Prolactin regulates ZnT 2 expression through the JAK 2 / STAT 5 1 signaling pathway in mammary cells

28 The zinc transporter ZnT2 (SLC30A2) plays an important role in zinc secretion into milk during 29 lactation. The physiological process of mammary gland secretion is regulated through complex 30 integration of multiple lactogenic hormones. Prolactin plays a primary role in this regulation 31 through the activation of various signaling cascades including Jak2/STAT5, MAPK, p38 and 32 PI3K. The precise mechanisms which regulate the transfer of specific nutrients such as zinc into 33 milk are not well understood. Herein we report that prolactin increased ZnT2 abundance 34 transcriptionally in cultured mammary epithelial (HC11) cells. To delineate the responsible 35 mechanisms, we first determined that prolactin-mediated ZnT2 induction was inhibited by pre36 treatment with Jak2 inhibitor AG490, but not by the MAPK inhibitor PD98059. Using a 37 luciferase reporter assay, we demonstrated that ZnT2 promoter activity was increased by 38 prolactin treatment which was subsequently abolished by expression of a dominant-negative 39 STAT5 construct, implicating the Jak2/STAT5 signaling pathway in the transcriptional regulation 40 of ZnT2. Two putative consensus STAT5 binding sequences in the ZnT2 promoter were 41 identified (GAS1:-674 to -665 and GAS2:-377 to -368). Mutagenesis of the proximal GAS2 42 element resulted in complete abrogation of PRL-induced ZnT2 promoter activity. The promoter 43 incorporating the distal GAS1 mutation was only able to respond to very high PRL concentrations. 44 Results from both the mutagenesis and gel shift assays indicated that a co-operative relationship 45 exists between GAS1 and GAS2 for PRL-induced activation; however, the proximal GAS2 plays 46 a more critical role in STAT5-bound signal transduction as compared to the GAS1 element. 47 Finally, chromosome immunoprecipition assay further confirmed that prolactin activates STAT5 48 binding to the ZnT2 promoter in vivo. Taken together, these results illustrate that prolactin 49 regulates the transcription of ZnT2 through activation of the Jak2/STAT5 signaling pathway to 50 provide optimal zinc for secretion into milk during lactation. 51