Sepsis causes acute kidney injury (AKI) in critically ill patients, although the pathophysiology remains unclear. The receptor-interacting protein kinase-3 (RIPK3), a cardinal regulator of necroptosis, has recently been implicated in the pathogenesis of human disease. In mice subjected to polymicrobial sepsis, we demonstrate that RIPK3 promotes sepsis-induced AKI. Utilizing genetic deletion and biochemical approaches in vitro and in vivo, we identify a potentially novel pathway by which RIPK3 aggravates kidney tubular injury independently of the classical mixed lineage kinase domain-like protein-dependent (MLKL-dependent) necroptosis pathway. In kidney tubular epithelial cells, we show that RIPK3 promotes oxidative stress and mitochondrial dysfunction involving upregulation of NADPH oxidase-4 (NOX4) and inhibition of mitochondrial complex I and -III, and that RIPK3 and NOX4 are critical for kidney tubular injury in vivo. Furthermore, we demonstrate that RIPK3 is required for increased mitochondrial translocation of NOX4 in response to proinflammatory stimuli, by a mechanism involving protein-protein interactions. Finally, we observed elevated urinary and plasma RIPK3 levels in human patients with sepsis-induced AKI, representing potential markers of this condition. In conclusion, we identify a pathway by which RIPK3 promotes kidney tubular injury via mitochondrial dysfunction, independently of MLKL, which may represent a promising therapeutic target in sepsis-induced AKI.

SummaryWe have utilized retinoic acid receptor c (gamma) knockout (RARc 2/2 ) embryonic stem (ES) cells as a model system to analyze RARc mediated transcriptional regulation of stem cell differentiation. Most of the transcripts regulated by all-trans retinoic acid (RA) in ES cells are dependent upon functional RARc signaling. Notably, many of these RA-RARc target genes are implicated in retinoid uptake and metabolism. For instance, Lrat (lecithin:retinol acyltransferase), Stra6 (stimulated by retinoic acid 6), Crabp2 (cellular retinoic acid binding protein 2), and Cyp26a1 (cytochrome p450 26a1) transcripts are induced in wild type (WT), but not in RARc 2/2 cells. Transcripts for the transcription factors Pbx1 (pre-B cell leukemia homeobox-1), Wt1 (Wilm's tumor gene-1), and Meis1 (myeloid ecotropic viral integration site-1) increase upon RA treatment of WT, but not RARc 2/2 cells. In contrast, Stra8, Dleu7, Leftb, Pitx2, and Cdx1 mRNAs are induced by RA even in the absence of RARc. Mapping of the epigenetic signature of Meis1 revealed that RA induces a rapid increase in the H3K9/ K14ac epigenetic mark at the proximal promoter and at two sites downstream of the transcription start site in WT, but not in RARc 2/2 cells. Thus, RA-associated increases in H3K9/K14ac epigenetic marks require RARc and are associated with increased Meis1 transcript levels, whereas H3K4me3 is present at the Meis1 proximal promoter even in the absence of RARc. In contrast, at the Lrat proximal promoter primarily the H3K4me3 mark, and not the H3K9/K14ac mark, increases in response to RA, independently of the presence of RARc. Our data show major epigenetic changes associated with addition of the RARc agonist RA in ES cells.

Mammalian Twist1, a master regulator in development and a key factor in tumorigenesis, is known to repress transcription by several mechanisms and is therefore considered to mediate its function mainly through inhibition. A role of Twist1 as transactivator has also been reported but, so far, without providing a mechanism for such an activity. Here we show that heterodimeric complexes of Twist1 and E12 mediate E-boxdependent transcriptional activation. We identify a novel Twist1 transactivation domain that coactivates together with the less potent E12 transactivation domain. We found three specific residues in the highly conserved WR domain to be essential for the transactivating function of murine Twist1 and suggest an ␣-helical structure of the transactivation domain.When the human genome was sequenced, one of the surprising results was the low number of genes, summing up to only about one-tenth of the expected number. Explanations for this apparent lack of genes were found in differential gene splicing and protein modification as well as in the combinatorial use of regulating signals and transcription factors. A third explanation, multifunctional proteins, has so far received little attention. Here we show that the basic helix-loop-helix (bHLH) 2 protein Twist1, a known transcriptional inhibitor, can also function as an activator, depending on the regulatory sequences of the target genes. Identification of this additional molecular mechanism may aid in explaining the pleiotropic effects of Twist1 in development and in understanding the complex phenotype of the human Saethre-Chotzen syndrome, which is caused by haploid insufficiency of Twist1.Tissue-specifically expressed bHLH transcription factors are important regulators during embryonic development and postnatal life. They mediate their function through binding to DNA elements of the NCANNTGN consensus sequence termed E-boxes (1, 2). The evolutionarily conserved molecular mechanisms leading to DNA binding have been firmly established. In brief, two amphipathic ␣-helices connected by a loop region form the HLH motif, a protein interaction domain through which bHLH factors form homo-or heterodimers. A region of basic residues N-terminal to the HLH motif is necessary for DNA binding. Members of a class of ubiquitously expressed bHLH factors termed E-proteins serve as activating dimerization partners for tissue-specific bHLH factors (3, 4). In general, single E-boxes are sufficient for bHLH responsiveness, yet cooperative binding to dual E-boxes has been observed (5). Id proteins constitute a specific class of inhibitory HLH factors, which are unable to bind DNA due to a lack of basic regions (6). The Id proteins consequently function as negative regulators.Although dimerization and DNA binding are mechanistically similar for all bHLH proteins, the transactivational mechanisms are often of different evolutionary origin. The closely related myogenic bHLH factors Myf5 and MyoD1 both up-regulate muscle-specific genes such as muscle creatine kinase, yet the sequence...

Polycomb proteins play key roles in mediating epigenetic modifications that occur during cell differentiation. The Polycomb repressive complex 2 (PRC2) mediates the tri-methylation of histone H3 lysine 27 (H3K27me3). In this study, we identify a distinguishing feature of two classes of PRC2 target genes, represented by the Nr2F1 (Coup-TF1) and the Hoxa5 gene, respectively. Both genes are transcriptionally activated by all-trans retinoic acid (RA) and display increased levels of the permissive H3K9/K14ac and tri-methylated histone H3 lysine 4 epigenetic marks in response to RA. However, while in response to RA the PRC2 and H3K27me3 marks are greatly decreased at the Hoxa5 promoter, these marks are initially increased at the Nr2F1 promoter. Functional depletion of the essential PRC2 protein Suz12 by short hairpin RNA (shRNA) technology enhanced the RA-associated transcription of Nr2F1, Nr2F2, Meis1, Sox9 and BMP2, but had no effect on the Hoxa5, Hoxa1, Cyp26a1, Cyp26b1 and RARβ2 transcript levels in wild-type embryonic stem cells. We propose that PRC2 recruitment attenuates the RA-associated transcriptional activation of a subset of genes. Such a mechanism would permit the fine-tuning of transcriptional networks during differentiation.

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