Wang Fang, van Baal Jürgen, Ma Lu, Gao Xuejun, Dijkstra Jan, Bu Dengpan
State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
Animal Nutrition Group, Wageningen University and Research, Wageningen, 6708, WD, the Netherlands.
Anim Nutr. 2025 Feb 15;21:129-139. doi: 10.1016/j.aninu.2025.01.003. eCollection 2025 Jun.
Amino acids (AA), including methionine (Met) and leucine (Leu), stimulate milk synthesis in bovine mammary epithelial cells (BMEC) via activation of protein kinase mechanistic target of rapamycin (mTOR). In this study, we further explored the potential role of myotonic dystrophy-related CDC42-binding kinase alpha (MRCKα), previously identified as a critical mediator of prolactin-stimulated milk synthesis in BMEC. Administering different doses (0, 0.2, 0.4, 0.6, 0.8 mM) of Met or Leu to a primary BMEC culture showed that 0.6 mM was the optimal dose for stimulating β-casein production with both AA. At this dose, Met and Leu independently evoked higher ( < 0.05) protein levels of β-casein, MRCKα and sterol regulatory element-binding protein 1 (SREBP1), and increased ( < 0.05) phosphorylation of mTOR (Ser) and phosphatidylinositol 3-kinase (PI3K) (Tyr) after 24 h. The stimulatory effects of both AA on relative protein level of β-casein, phosphorylation of mTOR, and phosphorylation of protein kinase B (PKB) (Thr), were blocked by silencing MRCKα expression ( < 0.05). Whereas, that on the phosphorylation of PI3K remained intact ( = 0.385). Inhibiting PI3K with LY294002 blocked Met- and Leu-induced protein expression of MRCKα and β-casein and phosphorylation of mTOR ( < 0.05). Overexpression of MRCKα increased protein levels of β-casein and phosphorylation of mTOR, which was prevented by PKB inhibitor MK2206 ( < 0.05). Our results indicate that MRCKα is a key mediator of the Met- and Leu-induced signaling cascade, acting downstream of PI3K and upstream of PKB to regulate β-casein synthesis in BMEC.
包括蛋氨酸（Met）和亮氨酸（Leu）在内的氨基酸（AA）通过激活蛋白激酶雷帕霉素机制性靶标（mTOR）刺激牛乳腺上皮细胞（BMEC）中的乳汁合成。在本研究中，我们进一步探讨了强直性肌营养不良相关的CDC42结合激酶α（MRCKα）的潜在作用，该激酶先前被确定为BMEC中催乳素刺激乳汁合成的关键介质。对原代BMEC培养物施用不同剂量（0、0.2、0.4、0.6、0.8 mM）的Met或Leu表明，0.6 mM是两种AA刺激β-酪蛋白产生的最佳剂量。在此剂量下，Met和Leu在24小时后分别引起β-酪蛋白、MRCKα和固醇调节元件结合蛋白1（SREBP1）的蛋白水平升高（<0.05），并增加（<0.05）mTOR（Ser）和磷脂酰肌醇3激酶（PI3K）（Tyr）的磷酸化。通过沉默MRCKα表达（<0.05），两种AA对β-酪蛋白相对蛋白水平、mTOR磷酸化和蛋白激酶B（PKB）（Thr）磷酸化的刺激作用均被阻断。然而，对PI3K磷酸化的刺激作用保持不变（=0.385）。用LY294002抑制PI3K可阻断Met和Leu诱导的MRCKα和β-酪蛋白的蛋白表达以及mTOR的磷酸化（<0.05）。MRCKα的过表达增加了β-酪蛋白的蛋白水平和mTOR的磷酸化，这被PKB抑制剂MK2206阻断（<0.05）。我们的结果表明，MRCKα是Met和Leu诱导的信号级联反应的关键介质，在PI3K下游和PKB上游起作用，以调节BMEC中的β-酪蛋白合成。
