Multi-Level Regulation of Milk Protein Expression in the Mammary Gland

Milk is often refereed to as "nearly the perfect food" and protein is the most valuable gross component in milk.  Regulation of milk protein synthesis has been associated with anatomical and spatial adaptation of the mammary gland to the reproductive cycle and is performed at several levels.

The signal transducer and activator of transcription, Stat5, transduces extracellular cytokine and growth factor signals to the nucleus of mammary epithelial cells and thereby regulates gene transcription during pregnancy, lactation and weaning. Expression levels and states of activity of Stat5 in the mammary gland tissue were manipulated by introducing Stat5 variants as transgenes into the pronuclei of transgenic mice.  Overexpression and forced activation of Stat5 induced mammary gland development during pregnancy and delayed apoptosis-associated involution. β-casein synthesis and secretion into milk during lactation were enhanced and pup growth was accelerated. Delayed mammary glad maturation and induced apoptosis were associated with expression of Stat5 variant lacking its C-terminally transactivation domain. Interestingly, transgenic females over 8 months old, which expressed all Stat5 transgenic varients, were predisposed to tumor formation in the mammary gland. Undifferentiated carcinomas most frequently occurred in mice expressing the C-terminally truncated Stat5. The more differentiated papillary and micropapillary adenocarcinomas were primarily found in mice overexpressing the native and constitutively active transgenes.

Whereas caseins are the major milk proteins, β-lactoglobulin (BLG) could be considered the most characteristic of ruminants, due to its absence from human and murine milk, and its highest level in the ruminant whey.  We have defined a regulatory element, termed BLG-e1, in the distal region of the ovine BLG gene promoter. This 299-bp element lacks the established cis-regulatory sequences that affect milk-protein gene expression. Nevertheless, it alters the binding of downstream BLG sequences to histone H4 and the sensitivity of the histone-DNA complexes to trichostatin A treatment.  In mammary cells cultured under favorable lactogenic conditions, BLG-e1 acts as a potent, position-independent silencer of BLG/luciferase expression, and similarly affects the promoter activity of the mouse whey acidic protein (WAP) gene. Intragenic sequences upstream of BLG exon 2 reverse the silencing effect of BLG-e1 in vitro and in transgenic mice.

While transcriptin regulation of milk protein synthesis has been extensively studied, post-transcriptional regulation has been relatively uninvestigated.  In cultured mouse mammary CID-9 cells, we have studied the synergistic effect of insulin and prolactin on β-casein synthesis and showed that when added together, these hormones increase β-casein mRNA translation, reflected by a shift to larger polysomes. The poly(A) tract of the β-casein mRNA progressively increased with the addition of insulin and prolactin, indicating regulation of of cytoplasmatic poly(A) polymerase activity.

The notion that post trascriptional mechanism govern milk protein expression and accumulation in milk has been suported by our studies on the role of amino acids (AA) on milk protein expression. We have compared bovine and murine mammary epithelial cells and showed that the bovine cells are more sucestible to AA, perticularly leucine (Leu) manipulation. The sharper decrease in BLG synthesis in AA or Leu - deprived bovine cells has been associated with a more marked decrease in the phosphorylation of the translation factors 4E-BP1 and S6K1. This probably reflects a faster and more pronounced operation of the signaling aparatus in the bovine cells and a more rapid shutdown of translation in the face of AA insufficiency. Our finding that Leu sepcifically induced BLG translation and that rapamycin, an mTOR inhibitor, specifically reduced Leu and AA - stimulated translation intiation in a dose dependent manner suggest that direct signaling from AA to the translational machinary is involved in determining the rates of milk protein synthesis in mammary epithelial cells.