Down-regulation of Ca<sup>2+</sup>-activated K<sup>+</sup> channel K<sub>Ca</sub>3.1 in mouse pre-osteoblast cells treated with vitamin D receptor agonists

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  • Kito Hiroaki
    Dept. Pharmacol., Grad. Sch. Med. Sci., Nagoya City Univ., Japan Dept. Pharmacol., Kyoto Pharm. Univ., Japan
  • Morihiro Haruka
    Dept. Pharmacol., Kyoto Pharm. Univ., Japan
  • Sakakibara Yuka
    Dept. Pharmacol., Kyoto Pharm. Univ., Japan
  • Ohya Susumu
    Dept. Pharmacol., Grad. Sch. Med. Sci., Nagoya City Univ., Japan Dept. Pharmacol., Kyoto Pharm. Univ., Japan

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<p>[Background]</p><p>Bone tissue is a dynamic and living organ, constantly renewed through the process of bone remodeling. The intricate balance between bone-forming osteoblastic and bone-resorbing osteoclastic activity is vital for maintenance of normal bone homeostasis. Vitamin D (VD) plays important roles in calcium homeostasis, mineral metabolism, and bone development indirectly via control calcium absorption in the intestine and reabsorption in the kidney. However, several in vitro studies showed that VD can directly suppress the cell proliferation of mouse osteoblasts. The direct action of VD receptor (VDR) in osteoblasts is poorly understood. The intermediate-conductance Ca2+-activated K+ channel KCa3.1 regulates intracellular Ca2+ signaling pathways and is associated with cell proliferation in various types of cells including pre-osteoblasts. In the present study, we examined the effects of treatments with VDR agonists on the expression and activity of KCa3.1 in mouse pre-osteoblast MC3T3-E1 cells.</p><p>　[Methods] </p><p>Store-operated Ca2+ entry (SOCE) was measured in MC3T3-E1 cells by Ca2+ indicator, fura-2/AM. To induce SOCE, MC3T3-E1 cells were pretreated with 1 μM thapsigargin, a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase inhibitor, for 30 min in Ca2+ free solution and the Ca2+ store was depleted. Then, the bath solution was changed to the standard solution containing 2.2 mM Ca2+. Cell viability was measured by WST-1 assay. </p><p>[Results] </p><p>Treatments with VDR agonists for 48 h markedly decreased the expression levels of KCa3.1 transcripts and proteins in MC3T3-E1 cells, resulting in the significant inhibition of Ca2+ rises induced by DCEBIO, a specific KCa3.1 activator. Treatments with VDR agonists also significantly decreased the expression of several transcriptional regulators of KCa3.1 such as histone deacetylase 2 (HDAC2) and Fra-1 composed of activation protein 1. In addition, the pharmacological blockade of HDAC with vorinostat, a pan-HDAC inhibitor, significantly decreased the expression of KCa3.1. The knockdown of Fra-1 using siRNA decreased the expression of KCa3.1 mRNA without the suppression of HDAC2 mRNA. </p><p>　[Conclusion] </p><p>Our results suggest that KCa3.1 is a new downstream target of VDR signaling and the down-regulation of KCa3.1 through the transcriptional repression of KCa3.1 contribute, at least partly, to the antiproliferative effects of VDR agonists in mouse pre-osteoblasts.</p>

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