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First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01607

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A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis

¹ Laboratoire CRRET, CNRS UMR 7149, Université Paris 12-Val de Marne, 61 Avenue du Général de Gaulle, 94010 Créteil CEDEX, France
² Faculty of Science Semlalia, University Cadi Ayyat, BP 2390, Marrakech, Morocco
³ Division of Bioengineering and Environmental Health, Massachusetts Institute of technology, Cambridge, MA 02139, USA
⁴ ImClone Systems Incorporated, New York, NY 10014, USA
⁵ School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZB, UK
⁶ Department of Biochemistry194, University Medical Centre, NCMLS, PO Box 9101, 6500 HB Nijmegen, The Netherlands

View larger version (15K): [in a new window]   Fig. 1. Schematic structure of RGTA D120. The dextran derivative on the 1-6 glucose polymeric chain contains carboxymethyl (–CH2COO–) and sulfate residues (–SO3–) at degrees of substitution of 0.26 and 1.92, respectively. Three differently substituted glucosidic units are represented according to the nature of the group linked to the C2 position. For easier interpretation, these units were arranged in an arbitrary combination. R represents the possible substituted groups in the global C3 and C4 positions. The position of each group on the C-2 compared to C-3 + C-4 was also determined by analyzing the anomeric proton signal by 1H NMR (300 MHz).

View larger version (19K): [in a new window]   Fig. 2. Effect of RGTA on C2.7 cell growth. (A) Effect of different concentrations of RGTA (0.1 to 20 µg/ml) on cell proliferation. Cells were counted at day 4 and each value is the mean±s.d. of four cultures. (B) Growth of C2.7 myoblasts in the presence of heparin (10 µg/ml) or RGTA (0.5 µg/ml). DNA was measured in cellular extracts at the indicated times. Each point is the mean±s.d. of three determinations on three independent cultures.

View larger version (86K): [in a new window]   Fig. 3. Effect of RGTA on C2.7 cell differentiation. (A) C2.7 cells grown at high serum concentrations at day 4, without treatment (1) or in the presence of 0.5 µg/ml RGTA (2). C2.7 cells at 24 hours in differentiating medium (0.25% SVF + 0.25% SC) without treatment (3) or in the presence of 0.5 µg/ml RGTA (4). Arrows indicate myotubes in the cultures. (B) Subunits of creatine kinase (CK) and enzymatic activity at day 4 of C2.7 cell culture. Isoenzyme analysis was performed after agarose gel electrophoresis and the percentage of each subunit was calculated as described in Materials and Methods. CK activity is expressed as international units per dish. Bar, 100 µm.

View larger version (28K): [in a new window]   Fig. 4. Effect of RGTA on total sulfated GAG in C2.7 cellular extracts. Total sulfated GAG determinations were performed using the DMMB assay in proliferating and differentiating cells grown in the presence or absence of 0.5 µg/ml RGTA. The amounts of GAG were normalized by the amounts of DNA contained in each sample. Data are the mean±s.d. of duplicates or triplicates from four independent experiments. (A) Total GAG during proliferation. (B) Cell:medium ratio of total GAG during proliferation. (C) Total GAG during differentiation. (D) Cell:medium ratio of total GAG during differentiation. *P<0.05; **P<0.005; ***P<0.0005, when total GAG content or cell:medium ratios in RGTA-treated cells are compared with the respective controls.

View larger version (15K): [in a new window]   Fig. 5. Measurement of total GAG by HPLC on samples from proliferating or differentiating C2.7 cells. GAG extracts from proliferating and differentiating C2.7 cells were tagged with anthranilic acid and quantified by HPLC. Results were normalized to the amount of DNA in the samples. Data are expressed in arbitrary units of fluorescence (AUF)/µg DNA are the mean±s.d. of two independent experiments. **P<0.005; ***P<0.0005, when compared to AUF of the respective controls.

View larger version (22K): [in a new window]   Fig. 6. Arylsulfatase specific activity in differentiating C2.7 cells. Arylsulfatase A (A) and B (B) were assayed in extracts performed at the indicated times after a shift to low serum medium. Each value is the mean±s.d. of duplicated determinations performed with two independent cultures.

View larger version (24K): [in a new window]   Fig. 7. Analysis of enzymes involved in GAG metabolism by real-time PCR. Real-time PCR of EXT1 and 2, NDST1, epimerase, 2OST and 6OST1 as well as heparanase was performed with RNA extracts from differentiating control and RGTA-treated C2.7 cells. Values were normalized using the following reference genes: glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA polymerase II (RPII), TATA-Box binding protein (TBP) and -tubulin. Each value is the mean±s.e. of measurements performed on three independent cultures.

View larger version (15K): [in a new window]   Fig. 8. Changes in GAG composition induced by RGTA in proliferating and differentiating C2.7 cells. HS and CS were measured in cellular extracts of cultures from (A) proliferating and (B) differentiating cells grown in the presence or absence of RGTA added at 0.5 µg/ml. Ratios of HS or CS contents in RGTA-treated cells compared to controls were calculated.

View larger version (20K): [in a new window]   Fig. 9. ELISA assay of HS species extracted from differentiating C2.7 cells. GAG samples were prepared from differentiating controls and RGTA-treated cells of one representative culture. They were grafted on BSA and tested in an ELISA assay using different HS antibodies (RB4CD12, AO4BO5 and AO4F12). Each value is the mean of duplicate measurements.

View larger version (109K): [in a new window]   Fig. 10. Immunolocalization of HS in C2.7 myoblasts. Localization of HS subspecies was investigated using (A) RB4CD12, (B) AO4F12 and (C-E) AO4BO5 antibodies and confocal microscopy. Note the differential labelling of HS species either at the periphery of cells (RB4CD12) or at cell-to-cell contacts with AO4BO5 or AO4F12. These two antibodies labelled cell-to-cell contacts differentially. Areas indicated by arrows are described in the text. Panel E shows the boxed area in C. Bar, 10 µm.

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