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Extracellular matrix histone H1 binds to perlecan, is present in regenerating skeletal muscle and stimulates myoblast proliferation

¹ Centro de Regulación Celular y Patología, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, MIFAB, P. Universidad Católica de Chile, Santiago, Chile
² Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, PA 17822-2613 USA

View larger version (56K): [in a new window]   Fig. 1. Skeletal muscle cells synthesize an extracellular heparin-binding protein that increases in concentration during differentiation. (A) Proteins soluble in PBS, TX-100 and TX-100/KCl were sequentially extracted from C2C12 myoblasts. Proteins were analyzed by SDS-PAGE and stained with Coomassie blue (left panel) or subjected to a [35S]heparin ligand blot assay (right panel). (B) Heparin-solubilized proteins obtained from intact C2C12 cultures on various days of differentiation (days 0-6) were analyzed by SDS-PAGE. Gel loading was normalized on the basis of equivalent amounts of DNA per lane. The presence of the p33/30 doublet was detected by a [35S]heparin ligand blot assay. (C) Proteins solubilized in TX-100/KCl obtained from myoblasts were analyzed by SDS-PAGE and stained by Coomassie blue (left panel) or analyzed in a competition [35S]heparin ligand blot assay (right panel). [35S]heparin was co-incubated with 2 mg/ml of unlabeled heparin (Hep), chondroitin sulfate (CS) or dermatan sulfate (DS). An equivalent amount of total protein was loaded per lane. Molecular weight markers are shown on the left. Arrows on the right indicate the migration of p33/30.

View larger version (51K): [in a new window]   Fig. 2. The p33/30 purified by heparin-affinity chromatography from myotubes is histone H1. (A) Triton X-100/KCl fractions (T) were subjected to heparin-affinity chromatography, and the resulting unbound (U) and NaCl-eluted fractions (E1-11) were analyzed by SDS-PAGE and stained with Coomassie blue. The molecular weight markers and the NaCl gradient are shown. Arrows on the right indicate the migration of p33/30. (B) A 15 amino acid sequence was obtained from a HPLC-purified p33-derived peptide. High homology with sequences of various subclasses of histone H1 from mouse, human, rat and bovine are shown for the peptide. Sequences are given in the one-letter code for amino acids. Underlined amino acids correspond to identities with the p33 sequence (in bold). (C) TX-100/KCl, PBS/heparin extracts, heparin-affinity purified p33/30 and an acid-soluble nuclear fraction obtained from myotubes were separated by 12.5% (20% in the case of nuclear extract) SDS-PAGE and stained with Coomassie blue (left panel). Similar SDS gels were transferred onto nitrocellulose membranes, stained with a specific monoclonal anti-histone H1 antibody and detected by ECL (right panel). (D) C2C12 myoblasts were extracted in the presence of 2 mg/ml heparin (Hep), chondroitin sulfate (CS), dermatan sulfate (DS) or N-desulfated heparin (N-deS Hep). Equivalent amounts of total proteins were analyzed by western blot with a specific monoclonal antihistone H1 antibody, and detected by ECL. Molecular weight markers are shown on the left and the migration of p33/30 is indicated (arrows) on the right of each panel.

View larger version (30K): [in a new window]   Fig. 3. Histone H1 binds specifically to perlecan secreted by myotubes. A total proteoglycan-enriched fraction obtained from the conditioned media of myotubes was incubated with histone H1 immobilized on nitrocellulose membranes and then eluted with 8 M urea. Total, unbound and histone-H1-bound fractions were incubated with heparitinase and separated by SDS-PAGE. Western blots were performed using (A) an anti--heparan sulfate monoclonal antibody to reveal the core proteins of heparan sulfate proteoglycans and (B) specific anti-mouse perlecan antisera, and detected by ECL. The migration of perlecan (per) and glypican (gly) are indicated on the right, and molecular weight markers on the left of each panel.

View larger version (96K): [in a new window]   Fig. 4. A pool of histone H1 is localized to the myotube ECM and colocalizes with heparan sulfate proteoglycans. Inhibition of proteoglycan sulfation reduces ECM localization of histone H1. (Left and middle panels) Non-permeabilized myotubes were double-stained with anti-mouse perlecan (per) or anti-rat glypican (gly) antibodies together with an anti-human histone H1 (H1). Phase contrast micrographs of respective fields are also shown (pc). Merged images (H1+per and H1+gly) are also shown. As a control, permeabilized myotubes were stained with the anti-human histone H1 (pH1). TRITC-conjugated anti-rabbit IgG and FITC-conjugated anti-mouse IgG were used as secondary antibodies. (Right panel) C2C12 cultures were treated with sodium chlorate during differentiation and processed as described for the left panel. Bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 5. Histone H1 colocalizes with perlecan in regenerating skeletal muscle. Hamster tibialis anterior muscle was induced to regenerate for four (d4) or five days (d5) after a barium chloride injection. (A) Serial cryosections were stained with hematoxylin/eosin to visualize morphological progression. (B) Similar sections were double stained with monoclonal antibodies against histone H1 (H1) or embryonic myosin (em) together with a polyclonal anti-perlecan (per) antibodies. TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG were used as secondary antibodies. Nuclei in regeneration foci were visualized by Hoechst 33258 nuclear staining (Ho). As a control, cryosections of non-injected hamster muscle (ctrl) were similarly analyzed. Inserts correspond to the regions marked with a dotted square in the respective sections. Arrows indicate the colocalization of anti-histone H1 and anti-perlecan antibodies in the ECM. Bar, 25 µm.

View larger version (19K): [in a new window]   Fig. 6. Cell death may be related to accumulation of extracellular histone H1. (A) C2C12 cells were induced to differentiate. The culture medium was changed every 2 days. LDH activity was quantified in conditioned media harvested on the indicated days and in cell extracts. Extracellular LDH (in the extracellular space) is expressed as a percentage of the total enzyme. The insert shows cumulative LDH release, calculated using the data in Fig. 6A. (B) Necrosis (dashed bars) and apoptosis (closed bars) were determined by specific ELISA assays. Data are expressed as the absorbance at 410 nm per ng of DNA. (C) Heparin-displaced proteins from intact cells and TX-100/KCl extracts from the corresponding cells were subjected to [35S]heparin ligand blot assay. Densitometric analysis of heparin-displaced histone H1 is shown as the percentage of total histone H1. Data are plotted as a function of the differentiation period (days 0-6). Results correspond to the average and s.d. of two independent experiments.

View larger version (67K): [in a new window]   Fig. 7. Histone H1 induces myoblast proliferation via a heparan-sulfate-dependent mechanism. (A) Culture dishes were coated with nitrocellulose, and 10 µl droplets containing 2 µg of affinity-purified histone H1 obtained from myotubes (H1), laminin (LN), fibronectin (FN) or BSA were applied to the surface of the dishes and dried. C2C12 myoblasts were then incubated for 2 hours and examined by phase contrast microscopy. Photographs were taken at the border of protein-coated areas (upper panel, dashed lines) and 21 hours later, at the center of the protein-coated regions (lower panel). Bar, 10 µm. (B) The time course of the [3H]thymidine incorporation in myoblasts induced by the addition of the indicated concentrations of exogenous histone H1. (C) C2C12 cultures were incubated with increasing concentrations of histone H1 for 24 hours. [3H]thymidine incorporation was determined in the last 3 hours of incubation and expressed as fold increase over controls (without histone H1). (D) Myoblast cultures incubated for 24 hours with increasing concentrations of histone H1 in the absence or presence of heparin (20 ng/ml). Results correspond to the average and s.d. of two independent experiments. (E) Myoblasts were seeded on myotube ECM preincubated with anti-histone H1 (-H1) or anti-ß galactosidase (-ßgal) antibodies. After 24 hours, [3H]thymidine incorporation was determined and expressed as a percentage of maximum effect (control ECM=1521±250 cpm). The results correspond to the average and s.d. of two independent experiments (*P0.01).