蝙蝠在冬季蛰伏期高表达热激活瞬时受体电位通道（TRPs）却在夏季活跃期高表达冷激活TRPs

Yang-Yang Li; Qing-Yun Lv; Guan-Tao Zheng; Di Liu; Ji Ma; Gui-Mei He; Li-Biao Zhang; Shan Zheng; Hai-Peng Li; Yi-Hsuan Pan

doi:10.24272/j.issn.2095-8137.2021.209

蝙蝠在冬季蛰伏期高表达热激活瞬时受体电位通道（TRPs）却在夏季活跃期高表达冷激活TRPs

doi: 10.24272/j.issn.2095-8137.2021.209

李洋洋^{1, #},
吕青云^{1, #},
郑冠涛¹,
刘娣¹,
马骥²,
何桂梅³,
张礼标⁴,
郑珊¹,
李海鹏^5, ,,
潘逸萱^1, ,

1.
华东师范大学上海市脑功能基因组学重点实验室, 上海200062, 中国
2.
华东师范大学生命科学学院生命医学系, 上海200062, 中国
3.
华东师范大学生命科学学院崇明生态研究院, 上海200062, 中国
4.
广东省科学院动物研究所广东省动物保护与资源利用重点实验室, 广东省野生动物保护与利用公共实验室, 广东广州510260, 中国
5.
中国科学院上海营养与健康研究所中国科学院计算生物学重点实验室, 上海200031, 中国

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出版历程
- 收稿日期: 2021-10-21
- 录用日期: 2021-11-17
- 网络出版日期: 2021-11-17
- 刊出日期: 2022-01-18

Unexpected expression of heat-activated transient receptor potential (TRP) channels in winter torpid bats and cold-activated TRP channels in summer active bats

Yang-Yang Li^{1, #},
Qing-Yun Lv^{1, #},
Guan-Tao Zheng¹,
Di Liu¹,
Ji Ma²,
Gui-Mei He³,
Li-Biao Zhang⁴,
Shan Zheng¹,
Hai-Peng Li^{5
, ,},
Yi-Hsuan Pan^{1
, ,}

1.
Key Laboratory of Brain Functional Genomics of Shanghai and Ministry of Education, School of Life Sciences, East China Normal University, Shanghai 200062, China
2.
School of Life Science, Department of Biomedical Science, East China Normal University, Shanghai 200062, China
3.
Institute of Eco-Chongming, School of Life Sciences, East China Normal University, Shanghai 200062, China
4.
Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, Guangdong 510260, China
5.
CAS Key Laboratory of Computational Biology, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai 200031, China

Funds: This study was supported by the National Natural Science Foundation of China (31100273 to Y.H.P. and 91731304 to H.P.L.)

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Corresponding author: E-mail: yihsuanp@gmail.com; lihaipeng@picb.ac.cn

#Authors contributed equally to this work

摘要

摘要: 感知温度变化的能力对哺乳动物的生存至关重要。哺乳动物主要是通过温度敏感型瞬时受体电位通道（Thermosensitive transient receptor potential channels, Thermo-TRPs）感应温度变化。一些哺乳类动物为了度过寒冷的冬季并存活下来会进入冬眠状态，在此期间它们的体温有剧烈波动；然而，这些哺乳动物具有哪些潜在的温度感受（thermal response）调节机制还不清楚。我们运用实时荧光聚合酶链式反应和蛋白质免疫印记等方法，发现大足鼠耳蝠（Myotis ricketti）在冬季蛰伏期会高表达热激活通道TRPs（例如TRPV1和TRPV4），而在夏季活跃期则高表达冷激活TRPs（例如，TRPM8和TRPC5）。我们也发现小鼠在温度相对较高的条件（40 ^oC）下，会高表达冷激活TRPs（例如 Trpm8 和 Trpc5 ）。这些结果提示，小型哺乳动物在温暖或较热的状况下，会上调表达冷激活TRPs。通过结合位点分析，我们发现一些同源异型盒（HOX）转录因子（Transcription factor, TF）可以调节热激活和冷激活TRPs基因的表达，而POU（Pit-Oct-Unc）家族的一些转录因子则调节温热敏感和冷激活TRPs基因的表达。双荧光素酶报告基因检测结果表明，转录因子HOXA9，POU3F1和POU5F1调节 TRPC5 的表达，可见Thermo-TRPs基因是受到HOX和POU家族中多个转录因子在不同水平上的调控。这项研究揭示蝙蝠为了在冬眠中存活下来，而采取的温度感觉适应机制。
- 冬眠 /
- 蝙蝠 /
- 脑 /
- 温度敏感型瞬时受体电位通道
Abstract: The ability to sense temperature changes is crucial for mammalian survival. Mammalian thermal sensing is primarily carried out by thermosensitive transient receptor potential channels (Thermo-TRPs). Some mammals hibernate to survive cold winter conditions, during which time their body temperature fluctuates dramatically. However, the underlying mechanisms by which these mammals regulate thermal responses remain unclear. Using quantitative real-time polymerase chain reaction (qRT-PCR) and the Western blotting, we found that Myotis ricketti bats had high levels of heat-activated TRPs (e.g., TRPV1 and TRPV4) during torpor in winter and cold-activated TRPs (e.g., TRPM8 and TRPC5) during active states in summer. We also found that laboratory mice had high mRNA levels of cold-activated TRPs (e.g., Trpm8 and Trpc5) under relatively hot conditions (i.e., 40 °C). These data suggest that small mammals up-regulate the expression of cold-activated TRPs even under warm or hot conditions. Binding site analysis showed that some homeobox (HOX) transcription factors (TFs) regulate the expression of hot- and cold-activated TRP genes and that some TFs of the Pit-Oct-Unc (POU) family regulate warm-sensitive and cold-activated TRP genes. The dual-luciferase reporter assay results demonstrated that TFs HOXA9, POU3F1, and POU5F1 regulate TRPC5 expression, suggesting that Thermo-TRP genes are regulated by multiple TFs of the HOX and POU families at different levels. This study provides insights into the adaptive mechanisms underlying thermal sensing used by bats to survive hibernation.
- Hibernation /
- Bats /
- Brain /
- Thermo-TRPs
#Authors contributed equally to this work
注释:

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Figure 1. Expression of various Thermo-TRPs in bats under different physiological states

A–C: Relative mRNA levels of Thermo-TRPs are shown: heat-activated TRPV-1, -2, -3, and -4 (A), TRPM-2, -4, and -5 (B), and cold-activated TRPM8, TRPC5, and TRPA1 (C) of bats in torpid (T), 2 h after arousal (2 h), 24 h after arousal (24 h), and active (AC) states. D: Schematic: Torpid bats with higher abundance of heat- activated TRPs and active bats with higher abundance of warm-sensitive and cold-activated TRPs. E: Relative level of UCP1 mRNA. F, G: Western blotting of Thermo-TRPs (F), and relative amount of each target protein (G) in bats under different states. kDa represents molecular weight of proteins. H: Relative mRNA and protein levels of six Thermo-TRPs in bats under different states. Difference is significant (P<0.05) for values denoted with different letters.

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Figure 2. Transcription factors (TFs) that regulate Thermo-TRP gene expression

A–C: Schematic of TF binding of HOX, POU, FOX, SP, E2F, JUND, and ETS families to Thermo-TRP genes. h- and c-TRPs represent heat- and cold-activated TRPs, respectively (A). Other panels show relative mRNA levels of some TFs of HOX (B) and POU (C) families of bats in torpid (T), 2 h after arousal (2 h), 24 h after arousal (24 h), and active (AC) states. D, E: Western blotting results of several TFs are shown in (D), and their relative abundance in bats under various states is shown in (E). F, G: Relative mRNA and protein levels of six TFs are shown in (F) and six TFs and 10 TRPs are shown in (G). Difference is significant (P<0.05) for values denoted with different letters.

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Figure 3. Relative mRNA levels of Thermo-TRPs and TFs in temperature-stimulated mice

A, B: Relative mRNA abundance of 10 Thermo-TRPs (A) and several TFs in HOX and POU families (B) of mice (n=6 per temperature group). C: Body weights of mice treated at 4 °C, 25 °C, 37 °C, and 40 °C for 6 days. Statistical significance (^*: P=0.0041) of differences in body weight between 37 °C- and 40 °C-treated mice and between 4 °C- and 25 °C-treated mice was determined by two-way ANOVA. Difference is significant (P<0.05) for values denoted with different letters.

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Figure 4. Regulation of TRPC5 expression by HOXA9, POU3F1, and POU5F1

A: Schematic of plasmids pcDNA3.1⁽⁺⁾ and psiCHECK-2^TM. B: Relative luciferase activity of cells containing HOXA9 expression vector and reporter plasmid with Trpc5 promoter region. C, D: Relative luciferase activity of cells containing POU5F1 expression vector and reporter plasmid with Trpc5 3'-untranslated region (3'-UTR) (C) and that of cells containing POU3F1 expression vector and reporter plasmid with Trpc5 3'-UTR (D). Blank: Cells containing no plasmids. Positive control (pcheck2): Cells transfected with psiCHECK-2. pcDNA3.1NC: Cells transfected with empty vector pcDNA3.1⁽⁺⁾. Difference is significant (P<0.05) for values denoted with different letters.

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Table 1. List of 10 thermo-TRPs and their temperature thresholds for activation^#

H^*	Channel	Threshold temp for activation (°C)	Expression location	Physiological function
Hot-sensitive	TRPV subfamily
	TRPV1	>40	Brain, Skin, Bladder	Thermosensation, synaptic plasticity, pain sensation, inflammation, ischemia/reperfusion injury, body temperature regulation, metabolism (Baez et al., 2014; Castillo et al., 2018; Caterina et al., 1999; Dhaka et al., 2006; Güler et al., 2002; Huang et al., 2006; Jardín et al., 2017; Kashio, 2021; Nilius & Owsianik, 2011; Peier et al., 2002; Smith et al., 2002; Talavera et al., 2020; Vay et al., 2012)
	TRPV2	>50	CNS neurons, Heart, Various tissues	Thermosensation (noxious heat), phagocytosis, pain sensation, osmosensation (Castillo et al., 2018; Dhaka et al., 2006; Huang et al., 2006; Kashio, 2021; Nilius & Owsianik, 2011; Palkar et al., 2015; Peier et al., 2002; Smith et al., 2002; Vay et al., 2012)
	TRPV3	>30	Brain, Tongue, Gut, Skin keratinocytes	Thermosensation, nociception, wound healing, pain sensation, skin functions (Baez et al., 2014; Castillo et al., 2018; Dhaka et al., 2006; Huang et al., 2006; Kashio, 2021; Nilius & Owsianik, 2011; Peier et al., 2002; Smith et al., 2002)
	TRPV4	25–42	CNS neurons, Heart, Kidney, Inner ear, liver, Trachea, Fat, Salivary gland, Skin keratinocytes	Thermosensation, osmosensation, pain sensation, neural excitability, skin functions (Baez et al., 2014; Castillo et al., 2018; Dhaka et al., 2006; Güler et al., 2002; Huang et al., 2006; Kashio, 2021; Nilius & Owsianik, 2011; Vay et al., 2012)
	TRPM subfamily
	TRPM2	34–42	Brain, Bone marrow, Eye, Heart, Lymph nodes	Thermosensation, apoptosis, pain sensation, body temperature regulation, insulin secretion (Castillo et al., 2018; Kashio, 2021; Nilius & Owsianik, 2011; Zhong et al., 2016)
	TRPM4	15–35	Heart, Lung, CNS neurons, Gut	Thermosensation, releasing hormone, insulin secretion, chemosensory (Baez et al., 2014; Castillo et al., 2018; Kashio, 2021; Nilius & Owsianik, 2011)
	TRPM5	15–35	Tongue, Lung, Brain, Taste cells, Gut	Taste (sweet, bitter, umami), thermosensation, chemosensory, insulin secretion (Castillo et al., 2018; Kashio, 2021; Nilius & Owsianik, 2011; Talavera et al., 2005)
Cold-sensitive	TRPM8	≤25	Sensory dorsal root, Trigeminal ganglia neurons, Liver, Prostate, Testis	Thermosensation (cold), acrosome reaction, tumor growth, body temperature regulation (Baez et al., 2014; Castillo et al., 2018; Caterina et al., 1999; Dhaka et al., 2006, 2007; Güler et al., 2002; Huang et al., 2006; Kashio, 2021; Nilius & Owsianik, 2011; Peier et al., 2002; Vay et al., 2012)
	TRPC subfamily
	TRPC5	25–37	Brain	Brain development, thermosensation (Baez et al., 2014; Castillo et al., 2018; Kashio, 2021; Nilius & Owsianik, 2011; Zimmermann et al., 2011)
	TRPA subfamily
	TRPA1^$	≤17	Sensory dorsal root, Trigeminal ganglia neurons, Various tissues	Thermosensation (noxious cold), nociception, olfactory responses, pain sensation (Baez et al., 2014; Castillo et al., 2018; Dhaka et al., 2006; Huang et al., 2006; Jardín et al., 2017; Kashio, 2021; Nilius & Owsianik, 2011; Vay et al., 2012)
^#: Features of thermo-TRPs previously characterized in other mammalian species rather than bats. H^*: Classification of several TRPs in human body (Ezquerra-Romano & Ezquerra, 2017): hot- and cold-sensitive TRPs are denoted in orange and green, respectively. ^$: TRPA1 is a heat-activated sensor in non-mammalian species (Saito & Tominaga, 2015).

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