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

Abstract

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 192h 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 24h 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 24h postmilking, no change in STAT3 levels, and an increase in STAT3-P protein (barely detectable at the early time points) by 72h postmilking. Quantitative real-time reverse transcription PCR analysis showed SOCS1 and SOCS3 mRNA increased by 72h postmilking compared with 6h postmilking. The SOCS2 mRNA remained unchanged across the time series, whereas CIS decreased by 18h postmilking and remained lower compared with that at 6h postmilking until 72h postmilking. The IGF1 mRNA increased by 192h postmilking, whereas IGF2 mRNA decreased by 18h postmilking compared with 6h 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 192h postmilking in dairy cows, the involution process is still in the reversible phase, with quiescent mammary epithelial cells not yet in the senescent phase.

MicroRNA roles in signalling during lactation: an insight from differential expression, time course and pathway analyses of deep sequence data

“…Meanwhile the most significant specific DE miRNAs for GAL (miR-EIA23-25909) and INV (miR-2284j) stages have not been functionally validated in dairy mammary gland. Enrichments for target genes of stage specific DE miRNAs (Figure S3) showed importance of PTEN signalling pathway for LAC48, prolactin signalling pathway for GAL and INV64 and STAT3 pathway for INV stages65.…”

RNA-Seq reveals novel genes and pathways involved in bovine mammary involution during the dry period and under environmental heat stress

“…In our study, not IGF-1 but IGF1-R , IGFBP2 and IGFBP4 were upregulated in the mammary gland at D3 of involution compared with late lactation. Abruptly drying-off non-pregnant dairy cows at peak lactation increased apoptosis of the mammary gland (D3 to D8 after milk stasis), indicated by increased STAT3 and SOCS3 protein levels and decreased STAT5 gene expression 12 . However, IGF1 expression increased and IGFBP5, Akt and Akt-P protein concentrations did not change 12 .…”

“…Key candidate genes have been well characterized in rodent models. In dairy cattle, limited studies have utilized microarrays and qRT-PCR to evaluate the molecular events occurring in the mammary gland during a typical dry period of pregnant cows 5 , during forced involution of non-pregnant cows at peak lactation 10 , 12 , and during gradual involution of non-pregnant cows at peak lactation 11 . These studies report an overall upregulation of genes related to cell turnover, oxidative stress, tissue remodeling, and inflammation and downregulation of cell survival signaling and biosynthesis of milk constituents during involution and upregulation of cellular proliferation later during redevelopment.…”

The Multifaceted Role of STAT3 in Mammary Gland Involution and Breast Cancer

“…Bovine mammary involution is considered to encompass both a phase of cell death, but also an important period of epithelial cell renewal prior to the next lactation [ 55 , 56 , 57 ]. Interestingly, when involution is experimentally induced by cessation of milking in non-pregnant dairy cows that are close to the point of peak lactation in mid-lactation, increased phosphorylation of STAT3 is observed by 72 h [ 58 , 59 ]. As would be anticipated, the dynamics of the involution process in cows are strikingly different from that of rodents, and, following abrupt cessation of milking at mid lactation, milking can be reinitiated after seven days of non-milking, with a 91% milk yield recovery and milk composition similar to that observed prior to cessation of lactation [ 60 ].…”