Janus kinase 2 (JAK2) regulates prolactin-mediated chloride transport in mouse mammary epithelial cells through tyrosine phosphorylation of Na+-K+-2Cl- cotransporter.
Epithelial chloride (Cl-) transport is achieved by the coordinated action of symporters such as the Na+-K+-2Cl- cotransporter (NKCC1) and chloride channels such as the cystic fibrosis transmembrane conductance regulator (CFTR). As a secretory tissue, mammary epithelial cells are obvious candidates for such mechanisms, but Cl- transport and its hormonal regulation have been poorly delineated in mammary epithelial cells. We determined whether the mammary epithelial cell line, HC11, transports chloride and whether this was regulated by PRL, a hormone known to stimulate ion transport. HC11 cells express both CFTR and NKCC1. Exposure to PRL or PGE1 increased Cl- transport in HC11 cells. This was inhibited by the NKCC1 blocker, furosemide, and by the Cl- channel inhibitor, diphenylamine 2-carboxylate. Dose and time course of PRL action indicate that PRL had maximal effect on Cl- transport at 1 microg/ml and at 10 min of stimulation. Examination of the signaling pathways suggests that the PRL effect on Cl- transport does not involve an increase in [Ca2+]i or MAP kinase activity. RT-PCR analyses indicate that HC11 cells express mRNA for Janus kinase 1 (JAK1), JAK2, and signal transducer and activator of transcription 5 (STAT5) but not for JAK3. PRL treatment of HC11 cells increased phosphorylation of STAT5. The JAK2 inhibitor AG490 blocked phosphorylation of STAT5 and PRL-induced, but not PGE1-induced, Cl- transport. NKCC1, but not CFTR, is tyrosine phosphorylated in HC11 cells. PRL enhanced tyrosine phosphorylation of NKCC1, and this effect was attenuated by the JAK2 inhibitor AG490. These results are the first demonstrations of a role for tyrosine phosphorylation of NKCC1 and of the PRL-JAK2 cascade in the regulation of Cl- transport.
Selvaraj
N G
NG
Department of Physiology and Biophysics, University of Illinois at Chicago, 60612, USA.
Omi
E
E
Gibori
G
G
Rao
M C
MC
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D053614:Janus Kinase 2; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011388:Prolactin / Q000008:administration & dosage / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000032:analysis; D011981:Receptors, Prolactin / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D028021:Sodium-Potassium-Chloride Symporters; D015534:Trans-Activators / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D002352:Carrier Proteins; D002712:Chlorides; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011981:Receptors, Prolactin; D050799:STAT5 Transcription Factor; D028021:Sodium-Potassium-Chloride Symporters; D015534:Trans-Activators; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019000:Phosphotyrosine; D011388:Prolactin; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2