m6A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

Yabing Chen; Jing Wang; Dihui Xu; Zou Xiang; Jie Ding; Xiaoyu Yang; Dongmei Li; Xiaodong Han

doi:10.1080/15548627.2020.1720431

m⁶A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells

Autophagy. 2021 Feb;17(2):457-475. doi: 10.1080/15548627.2020.1720431. Epub 2020 Jan 31.

Authors

Yabing Chen^{1

2}, Jing Wang^{1

2}, Dihui Xu^{1

2}, Zou Xiang³, Jie Ding^{1

2}, Xiaoyu Yang⁴, Dongmei Li^{1

2}, Xiaodong Han^{1

2}

Affiliations

¹ Immunology and Reproduction Biology Laboratory & State Key Laboratory of Analytical Chemistry for Life Science, Medical School, Nanjing University, Nanjing, China.
² Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, China.
³ Department of Health Technology and Informatics, Faculty of Health and Social Sciences, The Hong Kong Polytechnic University, Hong Kong, China.
⁴ State Key Laboratory of Reproductive Medicine, Clinical Center of Reproductive Medicine, The First Hospital of Nanjing Medical University, Nanjing, China.

Abstract

Macroautophagy/autophagy is indispensable for testosterone synthesis in Leydig cells (LCs), and here we report a negative association between m⁶A modification and autophagy in LCs during testosterone synthesis. A gradual decrease of METTL14 (methyltransferase like 14) and an increase of ALKBH5 (alkB homolog 5, RNA demethylase) were observed in LCs during their differentiation from stem LCs to adult LCs. These events led to reduced mRNA methylation levels of N⁶-methyladenosine (m⁶A) and enhanced autophagy in LCs. Similar regulation of METTL14, ALKBH5, and m⁶A was also observed in LCs upon treatment with human chorionic gonadotropin (HsCG). Mechanistically, m⁶A modification promoted translation of PPM1A (protein phosphatase 1A, magnesium dependent, alpha isoform), a negative AMP-activated protein kinase (AMPK) regulator, but decreased expression of CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, beta), a positive AMPK regulator, by reducing its RNA stability. Thus, m⁶A modification resulted in reduced AMPK activity and subsequent autophagy inhibition. We further demonstrated that ALKBH5 upregulation by HsCG was dependent on enhanced binding of the transcriptional factor CEBPB (CCAAT/enhancer binding protein [C/EBP], beta) and the TFEB (transcription factor EB) to its gene promoter. Moreover, HsCG treatment decreased METTL14 by reducing its stability. Collectively, this study highlights a vital role of m⁶A RNA methylation in the modulation of testosterone synthesis in LCs, providing insight into novel therapeutic strategies by exploiting m⁶A RNA methylation as targets for treating azoospermatism and oligospermatism patients with reduction in serum testosterone.Abbreviations: 3-MA: 3-methyladenine; ACTB: Actin, beta; ALKBH5: alkB homolog 5, RNA demethylase; AMPK: AMP-activated protein kinase; BafA1: bafilomycin A_1; CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CEBPB: CCAAT/enhancer-binding protein (C/EBP), beta; ChIP: chromatin immunoprecipitation; FTO: fat mass and obesity associated; HsCG: human chorionic gonadotropin; HSD3B: 3β-hydroxysteroid dehydrogenase; LCs: Leydig cells; m⁶A: N⁶-methyladenosine; METTL14: methyltransferase like 14; METTL3: methyltransferase like 3; MTOR: mechanistic target of rapamycin kinase; PPM1A: protein phosphatase 1A, magnesium dependent, alpha isoform; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; ULK1: unc-51-like kinase 1; WTAP: Wilms tumor 1-associating protein; YTHDF: YTH N6-methyladenosine RNA binding protein.

Keywords: AMPK; Leydig cells; autophagy; m6A modification; testosterone synthesis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Autophagy / genetics*
Leydig Cells / metabolism*
Male
Methylation
Methyltransferases / metabolism*
Mice
Mice, Inbred BALB C
Signal Transduction / genetics
TOR Serine-Threonine Kinases / metabolism
Testosterone / biosynthesis*

Substances

Testosterone
Methyltransferases
TOR Serine-Threonine Kinases

Grants and funding

This work was supported by the National Natural Science Foundation of China (31901182, 31870492, 31971517 and 31670519) and Fundamental Research Funds for the Central Universities (0214-14380438).