Cell survival signaling in the bovine mammary gland during the transition from lactation to involution

In dairy cows, mammary gland involution, and thus a decline in milk production, occurs following peak lactation. To examine the cell signaling pathways regulating involution of the mammary gland, signal transducer and activator of transcription factors (STAT5 and 3), suppressors of cytokine signaling (SOCS1-3 and CIS), insulin-like growth factors (IGF1 and 2), and protein kinase B (Akt) were examined. Mammary involution was induced by termination of milking, and alveolar tissue was collected from 52 nonpregnant, primiparous, mid-lactation Holstein-Friesian cows killed at 0, 6, 12, 18, 24, 36, 72, and 192 h postmilking. Qualitative immunohistochemistry showed that activated (phosphorylated) STAT5-P was localized in nuclei of mammary epithelial cells at the early time points, with detection levels decreasing by 24 h postmilking. In contrast, STAT3-P was barely detectable at the early time points, with detection levels increasing following longer postmilking periods. This was supported by Western analysis, which showed a decline in STAT5 and STAT5-P protein levels by 24 h postmilking, no change in STAT3 levels, and an increase in STAT3-P protein (barely detectable at the early time points) by 72 h postmilking. Quantitative real-time reverse transcription PCR analysis showed SOCS1 and SOCS3 mRNA increased by 72 h postmilking compared with 6 h postmilking. The SOCS2 mRNA remained unchanged across the time series, whereas CIS decreased by 18 h postmilking and remained lower compared with that at 6 h postmilking until 72 h postmilking. The IGF1 mRNA increased by 192 h postmilking, whereas IGF2 mRNA decreased by 18 h postmilking compared with 6 h postmilking. The IGFBP5 mRNA and protein levels of Akt and Akt-P remained unchanged over the time series. These results show that reciprocal activation of STAT5 and STAT3 occurs at the onset of mammary gland involution in the bovine, albeit at a slower rate than in rodents. Mathematical modeling of the pathways indicated that activated STAT3 could block the STAT5 pathway by upregulating SOCS3. The regulation of IGF1-Akt signaling suggests that by 192 h postmilking in dairy cows, the involution process is still in the reversible phase, with quiescent mammary epithelial cells not yet in the senescent phase.