Generation of mice with a conditional and reporter allele for Tmem100

Activin receptor‐like kinase 1 (ACVRL1; ALK1) is predominantly expressed in arterial endothelial cells and plays an important role in angiogenesis. ACVRL1 mutations cause hereditary hemorrhagic telangiectasia (HHT), a genetic vascular disorder for which the underlying mechanism is poorly understood. We have found that expression of transmembrane protein 100 (Tmem100) is downregulated in the lung of Acvrl1‐deficient mice; however, its function is unknown. To elucidate the role of Tmem100 in vivo, we generated a conditional knockout allele for Tmem100 in which exon3, containing the entire coding sequence, was flanked by loxP sequences. The targeted allele also possessed a lacZ reporter cassette in intron2 for visualization of Tmem100 expression. We found that Tmem100 was predominantly expressed in arterial endothelial cells of developing embryos. The conditional and reporter allele will be a useful resource to investigate the in vivo role of Tmem100, especially in angiogenesis. genesis 48:673–378, 2010. © 2010 Wiley‐Liss, Inc.     

Knockdown of transmembrane protein 132A by RNA interference facilitates serum starvation-induced cell death in Neuro2a cells

Transmembrane protein 132A (TMEM132A) is a novel GRP78 binding protein that we recently discovered. However, the biological functions of TMEM132A are merely characterized because it does not encode any known structural domains. In this study, we down regulated intrinsic TMEM132A by RNA interference and identified a variety of genes that fluctuated during TMEM132A gene silencing using microarray analysis. TMEM132A-knockdown in Neuro2a cells caused neurite-like projection without any stimuli and enhanced the expression of ATF6 mRNA, an ER stress transducer, and GADD153 mRNA, a stress inducible gene. Under serum-deprived condition, TMEM132A-knockdown cells gradually retarded neurite-like projection and decreased cell viability. Moreover, TMEM132A knockdown markedly induced GADD153 expression due to serum starvation without affecting the level of cleaved caspase-3. Our data suggest that TMEM132A is an important factor of cell survival in regulating certain ER stress-related gene expression in neuronal cells.     

The cataract-associated protein TMEM114, and TMEM235, are glycosylated transmembrane proteins that are distinct from claudin family members

A novel gene, TMEM114, was annotated as a member of the claudin gene family and was subsequently associated as a cause of autosomal dominant cataract because of a translocation in its putative promoter. Our bioinformatic and molecular analyses of TMEM114, and the closely related TMEM235, demonstrate that these proteins are more closely related to members of the voltage dependent calcium channel gamma subunit family. TMEM114 and TMEM235 differed from claudins in terms of localisation in polarised epithelial cells and by the presence of N-linked glycans. By gene expression knockdown in Xenopus tropicalis we also demonstrate a role for Tmem114 in eye development.     

Endoplasmic Reticulum Stress Is Chronically Activated in Chronic Pancreatitis

The pathogenesis of chronic pancreatitis (CP) is poorly understood. Endoplasmic reticulum (ER) stress has now been recognized as a pathogenic event in many chronic diseases. However, ER stress has not been studied in CP, although pancreatic acinar cells seem to be especially vulnerable to ER dysfunction because of their dependence on high ER volume and functionality. Here, we aim to investigate ER stress in CP, study its pathogenesis in relation to trypsinogen activation (widely regarded as the key event of pancreatitis), and explore its mechanism, time course, and downstream consequences during pancreatic injury. CP was induced in mice by repeated episodes of acute pancreatitis (AP) based on caerulein hyperstimulation. ER stress leads to activation of unfolded protein response components that were measured in CP and AP. We show sustained up-regulation of unfolded protein response components ATF4, CHOP, GRP78, and XBP1 in CP. Overexpression of GRP78 and ATF4 in human CP confirmed the experimental findings. We used novel trypsinogen-7 knock-out mice (T^−/−), which lack intra-acinar trypsinogen activation, to clarify the relationship of ER stress to intra-acinar trypsinogen activation in pancreatic injury. Comparable activation of ER stress was seen in wild type and T^−/− mice. Induction of ER stress occurred through pathologic calcium signaling very early in the course of pancreatic injury. Our results establish that ER stress is chronically activated in CP and is induced early in pancreatic injury through pathologic calcium signaling independent of trypsinogen activation. ER stress may be an important pathogenic mechanism in pancreatitis that needs to be explored in future studies.