RESTORATION OF RESPONSIVENESS TO PHORBOL ESTER BY RECONSTITUTION OF AFUNCTIONAL NA K/CL COTRANSPORTER IN COTRANSPORTER-DEFICIENT BALB/C 3T3 CELLS/

Previous studies in this laboratory have implicated the membrane trans port protein Na/K/Cl cotransporter (NKCC1) as an important component o f the signaling pathways activated by phorbol esters in BALB/c 3T3 cel ls. The NKCC1 protein functions as a Na/K/Cl cotransporter in BALB/c 3 T3 cells and many other cell types. Loss of NKCC1 function has been as sociated with loss of mitogenic responsiveness to phorbol ester. Here we report that expression of a cloned NKCC1 cDNA fused to a tetracycli ne-regulated promoter in BALB/c 3T3 cells deficient in Na/K/Cl cotrans port activity (clone E12a cells) restored cotransport function. Compar ed with parental cotransport-deficient cells, transfected clones expre ssing the exogenous NKCC1 gene responded like typical BALB/c 3T3 cells to 12-O-tetradecanoyl phorbol-13-acetate: loop diuretic-sensitive Rb- 86(+) flux was inhibited, cell volume was decreased, and cell growth w as stimulated. These results support our previous conclusion that the loss of responsiveness of E12a cells to phorbol ester is caused by mut ation of the endogenous NKCC1 gene. (C) 1996 Wiley-Liss, Inc.