Methionine and leucine induce ARID1A degradation to promote mTOR expression and milk synthesis in mammary epithelial cells

Abstract

Amino acids can activate mTOR to promote milk synthesis in mammary epithelial cells (MECs), but the underlying molecular mechanism is still largely unknown. The objective is to investigate the regulatory mechanism of amino acids (Met and Leu) in stimulating mRNA expression of mTOR in MECs. We found that the protein abundance of AT-rich interaction domain 1A (ARID1A) was poorly expressed in mouse mammary gland tissues of lactating period. ARID1A knockdown and gene activation experiments detected whether ARID1A negatively regulated milk protein and fat synthesis in bovine MECs, cell proliferation and the expression and activation of mTOR. ChIP-PCR detected that ARID1A, H3K27ac, H3K27me3 and H3K4me3 all bound to the mTOR promoter at -548∼-793 nt. Knockdown or gene activation of ARID1A enhanced or weakened the binding of H3K27ac on the mTOR promoter, respectively. The stimulation of Met and Leu on mTOR expression and phosphorylation were eliminated by ARID1A gene activation. Furthermore, Met and Leu decreased the protein level of ARID1A through ubiquitination and proteasomal degradation. TRIM21 bound to ARID1A, and TRIM21 knockdown blocked the stimulation of Met and Leu on ARID1A degradation. In summary, these data reveal that ARID1A blocks Met and Leu signaling to mTOR gene transcription through inhibiting H3K27ac deposition on its promoter, and Met and Leu decrease ARID1A protein level through TRIM21-mediated ubiquitination and proteasomal degradation. Our findings uncover that Met and Leu promote mTOR expression for milk synthesis through the TRIM21-ARID1A signaling pathway, providing a novel theoretical basis for the application of amino acids in milk production.