Early embryonic lethality caused by disruption of the gene for choline kinase alpha, the first enzyme in phosphatidylcholine biosynthesis

Abstract

Choline kinase alpha (CK-alpha) 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-alpha with an embryonic stem cell line containing an insertional mutation in the gene for CK-alpha (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-alpha 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 approximately 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 approximately 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.

FIGURE 1. CK-α protein is decreased, but CK-β protein is unchanged in Chka^+/− mice

A, detection of wild-type and mutant alleles. SA, splice acceptor; pA, polyadenylation signal; black arrows, primer for genotyping. B, homogenates were prepared from livers and testes of Chka^+/− and Chka^+/+ mice, followed by the immunoblot of CK-α and CK-β. The results are representative of three independent experiments. PDI, protein disulfide isomerase.

FIGURE 2. Real-time PCR analysis of Chka and Chkb mRNA levels relative to cyclophilin mRNA in liver and testis

Relative Chka mRNA expression in liver (A) and testis (B); relative Chkb mRNA expression in liver (C) and testis (D). a, p < 0.001. Data are mean ± S.D. from four mice of each genotype.

FIGURE 3. Choline kinase activity in livers and testes in Chka^+/+ and Chka^+/− mice

Frozen tissues were homogenized and CK activity was assayed from the supernatant after high-speed centrifugation. Panels A and B present total CK activity. Panels C and D present the activities of the two isoforms of CK. It is assumed that activity not precipitated by α-specific antiserum was due to β/β homodimers, activity not precipitated by β-specific antiserum was due to α/α homodimers, and the remaining activity was from α/β heterodimers (3). a, p < 0.001. Data are mean ± S.D. from four mice of each genotype.

FIGURE 4. Concentrations of choline and phosphocholine in livers and testes from Chka^+/+ and Chka^+/− mice

Total lipids were extracted from tissue homogenates (1 mg of protein) and the aqueous phase dried under nitrogen gas. After resuspending with distilled water, the level of choline in samples was determined using Wako Phospholipids B test kit. To measure the amount of phosphocholine plus choline, 2 units/ml of alkaline phosphatase was added before the assay. The amount of phosphocholine was calculated by subtracting the amount of choline from the amount of phosphocholine plus choline (13). a, p < 0.001. Data are mean ± S.D. from analyses of at least four mice of each genotype.

FIGURE 5. PC and PE concentrations in livers and testes from Chka^+/− and Chka^+/+ mice

Lipids were extracted from homogenates (1 mg of protein) of livers (A) and testes (B), and the concentrations of PC and PE were determined by high-performance liquid chromatography. PC/PE ratio in liver (C) and testes (D) was calculated. a, p < 0.05. All data are mean ± S.D. from analyses of at least four mice of each genotype.

FIGURE 6. CT and PEMT activity in livers and testes from Chka^+/− and Chka^+/+ mice

Tissue homogenates (1 mg of protein) were prepared from livers and testes of adult Chka^+/− and Chka^+/+ mice. CT activity (A) was measured in both livers and testes. PEMT activity (B) was measured only in liver. Data are mean ± S.D. from at least three mice of each genotype.