The effect of 1,25-dihydroxyvitamin D₃ on the cytoskeleton of rat calvaria and rat osteosarcoma (ROS 17/2.8) osteoblastic cells

1,25-dihydroxy vitamin D₃ produces pronounced shape changes in fetal rat calvaria and osteosarcoma-derived (ROS 17/2.8) osteoblastic cells, characterized by retracting processes and cell rounding followed by aggregation of cells. The 1,25(OH)₂D₃ effect on ROS 17/2.8 morphology was determined morphometrically on scanning electron micrographs. The hormone effect was found to be dose dependent between 10⁻¹² and 10⁻⁹ M. The shape changes appeared 12 h after hormone (10⁻¹⁰ M) addition and were present in 80% of the ROS 17/2.8 cells and in 50% of the calvaria cells at 72 h. Cycloheximide at 1 μM, inhibited the hormone-dependent change in morphology. The 1,25(OH)₂D₃ effects were partially mimicked by 10⁻⁸ M 25(OH)D₃ but not by 10⁻¹⁰ M 25(OH)D₃ or 10⁻¹¹-10⁻⁸ M 24,25(OH)₂D₃. 1,25-dihydroxy vitamin D₃ also increased cell proliferation twofold at 14 days in serum-free medium.

1,25(OH)₂D₃ treatment produced changes in microfilament organization, visualized with rhodamine-conjugated phalloidin. Microfilaments were localized at the terminal attachment points and in the perinuclear region, and few if any, were seen in the retracting processes themselves. Estimation of cytoskeletal actin and myosin by gel electrophoresis of Triton X-100 nonextractable proteins showed a 30% reduction in these proteins in the hormone-treated cells. Microtubules visualized by indirect immunofluorescence showed no major changes in organization. Both colchicine and cytochalasin D altered the hormone-induced shape change, suggesting that both microfilaments and microtubules were required for this process.

Thus, 1,25(OH)₂D₃ had pronounced effects on cell shape in osteoblastic cells, probably via de novo protein synthesis. These changes lead to rearrangement of the cytoskeleton, primarily the microfilaments.