Genes to Cells

Autophagy is a self‐eating system conserved among eukaryotes, in which cellular components including organelles are entrapped into a double membrane structure called the autophagosome and then degraded by lysosomal hydrolases. In addition to its role in supplying amino acids in response to nutrient starvation, autophagy is involved in quality control to maintain cell health. Thus, inactivation of autophagy causes the formation of cytoplasmic protein inclusions, which comprise misfolded proteins and the accumulation of many degenerated organelles, resulting in liver injury, diabetes, myopathy and neurodegeneration. Furthermore, although autophagy has been considered nonselective, increasing evidence points to the selectivity of autophagy in sorting vacuolar enzymes and removal of aggregate‐prone proteins and unwanted organelles. Such selectivity allows diverse cellular regulation, similar to the ubiquitin proteasome pathway. In this review, we discuss the physiological roles of selective autophagy and their molecular mechanisms.

The superoxide‐producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity‐purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H‐dependent superoxide‐producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12‐myristate 13‐acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf‐recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi‐mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

The unfolded protein response (UPR) is an adaptive stress response that responds to the accumulation of unfolded proteins in the lumen of the endoplasmic reticulum (ER) and that adjusts the protein‐folding capacity to the needs of the cell. Perturbation of cellular lipids also activates the UPR. Lipid‐induced UPR has attracted much attention because it is associated with the pathology of some metabolic diseases. However, how the lipid‐induced UPR is activated remains unclear. We previously showed that palmitic acid treatment or knockdown of stearoyl‐CoA desaturase in HeLa cells promotes membrane lipid saturation and activates the UPR. In this study, we compared UPR activation by membrane lipid saturation with UPR activation by conventional ER stressors that cause the accumulation of unfolded proteins such as tunicamycin and thapsigargin. Membrane lipid saturation induced autophosphorylation of inositol‐requiring 1α (IRE1α) and protein kinase RNA‐like ER kinase, but not the conversion of activating transcription factor‐6α to the active form. A conventional ER stressor induced clustering of fluorescently tagged IRE1α fusion protein, but palmitic acid treatment did not, suggesting that IRE1α was activated without large cluster formation by membrane lipid saturation. Together, these results suggest membrane lipid saturation, and unfolded proteins activate the UPR through different mechanisms.

During embryonic lymphatic development, Prox1 homeobox transcription factor is expressed in a subset of venous blood vascular endothelial cells (BECs) in which COUP‐TFII orphan nuclear receptor is highly expressed. Prox1 induces differentiation of BECs into lymphatic endothelial cells (LECs) by inducing the expression of various LEC markers including vascular endothelial growth factor receptor 3 (VEGFR3). However, the molecular mechanisms of how transcriptional activities of Prox1 are regulated are largely unknown. In the present study, we show that COUP‐TFII plays important roles in the regulation of the function of Prox1. In BECs and LECs, Prox1 promotes the proliferation and migration toward VEGF‐C by inducing the expression of cyclin E1 and VEGFR3, respectively. Gain‐of‐function studies showed that COUP‐TFII negatively regulates the effects of Prox1 in BECs and LECs whereas loss‐of‐function studies showed that COUP‐TFII negatively and positively regulates Prox1 in BECs and LECs, respectively. We also show that endogenous Prox1 and COUP‐TFII physically interact in LECs and that both Prox1 and COUP‐TFII bind to the endogenous cyclin E1 promoter. These results suggest that COUP‐TFII physically and functionally interact during differentiation and maintenance of lymphatic vessels.

Much recent attention has been focused on Aurora C, the third member of the mammalian Aurora kinases family that plays significant roles in mitosis. We report here that using sensitive RT‐PCR to amplify the C‐terminal, we found that Aurora C is not only expressed highly in testis, but also among 16 other human tissues in a broad‐spectrum way. Aurora C, as a chromosomal passenger protein, is co‐localized with Aurora B and Survivin in mitotic cells. Aurora C can also be associated with Aurora B and Survivin in vivo and directly binds to Survivin but not Aurora B in vitro. Over‐expression of a catalytically inactive mutant of Aurora C impaired the localization of Aurora C to the spindle midzone and severely disturbed the cytokinesis, resulting in multinucleation, all of which are consistent with the results induced by the mutant of Aurora B. Furthermore, we provide evidence that Aurora C could rescue the multinucleate phenotype produced by Aurora B mutant, and vice versa. Overall, these findings demonstrate that Aurora C, a member of the chromosomal passenger complex, is required for cytokinesis.

Background: Densin‐180, a brain‐specific protein highly concentrated at the postsynaptic density (PSD), belongs to the LAP [leucine‐rich repeats and PSD‐95/Dlg‐A/ZO‐1 (PDZ) domains] family of proteins, some of which play fundamental roles in the establishment of cell polarity.

Results: To identify new Densin‐180‐interacting proteins, we screened a yeast two‐hybrid library using the COOH‐terminal fragment of Densin‐180 containing the PDZ domain as bait, and we isolated MAGUIN‐1 as a Densin‐180‐binding protein. MAGUIN‐1, a mammalian homologue of Drosophila connector enhancer of KSR (CNK), is known to interact with PSD‐95 and has a short isoform, MAGUIN‐2. The Densin‐180 PDZ domain bound to the COOH‐terminal PDZ domain‐binding motif of MAGUIN‐1. Densin‐180 co‐immunoprecipitated with MAGUIN‐1 as well as with PSD‐95 from the rat brain. In dissociated hippocampal neurones Densin‐180 co‐localized with MAGUINs and PSD‐95, mainly at neuritic spines. In transfected cells, Densin‐180 formed a ternary complex with MAGUIN‐1 and PSD‐95, whereas no association was detected between Densin‐180 and PSD‐95 in the absence of MAGUIN‐1. MAGUIN‐1 formed a dimer or multimer via the COOH‐terminal leucine‐rich region which is present in MAGUIN‐1 but not in ‐2. Among the PDZ domains of PSD‐95, the first was sufficient for interaction with MAGUIN‐1.

Conclusion: These results suggest that the potential to dimerize or multimerize allows MAGUIN‐1 to bind simultaneously to both Densin‐180 and PSD‐95, leading to the ternary complex assembly of these proteins at the postsynaptic membrane.

Background MST1 is an upstream kinase of the JNK and p38 MAPK pathways whose expression induces apoptotic morphological changes such as nuclear condensation. During apoptosis, caspase cleavage of MST1 removes a C‐terminal regulatory domain, increasing the kinase activity of the MST1 N‐terminal domain. Downstream pathways of MST1 in the induction of apoptosis remain to be clarified.

Results In this study, we found that the expression of MST1 resulted in caspase‐3 activation. Therefore, MST1 is not only a target of caspases but also an activator of caspases. This caspase activation and apoptotic changes occur through JNK, since the co‐expression of a dominant‐negative mutant of JNK inhibited MST1‐induced morphological changes as well as caspase activation. In contrast, neither a dominant‐negative p38 nor the p38 inhibitor SB203580 inhibited them. MST1 induced nucleosomal DNA fragmentation, which was suppressed by caspase inhibitors or ICAD (Inhibitor of Caspase‐Activated DNase). Surprisingly, however, other changes such as membrane blebbing and chromatin condensation were not inhibited by caspase inhibitors.

Conclusion These results suggest that MST1 most likely promotes two events through JNK activation; first, MST1 induces the activation of caspases, resulting in CAD‐mediated DNA fragmentation, and second, MST1 induces chromatin condensation and membrane blebbing without utilizing downstream caspases.

Mef2c protein is one of the MADS‐box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that theMef2cgene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants ofMef2cduring neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that twoMef2cmRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably,Mef2cisoforms containing exon β specifically appeared in the neural cell stage. Because most transcripts contain exon β in the neural cell stage and in the brain, this suggests that the alternative splicing of exon β is highly regulated. Among known regulators, Fox‐1 was specifically expressed in the neural cell stage in correlation withMef2cexon β. Fox‐1 promoted exon β inclusion in transfection experiments usingMef2cβ minigene. Moreover, we found that the promotion required RNA‐binding activity of Fox‐1 and GCAUG sequence located in adjacent intron of exon β. Taken together, our results suggest that Fox‐1, expressed specifically in the neural cell stage, promotedMef2cexon β inclusion via the GCAUG.