Involvement of CaV3.1 T-type calcium channels in cell proliferation in mouse preadipocytes
Involvement of CaV3.1 T-type calcium channels in cell proliferation in mouse preadipocytes
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DOI：
10.1152/ajpcell.00488.2009
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发表时间：
2010-06-01
影响因子：
5.5
通讯作者：
Nakajima, Toshiaki
中科院分区：
文献类型：
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作者：
Oguri, Atsushi;Tanaka, Tomofumi;Nakajima, Toshiaki
[10]李文，李文，李文. Ca（V）3.1 T型钙通道参与小鼠前脂肪细胞的细胞增殖美国生理学杂志细胞生理学298：C1414-C1423，2010年。首次发表于2010年3月24日; doi：10.1152/ajpcell.00488.2009。电压门控性钙通道（Ca 2 + channels，Ca-V）广泛存在于多种细胞中，在细胞增殖等生理功能的调节中发挥重要作用。然而，在前脂肪细胞中，Ca-V的分子身份和功能仍然未被探索。因此，全细胞电压钳技术，常规/定量实时RT-PCR，Western blot，小干扰RNA（siRNA）实验，和免疫组化分析应用于小鼠原代培养的前脂肪细胞以及小鼠3 T3-L1前脂肪细胞。同时观察了Ca-V阻断剂对细胞增殖和细胞周期的影响。3 T3-L1前脂肪细胞的全细胞记录显示低阈值Ca-V，可被米贝拉地尔、Ni 2+（IC 50为200 μ M）和NNC 55 -0396抑制。免疫组化和Western blot分析表明，在Ca-V转录本（α（1A）-α（1 I））中，α（1G）mRNA的表达占优势，并得到α（1G）基因编码的Ca（V）3.1蛋白表达的支持。针对α（1G）的siRNA显著抑制Ca（V）。在原代培养的小鼠前脂肪细胞中也观察到α（1G）mRNA的显性表达和Ca（V）3.1蛋白的表达。α（1G）mRNA和Ca（V）3.1蛋白表达水平在分化的脂肪细胞中显著降低。米贝拉地尔（NNC 55 -0396）是一种选择性T型Ca-V阻滞剂，可抑制细胞对血清的增殖反应，但不抑制地尔硫卓。NNC 55 -0396和靶向α（1G）的siRNA也可阻止细胞周期进入/进展。目前的研究表明，由α（1G）亚型编码的Ca（V）3.1 T型Ca 2+通道是小鼠前脂肪细胞中的主导Ca-V，并可能在调节前脂肪细胞增殖中发挥作用，这是脂肪组织发育的关键步骤。
Oguri A, Tanaka T, Iida H, Meguro K, Takano H, Oonuma H, Nishimura S, Morita T, Yamasoba T, Nagai R, Nakajima T. Involvement of Ca(V)3.1 T-type calcium channels in cell proliferation in mouse preadipocytes. Am J Physiol Cell Physiol 298: C1414-C1423, 2010. First published March 24, 2010; doi:10.1152/ajpcell.00488.2009.-Voltage- gated Ca2+ channels (Ca-V) are ubiquitously expressed in various cell types and play vital roles in regulation of cellular functions including proliferation. However, the molecular identities and function of Ca-V remained unexplored in preadipocytes. Therefore, whole cell voltage-clamp technique, conventional/quantitative real-time RT-PCR, Western blot, small interfering RNA (siRNA) experiments, and immunohistochemical analysis were applied in mouse primary cultured preadipocytes as well as mouse 3T3-L1 preadipocytes. The effects of Ca-V blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold Ca-V, which could be inhibited by mibefradil, Ni2+ (IC50 of 200 mu M), and NNC55-0396. Dominant expression of alpha(1G) mRNA was detected among Ca-V transcripts (alpha(1A)-alpha(1I)), supported by expression of Ca(V)3.1 protein encoded by alpha(1G) gene, with immunohistochemical studies and Western blot analysis. siRNA targeted for alpha(1G) markedly inhibited Ca-V. Dominant expression of alpha(1G) mRNA and expression of Ca(V)3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of alpha(1G) mRNA and Ca(V)3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type Ca-V blocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for alpha(1G) also prevented cell cycle entry/progression. The present study demonstrates that the Ca(V)3.1 T-type Ca2+ channel encoded by alpha(1G) subtype is the dominant Ca-V in mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development.