Regulation of inositol biosynthesis and consequences of inositol depletion

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Inositol is essential for the viability of eukaryotic cells. Myo-inositol is the precursor of all inositol compounds, which play pivotal roles in cell signaling and metabolism. Consistent with its importance, perturbation of inositol homeostasis is associated with pathologies as diverse as neurological and psychiatric illnesses, myopathies, cancer, and diabetes. Furthermore, inositol depletion is a hypothesized therapeutic mechanism of action of drugs used to treat bipolar disorder, a devastating psychiatric illness that affects ~2% of the population. In this light, it is striking that very little is known about the regulation of inositol homeostasis in human cells. Our research seeks to determine how inositol synthesis is regulated and how inositol deprivation affects essential cellular functions in human cells. Toward this end, we identified the first negative transcriptional regulator of inositol synthesis in mammalian cells – inositol hexakisphosphate kinase 1 (IP6K1). IP6K1 represses expression of ISYNA1, the gene coding for the rate-limiting enzyme of inositol synthesis, myo-inositol-3-P synthase (MIPS). Further, we demonstrated that binding of IP6K1 to phosphatidic acid (PA) is required for nuclear localization of IP6K1 and repression of MIPS expression. Our first project will rigorously dissect the mechanism of regulation of ISYNA1 by IP6K1. To elucidate the consequences of inositol deprivation, we constructed an ISYNA1 knock-out (ISYNA1-KO) human cell line, which cannot synthesize inositol. Inositol-deprived ISYNA1- KO cells exhibit profound alteration of lipid homeostasis and expression of genes involved in stress signaling. Specifically, we observed an increase in ceramides and increased expression of genes that mediate the unfolded protein response (UPR), a stress response that is activated by ceramides. Our second project will test the hypothesis that inositol deprivation results in activation of the UPR pathway by upregulating ceramides. Intriguingly, inositol-deprived cells exhibited increased levels of monolysocardiolipin (MLCL), which is the biochemical hallmark of the mitochondrial disorder Barth syndrome. Consistent with mitochondrial dysfunction, we observed increased activation of ERK, a promoter of mitochondrial fission, in inositol-starved cells. The third focus of our studies will test the working hypothesis that inositol deprivation perturbs mitochondrial function. Successful completion of the proposed studies will lead to the first molecular model of regulation of inositol synthesis in mammalian cells and demonstrate how inositol deprivation affects the cellular stress response and mitochondrial function. This knowledge will have important implications for understanding inositol homeostasis as well as the therapeutic mechanisms of inositol-depleting mood stabilizing drugs.