Role of inositol polyphosphates in programmed cell death.

The role of inositol polyphosphates (InsPs) in the mediation of cellular apoptosis was investigated in mouse MC3T3 osteoblastic cell line. Extracellular administration of InsP(4), InsP(5), and InsP(6) increased apoptosis in a dose-dependent manner. InsP(6) was more potent than InsP(5) and InsP(4) in promoting apoptosis. Inositol hexasulfate (InsS(6)), a structural analog of InsP(6), was used to determine specificity of InsP(6)-induced apoptosis as measured by acridine orange/ethidium bromide, flow cytometry, and DNA degradation. In order to study the effects of endogenous InsPs on apoptosis, we used NaF and antimycin A as treatment agents to manipulate intracellular levels of InsPs. NaF is known to increase levels of higher InsPs by inhibiting InsPs phosphatases, a process that is reversed by antimycin A because InsPs kinases are inhibited as a result of depletion of cellular ATP pools. Apoptosis was induced in MC3T3 cells in a NaF dose- and time-dependent manner. Approximately 50% apoptosis was observed at 1 mM NaF in 8 h. Prior treatment with 10 microM antimycin A for 30 min significantly reduced the NaF-induced apoptosis as compared with its control. Additionally, we measured changes in AKT phosphorylation, cleavage of caspase-3 and caspase-9, and release of cytochrome C from mitochondria into cytosol. These changes coincided with total cellular InsPs under similar conditions. The data indicated that NaF-induced changes in apoptotic markers could be due to an increased endogenous InsPs that were partially reversed by antimycin A treatment.