Methionine promotes cell proliferation in bovine mammary epithelial cells via the ATF4-GSK3β signalling pathway

Increasing the methionine (Met) supply to lactating cows will increase mammary cell proliferation. Activating transcription factor 4 (ATF4) is a regulator of cellular amino acid sensing, linking nutrient signals to cellular anabolism; however, its role in regulating bovine mammary epithelial cell (BMEC) proliferation remains largely unknown. In this study, ATF4 expression paralleled that of Cyclin D1 in mammary tissues from dairy cows at different developmental stages (puberty, lactation and dry period), suggesting that ATF4 is associated with cell proliferation. In BMECs, knockdown of ATF4 increased the number of cells arrested in the G1 phase (∼11%; P < 0.05; control) and downregulated Cyclin D1 expression (∼40%; P < 0.05; control), indicating that ATF4 is required for cell proliferation. Treatment of BMECs with increasing concentrations of Met (0, 0.3, 0.6, 0.9, 1.0 and 1.2 mM) showed that 0.6 mM Met enhanced mammalian target of rapamycin (mTOR) phosphorylation and increased protein expression of ATF4 and Cyclin D1, without activating canonical endoplasmic reticulum stress markers. Inhibition assays demonstrated that mTOR is required for Met-induced ATF4 activation. Functional studies further revealed that ATF4 is a key mediator of Met-regulated cell proliferation. Mechanistically, Met facilitated the physical interaction between ATF4 and glycogen synthase kinase-3β (GSK3β), promoting GSK3β phosphorylation at Ser9 and subsequently increasing Cyclin D1 expression. Collectively, these findings indicate that Met promotes BMEC proliferation through the mTOR-ATF4-GSK3β signalling pathway, providing a potential nutritional strategy to enhance mammary cell proliferation in dairy cows.