|Home | About | Journals | Submit | Contact Us | Français|
Inositol can be obtained through the diet or synthesized by some cells from glucose-6-phosphate (Daughaday et al., 1955; Holub, 1986). Through the actions of kinases and phosphatases, inositol can be phosphorylated and dephosphorylated to form a large number of intermediates in a complex metabolic pathway. With six positions available for phosphorylation there are roughly 70 potential intermediates, including both the lipid phosphatidylinositols and the soluble forms of inositol, several of which can contain pyrophosphate bonds (Irvine et al., 2001).
Inositol, 1,3,4-trisphosphate 5/6-kinase (ITPK1, E.C. 126.96.36.199) plays a pivotal role in inositol metabolism, is conserved from plants to humans, and is even found in Entamoeba histolytica (Field et al., 2000; Wilson et al., 1996; Wilson et al., 1997). The known phosphoryl transferase activities of ITPK1 are shown diagrammatically in Fig. 1. The substrate inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) sits at a major branch point in the inositol metabolic pathway. It can be phosphorylated by ITPK1 in either the D5 or the D6 position of the inositol ring to form inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4 or inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4), respectively (Fig. 1A). Either tetrakisphosphate (IP4) product can also serve as a substrate in an isomerization reaction (Ho et al., 2002; Miller et al., 2005). In addition, ITPK1 has also been shown to reversibly phosphorylate another IP4 isomer, inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4), to form inositol pentakisphosphate (IP5) (Fig. 1B) (Chamberlain et al., 2007; Ho et al., 2002; Miller et al., 2005; Yang et al., 2000).
The formation of Ins(1,3,4,6)P4 by ITPK1 represents the rate-limiting step in the formation of the higher phosphorylated forms of inositol in mammalian cells (Verbsky et al., 2005) including IP5, IP6, and the pyrophosphates diphosphoinositol-pentakisphosphate (IP7) and bisdiphosphoinositol tetrakisphosphate (IP8), shown diagrammatically in Fig. 2.
In recent years, a number of functions for the more highly phosphorylated forms of inositol have come to light. IP6 has been shown to be an integral component of three proteins: adenosine deaminase (E.C.188.8.131.52) (Macbeth et al., 2005), arrestin (Milano et al., 2006), and the receptor for the plant hormone auxin (Tan et al., 2007). In addition to these structural roles, IP6 has been implicated in a wide range of cellular processes, including mRNA export from the nucleus (Alcazar-Roman et al., 2006; Feng et al., 2001; Weirich et al., 2006; York et al., 1999), regulation of transcription (Odom et al., 2000), increasing the ATPase activity of the dead box protein Dbp5 (E.C.3.6.1.-) (Alcazar-Roman et al., 2006), binding to the clathrin assembly proteins AP2 and AP180, inhibiting clathrin cage assembly in vitro (Chadwick et al., 1992; Norris et al., 1995; Theibert et al., 1991; Ye et al., 1995), inhibiting serine and threonine protein kinases (Larsson et al., 1997), stimulating protein kinases including casein kinase-2 (E.C.184.108.40.206) (Hilton et al., 2001; Solyakov et al., 2004), regulating nonhomologous DNA end joining of double strand breaks (Hanakahi et al., 2000; Hanakahi et al., 2002), and endocytosis (Hoy et al., 2002). Most recently, IP6 has been shown to activate the cysteine protease domains of both the Vibrio cholera RTX (repeats in toxin) (Lupardus et al., 2008; Prochazkova et al., 2008) and the Clostridium difficile A and B toxins (Pruitt et al., 2009).
To underscore the importance of IP6 in cells, deletion of the murine genes encoding the two other kinases leading to the synthesis of IP6 results in early embryonic lethality, suggesting that IP6 is essential for life (Frederick et al., 2005; Verbsky et al., 2005; Wilson et al., 2009).
We have generated a genetically engineered mouse harboring a hypomorphic allele in Itpk1 (Wilson et al., 2009). The β-galactosidase (βgal) containing gene trap cassette is inserted in the second intron of the Itpk1 gene, and by alternative splicing produces both an Itpk1-βgal fusion transcript as well as a wild type, full-length transcript. RT-PCR using mRNA extracted from brain tissue of wild type animals as well as those heterozygous and homozygous for the gene trap allele show that all animals produce mRNA for Itpk1 (Fig. 3). RT-PCR of L19, a ribosomal protein, is shown as a loading control. The levels of Itpk1 mRNA vary in both the heterozygotes and homozygotes, consistent with varying levels of ITPK1 protein produced by these animals (Wilson et al., 2009).
We have previously reported that Itpk1 is widely expressed throughout the nervous system in developing embryos and in adult mice (Wilson et al., 2009). Using Xgal staining, we have also analyzed Itpk1expression in other mouse tissues. Expression was seen in the vascular smooth muscle cells in arterioles of kidney (Fig. 4A-C), thorax (Fig. 4D), and gut (Fig. 4E-F). In the digestive tract, expression is seen in serosal cells (Fig. 4E), visceral smooth muscle cells (Fig. 4F-H), and crypt cells (Fig. 4I). Expression was also evident in the smooth muscle cells in the vas deferens (Fig. 4J), neuroepithelial derivatives including brainstem neurons (Fig. 4K), and pia mater (Fig. 4L).
Between embryonic day (E) 9.5 and E11.5, 12% of embryos resulting from mating animals homozygous for the gene trap allele have some form of neural tube defect (NTD), either spina bifida, exencephaly, or both (Wilson et al., 2009). The severity of the NTDs is quite variable, ranging from the mild caudal defects (Fig 5D), to moderately severe cranial defects (Fig. 5A-B) to cases of exencephaly that would be incompatible with life (Fig. 5C). The animals used in these experiments are of a mixed genetic background (C57Bl/6 × 129(P2)Ola). Animals are currently being backcrossed to pure genetic backgrounds representing each of the parental strains to determine whether genetic background plays a role in the incidence of NTDs, as has been shown to be the case for NTDs in the curly tail mouse, a mouse model for folic acid-resistant NTDs (Letts et al., 1995; Neumann et al., 1994; Van Straaten et al., 2001). Defects in neural tube development have also been shown to occur in mice harboring deletion of PIP5KIγ (Wang et al., 2007) further establishing the role of inositol signaling in neuronal development.
The NTDs observed in the Itpk1 hypomorphs likely represent an example of folate-resistant NTDs. In humans, folic acid supplementation provides a 70% protective effect (Wald, 1991) leaving 30% which are folate-resistant. There are several animal models for folate-resistant NTDs. In some of these, the level of inositol affects the prevalence of the NTDs (Cockroft, 1988; Cockroft et al., 1992; Copp et al., 2003; Reece et al., 1997). In order to determine whether the NTDs seen in the Itpk1 hypomorphs result from a perturbation in inositol metabolism, animals will be fed diets containing varying levels of inositol and the prevalence of NTDs will be examined. To determine the level of inositol in the mouse chow as well as in the mice, to confirm that the levels of inositol are being altered, it is necessary to be able to assay for inositol concentrations. The enzyme inositol dehydrogenase (E.C.220.127.116.11) converts myo-inositol to scyllo-inosose in an NAD+-dependent oxidation reaction (Ramaley et al., 1979; Weissbach, 1965). Using purified solutions of inositol in a room temperature reaction containing 50 μg/ml of inositol dehydrogenase (Sigma Aldrich), the generation of NADH is measured by following the absorbance at 340 nm (Fig. 6A). Reactions reach their maximum absorbance by 70 minutes, as the A340 does not increase at time points beyond this time. To ensure that reactions go to completion, the A340 is read after 120 minutes. The assay is linear up to 0.25 mM inositol (Fig. 6B).
For determination of inositol content in food (Harlan Teklad), samples were homogenized and extracted as described (Kawa et al., 2003). In addition to the standard mouse chow used by the Washington University Division of Comparative Medicine, two diet samples were obtained from Harlan Teklad (Madison, WI) for assay. Both test diets were based on the AIN-93 nutritionally complete rodent diet (Reeves et al., 1993). The level of inositol in the standard diet was 0.8 mg inositol/kg chow. A diet containing soybean oil (70 g/kg; TD.06706) and a diet containing 7% corn oil (TD.09146) both had reduced levels of inositol: 0.095 mg/kg and 0.072 mg/kg, respectively. We are currently assaying other test diets in order to obtain a relatively inositol free diet.
ITPK1 is the rate limiting enzyme in the pathway leading to formation of the highly phosphorylated inositol phosphates including IP6 and the inositol pyrophosphates. One or more of these metabolites are essential for life as deletion of either of the kinases that form IP5 or IP6 in mice results in embryonic lethality. We have produced mice harboring a hypomorphic allele for Itpk1, and mice homozygous for this gene trap allele produce low but detectable levels of active enzyme. We have studied the expression of Itpk1 in various tissues and found that the enzyme is highly expressed in smooth muscle of vessels and other tissues. In addition, these mice have neural tube defects in 12% of homozygous embryos. Since the levels of enzyme expression vary greatly in homozygous animals, we speculate that relative deficiency of one or more inositol phosphates accounts for these defects. We plan to feed an inositol deficient diet or one with supplemental inositol to animals to demonstrate altered prevalence of neural tube defects.
We thank Jun Zou, Jasna Marjanovic, and Marina Kisseleva for helpful discussions. This work was supported by grants from the National Institutes of Health (HL-16634-43 to P.W.M.; DK07618, to D.B.W.; and DK52574, to the Histology Core Facility) and the Children's Discovery Institute (MD-II-2009-174, to M.P.W.).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.