RELEASING THE SECOND MESSENGERS:
© 2002 Garland Science/Taylor & Francis Books
In this common pathway, activated phospholipase C-β hydrolyzes the inositide PI 4,5-bisphosphate to release diacylglyerol and inositol 1,4,5 trisphosphate. IP3 opens specific Ca2+ channels releasing the ions from the lumen of the endoplasmic reticulum. Diacylglycerol can be further cleaved to release arachidonic acid, a signaling molecule needed for the synthesis of other messengers such as prosta-glandins, or it can activate protein kinase C, a calcium-dependent kinase. (Adapted from B. Alberts et al.
Inside and out, cells teem with signaling activity. And inositides – particularly inositol phosphates and inositol lipids – are everywhere, mediating everything from ion-channel function to vesicle trafficking, apoptosis, cell growth, motility, and differentiation.1 Almost every cell uses one of the phosphoinositide signaling cascades: Receptors trigger the hydrolysis of the lipid phosphatidylinositol 4,5-bisphosphate to yield the second messengers, inositol...
QUALITY OVER QUANTITY
Jordi Folch isolated the first inositide, diphospho-inositide, in 1949. Four years later, Lowell and Mabel Hokin demonstrated that cholinergic agonists increased radio-labeled phosphate uptake into phosphatidylinositols.134 Over the years, the number of inositol phosphates and inositol lipids expanded dramatically, yet few have well-defined roles.
"Manuscripts frequently open with the authors' estimate of how many inositol phosphates there are, followed by an expression of dismay at how few have known biological functions," says Steve Shears at the National Institute of Environmental Health Sciences. "This may not be appropriate." Among other reasons, many enzymes that metabolize inositol phosphates are promiscuous, limiting their ability to regulate levels of a particular inositide.
Shears suggests that relatively few inositol phosphates may elicit biological responses. In 1983, Hanspeter Streb reported that IP3 mobilized calcium from the endoplasmic reticulum, demonstrating its role as a second messenger.4 Eleven years later, Shears' group showed that inositol 3,4,5,6-tetrakisphosphate (IP4) inhibits chloride channels.5 More recently, researchers have shown growing interest in the roles played by IP7 and IP8. Shears argues that other inositol phosphates might represent metabolic intermediates and inactive precursor pools. The number of "truly functional" inositol phosphates, he says, "is a major issue that still needs resolving."
Inositol's functional diversity raises another issue: how cells maintain signaling specificity. The multiplicity of cytoskeletal-protein motifs that recognize specific inositol lipids may hold part of the answer. Tamas Balla, of the National Institute of Child Health and Human Development, says this multiplicity challenges the view that inositol lipids diffuse away from their production site to exert their effects. Instead, inositol lipids may modulate protein-protein and protein-lipid interactions at the membrane-cytoplasm interface. In particular, inositol lipids may regulate the transition between active and inactive conformations.
At Bristol University, UK, Peter Cullen formulates his "working hypothesis" in terms of phosphoinositides' ability to function as membrane scaffolds regulating the spatial and temporal function of protein-signaling complexes. "These complexes are extremely dynamic," he says. "They form and dissociate based on the presence or absence of their required phosphoinositides."
Phosphorylation contributes to the inositides' ability to maintain signaling specificity. Bulky phosphate groups impose geometric constraints on ligand-protein interactions. Moreover, phosphate groups form multiple ionic and hydrogen bonds with proteins. "Inositol phosphates and inositol lipids regulate many different cellular activities, without there being unity of function," says Shears.
VISUALIZING THE PATHWAYS
Imaging methods have opened a new dimension in inositol research. Investigators have used fluorescent dyes to image calcium flux in single cells for many years. Visualizing upstream phosphoinositide signaling, however, became possible only in the past three or four years, says Steve Nahorski, University of Leicester, UK. New approaches use green fluorescent protein (GFP) biosensors, which translocate in response to changes in inositol lipids or protein kinase C.2
"These techniques have allowed detection of various aspects of inositol lipid signaling in near-real time, in intact, single whole cells," Nahorski says. Researchers can now view signaling within specific cells in extremely heterogenous tissues, such as neurons in the central nervous system. Moreover, novel visualization techniques established that IP3 variations can drive complex wave patterns and oscillations in calcium levels producing diverse and graded signals.2 "In the future, I expect that the methods will identify signaling within microdomains of differentiated cells," Nahorski comments.
THE NUCLEAR OPTION
Real-time microdomain imaging should aid investigations into the key roles played by nuclear inositol lipids. For example, elevated levels of insulin-like growth factor 1 (IGF-1) contribute to uncontrolled proliferation in some malignancies. Nuclear phosphoinositide-specific phospholipase C-β1 (PI-PLCβ1) seems to mediate IGF-1's mitogenic effect in some cell lines. GFP biosensors overcome fixation problems and poor antibody penetration that dogged early nuclear-inositide research.1
Nuclear and cytoplasmic inositides can differ markedly. Agonists that don't affect membrane inositol function may stimulate nuclear inositides, and vice versa. Agonists affecting both compartments tend to show temporal discordance. "Nuclear inositol lipids may not just act as a source for second messengers, but they may directly influence mRNA splicing or even nuclear structure," comments Bologna's Martelli.
Nuclear inositol lipid metabolism may occur in microdomains. "The appreciation that the nucleus is extremely compartmentalized, even if there are no visible membrane structures inside, is one of the milestones in cell biology," Martelli comments. Some researchers believe that abundant nuclear proteins, such as lamin B and topoisomerase II, are analogous to the cytoskeleton.1 Research now focuses on determining if nuclear inositol lipids remain in one place or move between microdomains.
Given their ubiquitous presence in cells, perhaps it's not surprising that inositides offer tempting therapeutic targets. For example, Marco Falasca, University College London, recently found that inositol 1,3,4,5,6-pentakisphosphate (IP5) possesses antitumor activity in ovarian, lung, and breast cancer cell lines.6 IP5 also sensitizes these cell lines to chemotherapeutic drugs.
IP5 seems to inhibit a signal-transduction pathway involving phosphatidylinositol 3 kinase (PI3K) and another kinase, Akt. Inhibiting PI3K also enhances apoptosis induced by conventional anticancer drugs. "We believe that [IP5] is a very promising agent," Falasca says. They are currently investigating potential synergy between IP5 and chemotherapeutics, and they plan to develop more potent and specific inhibitors of the PI3K-Akt pathway.
Shears contributed to studies showing that IP4 inhibits chloride-channel conductance, thereby regulating salt and fluid secretion by epithelial cells.5 Antagonizing IP4 in airway epithelial cells could offer relief to patients with cystic fibrosis by thinning the sticky mucous that promotes infection and inflammation. Shears' collaborators at the Seattle-based biotech company, Inologic, developed an inositol polyphosphate ion-channel modulator that will shortly enter Phase I clinical trials. "It's truly rewarding to see my contributions to a scientific discovery potentially having a therapeutically beneficial outcome," Shears says.
Shears predicts that inositol pyrophosphates "will soon become a very prominent and popular field of research," exemplifying how the roles of inositides continue to expand. IP7 and IP8 cram seven and eight phosphates respectively around the inositol ring despite considerable electrostatic and steric constraints. "There is an expectation in the field that IP7 and IP8 play key, fundamental roles in cells," Shears says.
Indeed, Solomon Snyder at Johns Hopkins Medical School in Baltimore suggests that inositol pyrophosphates may emerge as the most important inositol phosphate signaling molecules. Snyder suspects IP7 turns over more rapidly than other inositol phosphates, and according to unpublished work, is a major phospho-rylator of mammalian and yeast proteins. He adds that pyrophosphates play a key role in vesicle turnover.
Steve Safrany and colleagues at the University of Dundee, Scotland, suggest that inositol diphosphates bind and inhibit various protein-trafficking proteins and contribute to mRNA export from the nucleus. His group purified and cloned diphosphoinositol polyphosphate phosphohydrolase (DIPP), a key enzyme in inositol pyrophosphate metabolism. Safrany adds that the genes encoding DIPP-3a and DIPP-3b (
Inositides' function and chemical diversity may seem daunting, but given their ubiquitous role in determining cell function, more and more researchers must grapple with this diversity. Says Balla: "There is hardly any research area that does not, at one point, discover that its favorite molecule or process is not regulated by inositol lipids or phosphates."