Medium-chain acyl-Coenzyme A dehydrogenase (MCAD) is involved in the initial step of mitochondrial fatty acid β-oxidation (FAO). Loss of function results in MCAD deficiency, a disorder that usually presents in childhood with hypoketotic hypoglycemia, vomiting and lethargy. While the disruption of mitochondrial fatty acid metabolism is the primary metabolic defect, secondary defects in mitochondrial oxidative phosphorylation (OXPHOS) may also contribute to disease pathogenesis. Therefore, we examined OXPHOS activity and stability in MCAD-deficient patient fibroblasts that have no detectable MCAD protein. We found a deficit in mitochondrial oxygen consumption, with reduced steady-state levels of OXPHOS complexes I, III and IV, as well as the OXPHOS supercomplex. To examine the mechanisms involved, we generated an MCAD knockout (KO) using human 143B osteosarcoma cells. These cells also exhibited defects in OXPHOS complex function and steady-state levels, as well as disrupted biogenesis of newly-translated OXPHOS subunits. Overall, our findings suggest that the loss of MCAD is associated with a reduction in steady-state OXPHOS complex levels, resulting in secondary defects in OXPHOS function which may contribute to the pathology of MCAD deficiency.

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During the stress response and metabolic fasting, glucocorticoids acting via the glucocorticoid receptor (GR) stimulate hepatic glucose production by activating specific gluconeogenic enzyme target genes. To characterize novel direct GR-regulated hepatic target genes under glucocorticoid control, we performed a whole genome gene expression microarray using dexamethasone-treated GR-null mice. Strongly induced previously characterized genes included phosphoenolpyruvate carboxykinase, serine dehydratase, tyrosine oxygenase, lipin 1, metallothionine, and cdkn1A. Novel induced genes included Ddit4, Fkbp5, Megf9, Sult1e1, and Sult1d1, and all were verified by real-time PCR. Sult1d1, a sulfotransferase, is a member of a large superfamily of detoxification enzymes and has an important role in the inactivation of endogenous dopamine-derived compounds, including the catecholamines. Treatment of primary mouse hepatocytes with dexamethasone for 6 h dramatically increased Sult1d1 mRNA levels, whereas cotreatment with RU-486, a GR antagonist, blocked induction by dexamethasone. Sult1d1 mRNA levels were also increased by dexamethasone in the kidney, a major site of Sult1d1 synthesis. Sult1d1 mRNA was localized by in situ hybridization to renal collecting ducts and was rapidly induced by glucocorticoids in renal inner medullary collecting duct (IMCD3) cells. Hepatic and renal Sult1d1 enzymatic activity was significantly induced in vivo in wild-type mice 6 h after dexamethasone treatment. Chromatin immunoprecipitation assay analysis upstream of the Sult1d1 gene promoter identified a glucocorticoid response element close to the neighboring glucocorticoid-responsive estrogen sulfotransferase Sult1e1 gene, indicating that both genes potentially share a common glucocorticoid response element. These results suggest that Sult1d1 in mice is directly induced by glucocorticoids and may attenuate elevated catecholamine activity during the stress response.

Glucocorticoids (GCs) have essential roles in the regulation of development, integrated metabolism, and immune and neurological responses, and act primarily via the glucocorticoid receptor (GR). In most cells, GC treatment results in down-regulation of GR mRNA and protein levels via negative feedback mechanisms. However, in GC-treated thymocytes, GR protein levels are maintained at a high level, increasing sensitivity of thymocytes to GCs, resulting in apoptosis termed glucocorticoid-induced cell death (GICD). CD4(+)CD8(+) double-positive thymocytes and thymic natural killer T cells in particular are highly sensitive to GICD. Although GICD is exploited via the use of synthetic GC analogues in the treatment of hematopoietic malignancies, the intracellular molecular pathway of GICD is not well understood. To explore GICD in thymocytes, the authors performed whole genome expression microarray analysis in mouse GR exon 2 null vs wild-type thymus RNA 3 hours after dexamethasone treatment. Identified and validated direct GR targets included P21 and Bim, in addition to an important transcriptional regulator Nfil3, which previously has been associated with GICD and is essential for natural killer cell development in vivo. Immunostaining of NFIL3 in whole thymus localized NFIL3 primarily to the medullary region, and double labeling colocalized NFIL3 to apoptotic cells. In silico analysis revealed a putative GC response element 5 kb upstream of the Nfil3 promoter that is strongly conserved in the rat genome and was confirmed to bind GR by chromatin immunoprecipitation. The knockdown of Nfil3 mRNA levels to 20% of normal using specific small interfering RNAs abrogated GICD, indicating that NFIL3 is required for normal GICD in CTLL-2 T cells.

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

In eukaryotic cells, messenger RNA (mRNA) molecules are exported from the nucleus to the cytoplasm, where they are translated. The highly conserved protein nuclear RNA export factor1 (Nxf1) is an important mediator of this process. Although studies in yeast and in human cell lines have shed light on the biochemical mechanisms of Nxf1 function, its contribution to mammalian physiology is less clear. Several groups have identified recurrent NXF1 mutations in chronic lymphocytic leukemia (CLL), placing it alongside several RNA-metabolism factors (including SF3B1, XPO, RPS15) whose dysregulation is thought to contribute to CLL pathogenesis. We report here an allelic series of germline point mutations in murine Nxf1. Mice heterozygous for these loss-of-function Nxf1 mutations exhibit thrombocytopenia and lymphopenia, together with milder hematological defects. This is primarily caused by cell-intrinsic defects in the survival of platelets and peripheral lymphocytes, which are sensitized to intrinsic apoptosis. In contrast, Nxf1 mutations have almost no effect on red blood cell homeostasis. Comparative transcriptome analysis of platelets, lymphocytes, and erythrocytes from Nxf1-mutant mice shows that, in response to impaired Nxf1 function, the cytoplasmic representation of transcripts encoding regulators of RNA metabolism is altered in a unique, lineage-specific way. Thus, blood cell lineages exhibit differential requirements for Nxf1-mediated global mRNA export.

Mitochondria play an integral role in cell death signaling, yet how mitochondrial defects disrupt this important function is not well understood. We have used a mouse L-cell fibroblast model harboring Rattus norvegicus mtDNA (Rn xenocybrids) to examine the effects of multiple oxidative phosphorylation (OXPHOS) defects on reactive oxygen species (ROS) generation and cell death signaling. Blue native-PAGE analyses of Rn xenocybrids revealed defects in OXPHOS complex biogenesis with reduced steady-state levels of complexes I, III and IV. Isolated Rn xenocybrid mitochondria exhibited deficiencies in complex II+III and III activities, with CIII-stimulated ROS generation 66% higher than in control mitochondria. Rn xenocybrid cells were resistant to staurosporine-induced cell death, but exhibited a four-fold increase in sensitivity to ceramide-induced cell death that was caspase-3 independent and did not induce chromosomal DNA degradation. Furthermore, ceramide directly inhibited Rn xenocybrid complex II+III activity by 97%, although this inhibition could be completely abolished by exogenous decylubiquinone. Ceramide also induced a further increase in ROS output from Rn xenocybrid complex III by 42%. These results suggest that the interaction of ceramide with OXPHOS complex III is significantly enhanced by the presence of the xenotypic Rattus cytochrome b in complex III, likely due to the increased affinity for ceramide at the ubiquinone binding site. We propose a novel mechanism of altered mitochondrial cell death signaling due to mtDNA mutations whereby ceramide directly induces OXPHOS complex ROS generation to initiate cell death pathways.

The nuclear pore complex (NPC) is a multi-protein complex that regulates the trafficking of macromolecules between the nucleus and cytoplasm. Genetic variants in components of the NPC have been shown to cause a range of neurological disorders, including intellectual disability and microcephaly. Translocated promoter region, nuclear basket protein (TPR) is a critical scaffolding element of the nuclear facing interior of the NPC. Here we present two siblings with biallelic variants in TPR who present with a phenotype of microcephaly, ataxia and severe intellectual disability. The variants result in a premature truncation variant, and a splice variant leading to a 12-amino acid deletion respectively. Functional analyses in patient fibroblasts demonstrate significantly reduced TPR levels, and decreased TPR-containing NPC density. A compensatory increase in total NPC levels was observed, and decreased global RNA intensity in the nucleus. The discovery of variants that partly disable TPR function provide valuable insight into this essential protein in human disease, and our findings suggest that TPR variants are the cause of the siblings’ neurological disorder.