Restoration of responsiveness to phorbol ester by reconstitution of a functional Na/K/Cl cotransporter in cotransporter-deficient BALB/c 3T3 cells.
Previous studies in this laboratory have implicated the membrane transport protein Na/K/Cl cotransporter (NKCC1) as an important component of the signaling pathways activated by phorbol esters in BALB/c 3T3 cells. The NKCC1 protein functions as a Na/K/Cl cotransporter in BALB/c 3T3 cells and many other cell types. Loss of NKCC1 function has been associated with loss of mitogenic responsiveness to phorbol ester. Here we report that expression of a cloned NKCC1 cDNA fused to a tetracycline-regulated promoter in BALB/c 3T3 cells deficient in Na/K/Cl cotransport activity (clone E12a cells) restored cotransport function. Compared with parental cotransport-deficient cells, transfected clones expressing the exogenous NKCC1 gene responded like typical BALB/c 3T3 cells to 12-O-tetradecanoylphorbol-13-acetate: loop diuretic-sensitive 86Rb+ flux was inhibited, cell volume was decreased, and cell growth was stimulated. These results support our previous conclusion that the loss of responsiveness of E12a cells to phorbol ester is caused by mutation of the endogenous NKCC1 gene.
Guo
Y
Y
Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096, USA.
O'Brien
T G
TG
D016475:3T3 Cells; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D048429:Cell Size / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis / Q000187:drug effects*; D011493:Protein Kinase C / Q000502:physiology*; D011994:Recombinant Proteins; D012413:Rubidium; D012754:Sharks; D015398:Signal Transduction; D028021:Sodium-Potassium-Chloride Symporters; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D011994:Recombinant Proteins; D028021:Sodium-Potassium-Chloride Symporters; D011493:Protein Kinase C; D012413:Rubidium; D013755:Tetradecanoylphorbol Acetate