Signal Transducer And Activator Of Transcription5a Regulates Goat Mammary Gland Epithelial Cells Apoptosis

The signal transducer and activator of transcription5a (Stat5a) regulates genesinvolved in proliferation and survival and plays a pivotal role in the regulation ofmammary gland function during pregnancy, lactation, and involution. However, thereis little information about the effects of Stat5a in goat mammary gland epithelial cells(GMECs) apoptosis. Parathyroid hormone-related protein (PTHrP) is a key regulatorin cellular calcium transport, mammary gland development and breast tumor biology.Although both Stat5a and PTHrP are known to participate in breast development, cellproliferation and apoptosis of the mammary gland epithelial cells, the interactionsbetween them in the apoptosis of GMECs are still unknown. In the present study, weshow for the first time that Stat5a suppresses PTHrP expression in GMECs andPTHrP protects cells from apoptosis. We propose that Stat5inhibition may trigger cellapoptosis by regulating the expression of PTHrP.(1) The cDNA of goat Stat5a (including full length coding sequence) was3165bp, which included2385bp of open reading frame (ORF) encoding a polypeptide of794amino acids with a putative molecular weight of90693Da, and an isoelectricpoint (pI) of5.92. The phylogenetic relationship analysis revealed that the deducedamino acid sequence of goat Stat5a has the highest degree of sequence similarity withsheep, while there is the furthest distance from Danio rerio. It showed that Stat5a isexpressed in a wide range of tissues, with the highest mRNA level observed inmammary gland (141.08-fold) and the lowest levels were detected in kidney (thecalibrator).(2) The GMECs treated with the Stat5inhibitor pimozide for18h, thenQRT-PCR revealed a decrease in4key Stat5a target genes expression, includingpro-growth and anti-apoptotic genes Bcl-xl, CyclinD1, Pim1and Mcl1. We measuredPTHrP mRNA levels in GMECs treated with pimozide for18h, and we found pimozide led to a dose-dependent increase of PTHrP levels. In GMECs, pimozidedecreased Bcl-2expression and cell proliferation, and induced apoptotic, and thisdownregulation was promoted by PTHrP overexpression and simultaneously inhibitedby siPTHrP.(3) We measured PTHrP mRNA levels in GMECs treated with CaCl2and wefound CaCl2led to a dose-dependent increase of PTHrP levels. In GMECs, CaCl2decreased Bcl-2expression and cell proliferation, and induced apoptotic, and thisdownregulation was promoted by PTHrP overexpression and simultaneously inhibitedby siPTHrP.(4) Pimozide significantly increased the expression of MMP-9and GMECstreated with PRL represented opposite results. Moreover, the MMP-9levels showed asimilar variation trend in GMECs treated with4mM CaCl2. We further examinedwhether PTHrP could regulate the expression of MMP-9in GMECs. We found PTHrPoverexpression was associated with an increase in the activity of MMP-9and siPTHrPinhibited MMP-9mRNA level in GMECs.