Identification of 1,25-dihydroxyvitamin D3 receptors and activities in muscle

• Published in: The Journal of biological chemistry Vol. 260; no. 15; pp. 8882 - 8891
• Main Authors: Simpson, R U, Thomas, G A, Arnold, A J
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.07.1985; American Society for Biochemistry and Molecular Biology
• Subjects: activity; Animals; calcitriol; Calcitriol - metabolism; Calcitriol - pharmacology; Calcium - metabolism; Cell Differentiation; Cell Division - drug effects; Cells, Cultured; Chromatography, Gel; Computers; DNA - biosynthesis; Mice; muscle tissues; Muscles - analysis; Muscles - cytology; Muscles - drug effects; Radioligand Assay; Rats; receptors; Receptors, Calcitriol; Receptors, Steroid - analysis; Tritium; Vitamin D Deficiency - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)39433-4

Cytosols from cultured myoblast cells (G-8 and H9c2) prepared in high salt (0.3 M KCl) possesses receptor like proteins for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) that sediment in the 3.2 S region of sucrose gradients. These receptors were characterized as having high affinity (Kd less than 0.1 nM) for 1,25-(OH)2D3 and are in low capacity (less than 80 fmol/mg of cytosol protein). Analog competition for receptor binding revealed that 1,25-(OH)2D3 was more potent than 24,25-(OH)2D3, or 25-(OH)2D3 for displacement of 1,25-(OH)2[3H]D3 from these 3.2 S region sedimenting receptors. Furthermore, the receptor proteins had affinity for DNA and eluted from Sephacryl S-200 as a macromolecule with Stokes radius (Rs) of 32 A. High salt cytosol from collagenase-dispersed skeletal muscle cells was also found to possess a 3.2 S 1,25-(OH)2D3 receptor-like protein. The 1,25-(OH)2D3 receptor concentration in both G-8 and H9c2 myoblast lines was found to down-regulate by 50-70% when cells were stimulated to differentiate to myotubes by lowering fetal calf serum to 5% of the medium. Moreover, we demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner. 1,25-(OH)2D3 was more potent at inhibiting cell proliferation in cells grown in 5% serum than in 20% serum. The data suggest that 1,25-(OH)2D3 can act directly on muscle myoblast via a 1,25-(OH)2D3 receptor that is similar to those found in intestine and bone. The data support the possibility that muscle is a target tissue for 1,25-(OH)2D3 and the hormone may act to initiate terminal differentiation of myoblast cells.