Prolactin regulates ZNT2 expression through the JAK2/STAT5 signaling pathway in mammary cells

Linxi Qian, Veronica Lopez, Young Ah Seo, Shannon Kelleher

Research output: Contribution to journalArticle

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Abstract

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, mitogen-activated protein kinase (MAPK), p38, and phosphatidylinositol 3-kinase (PI3K). The precise mechanisms that 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 pretreatment with the Jak2 inhibitor AG490 but not by the MAPK inhibitor PD-98059. 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 cooperative relationship exists between GAS1 and GAS2 for PRL-induced activation; however, the proximal GAS2 plays a more critical role in STAT5-mediated signal transduction compared with 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 assist in providing optimal zinc for secretion into milk during lactation.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume297
Issue number2
DOIs
StatePublished - Aug 1 2009

Fingerprint

Prolactin
Breast
Zinc
Milk
Lactation
Mutagenesis
Phosphatidylinositol 3-Kinase
Physiological Phenomena
p38 Mitogen-Activated Protein Kinases
Human Mammary Glands
Protein Kinase Inhibitors
Mitogen-Activated Protein Kinases
Luciferases
Signal Transduction
Chromosomes
Gels
Epithelial Cells
Hormones
Food
Mutation

All Science Journal Classification (ASJC) codes

  • Physiology
  • Cell Biology

Cite this

Qian, Linxi ; Lopez, Veronica ; Seo, Young Ah ; Kelleher, Shannon. / Prolactin regulates ZNT2 expression through the JAK2/STAT5 signaling pathway in mammary cells. In: American Journal of Physiology - Cell Physiology. 2009 ; Vol. 297, No. 2.
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abstract = "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, mitogen-activated protein kinase (MAPK), p38, and phosphatidylinositol 3-kinase (PI3K). The precise mechanisms that 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 pretreatment with the Jak2 inhibitor AG490 but not by the MAPK inhibitor PD-98059. 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 cooperative relationship exists between GAS1 and GAS2 for PRL-induced activation; however, the proximal GAS2 plays a more critical role in STAT5-mediated signal transduction compared with 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 assist in providing optimal zinc for secretion into milk during lactation.",
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Prolactin regulates ZNT2 expression through the JAK2/STAT5 signaling pathway in mammary cells. / Qian, Linxi; Lopez, Veronica; Seo, Young Ah; Kelleher, Shannon.

In: American Journal of Physiology - Cell Physiology, Vol. 297, No. 2, 01.08.2009.

Research output: Contribution to journalArticle

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