Inositol polyphosphates are not increased by overexpression of Ins(1,4,5)P3 3-kinase but show cell-cycle dependent changes in growth factor-stimulated fibroblasts

Abstract

NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.