CRTC2 Is a Key Mediator of Amino Acid-Induced Milk Fat Synthesis in Mammary Epithelial Cells

Abstract

Amino acids can stimulate milk fat synthesis, but the underlying molecular mechanism is still largely unknown. In this study, we studied the regulatory role and corresponding molecular mechanism of cAMP response element-binding protein-regulated transcription coactivator 2 (CRTC2) in amino acid-induced milk fat synthesis in mammary epithelial cells. We showed that leucine and methionine stimulated CRTC2 but not p-CRTC2(Ser171) expression and nuclear localization in cow mammary epithelial cells. Knockdown of CRTC2 decreased milk fat synthesis and sterol regulatory element binding protein 1c (SREBP-1c) expression and activation, whereas its overexpression had the opposite effects. Neither knockdown nor overexpression of CRTC2 affected β-casein synthesis and phosphorylation of the machanistic target of rapamycin (mTOR), suggesting that CRTC2 only regulates milk fat synthesis. CRTC2 knockdown abolished the stimulation of leucine and methionine on SREBP-1c expression and activation. Knockdown or overexpression of CRTC2 did not affect the protein level of cAMP-response element-binding protein (CREB) and its phosphorylation but decreased or increased the binding of p-CREB to the promoter of SREBP-1c gene and its mRNA expression, respectively. Mutation of Ser171 of CRTC2 did not alter the stimulation of CRTC2 on SREBP-1c expression and activation, further suggesting that CRTC2 functions in the nucleus. mTOR inhibition by rapamycin totally blocked the stimulation of leucine and methionine on CRTC2 expression. The expression of CRTC2 was dramatically higher in the mouse mammary gland of lactation period, compared with that of the dry and puberty periods, whereas p-CRTC2(Ser171) was not changed, further supporting that CRTC2 is a key transcription coactivator for milk fat synthesis. These results uncover that CRTC2 is a key transcription coactivator of amino acid-stimulated mTOR-mediated milk fat synthesis in mammary epithelial cells.

Keywords: SREBP-1c; cAMP-response element-binding protein; cAMP-response element-binding protein-regulated transcription coactivator 2; leucine; mTOR; mammary epithelial cells; methionine; milk fat synthesis.