Abstract
Osteoblast Ca2+ channels play a fundamental role in controlling intracellular and systemic Ca2+ homeostasis. A reverse transcription-polymerase chain reaction strategy was used to determine the molecular identity of voltage-sensitive calcium channels present in ROS 17/2.8 osteosarcoma cells. The amino acid sequences encoded by the two resultant PCR products matched the alpha1C-a and the alpha1C-d isoforms. The ability of 1, 25-dihydroxyvitamin D3 (1,25(OH)2D3) and structural analogs to modulate expression of voltage-sensitive calcium channel mRNA transcripts was then investigated. ROS 17/2.8 cells were cultured for 48 h in the presence of either 1,25(OH)2D3,1,24-dihydroxy-22-ene-24-cyclopropyl D3 (analog BT) or 25-hydroxy-16-ene-23-yne-D3 (analog AT), and the levels of mRNA encoding alpha1C were quantitated using a competitive reverse transcription-polymerase chain reaction assay. We found that 1, 25(OH)2D3 and analog BT reduced steady state levels of alpha1C mRNA. Conversely, the Ca2+-mobilizing analog AT did not alter steady state levels of voltage-sensitive calcium channel mRNA. Since analog BT, but not analog AT, binds and transcriptionally activates the nuclear receptor for 1,25(OH)2D3, these findings suggest that the down-regulation of voltage-sensitive calcium channel mRNA levels may involve the nuclear receptor.
Highlights
The balance between osteoclastic bone resorption and osteoblastic bone formation determines skeletal mass and composition. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3)1 has long been appreciated as a hormonal modulator of osteoblast function and bone remodeling. 1,25(OH)2D3, classically considered a resorptive hormone, has the paracrine effects on osteoclasts that are believed to be mediated through separate membrane and nuclear osteoblastic 1,25(OH)2D3 receptor systems [1]
Since analog BT, but not analog AT, binds and transcriptionally activates the nuclear receptor for 1,25(OH)2D3, these findings suggest that the down-regulation of voltage-sensitive calcium channel mRNA levels may involve the nuclear receptor
We previously reported the use of analogs that preferentially stimulate either nVDR-mediated or membrane-initiated pathways and showed that Ca2ϩ influx is not required for the up-regulation of osteopontin and osteocalcin mRNA levels [10]
Summary
1,25(OH)2D3, 1,25-dihydroxyvitamin D3; VSCC(s), voltage-sensitive calcium channel(s); nVDR, nuclear receptor for 1,25-dihydroxyvitamin D3; RT, reverse transcription; PCR, polymerase chain reaction; analog BT, 1,24-(OH)2-22-ene-24-cyclopropyl-D3; analog AT, 25(OH)-16-ene-23-yne-D3; bp, base pairs. We previously reported the use of analogs that preferentially stimulate either nVDR-mediated or membrane-initiated pathways and showed that Ca2ϩ influx is not required for the up-regulation of osteopontin and osteocalcin mRNA levels [10]. L-type voltage-sensitive calcium channels (VSCCs) on the plasma membranes of diverse cell types play a critical role in calcium signaling and cellular function Excitable tissues such as skeletal muscle [11], cardiac muscle [12], and brain [13] express high levels of VSCCs that control excitation-contraction coupling and excitation-secretion coupling and initiate calcium-signaling events involved in intracellular signal transduction. L-type VSCCs have been identified in other tissues including kidney [14], pancreas [15], and vascular smooth muscle [16] These highly conserved channels consist of five distinct subunits (␣1, ␣2, , ␥, and ␦) encoded by four genes (reviewed by Catterall [17]). It was found that 1,25(OH)2D3 down-regulates the ␣1C transcript levels at a physiological dose (1 nM), and vitamin D analog studies suggest that this downregulation involves the nVDR
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
