Abstract
The mitochondrial antioxidant enzyme manganese superoxide dismutase (Mn-SOD) is crucial in maintaining cellular and organismal homeostasis. Mn-SOD expression is tightly regulated in a manner that synchronizes its cytoprotective functions during inflammatory challenges. Induction of Mn-SOD gene expression by the proinflammatory cytokine IL-1beta is mediated through a complex intronic enhancer element. To identify and characterize the transcription factors required for Mn-SOD enhancer function, a yeast one-hybrid assay was utilized, and two CCAAT enhancer-binding protein (C/EBP) members, C/EBP beta and C/EBP delta, were identified. These two transcription factors responded to IL-1beta treatment with distinct expression profiles, different temporal yet inducible interactions with the endogenous Mn-SOD enhancer, and also opposite effects on Mn-SOD transcription. C/EBP beta is expressed as three isoforms, LAP* (liver-activating protein), LAP, and LIP (liver-inhibitory protein). Our functional analysis demonstrated that only the full-length C/EBP beta/LAP* served as a true activator for Mn-SOD, whereas LAP, LIP, and C/EBP delta functioned as potential repressors. Finally, our systematic mutagenesis of the unique N-terminal 21 amino acids further solidified the importance of LAP* in the induction of Mn-SOD and emphasized the crucial role of this isoform. Our data demonstrating the physiological relevance of the N-terminal peptide also provide a rationale for revisiting the role of LAP* in the regulation of other genes and in pathways such as lipogenesis and development.
Highlights
The present studies have focused on the regulation of MnSOD gene expression by proinflammatory cytokines, IL-1
Our results have demonstrated the central importance of specific C/EBP  isoforms and another family member, C/EBP ␦, in the IL-1-dependent regulation of MnSOD
We have shown that full-length C/EBP /LAP*, LAP, and LIP, as well as C/EBP ␦ protein levels, are rapidly induced by treatment with IL-1
Summary
Our results have demonstrated the central importance of specific C/EBP  isoforms and another family member, C/EBP ␦, in the IL-1-dependent regulation of MnSOD. Our data have elucidated the specific role of LAP* as the only isoform of C/EBP  that serves as a specific transcriptional activator of Mn-SOD gene expression. We found that mediumlength C/EBP /LAP when overexpressed could inhibit IL-1dependent induction of Mn-SOD, as was true for the established dominant negative isoform LIP and the other family member identified by one-hybrid analysis, C/EBP ␦.
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