Abstract
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.
Highlights
Cytosols from culturedmyoblast cells(6-8and H9c2) gest that a form of the avian receptor exists as a 63,000
We tion of theG-8 myoblastcells in adose-dependent report that receptor concentrationsin myoblast cells manner. 1,25-(OH)& was more potent at inhibiting decreased after terminal differentiation of these cells to the cell proliferation in cells grown in 5% serum than in fused myotube form
Lines-Analysis of 1,25-(OH)2D3binding to cytosol prepared in TKED buffer from cultured skeletal (G-8) myoblast cells revealed a single binding component in the3.2 S region of the sucrose gradient (Fig. lA).When cytosol was incubated with 1,25-(OH)2[3H]D3(1 nM) in the presence of 10-fold excess nonradioactive 1,25-(OH)2D3,the 3.2 S region sedimenting binding component was no longer evident
Summary
10’’Bq) was purchased from Amersham Corp. (Chicago, IL).Nonradioactive 24(R),25-dihydroxyvitaminDS [24(R),25-(OH)zD3]and. D3; 25-(OH)D3,25-hydroxyvitamin Ds;24;25-(OH)zD3, 24,25-dihy- Preparation of Muscle Cell Cytosol-The cells from a confluent droxyvitamin DB;FCS, fetal bovine calf serum; PBS, phosphate- culture were detached from flasks with 10 mM EDTA in phosphatebuffered saline; DMEM, Dulbecco’s modifiedEagle’s medium. Bound hormone was separated from free by absorption of free hormone to dextrancoated charcoal as described above and 0.5-ml aliquots were withdrawn and assayed for radioactivity In these experiments specific binding was 40-50% of total binding. Statistical analysis was performed using the two-tailed Student's t test
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