HDAC2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma.

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.

Identification of a consensus element recognized and cleaved by IRE1 alpha.

IRE1α is an endoplasmic reticulum (ER)-located transmembrane RNase that plays a central role in the ER stress response. Upon ER stress, IRE1α is activated and cleaves specific exon-intron sites in the mRNA encoding the transcription factor X-box-binding protein 1 (XBP1). In addition, previous studies allow us to predict that IRE1α targets several RNAs other than the XBP1. In fact, we have identified CD59 mRNA as a cleavage target of IRE1α. However, it is not yet clear how IRE1α recognizes and cleaves target RNAs. To address this question, we devised a unique method that combines an in vitro cleavage assay with an exon microarray analysis, and performed genome-wide screening for IRE1α cleavage targets. We identified 13 novel mRNAs as candidate IRE1α cleavage targets. Moreover, an analysis of the novel cleavage sites revealed a consensus sequence (CUGCAG) which, when accompanied by a stem-loop structure, is essential for IRE1α-mediated cleavage. The sequence and structure were also conserved in the known IRE1α cleavage targets, CD59 and XBP1. These findings provide the important clue to understanding the molecular mechanisms by which IRE1α recognizes and cleaves target RNAs.

Function of yeast and amphioxus tRNA ligase in IRE1alpha-dependent XBP1 mRNA splicing.

During their maturation step, transfer RNAs (tRNAs) undergo excision of their introns by specific splicing. Although tRNA splicing is a molecular event observed in all domains of life, the machinery of the ligation reaction has diverged during evolution. Yeast tRNA ligase 1 (TRL1) is a multifunctional protein that alone catalyzes RNA ligation in tRNA splicing, whereas three molecules [RNA ligase (RNL), Clp1, and PNK/CPDase] are necessary for RNA ligation in tRNA splicing in amphioxi. RNA ligation not only occurs in tRNA splicing, but also in yeast HAC1 mRNA splicing and in animal X-box binding protein 1 (XBP1) mRNA splicing under conditions of endoplasmic reticulum (ER) stress. Yeast TRL1 is known to function as an RNA ligase for HAC1 mRNA splicing, whereas the RNA ligase for XBP1 mRNA splicing is unknown in animals. We examined whether yeast and amphioxus RNA ligases for tRNA splicing function in RNA ligation in mammalian XBP1 splicing. Both RNA ligases functioned in RNA ligation in mammalian XBP1 splicing in vitro. Interestingly, Clp1, and PNK/CPDase were not necessary for exon-exon ligation in XBP1 mRNA by amphioxus RNL. These results suggest that RNA ligase for tRNA splicing might therefore commonly function as an RNA ligase for XBP1 mRNA splicing.

Positive contribution of ERdj5/JPDI to endoplasmic reticulum protein quality control in the salivary gland.

In eukaryotic cells, most membrane and secretory proteins are modified post-translationally in the ER (endoplasmic reticulum) for correct folding and assembly. Disulfide-bond formation is one of the important modifications affecting folding and is catalysed by the PDI (protein disulfide isomerase) family proteins. ERdj5 [also known as JPDI (J-domain-containing PDI-like protein)] is a member of the PDI family proteins and has been reported to act as a reductase in ERAD (ER-associated degradation). However, the role of ERdj5 at the whole-body level remains unclear. Therefore in the present study we generated ERdj5-knockout mice {the mouse gene of ERdj5 is known as Dnajc10 [DnaJ (Hsp40) homologue, subfamily C, member 10]} and analysed them. Although ERdj5-knockout mice were viable and healthy, the ER stress response was activated in the salivary gland of the knockout mice more than that of control mice. Furthermore, in ERdj5-knockout cells, the expression of exogenous ERdj5 mitigated the ER stress caused by overproduction of alpha-amylase, which is one of the most abundant proteins in saliva and has five intramolecular disulfide bonds. This effect was dependent on the thioredoxin-like motifs of ERdj5. Thus we suggest that ERdj5 contributes to ER protein quality control in the salivary gland.

CEP164 Deficiency Causes Hyperproliferation of Pancreatic Cancer Cells.

Primary cilia are hair-like projections that protrude from most mammalian cells and mediate various extracellular signaling pathways. Pancreatic ductal adenocarcinoma (PDAC) cells are known to lose their primary cilia, but the relevance of this phenomenon remains unclear. In this study, we generated PDAC-originated Panc1 cells devoid of primary cilia by mutating a centriolar protein, centrosomal protein 164 (CEP164), which is required for ciliogenesis. CEP164 depletion enhanced the clonogenicity of Panc1 cells, along with chemically induced elimination of primary cilia, suggesting that a lack of these organelles promotes PDAC cells proliferation. In addition, the loss of CEP164 altered the cell cycle progression irrespective of absence of primary cilia. We found that CEP164 was co-localized with the GLI2 transcription factor at the mother centriole and controlled its activation, thus inducing Cyclin D-CDK6 expression. Furthermore, CEP164-mutated Panc1 cells were significantly tolerant to KRAS depletion-dependent growth inhibition. This study suggests that CEP164 deficiency is advantageous for PDAC cells proliferation due to not only lack of ciliation but also cilia-independent GLI2-Cyclin D/CDK6 activation, and that CEP164 is a potential therapeutic target for PDAC.

A transgenic mouse model for monitoring oxidative stress.

Oxidative stress conditions enhance the production of reactive oxygen species resulting from a variety of stimuli, and are associated with various human diseases, including neurodegenerative disorders, inflammation, and various cancers. Though such associations have been closely studied using animal models, there has been no in vivo system for monitoring oxidative stress. We have developed an oxidative stress indicator that is dually regulated by induction at the transcriptional level, and by protein stabilisation at the post-translational level in Keap1-Nrf2 pathway. In vitro, our indicator elicited an intense and specific signal to oxidative stress among various agents, in a Keap1-Nrf2-dependent manner. Moreover, the transgenic animal expressing the indicator exhibited significant signals upon oxidative stress. These results indicate the usefulness of our system as an indicator of oxidative stress both in vitro and in vivo.

Site-specific cleavage of CD59 mRNA by endoplasmic reticulum-localized ribonuclease, IRE1.

IRE1, an ER-localized transmembrane-RNase, plays a central role in ER stress response. Upon ER stress, IRE1 induces various adaptive genes through the processing of mRNA encoding the transcription factor XBP1. Moreover, it was recently reported that in fly IRE1 attenuates the expression of several genes by cleaving mRNAs, but it has been unclear whether such a mechanism also exists in mammal. In this study, we searched for IRE1alpha-cleaved mRNAs in mammalian cells and identified human CD59 (complement defense 59) mRNA as a novel cleavage target. In addition, the expression of CD59 was significantly attenuated by overexpression of IRE1alpha or ER stress. These results suggest that IRE1alpha-mediated mRNA cleavage functions even in mammals as a common system to regulate gene expression.