Baldi Carolina, Vazquez Guillermo, Calvo Juan Carlos, Boland Ricardo
Departamento de Biología, Bioquímica and Farmacia, Universidad Nacional del Sur, Bahía Blanca, Argentina.
J Cell Biochem. 2003 Sep 1;90(1):197-205. doi: 10.1002/jcb.10612.
In ROS 17/2.8 rat osteoblastic-like cells a capacitative Ca(2+) entry (CCE) pathway operates which is activated by either 1alpha,25-dihydroxy-vitamin D3 (1alpha,25(OH)(2)D3 or thapsigargin (Tpg)-induced depletion of Ca(2+) stores. In view of recent evidence favoring a role for transient receptor potential (TRP) proteins in mediating CCE, we investigated if channels involved in the 1alpha,25(OH)(2)D3-sensitive CCE in rat osteoblasts were related to an endogenous TRP-canonical (TRPC) isoform homologue. By reverse transcription (RT)-PCR using mRNA from ROS 17/2.8 cells and primers based on conserved regions within the mammalian TRPC3/6/7 subfamily, two fragments were amplified of 390 and 201 bp with 100 and 94% sequence identity, respectively, with human TRPC3. Northern blot analysis showed the presence of a 3.5 kb transcript and both immunobloting and immunocytochemistry using a specific anti-TRPC3 antibody confirmed endogenous expression of a TRPC3-like protein ( approximately 110 kDa) with membrane localization. In ROS 17/2.8 cells intranuclearly microinjected with anti-TRPC3 antisense oligodeoxynucleotides (ODN), both the initial rate and magnitude of CCE activated by either 1alpha,25(OH)(2)D3 or Tpg were markedly reduced, whereas no changes were detected in control-injected cells. The present findings constitute the first evidence to date suggesting that an endogenous TRPC3-like protein is functionally involved in the CCE route activated by 1alpha,25(OH)(2)D3 in a secosteroid target cell. We anticipate TRPC3 as a candidate for mediating store-operated non-selective cation entry into osteoblasts.
在ROS 17/2.8大鼠成骨样细胞中，存在一种容量性Ca(2+)内流（CCE）途径，该途径可被1α,25-二羟基维生素D3（1α,25(OH)2D3）或毒胡萝卜素（Tpg）诱导的Ca(2+)储存耗竭所激活。鉴于最近有证据表明瞬时受体电位（TRP）蛋白在介导CCE中起作用，我们研究了大鼠成骨细胞中参与1α,25(OH)2D3敏感CCE的通道是否与内源性TRP-经典型（TRPC）同工型同源物有关。通过使用来自ROS 17/2.8细胞的mRNA和基于哺乳动物TRPC3/6/7亚家族保守区域的引物进行逆转录（RT）-PCR，扩增出了两个片段，分别为390和201 bp，与人类TRPC3的序列同一性分别为100%和94%。Northern印迹分析显示存在一个3.5 kb的转录本，使用特异性抗TRPC3抗体进行的免疫印迹和免疫细胞化学均证实了一种具有膜定位的TRPC3样蛋白（约110 kDa）的内源性表达。在核内显微注射了抗TRPC3反义寡脱氧核苷酸（ODN）的ROS 17/2.8细胞中，由1α,25(OH)2D3或Tpg激活的CCE的初始速率和幅度均显著降低，而在注射对照的细胞中未检测到变化。目前的研究结果构成了迄今为止的首个证据，表明内源性TRPC3样蛋白在类固醇靶细胞中功能性地参与了由1α,25(OH)2D3激活的CCE途径。我们预期TRPC3是介导储存操纵的非选择性阳离子进入成骨细胞的候选分子。
