Intracellular signaling of phosphorylated insitol compounds : A study in pancreatic beta-cells and hippocampal neurons

Sammanfattning: The family of phosphorylated inositol compounds consists of soluble cytosolic inositol phosphates and insoluble inositol phospholipids, localized in cellular membranes and the nuclear matrix. Although inositol phospholipids only account for 5-10% of total plasma membrane lipids, these compounds and their inositol phosphate derivatives play an important role in a broad range of cellular processes including intracellular Ca 2+ signalling, ion channel regulation, vesicle trafficking, both exo- and endocytosis, cell growth and apoptosis. Stimulation of plasma membrane receptors with neurotransmitters, hormones, and growth factors evokes the hydrolysis of inositol phospholipids, principally phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2) and hence generation of a series of inositol polyphosphates. Among them, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), as a Ca2+ mobilizing second messenger, is the best-characterized. However, much less is known about the biochemical pathways and intracellular signaling of other inositol polyphosphates in mammalian cells. One important finding has been novel roles for inositol hexakisphosphate (InsP6) in pancreatic betacells, particularly in the regulation of voltage-gated Ltype Ca channels, exo- and endocytosis. However, the influence of InsP6 on L-type Ca2+ channels is not limited to beta-cells. In this thesis it was shown that in hippocampal neurons, InsP6 also increases L-type Ca 2+ channel activity by activation of the adenylyl cyclase (Apolipoprotein kinase A (PKA) cascade. InsP6 stimulates AC without influencing cAMP phosphodiesterase, resulting in activation of PKA and thereby selective enhancement of L-type Ca2+ channel activity. These data suggest that InsP6 increases L-type Ca 2+ channel activity by facilitation of the phosphorylation of PKA phosphorylation sites. Furthermore, InsP6 Concentration is elevated in activated hippocampal neurons, suggesting a relevant physiological role. We have also shown that higher inositol polyphosphates can act as a source for important second messengers, independent of the hydrolysis of Ptdlns(4,5)P2. Thus, Ins(1,4,5)P3 can be derived from dephosphorylation of Ins(1,3,4,5,6)P5 and InsP6 by cytosolic multiple inositol polyphosphate phosphatase (cyt-MIPP), a truncated cytosolic form of the largely ER-confined MIPP. Cyt-MIPP was expressed in a poorly-responsive beta-cell line (HIT M2.2.2) with an abnormally low basal [Ca2+]i. The low basal [Ca2+]i of these cells was raised to normal levels in cyt-MIPP expressing HIT M2.2.2 cells (35 nM to 115 nM). Cyt-MIPP expression also led to a markedly enhanced glucoseinduced C2+-response, indicating that basal [Ca2+]i is an important modulator of Ca2+signalling in the pancreatic beta-cell. Expression of cyt-MIPP in HIT M2.2.2 cells also led to a significant decrease in cell growth and increased cell volume. In vitro and in intact cells, cyt-MIPP attacks not only its known inositol polyphosphate substrates but also phosphatidylinositol 3,4,5-trisphosphate (PtdIns (3,4,5)P3), a mitogenic signal. Thus cyt-MIPP shares some properties with PTEN, a tumor suppressor and PtdIns(3,4,5)P3 phosphatase, suggesting that the reduced PtdIns(3,4,5)P3 concentration contributes to growth-inhibition by cyt-MIPP. In contrast, full length MIPP increases cell growth, suggesting divergent phenotypes dependent on the cellular localization of this enzyme. A detailed study of Ptdlns(3,4,5)P3 and other PI 3-kinase products in normal HIT T15 cells revealed that 1. the high level of PtdIns(3,4,5)P3 in beta-cells in a previous study was an artifact, 2. phosphatidyl-inositol 3,5-bisphosphate (Ptdlns(3,5)P2) is present in beta-cells and together with Ptdlns(3,4,5)P3 and Ptdlns(3,4)P2 increase in response to glucose stimulation alone, 3. the glucosestimulated rise in 3 -phosphorylated lipids was secondary to insulin secretion. Our data suggest that the effect of glucose on 3 -phosphorylated inositol lipids is through an insulindependent positive feedback loop. This study demonstrates important new intracellular signaling roles for phosphorylated inositol compounds in the regulation of both neurons and pancreatic beta-cells.