Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 23:7:45155.
doi: 10.1038/srep45155.

TRPM8 in the negative regulation of TNFα expression during cold stress

Xin-Pei Wang  1 , Xuan Yu  1 , Xiao-Jin Yan  1 , Fan Lei  2 , Yu-Shuang Chai  1 , Jing-Fei Jiang  1 , Zhi-Yi Yuan  1 , Dong-Ming Xing  1 , Li-Jun Du  1
Affiliations

TRPM8 in the negative regulation of TNFα expression during cold stress

Xin-Pei Wang et al. Sci Rep. .

Abstract

Transient Receptor Potential Melastatin-8 (TRPM8) reportedly plays a fundamental role in a variety of processes including cold sensation, thermoregulation, pain transduction and tumorigenesis. However, the role of TRPM8 in inflammation under cold conditions is not well known. Since cooling allows the convergence of primary injury and injury-induced inflammation, we hypothesized that the mechanism of the protective effects of cooling might be related to TRPM8. We therefore investigated the involvement of TRPM8 activation in the regulation of inflammatory cytokines. The results showed that TRPM8 expression in the mouse hypothalamus was upregulated when the ambient temperature decreased; simultaneously, tumor necrosis factor-alpha (TNFα) was downregulated. The inhibitory effect of TRPM8 on TNFα was mediated by nuclear factor kappa B (NFκB). Specifically, cold stress stimulated the expression of TRPM8, which promoted the interaction of TRPM8 and NFκB, thereby suppressing NFκB nuclear localization. This suppression consequently led to the inhibition of TNFα gene transcription. The present data suggest a possible theoretical foundation for the anti-inflammatory role of TRPM8 activation, providing an experimental basis that could contribute to the advancement of cooling therapy for trauma patients.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Alteration of core temperature and the expression levels of TRPM8, TRPA1, NFκB and TNFα in mouse brains under cold conditions.
(A) Core body temperature under cold conditions (4 °C). (B) The mRNA expression levels of TRPM8, TRPA1, NFκB and TNFα. (C,D) The protein expression levels of TRPM8, TRPA1, NFκB and TNFα. Data are shown as the mean ± S.D. from 12 mice in each group. ##v.s. the control (zero hour), P < 0.01; #P < 0.05.
Figure 2
Figure 2. Expression of TRPM8, TRPA1, NFκB and TNFα in PC12 cells under cold conditions.
(A) Intracellular Ca2+ in the cells under cold conditions (4 °C). The Ca2+ concentration in the cytoplasm at 4 °C is higher than that at 37 °C. (B) The mRNA expression levels of TRPM8, TRPA1, NFκB and TNFα. (C,D) The protein expression levels of TRPM8, TRPA1, NFκB and TNFα. Data are shown as the mean ± S.D. from three experiments. #P < 0.05; ##P < 0.01, v.s. the control (zero time).
Figure 3
Figure 3. Expression of TRPM8, TRPA1, NFκB and TNFα in PC12 cells with Trpm8 knockdown under cold conditions (4 °C).
(A) Construction of a Trpm8 (Dylight 649) knockdown stable cell line. WT represents wild type cells. KD signifies the Trpm8 knockdown cells. (B,C) Protein expression levels of TRPM8, TRPA1, NFκB and TNFα. NS: no significance. Data are shown as the mean ± S.D. from three experiments. #P < 0.05; ##P < 0.01, v.s. the control (zero time).
Figure 4
Figure 4. Confocal imaging of TRPM8, NFκB and TNFα expression in PC12 cells.
(A) The expression of TRPM8, NFκB and TNFα in wild type cells and Trpm8 knockdown cells at 37 °C and 4 °C. KD signifies Trpm8 knockdown cells. (B) In wild type cells, TRPM8 was upregulated and NFκB and TNFα were downregulated under cold conditions. (C) In KD cells, TRPM8 showed weak expression and NFκB and TNFα expression levels were increased. (D) Co-localization of TRPM8 and TNFα in the cytoplasm (cold condition and 500 nM menthol). Data are shown as the mean ± S.D. from three experiments. *P < 0.05; **P < 0.01.
Figure 5
Figure 5. Co-localization of TRPM8 and NFκB in PC12 cells under cold conditions.
(A) Immunofluorescence assay image of TRPM8 and NFκB in the cytoplasm. WT represents wild type cells. KD represents Trpm8 knockdown cells. (B) The results of co-immunoprecipitation (CoIP) of endogenous TRPM8 and NFκB using NFκB antibodies. Western blot analysis was carried out to detect TRPM8 and NFκB. (C) Reverse CoIP confirmed interaction between NFκB and TRPM8. CoIPs were also performed with TRPM8 antibodies. Western blot analysis was carried out by TRPM8 and NFκB. Data are shown as the mean ± S.D. from three experiments. *P < 0.05; **P < 0.01.
Figure 6
Figure 6. Protein expression of NFκB in PC12 cells under cold conditions (4 °C).
(A,B) NFκB in the cytoplasm and in the nucleus. (A) WT represents wild type cells. (B) KD represents Trpm8 knockdown cells. (C,D) Kinetic expression levels of NFκB in both WT and KD cells. C-P65 represents NFκBp65 in the cytoplasm. N-P65 represents NFκBp65 in the nuclei. (E–H) The mRNA expression levels of NFκB and TNFα after JSH-23 (8 μM), the inhibitor of NFκB, was applied. (E,F) WT cells. (G,H) KD cells. Data are shown as the mean ± S.D. from three experiments. **P < 0.01, v.s. the control (zero time).
Figure 7
Figure 7. Protein expression of TRPM8 and TNFα in mouse brain with cerebral ischemia-reperfusion (CIR).
Brain cooling means putting the anesthetized mouse brain on the artificial ice. Normal means the room temperature (25 °C). Data are shown as the mean ± S.D. from five mice in each group. #P < 0.05; ##P < 0.01, v.s. the normal control. *P < 0.05; **P < 0.01, v.s. the CIR.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Raddatz N., Castillo J. P., Gonzalez C., Alvarez O. & Latorre R. Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8). J. Biol. Chem. 289(51), 35438–35454 (2014). - PMC - PubMed
    1. Bidaux G. et al.. Functional and modeling studies of the transmembrane region of the TRPM8 channel. Biophys. J. 109(9), 1840–1851 (2015). - PMC - PubMed
    1. de Jong P. R. et al.. TRPM8 on mucosal sensory nerves regulates colitogenic responses by innate immune cells via CGRP. Mucosal Immunol. 8(3), 491–504 (2015). - PMC - PubMed
    1. Grolez G. P. & Gkika D. TRPM8 puts the chill on prostate cancer. Pharmaceuticals (Basel) 9(3), doi: 10.3390/ph9030044 In press (2016). - DOI - PMC - PubMed
    1. Johnson C. D. et al.. Transient receptor potential melastatin 8 channel involvement in the regulation of vascular tone. Am. J. Physiol. Heart Circ. Physiol. 296(6), H1868–H1877 (2009). - PMC - PubMed

Publication types