen and calcium deposition in cultured chick chondrocytes[END_REF]Wu et al., 1989;[START_REF] Gerstenfeld | Gene expression and extracellular matrix ultrastructure of a mineralizing chondrocyte cell culture system[END_REF]Shapiro et al., 1991;[START_REF] Sullivan | Ascorbate modulation of chondrocyte gene expression is independent of its role in collagen secretion[END_REF][START_REF] Leboy | Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid[END_REF][START_REF] Farquharson | Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D[END_REF]]. [START_REF] Ballock | TGF-beta1 prevents hypertrophy of epiphyseal chondrocytes: regulation of gene expression for cartilage matrix proteins and metalloproteases[END_REF] showed that chondrocytes isolated from the reserve zone of the epiphyses of neonatal rats and cultured as a three-dimensional cell pellet with fresh ascorbic acid acquired a hypertrophic phenotype by day 28 with expression of type X collagen and inhibition of type II colla-gen. This terminal differentiation was inhibited by TGF beta-1. In constrast, [START_REF] Kirsch | Ascorbate independent differentiation of human chondrocytes in vitro: simultaneous expression of types I and X collagen and matrix mineralisation[END_REF] have shown that in the absence of ascorbic acid fetal human chondrocytes in long-term culture in suspension over agarose differentiate into so-called 'post-hypertrophic' chondrocytes, which synthesize types I and X collagen. Under these culture conditions, addition of ascorbic acid during the initial phase inhibited the passage to hypertrophic cells, perhaps due to production of radicals to which human chondrocytes may be more sensitive than bovine, rat, or chicken chondrocytes [START_REF] Tschan | Resting chondrocytes in culture survive without growth factors, but are sensitive to toxic oxygen metabolites[END_REF].

Retinoic acid is one of the more biologically active derivatives of retinol (vitamin A). It functions as an important regulatory signalling molecule for cell growth and differentiation both during embryogenesis and in the adult animal [START_REF] Gudas | Retinoids and vertebrate development[END_REF]. It regulates the expression of genes directly through interactions with DNAbinding retinoic acid receptors, the gamma subtype of which is found predominantly in cartilage [START_REF] Kastner | Murine isoforms of retinoic acid receptor gamma with specific patterns of expression[END_REF]. We have shown here that addition of high doses of retinoic acid (0.5 and 10 µM) to a culture medium in the absence of fresh ascorbic acid does not modify the metabolic activity or the phenotype of fetal bovine epiphyseal chondrocytes in high-density culture. Addition of retinoic acid in combination with ascorbic acid decreased the cell proliferation induced by ascorbic acid and suppressed the high level of total protein and collagen II and I synthesis observed at day 6. The synthesis of types I and X collagen was delayed but these two collagens were present at day 30. The presence of type X collagen, a marker of maturation, was again not associated with hypertrophy of the cells, as already shown with ascorbic acid without retinoic acid. Interestingly, with 10 µM retinoic acid ϩ ascorbic acid, we observed a striking increase in alkaline phosphatase activity in comparison with that seen with each vitamin alone (15-25-fold increase at days 23 and 30). At day 30, no mineralization of the matrix was found, probably because beta-glycerophosphate, a well-known activator of calcification, was not added to the culture medium [START_REF] Chung | Mechanism of action of beta-glycerophosphate on bone cell mineralization[END_REF][START_REF] Coe | Matrix mineralization in hypertrophic chondrocyte cultures. Beta glycerophosphate increases type X collagen messenger mRNA and the specific activity of pp60 c-src kinase[END_REF].

Previous studies have also shown that retinoic acid inhibits the expression of the genes coding for cartilage matrix proteins such as type II collagen [START_REF] Benya | Modulation of the rabbit chondrocyte phenotype by retinoic acid terminates type II collagen synthesis without inducing type I collagen: the modulated phenotype differs from that produced by subculture[END_REF][START_REF] Horton | Retinoic acid rapidly reduces cartilage matrix synthesis by altering gene transcription in chondrocytes[END_REF]. It has also been shown that retinoic acid induces maturation and matrix mineralization specifically in pre-hypertrophic cells, isolated from the cephalic portion of the chicken sternum or from growth plate cartilage [START_REF] Oettinger | Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures[END_REF]Pacifici et al., 1991a,b;Iwamoto et al., 1993a[START_REF] Iwamoto | Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization[END_REF][START_REF] Wu | Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing plate chondrocytes[END_REF]; however, alkaline phosphatase and mineralization were not induced in immature chondrocytes isolated from the caudal region of the chick embryo sternum in the presence of 35 or 100 nM retinoic acid, with or without ascorbic acid [Pacifici et al., 1991b;Iwamoto et al., 1993a[START_REF] Iwamoto | Retinoic acid is a major regulator of chondrocyte maturation and matrix mineralization[END_REF]. Thus, our results demonstrate for the first time that combined treatment with ascorbic acid and high doses of retinoic acid is necessary to induce both maturation of fetal epiphyseal chondrocytes and stimulation of alkaline phosphatase activity.

Regulation of the gene expression of alkaline phosphatase by retinoic acid (1 µM) has been demonstrated at both the transcriptional and post-transcriptional level [START_REF] Zhou | Novel action of retinoic acid. Stabilization of newly synthesized alkaline phosphatase transcripts[END_REF]. Other mechanisms of action of retinoids in chondrocytes include modulation of their integrinmediated attachment to extracellar matrix proteins [START_REF] Loeser | Modulation of integrin-mediated attachment of chondrocytes to extracellular matrix proteins by cations, retinoic acid, and transforming growth factor beta[END_REF][START_REF] Sanchez | The role of cell adhesion in retinoic acid-induced modulation of chondrocyte phenotype[END_REF] and up-regulation of metalloprotease genes [START_REF] Ballock | Inhibition of the chondrocyte phenotype by retinoic acid involves upregulation of metalloprotease genes independent of TGF-beta[END_REF][START_REF] Nie | Retinoic acid treatment elevates matrix metalloproteinase-2 protein and mRNA levels in avian growth plate chondrocyte cultures[END_REF]]. The importance of retinoic acid in regulating protein synthesis by fetal epiphyseal chondrocytes is well demonstrated by the numerous modifications induced in the pattern of secreted proteins, at both the quantitative and qualitative levels. Two-dimensional electrophoresis is a powerful technique for analysing complex mixtures of proteins, and specific differences in map composition were revealed when extracellular proteins derived from fibroblastic and osteoblastic cells were compared [START_REF] Hankey | Two-dimensional polyacrylamide gel electrophoresis reveals differences between osteoblast and fibroblast extracellular proteins[END_REF]. This technique was used by [START_REF] Mathieu | Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional gel electrophoresis, western blotting and microsequencing[END_REF] to identify novel proteins as markers of osteoblast differentiation. In our laboratory, changes in the chondrocyte phenotype were recorded in two-dimensional patterns after induction by retinoic acid as a decrease in type II collagen synthesis and expression of the small proteoglycan, decorin [Freyria et al., 1995b]. In the present study, major changes occurred on day 13, when type I collagen was strongly expressed in culture with ascorbic acid and suppressed by retinoic acid. This inducer further counteracted the effect of ascorbic acid on the expression of various proteins in the extracellular compartments. In the culture grown without ascorbic acid, in which retinoic acid delayed maturation of the chondrocytes, little variation was seen in the level of secreted proteins, suggesting that the mechanisms for this effect involve few extracellular proteins. Another important inhibitor of the maturation of fetal bovine epiphyseal chondrocytes induced by ascorbic acid in our culture conditions is DHCB. After addition of 10 or 20 µM DHCB to the culture medium, with or without fresh ascorbic acid, chondrocytes maintained their round morphology and showed a dramatic decrease in total and collagen protein synthesis, leading to a low deposition of extracellular matrix and partial detachment of the cells. Synthesis of types I and X collagen and alkaline phosphatase activity were completely inhibited, demonstrating that a cartilage phenotype with a low level of type II collagen synthesis and a pattern of secreted proteins is maintained after 13 days. This is very similar to the pattern obtained without ascorbic acid. It was reported previously that DHCB, a microfilament-modifying agent, enhances the expression of chondroblastic markers by rat mesenchymal cells cultured with cartilage-inducing factor [START_REF] Rosen | Differentiation of rat mesenchymal cells by cartilage-inducing factor. Enhanced phenotypic expression by dihydrocytochalasin B[END_REF] or can cause restoration of type II collagen synthesis by dedifferentiated chondrocytes [START_REF] Benya | Microfilament modification by dihydrocytochalasin B causes retinoic acid-modulated chondrocytes to reexpress the differenti-ated collagen phenotype without a change in shape[END_REF][START_REF] Brown | Alterations in chondrocyte cytoskeletal architecture during phenotypic modulation by retinoic acid and dihydrocytochalasin B-induced reexpression[END_REF][START_REF] Benya | Dihydrocytochalasin B enhances transforming growth factor-beta-induced reexpression of the differentiated chondrocyte phenotype without stimulation of collagen synthesis[END_REF] indicating a potential role of the cytoskeleton in the maintenance of the phenotype. The disruption of the cytoskeleton microfilament by DHCB has been also reported to inhibit beta-1 integrin increased-expression by TGF-beta-1 in articular chondrocytes [START_REF] Loeser | Expression of beta1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage[END_REF]. Cytochalasin B was reported to inhibit the transport of dehydroascorbic acid in human neutrophils through the inhibition of the hexose transport system [START_REF] Vera | Mammalian facilitative hexose transporters mediate the transport of dehydroascorbic acid[END_REF]. Similar mechanisms may be involved, in the bovine epiphyseal chondrocytes, regarding the inhibition of ascorbic acid effects by DHCB.

In conclusion, the phenotype of fetal bovine epiphyseal chondrocytes appears to be modified readily in high-density culture by the addition of inducers such ascorbic acid, retinoic acid and DHCB. Depending on the culture conditions, cells showed a maintained, a partially dedifferentiated or a terminally differentiated phenotype with expression of type X collagen, with or without high alkaline phosphatase activity. Our two-dimensional PAGE analyses have also shown that the changes in the chondrocyte phenotype are accompanied by extensive changes in the pattern of excreted proteins.

Complete identification of these proteins would be useful for defining the mechanisms involved more precisely.

Fig. 1 .

 1 Fig. 1. Phase-contrast photomicrographs of fetal bovine epiphyseal chondrocytes treated with and without retinoic acid. Cultures were observed after 23 days (a-d) and 30 days (e-h) of culture: control (a,e), ascorbic acid-treated (c,g), 10 µM retinoic acid-treated (b,f) and ascorbic acid ϩ 10 µM retinoic acidtreated chondrocytes (d,h); ϫ 110.

Fig. 2 .

 2 Fig. 2. Phase-contrast and scanning and transmission electron micrographs of fetal bovine epiphyseal chondrocytes treated with and without 10 µM dihydrocytochalasin B (DHCB) after 13 days of culture. No fresh ascorbic acid was added during this period. Chondrocytes in control culture are shown in a, b, c and in the DHCB-treated culture in d, e, f. Note the change in morphology and abundance of the pericellular matrix when comparing the controls a and b with DHCB-treated d and e. ϫ 142 (a,d) and ϫ 10,000 (b,e). Chondrocytes are surrounded by fine and striated fibrils (c,f); ϫ 21,500.

Fig. 3 .

 3 Fig. 3. Transmission electron micrographs of fetal bovine epiphyseal chondrocytes treated with 10 µM retinoic acid for 23 days. Cells are shown in a-d and the extracellular matrix in e-h; control cultures are shown in a and e, retinoic acid-treated cultures in b and f, ascorbic acid-treated culture in c and g and ascorbic acid ϩ retinoic acid-treated chondrocytes in d and h. ϫ 2500. Chondrocytes in the various culture conditions are surrounded by fine and striated fibrils (e-h); ϫ 49,200.

Fig. 4 .

 4 Fig.4. Effects of retinoic acid (RA) alone and with ascorbic acid (Asc) on the uptake of 35 S-methionine into newly synthesized total (a) and pepsin-resistant proteins (b) and on the levels of types II (c) and I (d) collagen. Fetal bovine epiphyseal chondrocytes were grown in high-density culture in the presence or absence of 0.5 or 10 µM RA and/or Asc. Media and cell layers were collected on days 6, 13, and 30 after labelling for the last 14 h of each time and submitted to limited pepsin digestion. Samples were precipitated with alcohol, and aliquots equivalent to the same volume of extract were counted in

Fig. 5 .

 5 Fig. 5. Effects of dihydrocytochalasin B (DHCB) alone and in combination with ascorbic acid (Asc) on the uptake of 14 Cproline into newly synthesized total (a) and pepsin-resistant proteins (b) and on the levels of types II (c) and I (d) collagen. Fetal bovine epiphyseal chondrocytes were grown in highdensity culture in the presence or absence of 3, 10, or 20 µM

Fig. 6 .

 6 Fig.6. Immunoblot analysis of the effects of ascorbic acid (Asc) and retinoic acid (RA) on type X collagen deposition in the cell layers of chondrocytes. Fetal bovine epiphyseal chondrocytes were grown in high-density culture in the presence or absence of 0.5 or 10 µM RA and/or Asc. Cell layers were collected on days 13 and 30. The pepsin-digested collagens were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride mem-

TABLE I . Effects of Ascorbic Acid (Asc) and Retinoic Acid (RA) on Alkaline Phosphatase Activity (mU/10 6 cells) in Fetal Bovine Epiphyseal Chondrocytes During 30 Days in Culture a

 I Chondrocytes were grown in high-density culture in the presence or absence of 0.5 and 10 µM RA and/or Asc. Cell layers were collected on days 13, 23, and 30 and alkaline phosphatase activity was measured. Values expressed in milliunit per million of cells (mU/10 6 cells) represent the average levels Ϯ SD for three to five independent experiments.

	Days of	RA 0 µM	RA 0.5 µM	RA 10 µM
	treatment	Asc (Ϫ)	Asc (ϩ)	Asc (Ϫ)	Asc (ϩ)	Asc (Ϫ)	Asc (ϩ)
	13	135 Ϯ 24	167 Ϯ 33	187 Ϯ 47	531 Ϯ 357	378 Ϯ 113	1308 Ϯ 232
	23	261 Ϯ 30	200 Ϯ 81	111 Ϯ 13	519 Ϯ 112	354 Ϯ 170	3148 Ϯ 292*
	30	126 Ϯ 23	55 Ϯ 17	155 Ϯ 8	214 Ϯ 27	656 Ϯ 182	3565 Ϯ 89*

a *P Ͻ 0.02 when untreated cultures were compared with treated cultures.

Freyria et al.

ACKNOWLEDGMENTS

We thank Dr G. Gibson (Breeche Research Laboratory, Henry Ford Hospital, Detroit, MI) for his generous gift of anti-type X collagen and Dr J. Guidollet (INSERM U 189, Hopital Lyon-Sud, Lyon, France) for assistance with measuring alkaline phosphatase activity. We also thank R. Willems and R. Pillot for expert technical assistance and C. Van Herrewege and A. Bosch for assistance in preparing the manuscript.

BMH4-CT95-0396; Grant sponsor: Rho ˆne-Alpes Re ´gion; Grant number: L094120401.


Audrey Gabelle
email: gabelle@chu-montpellier.fr

Stéphane Roche

Christian Gény

Karim Bennys

Pierre Labauge

Yannick Tholance

Isabelle Quadrio

Laurent Tiers

Baptiste Gor

Chloé Chaulet

Alain Vighetto

Bernard Croisile

Pierre Krolak-Salmon

Jacques Touchon

Armand Perret-Liaudet

Sylvain Lehmann
email: s-lehmann@chu-montpellier.fr

A Gabelle

Correlations between soluble α/β forms of amyloid precursor protein and Aβ38, 40, and 42 in human cerebrospinal fluid

Keywords:

Cerebrospinal fluid (CSF) biomarkers are now widely used for diagnosis of Alzheimer disease (AD) in atypical clinical forms, for differential and early diagnosis, or for stratification of patients in clinical trials. Among these biomarkers, different forms of amyloid peptides (Aβ) produced by the cleavage of a transmembrane precursor protein called APP (amyloid precursor protein) have a major role. Aβ peptides exist in different length the most common ones having 40 (Aβ40), 42 (Aβ42), or 38 (Aβ38) amino acids in length. APP processing by gamma-secretase releases also an amino-terminal secreted fragment called sAβPP-beta while an alternative nonamyloidogenic cleavage of APP, through an alpha-secretase, liberates another fragment called sAβPP-alpha. To decipher the molecular and pathological mechanisms leading to the production and the detection of these entities is essential for the comprehension and the prevention of AD. In this report, we present the results of the

Introduction

Alzheimer disease (AD) is a neurodegenerative disorder characterized by significant cognitive deficits, language troubles, behavior changes and loss of functional autonomy. As the major cause of dementia in the elderly population, AD is approaching epidemic proportions in the Western industrialized countries due to aging of the population. AD is becoming a major public health issue with the increasing burden on caregivers and on the health care system. AD characteristic neuropathological lesions are represented by amyloid plaques and neurofibrillary tangles which coexist in the brain. The diagnosis of AD based on clinical, neuropsychological, morphological, as well as functional neuroimaging can remain dubious in a considerable number of cases delaying the diagnosis and the medical care. These data are already powerful but lead only to probability diagnosis. Recent new sets of diagnosis criteria including specific morphological (volumetric MRI), functional imagery (PET scan), and CSF biomarkers (total and phosphorylated Tau (P-tau) proteins and Aβ peptides) have been proposed to optimize the diagnosis [START_REF] Dubois | Research criteria for the diagnosis of Alzheimer's disease: revising the NINCDS-ADRDA criteria[END_REF]. When comparing AD patients to healthy controls, CSF concentrations of Tau and P-tau were reported to be increased, while Aβ1-42 levels were decreased [START_REF] Blennow | CSF markers for incipient Alzheimer's disease[END_REF]. Numerous studies have analysed the potential of CSF biomarkers in terms of positive diagnosis, differential diagnosis, and prognosis as predictive marker of the conversion (mild cognitive impairment (MCI) to AD) [START_REF] Gabelle | CSF biomarkers: proteomics investigations and clinical applications in neurodegenerative disorders[END_REF][START_REF] Herukka | CSF Abeta42, Tau and phosphorylated Tau, APOE epsilon4 allele and MCI type in progressive MCI[END_REF][START_REF] Mattsson | CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive impairment[END_REF]. In combination and using several published ratios, these biomarkers reached a sensitivity and specificity for positive AD diagnosis beyond 80% [START_REF] Ibach | Cerebrospinal fluid tau and beta-amyloid in Alzheimer patients, disease controls and an age-matched random sample[END_REF]. We obtained similar results in our routine practice on more than 350 patients (unpublished results) using as cutoff values for P-tau and IATI (Innogenetics Aβ/Tau index), 60 and 0.8, respectively. The future development of therapeutic strategies aiming at slowing down or even blocking the neurodegenerative process could however benefit from earlier and more sensitive diagnosis AD biomarkers. Recently, [START_REF] Lewczuk | Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study[END_REF] brought to light the interest of CSF soluble amyloid precursor proteins (sAPP) as novel potential biomarkers in the positive diagnosis of AD. sAPPs are generated from APP following α and β cleavages [START_REF] Kang | The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor[END_REF]. The nonamyloidogenic pathway that cleaves off APP in position α (within the sequence corresponding to Aβ) leads to the α-secretase-cleaved soluble APP (sAPPα) fragment. The βsecretase action on APP leads to the β-secretase-cleaved soluble APP (sAPPβ). This β-secretase activity (BACE), combined with γ-secretase cleavages releases the three major peptide fragments Aβ40, Aβ42, and Aβ38 [START_REF] Wiltfang | Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation[END_REF] in a kinetics that is not yet fully elucidated. It is well known that most of the secreted Aβ fragments correspond to Aβ40, however, in AD, a prominent pathogenic role is given to Aβ42 that aggregates much more rapidly due to the presence of two additional hydrophobic amino acids, and represents the main constituent of senile plaques [START_REF] Iwatsubo | Neuronal and glial tau-positive inclusions in diverse neurologic diseases share common phosphorylation characteristics[END_REF]. While investigating presenilin mutations and γ-secretase inhibitors, [START_REF] Page | Generation of Abeta38 and Abeta42 is independently and differentially affected by familial Alzheimer disease-associated presenilin mutations and gamma-secretase modulation[END_REF] observed that while Aβ42 and Aβ40 productions were strongly related, Aβ38 generation by γsecretase was not dependent on the previous entities.

The objective of the current study was to investigate the relationship between sAPPs and Aβ fragments in different pathophysiological contexts. Our results confirmed the potential interest of sAPPs as AD biomarkers and provide important information on the complex correlation between the production of Aβ fragments and sAPPs.

Results

sAPPα/β correlation and values in NDD groups

A significant correlation (Fig. 1A; r = 0.87, P < 0.0001) was observed between the values of sAPPα and sAPPβ in our population, independently of the NDD biological status. When concentration of the biomarkers were compared between NDD groups, higher mean values were obtained in NDD+ (Figs. 1B andC) with a difference with NDD-significant for sAPPβ. These results were reminding of those of [START_REF] Lewczuk | Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study[END_REF] who analyzed a larger population and therefore obtained differences with lower P values.

The potential interest of sAPPs for dementia diagnosis could not be fully investigated in the present study that focused on the relationship between CSF biological values. However, receiver operating characteristic (ROC) curve of sAPPs for NDD+ resulted in cutoff values with sensitivity of 68.2% and specificity of 57.9% for sAPPα and 81.8% and 63.2% for sAPPβ. As illustrated Table 1, MMS status was different between NDD+ and NDD-while no significant difference was observed for the age or the CSF total protein content.

2.2.

Correlation between sAPPα/β and Aβ38, 40, and 42

Correlations between sAPPα/β and Aβ38, 40, and 42 were plotted, and Spearman factors were calculated for each pair of biomarkers (Fig. 2). Interestingly, we observed for the first time to our knowledge a positive correlation between sAPPs and Aβ38 or Aβ40 independently of the NDD status. This was particularly significant for sAPPβ and Aβ38 (r = 0.72, P < 0.0001) as well as for sAPPα and Aβ38 which was unexpected as this sAPP fragment is not generated in the same processing pathway as Aβ peptides. The significance of sAPP correlation was however lower with Aβ40 than with Aβ38 and was not significant at all with Aβ42 (compare Figs. 2 AD, BE, and CF). As Aβ42 and in a lesser extend Aβ40 are involved in AD pathogenesis and CSF levels of these biomarkers are known to be modified in the disease [START_REF] Blennow | CSF markers for incipient Alzheimer's disease[END_REF], the analysis was restricted to the NDD-group (Fig. 3). This resulted in an increase in correlation factors and a statistically significant correlation also between sAPPs and Aβ42.

Correlation between Aβ38, 40, and 42

Correlation between the levels of Aβ38, 40, and 42 were plotted, and Spearman factors were calculated for each pair of biomarkers (Figs. 4A, B, and C). Interestingly, we observed that Aβ38 and Aβ40 were strongly correlated (r = 0.90, P < 0.0001).

There was no correlation with Aβ42 in the total population; however, when correlation was restricted to the NDD-group (Fig. 4D), the correlation factor increased and was statistically significant for Aβ38 and Aβ42.

Discussion

In this report, we present in a series of neurochemical dementia diagnostic (NDD) positive and negative CSF samples the results of multiplex assays for soluble forms of amyloid precursor proteins (sAPPs) and Aβ peptides. Data were collected in two independent laboratories using similar and controlled preanalytic and analytic conditions [START_REF] Lehmann | Preanalytical guidelines for clinical proteomics investigation of biological fluids[END_REF]. Importantly, a recent investigation on sAPPα and Aβ CSF levels already confirmed the specificity of the detection assays used in the present study [START_REF] Lewczuk | Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study[END_REF]. This previous work revealed a surprising correlation between these two entities. We independently confirmed this correlation (Fig. 1) which was intriguing because it did not correspond to what would be intuitively expected. One could believe in fact that the production of sAPPβ would be inversely proportional to that of sAPPα since their metabolic pathways are exclusive. Moreover, these fragments have opposite impact (toxic vs. neuroprotective). To our knowledge, no precise metabolic regulation has been demonstrated between α and β APP cleavages in the literature, and further studies would be needed to explain this positive correlation.

We also observed a slight increase in sAPP levels in NDD+ samples compared to NDD-with a statistically significant difference only for sAPPβ (Fig. 1; P < 0.02). A higher level of sAPPβ in patients has been previously described [START_REF] Lewczuk | Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study[END_REF] but is still controversial [START_REF] Hock | Cerebrospinal fluid levels of amyloid precursor protein and amyloid beta-peptide in Alzheimer's disease and major depression-inverse correlation with dementia severity[END_REF][START_REF] Lannfelt | Decreased alpha-secretase-cleaved amyloid precursor protein as a diagnostic marker for Alzheimer's disease[END_REF][START_REF] Olsson | Measurement of alpha-and beta-secretase cleaved amyloid precursor protein in cerebrospinal fluid from Alzheimer patients[END_REF][START_REF] Palmert | Soluble derivatives of the beta amyloid protein precursor in cerebrospinal fluid: alterations in normal aging and in Alzheimer's disease[END_REF][START_REF] Zetterberg | Elevated cerebrospinal fluid BACE1 activity in incipient Alzheimer disease[END_REF]. In previous studies, for example, no significant increase of sAPP using Western Blotting or sandwich enzyme-linked immunosorbent assay has been observed in AD patients [START_REF] Olsson | Measurement of alpha-and beta-secretase cleaved amyloid precursor protein in cerebrospinal fluid from Alzheimer patients[END_REF][START_REF] Sennvik | Levels of alpha-and beta-secretase cleaved amyloid precursor protein in the cerebrospinal fluid of Alzheimer's disease patients[END_REF]. In familial AD, symptomatic patients with APP mutations had lower sAPPα [START_REF] Lannfelt | Decreased alpha-secretase-cleaved amyloid precursor protein as a diagnostic marker for Alzheimer's disease[END_REF] and low levels of this fragment were correlated to poor performance on neuropsychological tests [START_REF] Almkvist | Cerebrospinal fluid levels of alpha-secretase-cleaved soluble amyloid precursor protein mirror cognition in a Swedish family with Alzheimer disease and a gene mutation[END_REF]. This discrepancy with recent data, including our, might be related to differences in population number, preanalytics or analytical methods. In addition, we lacked here a cognitively healthy control group which would be needed to really evaluate the clinical interest and relevance of sAPP measurement. In any case, a higher concentration of sAPP in neurochemical pathological group could be explained by several hypotheses. Firstly, the intrinsic amount of APP available for metabolic processing could be increased in patients with symptomatic or asymptomatic AD amyloid lesions. This hypothesis is supported by the observation from [START_REF] Lewczuk | Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study[END_REF] that sAPPs were higher in MCI/ NDD+ than in MCI/NDD-and that no significant difference has been detected between MCI/NDD+ and AD/NDD+. This infers that pathological processes and neurochemical alterations precede clinical symptoms by several years, giving rational for the use of CSF sAPP biomarkers. It was not the purpose of our study, which focussed on biological data, to evaluate the interest of sAPPβ for AD diagnosis. However, with an optimal cutoff, a sensitivity of 78.6% and a specificity of 62.5% were observed for the positive diagnosis of AD, the area under the ROC curve being at 0.68. One explanation for our data could be related to a feed back regulation of APP level by Aβ concentrations. As soluble Aβ42 levels decrease in AD this could result in a positive production signal for APP and/or for secretase activities as observed for BACE1 in patients with AD and MCI/AD [START_REF] Blennow | CSF biomarkers for Alzheimer's disease: use in early diagnosis and evaluation of drug treatment[END_REF][START_REF] Zhong | Levels of beta-secretase (BACE1) in cerebrospinal fluid as a predictor of risk in mild cognitive impairment[END_REF]. Finally, a modification of the degradation/ clearance of theses sAPP fragments in AD could also account for our results.

The second major outcome of our study relates to the intricate relationship between sAPP fragments and Aβ peptide levels in human CSF. A strong correlation was indeed observed between Aβ38 and sAPPβ and, in a lesser extent, with sAPPα or with Aβ40 (Fig. 2). This suggested that Aβ38 production was in direct relation with β secretase activity even in pathological situations. This result was of high interest with in mind the follow-up of the activity of the β-secretase in therapeutic strategies targeting this metabolic pathway. sAPPα and β being correlated, it made sense to also find a limited correlation between Aβ38 and sAPPα. Noteworthy, the correlation of sAPPβ with Aβ40 and Aβ42 was really significant only in NND-samples (Fig. 3). One interpretation of theses data is that the three peptides were physiologically generated in parallel through the normal processing of APP. In the presence of an amyloid process like AD, the peptides would have different fates. Aβ42 that is particularly prone to aggregation would therefore have its concentration modified in the disease [START_REF] Blennow | Alzheimer's disease[END_REF]. The differential behaviour of the different Aβ species was also illustrated by the strong correlation existing in all situations between Aβ38 and Aβ40 (Fig. 4), while Aβ42 was correlated to these fragments only in non pathological situations (NDD-). These results are important for hypothesis on APP processing and pathologic involvement. They suggest a strong physiological coregulation between the generations of the different Aβ entities which are all produced by the same secretase [START_REF] Vassar | beta-Secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE[END_REF]. This argued also against a nonspecific degradation/conversion of the longer Aβ peptides into the smaller ones. The decrease of Aβ42 observed in NDD+ patients (that have also increased P-tau levels) illustrated the strong involvement of this peptide in the pathology and the loss of correlation with the other peptides in presence of neuropathological changes. Our observations contribute to the biochemical rational for using ratio between Aβ42 and Aβ40 (or Aβ38) to improve AD diagnosis [START_REF] Welge | Combined CSF tau, p-tau 181 and amyloid-beta 38/40/42 for diagnosing Alzheimer's disease[END_REF][START_REF] Wiltfang | Amyloid beta peptide ratio 42/40 but not A beta 42 correlates with phospho-Tau in patients with low-and high-CSF A beta 40 load[END_REF]. In fact, as the baseline level of Aβ production seems variable between individuals leading to possible misdiagnosis, the fact that Aβ fragments have different fates allows using ratios independently of baseline production.

In conclusion, our correlations between sAPPs and Aβ peptides, as well as between Aβ peptides themselves enlightened the complex relationships between these molecular markers in both physiological and pathological situations. Our results are important for the further use of these analytes for AD diagnosis, as well as to validated cell biological hypotheses of APP processing and Aβ fragment production.

4.

Experimental procedures

Subjects and study design

Patients included in this study were selected in two independent cohorts of subjects referring to the Neurological Units of et al., 1984) were used. For frontotemporal lobar dementia (FTD), the diagnosis criteria were based on the Lund and Manchester criteria established in 1994 [START_REF] Groups | Clinical and neuropathological criteria for frontotemporal dementia. The Lund and Manchester Groups[END_REF] and revised by [START_REF] Mckhann | Clinical and pathological diagnosis of frontotemporal dementia: report of the Work Group on Frontotemporal Dementia and Pick's Disease[END_REF] and [START_REF] Neary | Frontotemporal dementia[END_REF]. The patients with Lewy body dementia were validated using the international clinical diagnosis consensus criteria [START_REF] Mckeith | Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium[END_REF]. The diagnosis status of the patients was discussed and confirmed during a multidisciplinary evaluation integrating imagery and clinical/neuropsychological data. Sixty CSF samples with possible and probable AD (n = 28), FTD (n = 27), LBD (n = 3), and other neurological disease (n = 2) were analyzed. As the purpose of the investigation was primarily the biological relationships between the studied analytes, rather than their diagnosis interest, the samples were divided as in [START_REF] Lewczuk | Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study[END_REF] between neurochemical dementia diagnosis-positive (NDD+) and negative (NDD-) using our routine P-tau and IATI (Innogenetics Aβ/Tau index) cutoff values of 60 and 0.8, respectively. A total of 22 samples complied with NDD+ conditions, the 38 remaining being in the NDD-group.

CSF samples and routine assays

Lumbar puncture (LP) was performed in standardized conditions. LP was carried out at the L3/L4 or L4/L5 interspace after exclusion of the potential contraindications. CSF samples were taken directly in similar polypropylene tubes to avoid variation in adsorption of biomarkers to the container surface. All samples were transferred to the biochemical laboratories (CHU Montpellier/Nimes, Hospices Civils de Lyon) in less than 4 hours. The CSF was centrifuged (1000 × g, 10 min, at 4-8 °C, without break), and the supernatant was aliquoted by 0.5 mL in polypropylene tubes before storage at -80 °C. The three routine CSF biomarkers Tau, P-tau 181 , and Aβ42 were determined with a standardized commercially available ELISA Kit (Innotest beta-amyloid 1-42 [START_REF] Vanderstichele | Standardization of measurement of beta-amyloid(1-42) in cerebrospinal fluid and plasma[END_REF], Innotest hTau-Ag [START_REF] Vandermeeren | Detection of tau proteins in normal and Alzheimer's disease cerebrospinal fluid with a sensitive sandwich enzyme-linked immunosorbent assay[END_REF], and Innotest Phosphotau(181P) [START_REF] Vanmechelen | Quantification of tau phosphorylated at threonine 181 in human cerebrospinal fluid: a sandwich ELISA with a synthetic phosphopeptide for standardization[END_REF], Innogenetics, Ghent, Belgium) in both laboratories. Quality control program organised by Hospices Civils de Lyon was used to study inter laboratories variability, using two CSF samples. The IATI score was calculated using the formula IATI = Aβ42 / (240 + 1.18 × tau) as described [START_REF] Hulstaert | Improved discrimination of AD patients using beta-amyloid(1-42) and tau levels in CSF[END_REF]. Routine CSF analyses included glucose, total protein and cytology. Only samples that were assessed as red cells count <2000/mm 3 were used for analyses.

4.3.

Multiplex assays of CSF sAPPα/β and Aβ38, Aβ40, Aβ42

Detection of these analytes was performed independently in the two laboratories using multiplex kits purchased from Meso Scale Discovery (ref: K11120E, K11148E). All reagents were provided with the kits. They contained a 96-well plate with two carbon electrodes precoated with either anti APP antibody 22C11 (sAPPα/β) or antibodies specific for Aβ38, Aβ40, Aβ42. The wells were incubated for 1 hour with the block solution (bovine serum albumin) and washed four times with the Tris Wash Buffer (TWB). For sAPPs duplex assay, 25 μl of standards (dilution of recombinant sAPPs) and samples were then added to all wells, and the plate was sealed and incubated for 1 hour at room temperature on an orbital shaker (300 rpm). At the end of the incubation, the wells were washed four times using the TWB. Detection antibody (sAPPα/β) was added at 25 μl per well, and the plate was sealed and incubated for 1 hour at room temperature on an orbital shaker (300 rpm). For Aβ triplex assay, 25 μl of standards (dilution of recombinant Aβ) and samples were added at the same time of addition of 25 μl of detection antibody (6E10) to all wells, and the plates were incubated during 2 hours. At the end of the incubation, the plate was washed four times as before. About 150 μl of the MSD Read Buffer T was added to each well, and the MSD plates were measured on the MSD Sector Imager 6000 plate reader. The raw data were measured as electrochemiluminescence signal (light) detected by photodetectors and analysed using the Discovery Workbench 3.0 software (MSD). A 4-parameter logistic fit curve was generated for each analyte using the standards and the concentration of each sample calculated. The intra-assay coefficient of variation ranged from 0.2% to 15.9%. The interlaboratory coefficient of variation ranged from 0.3% to 17%.

Statistical analysis

If not stated otherwise, the results were presented as medians and interquartile ranges. The Mann-Whitney-Wilcoxon test was used to test the significance of the difference between two sample groups. Correlations between measured values were analyzed with Spearman's correlation factor. Differences were considered significant if P < 0.05. Analyses were performed with MedCalc (3.0).

Fig. 1 -

 1 Fig. 1 -(A) Correlation between soluble amyloid precursor protein α (sAPPα) and soluble amyloid precursor protein β (sAPPβ) in cerebrospinal fluid (CSF) samples in whole population (patients with neurochemical dementia diagnostic (NDD) positive and negative results). A statistically significant correlation was observed with r = 0.87 and P < 0.001. (B and C) Comparison of the CSF sAPPα/β concentrations between the NDD+ and the NDD-groups. A difference in mean concentration was observed and was significant for sAPPβ (P = 0.0160).

Fig. 2 -

 2 Fig. 2 -Correlation plots between CSF soluble amyloid precursor protein α/β (sAPP α/β) and Aβ38 (A, D), Aβ40 (B, E), and Aβ42 (C, F) peptides in the whole population. A statistically significant correlation was present only for Aβ38 and Aβ40 (A, B, D, and E).

Fig. 3 -

 3 Fig. 3 -Correlation plots between CSF soluble amyloid precursor protein α/β (sAPP α/β) and Aβ40 (A, C) and Aβ42 (B, D) peptides in the NDD-population. A statistically significant correlation was present in particular for sAPPβ and Aβ40 (C).

  Montpellier/Nimes and Lyon for cognitive or behavioural troubles and who gave their consent to include their biological samples into a research biobanks in the field of dementia. All patients underwent a standardized clinical and paraclinical investigations including anamnestic, physical and neurological examination, screening laboratory tests, neuropsychological assessment, brain morphological imaging (CT or MRI) or functional imaging (SPECT) evaluations, and also CSF Tau, Ptau 181 , and Aβ42 analyses using Innogenetics™ ELISA kits. All the clinical diagnoses (38 NDD-= 26 DFT, 3 DCL, 7 DTA, 1 psy, and 1 other and 22 NDD+ = 1 DFT and 21 DTA) were validated an expert in the field of dementia and during consensus multidisciplinary evaluation. For AD, diagnosis criteria of the National Institute of Neurological Disease and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association (NINCDS/ADRDA) (McKhann

Fig. 4 -

 4 Fig. 4 -(A-D) Correlation plots between CSF Aβ38, Aβ40, and Aβ42 peptides. A strong correlation was observed in the whole population between Aβ38 and Aβ40 (A), while the correlation between Aβ38 and Aβ42 (D) was significant only in NDD-patients.

Table 1 -

 1 Demographic and biologic data of the NDD groups.

		MMS	Age	CSF Total Protein
	NND+	16.4 ± 6.4	66.5 ± 7.9	0.40 ± 0.19
	NND-	21.5 ± 6.3	67.3 ± 11.3	0.41 ± 0.14
	P value	<0.01	0.730	0.917

Please cite this article as: Gabelle, A., et al., Correlations between soluble α/β forms of amyloid precursor protein and Aβ38, 40, and 42 in human cerebrospinal fluid, Brain Res. (2010), doi:10.1016/j.brainres.2010.08.022

B R A I N R E S E A R C H X X ( 2 0 1 0 ) X X X -X XX Please cite this article as: Gabelle, A., et al., Correlations between soluble α/β forms of amyloid precursor protein and Aβ38, 40, and 42 in human cerebrospinal fluid, Brain Res. (2010), doi:10.1016/j.brainres.2010.08.022

Acknowledgments

This study was supported by the following grants by the EU grants cNEUPRO (contract no. LSHM-CT-2007-037950) and by the French Health Ministry (contract no. Rhônes Alpes PHRC-2004-27/15). We thank Pr. JP Cristol (Service of Biochemistry) and Pr. B Klein (Director of IRB) for their support.