Milk is the most important source of nutrition for the early development of newborns, providing nutrients such as milk fat, milk protein and lactose [1], [2], [3]. Mammary epithelial cells (MECs) are the basic unit of mammary gland for milk synthesis, and the milk synthesis ability of MECs is determined by milk fat and protein synthesis and proliferation ability of MECs [4,5]. Amino acids are not only substrates for milk protein synthesis, but also key signaling molecules and stimuli of milk fat and protein synthesis. For examples, circulating amino acids can promote milk protein synthesis through insulin-like growth factor-I (IGF-I), growth hormone (GH) and prolactin (PRL) [6]; Glutamine (Gln) can positively regulate cell growth and casein synthesis in bovine MECs through CYTHs/ARFGAP1-Arf1-mTORC1 pathway [7]; Methionine (Met) is one of the key regulatory factors in milk synthesis [8,9]. Met can stimulate the phosphorylation of glycyl tRNA synthetase (GlyRS) through the GPR87-CDC42/Rac1-MAP3K10 signal pathway, and the phosphorylated GlyRS then enter the nucleus to stimulate related expression of genes such as the mechanistic target of rapamycin (mTOR) for milk synthesis [10]. mTOR is a highly conserved serine/threonine kinase, which exists in two different multi-protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [11,12]. Among them, mTORC1 is a central hub controlling various cellular processes such as protein synthesis, lipid synthesis and cell proliferation [13,14]. It has long been confirmed that Met, lysine and other amino acids have stimulatory effects on mTOR protein activation (phosphorylation) [15,16]. Met can activate mTOR in bovine MECs through heterodimeric amino acid taste receptor (TAS1R1/TAS1R3) [17]. Our laboratory has proved that Met, leucine (Leu) and other amino acids can stimulate the mRNA expression of mTOR, which will lead to subsequent protein phosphorylation, through Brahma-related gene1 (BRG1), AT-rich binding domain 4B (ARID4B) and other nuclear factors to promote milk synthesis in and proliferation of MECs [18,19]. However, the detailed mechanism of amino acids regulating mTOR mRNA expression for milk synthesis is still poorly understood.

The DNA binding AT-rich interactive domain (ARID) family is a superfamily composed of 15 members (ARID1A, ARID1B, ARID2, ARID3A, ARID4B, etc.), widely existing in a large number of eukaryotes. ARIDs participate in many important biological processes, such as chromatin remodeling, cell cycle regulation, and transcriptional regulation [20,21]. ARID1B is one of the non-catalytic subunits of BAF chromatin remodeling complex, also known as BAF250b subunit. ARID1B is a nuclear protein which can bind to DNA or other proteins and thereby regulate the activity of BAF complex and gene transcription [22,23]. ARID1B plays an important role in neural development. ARID1B gene deletion will lead to autism spectrum disorder and intellectual disability [24]. The disappearance of BAF250 domain in ARID1B protein will lead to Coffin-Siris syndrome [25,26]. ARID1B is a typical nuclear tumor suppressor and can inhibit proliferation of many kinds of tumor cells [27,28]. ARID1B deletion will lead to accelerated proliferation of adrenal cells [29]. Although there are some reports on the function of ARID1B in normal and tumor cells, the molecular mechanism of ARID1B is still unclear, and whether ARID1B participates in amino acid regulation of milk synthesis is not known yet.

The protein levels of ARIDs can be regulated though ubiquitination and proteasome degradation. For examples, ARID1A can be ubiquitinated by an E3 ubiquitin ligase tripartite motif containing 32 (TRIM32) and degraded, which promotes the development of squamous cell carcinoma [30]. We previously found that Met and Leu can promote ARID1A ubiquitination degradation and mTOR gene transcription in bovine MECs [16]. However, it is still unknown whether ARID1B can affect the mTOR signaling and whether amino acids can induce ARID1B ubiquitination degradation.

We previously found through proteomic analysis (data not shown) that ARID1B might be associated with milk synthesis in MECs. In this study, we aim to explore the role and molecular mechanism of ARID1B in Met-stimulated milk synthesis in MECs.