Malik AI and Storey KB (2011), Transcriptional regulation of antioxidant enzym...

XB-ART-43444

Gene 2011 Oct 10;4852:114-9. doi: 10.1016/j.gene.2011.06.014.

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Transcriptional regulation of antioxidant enzymes by FoxO1 under dehydration stress.

Malik AI , Storey KB .

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Antioxidant defenses are an important part of adaptation to environmental stress for many organisms. This study analyzed responses to dehydration stress by manganese-dependent superoxide dismutase (MnSOD) and catalase, and the role of forkhead box class O type 1 (FoxO1) transcription factor in regulating their up-regulation, in selected tissues of the African clawed frogs, Xenopus laevis. Protein and mRNA levels of MnSOD and catalase were analyzed by immunoblotting and PCR. Analysis of FoxO1 included protein and phosphoprotein (FoxO1(ser245)) levels, nuclear versus cytoplasmic distribution, and FoxO1 binding to DNA. MnSOD protein increased significantly in the liver during dehydration whereas catalase rose in the liver and skeletal muscle. This was supported in liver by 1.5-2.2 fold increases in MnSOD and catalase mRNA levels. FoxO1 transcriptional activity was enhanced in liver of dehydrated versus control frogs as evidenced by: (a) 1.8-fold increase in FoxO1 protein in the nucleus, (b) strong decreases in inactive phosphorylated FoxO1(ser245) in total and nuclear extracts, and (c) a 57% increase in FoxO1 binding to DNA in nuclear extracts. The study documents up-regulation of MnSOD and catalase in frog organs during dehydration and indicates a role for FoxO1 in controlling expression of these genes in liver. Dehydration-rehydration has components of an ischemia-reperfusion event and the oxidative stress that this generates appears to be effectively addressed, at least in X. laevis liver, by enhanced production of antioxidant enzymes under FoxO1 regulation.

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Species referenced: Xenopus laevis
Genes referenced: cat.1 foxo1
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	Fig. 1. Effect of dehydration on X. laevis FoxO1. (A) Representative western blots showing levels of FoxO1 protein in nuclear and cytoplasmic extracts under control (C) and dehydrated (D) conditions in skeletal muscle and liver of X. laevis (3 independent samples are shown for each condition). Histogram summarizes the mean data. (B) Representative western blots and summary histogram showing the expression of phospho-FoxO1ser245 in whole liver extracts (total protein) and liver nuclear extracts from control versus dehydrated frogs. Data are means ± S.E.M., n = 3–5 independent trials on tissue from different animals. : Significantly different from the corresponding control value using the Student's t-test, P < 0.05.
	Fig. 2. Effect of dehydration on the expression of MnSOD protein and mRNA in X. laevis tissues. (A) Representative western blots of MnSOD protein levels in tissues from control (C) and dehydrated (D) frogs; immunoblot images for three independent samples are shown per tissue. Histogram shows normalized protein levels for control versus dehydrated conditions. (B) Representative bands on agarose gels showing PCR product levels of amplified MnSOD and actin mRNA for three preparations from X. laevis muscle and liver. Band intensities for MnSOD PCR product were normalized against the corresponding intensities of the α-actin band amplified from the same sample. Histogram shows normalized mRNA levels in muscle and liver. Data are means ± S.E.M., n = 3–5 independent trials on tissue from different animals. : Significantly different from the corresponding control, P < 0.05.
	Fig. 3. Effect of dehydration on the expression of catalase in X. laevis muscle and liver. (A) Representative western blots and summary histogram of catalase protein levels in X. laevis tissues. (B) Representative bands on agarose gels show PCR product levels of amplified catalase and α-actin for three preparations from X. laevis muscle and liver. Histogram shows normalized catalase mRNA levels. Other information as in Fig. 2.