Physiological levels of Stat5 DNA binding activity and protein in bovine mammary gland

Signal transducer and activator of transcription (Stat)5 has been implicate d in the signal transduction pathways of several factors that are lactogeni c or galactopoeitic in mammary cells, including prolactin, GH, and IGF-I. D ata from cell or explant culture support the concept that Stat5 may represe nt part of a common route by which different extracellular signals converge and are transduced into the cell. There are few data on Stat5 activity and level in vivo, and we set out to determine whether physiological stimuli o f milk synthesis, including GH, GH-releasing factor, and milking frequency, would be associated with alterations in Stat5 activity or protein. We meas ured Stat5 DNA binding activity using electrophoretic mobility shift assay and Stat5 protein by Western blot in bovine mammary tissue obtained by biop sy or slaughter. Stat5 activity was absent in nonlactating, nonpregnant cow s and was present in late pregnancy and throughout lactation. Stat5 activit y varied considerably among cows at similar stages of lactation. Mammary St at5 activity and protein were determined in hormone-treated lactating cows and mammary quarters of cows milked at different frequencies. Infusion of G H and GH-releasing factor for 2 mo significantly raised levels of milk prod uction and depressed mammary Stat5 activity without influencing Stat5 prote in abundance. Mammary Stat5 was also influenced by milking frequency; once- daily milking reduced milk production, Stat5 activity, and protein abundanc e compared with twice-daily milking. Analysis of mammary Stat5 in relation to milk protein concentration in pooled data from lactating cows indicated that Stat5 activity was correlated (r = 0.505, P < 0.05) with average milk protein concentration and not related to milk protein yield (P > 0.05). The se results show that both Stat5 protein and Stat5 activity are modulated by different physiological signals in vivo and suggest that Stat5 lies within in the zone where signal transduction cascades from a variety of factors a re convergent. Further work is required to clarify the role of Stat5 in rel ation to other factors in regulation of milk protein gene expression.