Impaired expression of defence genes against oxidative stress leading to excess ROS production in Alx3-deficient embryos under hyperglycaemic conditions could reflect a possible role for ALX3 in the regulation of the transcriptional transactivation of these genes. Consistent with this hypothesis, we found that maternal diabetes results in increased expression of Alx3 mRNA in wild type embryos (Fig. 4A), and that the levels of ALX3 protein were elevated when primary MEM cells were cultured in the presence of high rather than low glucose (Fig. 4B,C), indicating that Alx3 is a glucose-responsive gene in mouse embryos. However, in silico analysis (http://www.dcode.org) of 10 kb regions of the promoters of the genes encoding MnSOD, catalase, Gpx1, and Hif1α did not predict the existence of binding sites for ALX3, characterized by the existence of a consensus TAAT motif45. We then argued that ALX3 could regulate the expression of these genes indirectly, by stimulating the expression of key transcription factors known to activate the oxidative stress defence response such as Nrf2 46, Foxo1 and Foxo4 16, 47, 48.Figure 4Regulation of the Foxo1 promoter by ALX3. (A) Quantitative RT-PCR of Alx3 mRNA in embryos from non-diabetic (ND) or diabetic (D) wild type mice (n = 7 per group). (B) Western blot showing ALX3 in primary MEM cells cultured in the indicated concentrations of glucose. (C) Densitometric measurements of ALX3 bands relative to actin bands from three western blots similar to that shown in B. D) Quantitative RT-PCR of Nrf2, Foxo1 or Foxo4 mRNAs, extracted from embryos of non-diabetic (ND) or diabetic (D) wild type (white bars) or Alx3-deficient (black bars) mice; n.s., non-significant (n = 7 per group). (E) ALX3-binding site and its position in the mouse Foxo1 promoter. (F) Electrophoretic mobility shift assays showing the binding of proteins from nuclear extracts of primary MEM cells to an oligonucleotide probe containing the sequence indicated in E. The absence (−) or presence of competing or nonspecific competing (NSC) oligonucleotides at the indicated fold molar excess, or of an ALX3 antibody or control IgG, is depicted on top. Arrows indicate complexes containing ALX3. (G,H) Relative luciferase activity elicited in Hela (G) or primary Alx3-defcient MEM (H) cells co-transfected with the reporter plasmids Foxo1T81Luc or control pT81Luc, and an ALX3 expression plasmid (n = 5). (I) Quantitative PCR amplification of Foxo1 chromatin immunoprecipitated with anti-ALX3 antiserum or with control non-immune rabbit serum (NRS) from primary MEM cells isolated from wild type mice and cultured in the presence of the indicated concentrations of glucose. Horizontal lines represent the mean (n = 4). (J) Electrophoretic mobility shift assays showing binding of nuclear extracts from primary MEM cells cultured at the indicated concentrations of glucose to the Foxo1 promoter site indicated in E. Arrows indicate the presence of ALX3. (K) Western blot showing FOXO1 in primary wild type (left panels) or Alx3-deficient (right panel) MEM cells cultured in the indicated concentrations of glucose. A representative example of three independent experiments with similar results is depicted. Uncropped blots are provided in Supplementary Fig. S5. Except in I, all values represent mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathop{+}\limits_{-}$$\end{document}+− s.e.m. *P < 0.05, **P < 0.01, Student’s t-test.