Fujita Takanori, Liu Yu, Higashitsuji Hiroaki, Itoh Katsuhiko, Shibasaki Koji, Fujita Jun, Nishiyama Hiroyuki
Department of Urology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan; Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan.
Biochem Biophys Res Commun. 2018 Jan 1;495(1):935-940. doi: 10.1016/j.bbrc.2017.11.136. Epub 2017 Nov 21.
Cold-inducible RNA-binding protein (CIRP), RNA-binding motif protein 3 (RBM3) and serine and arginine rich splicing factor 5 (SRSF5) are RNA-binding proteins that are transcriptionally upregulated in response to moderately low temperatures and a variety of cellular stresses in mammalian cells. Induction of these cold-inducible proteins (CIPs) is dependent on transient receptor potential (TRP) V4 channel protein, but seems independent of its ion channel activity. We herein report that in addition to TRPV4, TRPV3 and TRPM8 are necessary for the induction of CIPs. We established cell lines from the lung of TRPV4-knockout (KO) mouse, and observed induction of CIPs in them by western blot analysis. A TRPV4 antagonist RN1734 suppressed the induction in wild-type mouse cells, but not in TRPV4-KO cells. A TRPV3 channel blocker S408271 and a TRPM8 channel blocker AMTB as well as siRNAs against TRPV3 and TRPM8 suppressed the CIP induction in mouse TRPV4-KO cells and human U-2 OS cells. A TRPV3 channel agonist 2-APB induced CIP expression, but camphor did not. Neither did a TRPM8 channel agonist WS-12. These results suggest that TRPV4, TRPV3 and TRPM8 proteins, but not their ion channel activities are necessary for the induction of CIPs at 32 °C. Identification of proteins that differentially interact with these TRP channels at 37 °C and 32 °C would help elucidate the underlying mechanisms of CIP induction by hypothermia.
冷诱导RNA结合蛋白（CIRP）、RNA结合基序蛋白3（RBM3）和富含丝氨酸和精氨酸的剪接因子5（SRSF5）是RNA结合蛋白，在哺乳动物细胞中，它们会因适度低温和多种细胞应激而转录上调。这些冷诱导蛋白（CIPs）的诱导依赖于瞬时受体电位（TRP）V4通道蛋白，但似乎与其离子通道活性无关。我们在此报告，除了TRPV4外，TRPV3和TRPM8对于CIPs的诱导也是必需的。我们从TRPV4基因敲除（KO）小鼠的肺中建立了细胞系，并通过蛋白质印迹分析观察到其中CIPs的诱导情况。TRPV4拮抗剂RN1734抑制野生型小鼠细胞中的诱导，但不抑制TRPV4-KO细胞中的诱导。TRPV3通道阻滞剂S408271和TRPM8通道阻滞剂AMTB以及针对TRPV3和TRPM8的小干扰RNA（siRNAs）抑制了小鼠TRPV4-KO细胞和人U-2 OS细胞中的CIP诱导。TRPV3通道激动剂2-APB诱导CIP表达，但樟脑未诱导。TRPM8通道激动剂WS-12也未诱导。这些结果表明，TRPV4、TRPV3和TRPM8蛋白而非其离子通道活性是在32°C诱导CIPs所必需的。鉴定在37°C和32°C与这些TRP通道有差异相互作用的蛋白质将有助于阐明低温诱导CIPs的潜在机制。
