Targeting TRPV1-mediated autophagy attenuates nitrogen mustard-induced dermal toxicity

Mingliang Chen,Haoyue Deng,Zhongmin Zou,Jin Cheng,Yuanpeng Zhao,Xunhu Dong,Xiuwu Bian,Yan Sai,Feng Ye,Jiqing Zhao,Guorong Dan

doi:10.1038/s41392-020-00389-z

Mingliang Chen, Haoyue Deng + Show 9 more

Open Access

https://doi.org/10.1038/s41392-020-00389-z

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Abstract

Nitrogen mustard (NM) causes severe vesicating skin injury, which lacks effective targeted therapies. The major limitation is that the specific mechanism of NM-induced skin injury is not well understood. Recently, autophagy has been found to play important roles in physical and chemical exposure-caused cutaneous injuries. However, whether autophagy contributes to NM-induced dermal toxicity is unclear. Herein, we initially confirmed that NM dose-dependently caused cell death and induced autophagy in keratinocytes. Suppression of autophagy by 3-methyladenine, chloroquine, and bafilomycin A1 or ATG5 siRNA attenuated NM-induced keratinocyte cell death. Furthermore, NM increased transient receptor potential vanilloid 1 (TRPV1) expression, intracellular Ca2+ content, and the activities of Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), unc-51-like kinase 1 (ULK1), and mammalian target of rapamycin (mTOR). NM-induced autophagy in keratinocytes was abolished by treatment with inhibitors of TRPV1 (capsazepine), CaMKKβ (STO-609), AMPK (compound C), and ULK1 (SBI-0206965) as well as TRPV1, CaMKKβ, and AMPK siRNA transfection. In addition, an mTOR inhibitor (rapamycin) had no significant effect on NM-stimulated autophagy or cell death of keratinocytes. Finally, the results of the in vivo experiment in NM-treated skin tissues were consistent with the findings of the in vitro experiment. In conclusion, NM-caused dermal toxicity by overactivating autophagy partially through the activation of TRPV1-Ca2+-CaMKKβ-AMPK-ULK1 signaling pathway. These results suggest that blocking TRPV1-dependent autophagy could be a potential treatment strategy for NM-caused cutaneous injury.

Highlights

Nitrogen mustard (NM) is a chemical alkylating agent that was first used as a chemical warfare agent in World War I
ATP levels were detected in NM-treated keratinocytes, we found that NM slightly but not significantly increased ATP levels (Supplementary Fig. S5a), indicating that NM-activated AMPK in an ATP independent manner in keratinocytes
reactive oxygen species (ROS) levels were detected by dichloro-dihydro-fluorescein diacetate (DCFHDA) staining, and we found that NM markedly increased ROS levels in keratinocytes (Supplementary Fig. S5f)

Summary

Introduction

Nitrogen mustard (NM) is a chemical alkylating agent that was first used as a chemical warfare agent in World War I. Based on their alkylating effects, many kinds of NM derivatives, including mechlorethamine, chlorambucil, and melphalan have been widely used in the clinic against various tumors, such as lymphoma, leukemia, and multiple myeloma.[1] the clinical utility of NM is limited by its dose-dependent side effects especially cutaneous toxicity.[1] NM inflicts incapacitating skin injuries characterized by blistering, inflammation, induration, and edema.[2,3] There is currently no effective therapy for NM-caused dermal toxicity, due to a lack of understanding of the associated mechanism(s). Limited information is available regarding the potential contribution of autophagy to NM-induced dermal toxicity and the underlying mechanisms

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