doi: 10.1161/01.ATV.0000225290.10643.ea
© 2006 American Heart Association, Inc.
Letter to the Editor |
Inhibition of Extracellular Signal–Regulated Kinase in Liver of Hyperhomocysteinemic Mice
EA 3508, Université Paris 7-Denis Diderot
Service de Biochimie, Hopital Europeen Georges Pompidou
EA 3508, Université Paris 7-Denis Diderot, Paris, France
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 cAMP-response 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 high-performance 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 µg 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; Table), showing a 40-fold increase in tHcy (Table). To determine whether this decrease was attributable to the loss of CBS expression or to the resultant hyperhomocysteinemia, the activation of ERK was also examined in liver of Cbs+/– mice fed a hyperhomocysteinemic diet7 compared with the activation in liver of Cbs+/– mice. At weaning, mice were fed a standard diet supplemented with 0.5% L-methionine in drinking water for 3 months. Such a diet led to a 4-fold increase in tHcy (Table) and decreased ERK activation by 30% (Figure, b; Table). These results show that hyperhomocysteinemia inactivates ERK in mouse liver.
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Activated MAP kinases can be inactivated by MAP kinase phosphatases (MKPs). Among these MKPs, MKP-1 is inducible by a variety of cellular stresses, including oxidative stress, at mRNA levels.8 As oxidative stress has been found to be promoted by hyperhomocysteinemia in liver of CBS-deficient mice,9 we have also analyzed the gene expression of MKP-1. The mRNA levels of MKP-1 were assessed by real-time quantitative reverse transcription-PCR (Q-PCR), using Light Cycler FastStart DNA Master SYBR Green I Kit (Roche Diagnostics), as described.9 The primer-annealing temperature was 65°C. The mouse superoxide dismutase-1 (SOD1) mRNA was used as an endogenous control.7 
Cp analysis of the results allows to assess the ratio of the target mRNA versus SOD1 mRNA. MKP-1 mRNA levels in livers of Cbs+/– mice were similar from Cbs+/+ mice (Table), in agreement with ERK activation. However, MKP-1 mRNA levels not only in the livers of Cbs–/– mice but also in the liver of Cbs+/– mice fed a hyperhomocysteinemic diet were higher than that of Cbs+/+ mice and Cbs+/– mice (Table). These results emphasize the role of hyperhomocysteinemia on MKP-1 expression, which may be contributing to the inhibition of ERK phosphorylation in liver.
Taken together, our results support the view of Woo et al1 that ERK kinase signaling pathway is not involved in Hcy-induced CREB activation in hepatocytes. Moreover, our results demonstrate that some signal transduction pathways, like ERK, can be differentially regulated according to the tissue which is affected in hyperhomocysteinemic mice.
Acknowledgments
We thank Dr N. Maeda (Department of Pathology, University of North Carolina, Chapel Hill) for providing heterozygous Cbs+/– mice and Aurélie Ledru for expert technical assistance.
Sources of Funding
This work was supported by the European Union Grant (QLRT-2001-00816) and a grant "Vin et Santé."
Disclosure(s)
None.
References
1. Woo CW, Siow YL, OK. Homocysteine activates cAMP-response element binding protein in HepG2 through cAMP/PKA signaling pathway. Arterioscler Thromb Vasc Biol. 2006; 26: 1043–1050.
2. Mudd SH, Levy HL, Kraus JP. Disorders of transsulfuration. In The Metabolic and Molecular Bases of Inherited Disease. Scriver CR, Beaudet AL, Sly WS, Valle D, Childs B, Kinzler KW, Vogelstein B, eds. New York: McGraw Hill Inc; 2001: 2007–2056.
3. Shaywitz AJ, Greenberg ME. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem. 1999; 68: 821–861.[CrossRef][Medline] [Order article via Infotrieve]
4. Robert K, Pages C, Ledru A, Delabar J, Caboche J, Janel N. Regulation of extracellular signal-regulated kinase by homocysteine in hippocampus. Neuroscience. 2005; 133: 925–935.[Medline] [Order article via Infotrieve]
5. Watanabe M, Osada J, Aratani Y, Kluckman K, Reddick R, Malinow MR, Maeda N. Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia. Proc Natl Acad Sci U S A. 1995; 92: 1585–1589.
6. Fortin LJ, Genest J. Measurement of homocyst(e)ine in the prediction of arteriosclerosis. Clin Biochem. 1995; 28: 155–162.[CrossRef][Medline] [Order article via Infotrieve]
7. Janel N, Robert K, Chabert C, Ledru A, Gouedard C, Barouki R, Delabar JM, Chasse JF. Mouse liver paraoxonase-1 gene expression is downregulated in hyperhomocysteinemia. Thromb Haemost. 2004; 92: 221–222.[Medline] [Order article via Infotrieve]
8. Xu Q, Konta T, Nakayama K, Furusu A, Moreno-Manzano V, Lucio-Cazana J, Ishikawa Y, Fine LG, Yao J, Kitamura M. Cellular defense against H2O2-induced apoptosis via MAP kinase-MKP-1 pathway. Free Radic Biol Med. 2004; 36: 985–993.[CrossRef][Medline] [Order article via Infotrieve]
9. Robert K, Nehme J, Bourdon E, Pivert G, Friguet B, Delcayre C, Delabar JM, Janel N. Cystathionine beta synthase deficiency promotes oxidative stress, fibrosis, and steatosis in mice liver. Gastroenterology. 2005; 128: 1405–1415.[CrossRef][Medline] [Order article via Infotrieve]
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