Submitted on November 18, 2008
Revised on May 27, 2009
Accepted on May 29, 2009
Prolactin regulates ZnT2 expression through the JAK2/STAT5 signaling pathway in mammary cells
Linxi Qian1, Veronica Lopez2, Young Ah Seo1, and Shannon L Kelleher2*
1 Pennsylvania State University
2 The Pennsylvania State University
* To whom correspondence should be addressed. E-mail: slk39{at}psu.edu.
The zinc transporter ZnT2 (SLC30A2) plays an important role in zinc secretion into milk during lactation. The physiological process of mammary gland secretion is regulated through complex integration of multiple lactogenic hormones. Prolactin plays a primary role in this regulation through the activation of various signaling cascades including Jak2/STAT5, MAPK, p38 and PI3K. The precise mechanisms which regulate the transfer of specific nutrients such as zinc into milk are not well understood. Herein we report that prolactin increased ZnT2 abundance transcriptionally in cultured mammary epithelial (HC11) cells. To delineate the responsible mechanisms, we first determined that prolactin-mediated ZnT2 induction was inhibited by pre-treatment with Jak2 inhibitor AG490, but not by the MAPK inhibitor PD98059. Using a luciferase reporter assay, we demonstrated that ZnT2 promoter activity was increased by prolactin treatment which was subsequently abolished by expression of a dominant-negative STAT5 construct, implicating the Jak2/STAT5 signaling pathway in the transcriptional regulation of ZnT2. Two putative consensus STAT5 binding sequences in the ZnT2 promoter were identified (GAS1:-674 to -665 and GAS2:-377 to -368). Mutagenesis of the proximal GAS2 element resulted in complete abrogation of PRL-induced ZnT2 promoter activity. The promoter incorporating the distal GAS1 mutation was only able to respond to very high PRL concentrations. Results from both the mutagenesis and gel shift assays indicated that a co-operative relationship exists between GAS1 and GAS2 for PRL-induced activation; however, the proximal GAS2 plays a more critical role in STAT5-bound signal transduction as compared to the GAS1 element. Finally, chromosome immunoprecipition assay further confirmed that prolactin activates STAT5 binding to the ZnT2 promoter in vivo. Taken together, these results illustrate that prolactin regulates the transcription of ZnT2 through activation of the Jak2/STAT5 signaling pathway to provide optimal zinc for secretion into milk during lactation.
