Early Embryonic Lethality Caused by Disruption of the Gene for Choline Kinase α, the First Enzyme in Phosphatidylcholine Biosynthesis

Choline kinase α (CK-α) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-α with an embryonic stem cell line containing an insertional mutation in the gene for CK-α (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-α is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka+/-) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by ∼30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka+/- mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka+/- mice. Triacylglycerol and cholesterol ester were elevated ∼2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka+/- and wild-type (Chka+/+) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine. Choline kinase α (CK-α) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-α with an embryonic stem cell line containing an insertional mutation in the gene for CK-α (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-α is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka+/-) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by ∼30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka+/- mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka+/- mice. Triacylglycerol and cholesterol ester were elevated ∼2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka+/- and wild-type (Chka+/+) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine. Phosphatidylcholine (PC) 3The abbreviations used are: PCphosphatidylcholineCKcholine kinaseCTCTP:phosphocholine cytidylyltransferasePEphosphatidylethanolaminePEMTphosphatidylethanolamine N-methyltransferaseGSTglutathione S-transferaseMEFmouse embryonic fibroblastsEembryonic day. is the primary phospholipid of eukaryotic cellular membranes and has a crucial role in structural maintenance of the lipid bilayer. In all mammalian tissues, PC is made via the Kennedy pathway (CDP-choline pathway), and the activity of CTP:phosphocholine cytidylyltransferase (CT) usually regulates flux through this pathway (1Vance D.E. Vance D.E. Vance J.E. Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier Science, Amsterdam2002: 205-232Google Scholar). In addition, phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine (PE) to PC and is a liver-specific alternative route for PC synthesis. Choline kinase (CK) catalyzes the first phosphorylation reaction in the CDP-choline pathway. Besides having an active role in PC biosynthesis, the activation of CK and the resulting increase in phosphocholine levels are both proposed as necessary events for fibroblasts to proliferate during stimulation by certain growth factors (e.g. platelet-derived growth factor, basic fibroblast growth factor) (2Cuadrado A. Carnero A. Dolfi F. Jimenez B. Lacal J.C. Oncogene. 1993; 8: 2959-2968PubMed Google Scholar). The induction of CK gene expression is also associated with certain cell stress and cell defense (3Aoyama C. Liao H. Ishidate K. Prog. Lipid Res. 2004; 43: 266-281Crossref PubMed Scopus (179) Google Scholar, 4Ishidate K. Enosawa S. Nakazawa Y. Biochem. Biophys. Res. Commun. 1983; 111: 683-689Crossref PubMed Scopus (26) Google Scholar). phosphatidylcholine choline kinase CTP:phosphocholine cytidylyltransferase phosphatidylethanolamine phosphatidylethanolamine N-methyltransferase glutathione S-transferase mouse embryonic fibroblasts embryonic day. CK has two isoforms, CK-α and CK-β, which are encoded by separate genes, Chka and Chkb, respectively (3Aoyama C. Liao H. Ishidate K. Prog. Lipid Res. 2004; 43: 266-281Crossref PubMed Scopus (179) Google Scholar). CK-α and CK-β have different tissue distribution that suggests a different function for each isoform. Recently Sher et al. (5Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) showed that disruption of Chkb in mice can cause severe muscular dystrophy in limbs. To provide insights into the role of CK-α in mice, we generated Chka-deficient mice. Whereas the heterozygous Chkα knock-out mice (Chka+/-) were phenotypically indistinguishable from wild-type mice, early embryonic lethality was observed in homozygous Chkα knock-out embryos. Choline Kinase α Knock-out Mice—A mouse embryonic stem cell line (XH252, strain 129/OlaHsd) with an insertional mutation in Chka was created in a gene-trapping program, BayGenomics (baygenomics.ucsf.edu/). The gene-trapping vector, pGT1Lxf, was designed to create an in-frame fusion between the 5′ exons of the trapped gene and a reporter, βgeo (a fusion of β-galactosidase and neomycin phosphotransferase II). Chka spans 12 exons on mouse chromosome 19. The insertional mutation in XH252 occurred in intron 5. Thus, the gene-trapped locus is predicted to yield a fusion transcript containing exons 1–5 of Chka and βgeo. The embryonic stem cells were injected into C57BL/6 blastocysts to create chimeric mice, which were bred with C57BL/6 mice to generate heterozygous (+/-) Chka-deficient mice. All mice had a mixed genetic background (∼50% C57BL/6 and ∼50% 129/OlaHsd). The mice were weaned at 21 days of age, housed in a barrier facility with a 12-h light-dark cycle, and fed chow containing 4.5% fat (Ralston Purina, St. Louis, MO). Genotyping—Chka genotypes were assessed by PCR analysis of genomic DNA extracted from mouse tails or embryos. Mouse tail genomic DNA was extracted by DNeasy tissue kit (Qiagen). Chka+/- mice were mated and monitored daily for plugs. Females with copulation plugs were considered to be at day 0.5 of gestation. Pregnant females were sacrificed at different times of gestation. The uterine horns were flushed with HEPES-buffered M2 culture medium, and embryos were transferred into individual Eppendorf tubes containing 5 μl of lysis buffer (0.05% SDS and 0.035 n NaOH). The samples were boiled for 3 min, and 2.5 μl of this mixture was used for PCR. A common forward primer: 5′-GATTGGACACTGAAGAGTTACG-3′ (exon 4) and two reverse primers 5′-GAGGATCTTGTGCAGTTGCTGA-3′ (exon 5) and 5′-GGGTCACAAGGTTCATATGGT-3′ (vector), as illustrated in Fig. 1A, were used to detect wild-type and mutant alleles, respectively. The amplified DNA fragments were separated by agarose gel electrophoresis. Quantitative Real-time PCR—Liver or testis (50–100 mg) was homogenized with a Polytron in TRIzol Reagent (Invitrogen) for 3 × 10 s on ice, and total RNA was extracted according to the manufacturer's instructions. RNA quality was confirmed on a 1.5% formaldehyde-agarose gel by measurement of the 28 S/18 S ribosomal RNA ratio. Total RNA (2 μg) was reverse-transcribed in a 20-μl volume containing oligo(dT)12–18 primer (Invitrogen), and Superscript II enzyme (Invitrogen) according to the manufacturer's instructions. Primers used for the reference gene, cyclophilin, were 5′-TCCAAAGACAGCAGAAAACTTTCG-3′ and 5′-TCTTCTTGCTGGTCTTGCCATTCC-3′. For CK-α, the 5′ and 3′-primers were 5′-GCCGACTGGAGCAGTTTATC-3′ and 5′-GCTCAAGAGGCAGGTTGTAA-3′. For CK-β, the 5′ and 3′ primers were 5′-CGGAGGAGCTGAGCGTTTG-3′ and 5′-TCTGCGAGAATGGCGAACATC-3′. The 25-μl PCR contained 100 ng of cDNA, 10 μl of Platinum Sybr Green qPCR Supermix UDG (Invitrogen), 1.6 μm cyclophilin primers, and 3.0 μm CK-α or CK-β primers. Real-time PCR was performed with a Rotor-Gene RG-3000 thermocycler (Corbett Research, Mortlake, NSW, Australia) with samples from three separate mice, and each sample was analyzed in triplicate. Data were normalized to the reference gene with the Pfaffl method (6Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25871) Google Scholar). Sample Collection—Livers, testes, and skeletal muscle from forelimb and hindlimb were frozen in liquid N2 after dissection. Blood was collected by cardiac puncture with instruments pretreated with EDTA. Plasma was separated by centrifugation at 2000 × g for 20 min in a refrigerated benchtop centrifuge. All samples were stored at -70 °C before use. Lipid Analysis—Tissues were homogenized with a Polytron in 5 volumes of 10 mm Tris-HCl (pH 7.2), 150 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 1:100 protease/inhibitor mixture (Sigma, P8340). Homogenates were centrifuged for 5 min at 600 × g and supernatant fluids collected. Protein was quantified (7Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) and total lipids were extracted from tissue homogenates and plasma (8Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar). Phospholipids were separated by high-performance liquid chromatography and quantified with an electron-light scattering detector (9McCluer R.H. Ullman M.D. Jungalwala F.B. Adv. Chromatogr. 1986; 25: 309-353PubMed Google Scholar). Phosphatidyldimethylethanolamine was used as an internal standard for quantification. Neutral lipids, including triacylglycerols, cholesterol, and cholesteryl esters, were measured by gas-liquid chromatography (10Lohninger A. Preis P. Linhart L. Sommoggy S.V. Landau M. Kaiser E. Anal. Biochem. 1990; 186: 243-250Crossref PubMed Scopus (31) Google Scholar). Choline Kinase Activity Assay—Tissue homogenates were centrifuged (348,000 × g for 15 min) at 4 °C and supernatant fractions (cytosol) prepared. CK activity was determined as described elsewhere with minor modifications (11Ishidate K. Nakazawa Y. Methods Enzymol. 1992; 209: 121-134Crossref PubMed Scopus (33) Google Scholar). The supernatant fractions were incubated in a final volume of 100 μl of reaction buffer that contained 0.1 m Tris-HCl (pH 8.75), 10 mm ATP-2Na, 15 mm MgCl2, and 0.25 mm [3H]choline chloride (10.5 μCi/ml) at 37 °C for 30 min. The reaction product, phosphocholine, was separated using an AG1-X8 (200–400 mesh, OH- form) column (Bio-Rad). To determine the activity of each CK isoform, supernatant fractions were treated with an antisera raised against GST (control), GST-CK-α, or GST-CK-β fusion proteins combined with protein A-Sepharose overnight at 4 °C, and the supernatant was used for CK assays (5Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Immunoblotting Analysis of CK-α and CK-β—Proteins (50 μg) from and testis homogenates were separated by on containing SDS and with or (a from K. and Protein was used as a measurement of and fractions were from homogenates of or The was performed as described by the conversion of into For measurement of (50 of of homogenates were incubated with and and the incorporation of was measured Vance D.E. J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). of Choline and lipids were extracted from tissue homogenates as described The was separated and with the amount of choline in samples was determined using Phospholipids To the amount of phosphocholine of was before the K. S. Y. Y. Y. J. Res. PubMed Scopus Google Scholar). The amount of phosphocholine was by the amount of choline from the amount of phosphocholine hepatocytes were as described Y. Vance D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). after the cells were three times with medium, and hepatocytes were for 30 min with 15 of and incubated for to 4 in an the of the the cells were and the quantified in the choline after of Mouse from mice were by and were in with S. P. E. B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Data are as The was used to the between two of Chka heterozygous mice to normal are and do not analysis of from (+/-) from 100 were wild-type were and was homozygous for a Chka The observed of wild-type to is with the predicted of on was in with and mice. suggests that the disruption of Chka in embryonic To determine the of embryonic embryos were collected from heterozygous at different days of gestation. of the embryonic and embryos were embryos be by PCR The that embryos between and of of from of not were in PCR not were in PCR in a of CK-α and CK-α Protein in Chka+/- the CK-α in Chka+/- mice but the CK-β was The expression of CK-α protein was also decreased in both and testis from Chka+/- mice The expression of CK-β was Choline Kinase Activity in Chka+/- with mice, Chka+/- mice have CK activity in both and testis CK-α and protein with CK-β C. A. Ishidate K. Biochem. J. PubMed Scopus Google Scholar). In CK activity of in skeletal muscle not and in of and Chka+/- mice, and in from and Chka+/- mice, The are with the that CK-β not CK-α CK activity in skeletal muscle (5Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Choline and in Chka+/- CK activity was we determined the of choline and a of choline and of phosphocholine was observed in both testis and from Chka+/- mice, with mice Analysis in Chka+/- the of choline and phosphocholine the amount of PC in Chka+/- mice resulting in a and in Chka+/- activity both and was not by the disruption of which is with the of PC levels in the for the decreased in is that be into PC by the activity in Chka+/- mice was with wild-type mice The of Phosphatidylcholine in Chka+/- is in and was of CK-β and activity in Chka+/- mice, we determined the of PC be we hepatocytes from Chka+/- and mice and incubated with [3H]choline to the of PC biosynthesis in The incorporation of choline into PC was from 1 to 4 and was between the incorporation with hepatocytes of heterozygous and wild-type mice. The not the in total CK activity in The were with the of between heterozygous and wild-type We also that was in the between Phosphatidylcholine in and of Chka+/- Mice—A PC in Chka+/- mice also be of a decreased of To this we measured the activity of enzymes involved in PC in enzyme was observed between Chka+/- and mice for and in livers and and testes and Neutral Lipid CK activity was decreased in the which the CDP-choline pathway has to of B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Biophys. PubMed Scopus Google we also analyzed cholesterol, and cholesteryl ester in plasma and as of plasma lipid and were between Chka+/- and mice in levels of and cholesteryl ester were between Chka+/- and mice for plasma levels of cholesteryl or cholesterol, or for levels of cholesterol lipid profiles in and plasma are n in a of CK-α and CK-β to Choline Kinase Activity in into the involved in the early embryonic lethality in Chka+/- mice, we analyzed choline kinase activity in and and of total CK activity in from wild-type mice, whereas the was by We have not from or embryos. the expression of in the suggests that early embryonic lethality is to the of CK-α activity in embryos. is the first to the role of CK-α in The from and testis of Chka+/- mice in the the that choline kinase is not usually the enzyme in the CDP-choline pathway for PC the lethality of the homozygous Chka knock-out embryos the role of CK-α in early Although CK-α is essential for early embryonic development, adult do not full expression of CK-α to a normal of The of Chka is the gene to be in PC biosynthesis in mice. The disruption was in the gene Vance D.E. S. A. PubMed Scopus Google Scholar) that the mice were fed a Vance D.E. J. Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar) or a L. Vance D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The was that a knock-out of the gene be embryonic at an early and was used to generate mice that had or in Yang C. B. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) or in C. Vance D.E. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). was in the of L. S. S. Biol. 25: PubMed Scopus Google Scholar) that disruption of the gene was during embryonic of the gene in mice was not embryonic but the mice and S. J.E. J. K. P. Biol. 2004; PubMed Scopus Google Scholar). A mutation in the CK-β gene to of the gene was in which the mice had and muscular dystrophy (5Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). the for PC biosynthesis via the CDP-choline pathway in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). two are in mice and gene is essential to be CK has kinase activity at in but in biosynthesis was in mice (5Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). kinase has and that enzyme CK activity Y. P. C. J. S. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). A knock-out of this gene in mice in with development in embryos Y. P. C. J. S. J. Biol. Chem. 2006; 281: Full Text Full Text PDF PubMed Scopus Google Scholar). In the we attenuated levels in Chka+/- mice that that CK-α to kinase activity in the gene cytidylyltransferase was resulting in embryonic lethality M.D. F. M. Biol. PubMed Scopus Google Scholar). other gene in the pathway has to the gene which from has and in embryonic lethality A. A. Vance J.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Chka for of Mouse the we that CK-α is essential for the early development of mouse embryos. embryos were recovered at the blastocyst stage, but not at indicating that the of CK-α activity to or to embryonic to A as an PC for cell and CK is involved in this The of choline is to in the of PC biosynthesis J. PubMed Scopus Google Scholar, K.A. J. Google Scholar, 2006; PubMed Scopus Google Scholar). development, is a in choline Biochem. J. PubMed Scopus Google Scholar). Plasma or choline are in the and in the adult PubMed Scopus Google Scholar). in choline during the first of in the and the Biochem. J. PubMed Scopus Google Scholar). levels of choline in the of choline to J. PubMed Scopus Google Scholar, 2006; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). Thus, choline during and development of the and mouse be In Chka+/- mice and is with choline but with the of Chka+/- mice not a with wild-type normal CK activity is not to in Chka+/- mice. the embryonic lethality in Chka+/- mice, we the of CK-α to total CK activity in CK-α activity to of the total CK activity in is that CK-α activity has a crucial role for early embryonic development in mice. that CK-β knock-out mice not during embryonic development (5Sher R.B. Aoyama C. Huebsch K.A. Ji S. Kerner J. Yang Y. Frankel W.N. Hoppel C.L. Wood P.A. Vance D.E. Cox G.A. J. Biol. Chem. 2006; 281: 4938-4948Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Thus, the Chka is the for early embryonic CK activation and an of phosphocholine is associated with cell (2Cuadrado A. Carnero A. Dolfi F. Jimenez B. Lacal J.C. Oncogene. 1993; 8: 2959-2968PubMed Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a decreased phosphocholine was observed in Chka+/- mice, is to phosphocholine in embryos. Thus, is that cell and be attenuated to CK-α, and PC biosynthesis and phosphocholine in embryonic fibroblasts be the for embryonic et al. J. PubMed Scopus Google Scholar) that in choline cause in mouse embryos in treated embryos and with an of choline In that they decreased biosynthesis and decreased incorporation of choline into PC and PC not be via in the of the choline J. PubMed Scopus Google which that the CDP-choline pathway is the route for PC biosynthesis in mouse embryos. Thus, is not to PC biosynthesis in embryos. are with the of J. PubMed Scopus Google Scholar) that choline and PC are crucial for the normal development of mouse and we that CK-α is also an for the of CK-α in we were to generate homozygous knock-out mice, we into the role of CK-α by the between and Chka+/- mice. we observed choline in Chka+/- mice, in both and The of choline is to the of choline from the in the and PC biosynthesis S. M. Biol. 2006; PubMed Scopus Google Scholar). we not a in the amount of Thus, that the amount of phosphocholine is not PC biosynthesis in Chka+/- mice. was also in PC in Chka+/- mice. in total CK activity in heterozygous the activity of the CDP-choline pathway in Chka+/- hepatocytes was the as in wild-type The of CK activity not or we not a increase in Chkb and CK-β protein expression in Chka+/- mice. to PC be to PC through the pathway. We not a in activity in in this to a decreased of PC to in livers from mice fed a Vance D.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. L. A. Vance D.E. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, an a of in Chka+/- mice to an cell The increase of and cholesteryl ester that disruption of CK-α in mice in a of and cholesteryl ester or in the plasma lipid profiles were not was In we Chka+/- mice with a for 3 but plasma activity was not The between the heterozygous and wild-type adult mice in the suggests that the amount of CK-α enzyme in wild-type and testis is to for CK have in Besides involved in PC biosynthesis, CK is also associated with cell stress and cell defense (3Aoyama C. Liao H. Ishidate K. Prog. Lipid Res. 2004; 43: 266-281Crossref PubMed Scopus (179) Google Scholar, K. Nakazawa Y. Methods Enzymol. 1992; 209: 121-134Crossref PubMed Scopus (33) Google Scholar, E. E. J. Physiol. PubMed Scopus Google Scholar). also that cell showed phosphocholine levels as a of CK activity Biol. PubMed Scopus Google Scholar, C. J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). that elevated phosphocholine is a common in cell from C.L. H. M.D. Res. Google Scholar). CK protein levels have to be in both and cell from with normal or primary respectively A. J. F. Lacal J.C. Oncogene. PubMed Scopus Google Scholar). on in CK is a and for and Thus, we had to generate mice, have to determine mice were to the development of certain We and for

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