Inhibition of extracellular signal-regulated kinase in liver of hyperhomocysteinemic mice.

Kinase in Liver of Hyperhomocysteinemic Mice To the Editor: We read with great interest the article by Woo et al,1 reporting the involvement of cAMP-dependent protein kinase A (cAMP-PKA) signaling pathway in activation of the transcription factor cAMPresponse element binding protein (CREB) during hyperhomocysteinemia in the liver. Hyperhomocysteinemia, defined as elevated homocysteine (Hcy) levels in blood, is not only associated with cardiovascular and cerebrovascular disorders, but is also accompanied by hepatic steatosis.2 As CREB plays an important role in the expression of genes involved in lipid metabolism, the work of Woo et al1 is of great importance to show the mechanism by which Hcy induces CREB activation. Among the signaling pathways important for activation of CREB, protein kinases such as PKA, extracellular signal regulated-kinase (ERK), and p38 mitogen-activating protein (p38 MAP) kinase are able to phosphorylate CREB leading to its activation.3 Woo et al1 demonstrated, by incubation of hepatocytes with inhibitor of protein kinases, that Hcy-induced CREB activation is mediated by PKA signaling pathway, but not by ERK pathway or p38 MAP kinase pathway. These results are very intriguing because we have showed the involvement of ERK and PKA signaling pathways in the Hcy-induced CREB activation in an ex vivo model of hippocampal slices.4 As we have also shown an activation of ERK and the downstream nuclear target CREB in the hippocampus of cystathionine beta synthase (CBS)-deficient mice, a murine model of hyperhomocysteinemia,4 the present work was designed to investigate whether activation of ERK also occurs in liver of CBS-deficient mice. Heterozygous CBS-deficient (Cbs / ) mice were bred to obtain homozygous CBS deficient (Cbs / ) mice. Genotyping for the targeted CBS allele was performed by polymerase chain reaction (PCR) analysis.5 At weaning, Cbs / mice were fed a standard diet supplemented with 1.592 g/kg choline chloride salt, necessary to their survival. The wild-type littermates (Cbs / ) were also fed the same supplemented chow to avoid differences attributable to the diet. At the time of sacrifice, blood samples were collected into tubes containing an 1/10 volume of 3.8% sodium citrate, placed on ice immediately, and plasma was isolated by centrifugation at 2500g for 15 minutes at 4°C. Plasma total Hcy (tHcy), defined as the total concentration of Hcy after quantitative reductive cleavage of all disulfide bonds, was assayed by using the fluorimetric highperformance liquid chromatography (HPLC) method described by Fortin and Genest.6 The liver was harvested, snap frozen, and stored at 80°C until use. A piece of the liver was homogenized and 30 g of protein per samples were subjected to immunoblot analysis using antibodies specific to phospho-ERK1 (Tyr 204; 1:100; Santa Cruz Biotechnology) and ERK1/2 (1:12000; Chemicon) as described.4 Even if ERK activation in Cbs / mice, showing a 2-fold increase in tHcy level (Table), was similar from Cbs / mice (Table), ERK activation was decreased by 50% in liver of Cbs / mice (Figure, a; Western immunoblots showing ERK activation in liver of (a) wild-type (Cbs / ) mice and homozygous (Cbs / ) mice, (b) heterozygous (Cbs / ) mice and heterozygous mice fed a hyperhomocysteinemic diet (Cbs / Met). Proteins were subjected to immunoblot analysis using antibodies specific to phospho-ERK. After stripping, the membranes were reprobed with anti-ERK antibody for the control. The blots are representative of 4 independent experiments (4 mice for each genotype).