Stimulation of calmodulin synthesis in proliferating myoblasts by 1,25-dihydroxy-vitamin D 3

Abstract

Myoblasts contain a receptor specific for 1,25-dihydroxy-vitamin D 3. Morphological data have indicated that the hormone stimulates both myoblast proliferation and fusion. The synthesis of myoblast proteins in response to the sterol was studied during the proliferating stage of the cells. Chick embryo myoblast primary cultures (precultured for 24 h in the presence of low levels of 1,25-dihydroxy-vitamin D 3 after isolation) were used. Labelling (2 h) of cells incubated in the absence and presence of 1,25-dihydroxy-vitamin D 3 (10 −10 M) for 1–12 h with [ 14C]leucine and [ 3H]leucine, respectively, followed by coelectrophoresis of double-labelled proteins on sodium dodecyl sulfate polyacrylamide gels showed that the sterol initially stimulates the synthesis of proteins of 60 kDa (1.2 h), 70 kDa (2.4 h) and 80 kDa (4 h). These changes were transient and between 6 and 12 h a protein of 19 kDa was induced. This protein was identified as calmodulin on the basis of its isoelectric point (p I 4.1), Ca 2+-dependent electrophoretic mobility, ability to bind 45Ca and to interact with an immobilized phenothiazine in a Ca 2+-dependent manner, and by means of immunoblotting with a specific anti-calmodulin antibody and 3′,5′-cyclic AMP phosphodiesterase activation assays. In agreement with these results, hybridization analysis with a specific cDNA probe showed increased calmodulin mRNA levels in myoblasts treated for 4–12 h with 1,25-dihydroxy-vitamin D 3. These changes were paralleled by a stimulation of [ 3H]thymidine incorporation into DNA suggesting that they may be involved in the mitogenic action of the hormone.