Janus Kinase 2 (JAK2) Regulates Prolactin-Mediated Chloride Transport in Mouse Mammary Epithelial Cells through Tyrosine Phosphorylation of Na+-K+-2Cl- Cotransporter

Abstract Using a conditional knockout approach, we previously demonstrated that the Janus kinase 2 (Jak2) is crucial for prolactin (PRL) signaling and normal mammary gland development. PRL is suggested to synchronously activate multiple signaling cascades that emerge on the PRL receptor (PRLR). This study demonstrates that Jak2 is essential for the activation of the signal transducer and activator of transcription 5 (Stat5) and expression of Cish (cytokine-inducible SH2-containing protein), a Stat5-responsive negative regulator of Jak/Stat signaling. However, Jak2 is dispensable for the PRL-induced activation of c-Src, focal adhesion kinase, and the MAPK pathway. Despite activation of these kinases that are commonly associated with proliferative responses, the ablation of Jak2 reduces the multiplication of immortalized mammary epithelial cells (MECs). Our studies show that signaling through Jak2 controls not only the transcriptional activation of the Cyclin D1 gene, but, more importantly, it regulates the accumulation of the Cyclin D1 protein in the nucleus by altering the activity of signal transducers that mediate the phosphorylation and subsequent nuclear export of Cyclin D1. In particular, the levels of activated Akt (protein kinase B) and inactive glycogen synthase kinase-3β (i.e. a kinase that regulates the nuclear export and degradation of Cyclin D1) are reduced in MECs lacking Jak2. The proliferation of Jak2-deficient MECs can be rescued by expressing of a mutant form of Cyclin D1 that cannot be phosphorylated by glycogen synthase kinase-3β and therefore constitutively resides in the nucleus. Besides discriminating Jak2-dependent and Jak2-independent signaling events emerging from the PRLR, our observations provide a possible mechanism for phenotypic similarities between Cyclin D1 knockouts and females lacking individual members of the PRLR signaling cascade, in particular the PRLR, Jak2, and Stat5.

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Dipeptide (Methionyl-Methionine) Transport and Its Effect on β-Casein Synthesis in Bovine Mammary Epithelial Cells

Cellular Physiology and Biochemistry ◽

10.1159/000492987 ◽

2018 ◽

Vol 49 (2) ◽

pp. 479-488 ◽

Cited By ~ 9

Author(s):

Caihong Wang ◽

Fengqi Zhao ◽

Jianxin Liu ◽

Hongyun Liu

Keyword(s):

Epithelial Cells ◽

Cell Viability ◽

Transport Properties ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Janus Kinase ◽

Initiation Factor ◽

Peptide Transporter ◽

Small Interference Rna ◽

Bovine Mammary Epithelial Cells

Background/Aims: The aim of this study was to investigate the transport properties and utilization of methionyl-methionine dipeptide (Met-Met) in β-casein (β-CN) synthesis in bovine mammary epithelial cells (BMECs). Methods: The transport properties were studied for the effects of time, pH, concentration, temperature and inhibitors using Met-Met-FITC in BMECs. BMECs were treated with different concentrations of Met-Met (0, 20, 40, 80, 120 and 160 µg/ml). In several experiments, the cells were treated with Janus kinase 2 (JAK2) inhibitor (tyrphostin AG-490, 50 µM) and mammalian target of rapamycin (mTOR) inhibitor (rapamycin, 100 ng/ml). Results: The uptake of Met-Met-FITC by BMECs was rapid during the first fifteen minutes and became saturated after 15 minutes. The transport of Met-Met-FITC in BMECs exhibited a Michaelis constant of 52.4 µM and maximum transport velocity of 14.8 pmol/min/mg protein. The uptake of Met-Met-FITC in BMECs was pH-dependent, peaked at pH 6.5 and was significantly inhibited by other peptides, including Met-Lys, Lys-Lys, Gly-Met, Gly-Leu and Met-Leu. Knocking down the peptide transporter 2 (PepT2) with small interference RNA markedly decreased Met-Met-FITC uptake. Met-Met concentration-dependently increased the PepT2 expression and β-CN synthesis in BMECs with an optimal concentration of 80 µg/ml. At 80 µg/ml, Met-Met also enhanced the cell viability and cyclin D1 expression and promoted cell cycle transition from G1 phase to S phase. In addition, 80 µg/ml Met-Met increased the mRNA abundance of JAK2 and signal transducer and activator of transcription 5 (STAT5) and enhanced the phosphorylation of JAK2, STAT5, mTOR, p70 ribosomal S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1. The inhibition of JAK2 and mTOR significantly decreased Met-Met-induced increase in cell viability and β-CN synthesis in BMECs. Conclusion: Our data elucidated the properties of peptide transporter and its effect on β-CN synthesis in BMECs. Met-Met, taken up by PepT2, enhances cell proliferation and promotes β-CN synthesis by activating JAK2-STAT5 and mTOR signaling pathways in BMECs.

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IL-4 and IL-13 Promote Proliferation of Mammary Epithelial Cells through STAT6 and IRS-1

International Journal of Molecular Sciences ◽

10.3390/ijms222112008 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12008

Author(s):

Wan-Ju Wu ◽

Sue-Hong Wang ◽

Chun-Chi Wu ◽

Yi-An Su ◽

Chin-Yin Chiang ◽

...

Keyword(s):

Cell Proliferation ◽

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Prolonged Treatment ◽

Signaling Crosstalk ◽

Promote Cell Proliferation ◽

Primary Mouse ◽

Cytokine Milieu

T helper (Th)2 cytokines such as interleukin (IL)-4 and IL-13 control immune function by acting on leukocytes. They also regulate multiple responses in non-hematopoietic cells. During pregnancy, IL-4 and IL-13 facilitate alveologenesis of mammary glands. This particular morphogenesis generates alveoli from existing ducts and requires substantial cell proliferation. Using 3D cultures of primary mouse mammary epithelial cells, we demonstrate that IL-4 and IL-13 promote cell proliferation, leading to enlargement of mammary acini with partially filled lumens. The mitogenic effects of IL-4 and IL-13 are mediated by STAT6 as inhibition of STAT6 suppresses cell proliferation and improves lumen formation. In addition, IL-4 and IL-13 stimulate tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). Prolonged treatment with these cytokines leads to increased IRS-1 abundance, which, in turn, amplifies IL-4- and IL-13-stimulated IRS-1 tyrosine phosphorylation. Through signaling crosstalk between IL-4/IL-13 and insulin, a hormone routinely included in mammary cultures, IRS-1 tyrosine phosphorylation is further enhanced. Lowering IRS-1 expression reduces cell proliferation, suggesting that IRS-1 is involved in IL-4- and IL-13-stimulated cell proliferation. Thus, a Th2-dominant cytokine milieu during pregnancy confers mammary gland development by promoting cell proliferation.

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Cripto Enhances the Tyrosine Phosphorylation of Shc and Activates Mitogen-activated Protein Kinase (MAPK) in Mammary Epithelial Cells

Journal of Biological Chemistry ◽

10.1074/jbc.272.6.3330 ◽

1997 ◽

Vol 272 (6) ◽

pp. 3330-3335 ◽

Cited By ~ 70

Author(s):

Subha Kannan ◽

Marta De Santis ◽

Matthias Lohmeyer ◽

David J. Riese ◽

Gilbert H. Smith ◽

...

Keyword(s):

Protein Kinase ◽

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Mitogen Activated Protein Kinase ◽

Mitogen Activated Protein

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An Extensive Survey of Tyrosine Phosphorylation Revealing New Sites in Human Mammary Epithelial Cells

Journal of Proteome Research ◽

10.1021/pr900044c ◽

2009 ◽

Vol 8 (8) ◽

pp. 3852-3861 ◽

Cited By ~ 40

Author(s):

Tyler H. Heibeck ◽

Shi-Jian Ding ◽

Lee K. Opresko ◽

Rui Zhao ◽

Athena A. Schepmoes ◽

...

Keyword(s):

Epithelial Cells ◽

Tyrosine Phosphorylation ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Human Mammary Epithelial Cells ◽

Extensive Survey ◽

Human Mammary Epithelial

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Nuclear Jak2 and Transcription Factor NF1-C2: a Novel Mechanism of Prolactin Signaling in Mammary Epithelial Cells

Molecular and Cellular Biology ◽

10.1128/mcb.02095-05 ◽

2006 ◽

Vol 26 (15) ◽

pp. 5663-5674 ◽

Cited By ~ 41

Author(s):

Jeanette Nilsson ◽

Gunnar Bjursell ◽

Marie Kannius-Janson

Keyword(s):

Transcription Factor ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Alternative Pathway ◽

Prolactin Receptor ◽

Mammary Epithelial ◽

Janus Kinase ◽

Prolactin Signaling ◽

Nuclear Factor 1 ◽

Classical Mechanism

ABSTRACT The classical mechanism by which prolactin transduces its signal in mammary epithelial cells is by activation of cytosolic signal transducer and activator of transcription 5 (Stat5) via a plasma membrane-associated prolactin receptor-Janus kinase 2 (Jak2) complex. Here we describe an alternative pathway through which prolactin via Jak2 localized in the nucleus activates the transcription factor nuclear factor 1-C2 (NF1-C2). Previous reports have demonstrated a nuclear localization of Jak2, but the physiologic importance of nuclear Jak2 has not been clear. We demonstrate that nuclear Jak2 regulates the amount of active NF1-C2 through tyrosine phosphorylation and proteasomal degradation. Our data also demonstrate a link between prolactin and p53 as well as the milk gene carboxyl ester lipase through nuclear Jak2 and NF1-C2. Hence, we describe a novel pathway through which nuclear Jak2 is subject to regulation by prolactin in mammary epithelial cells.

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