Ken Iseki

OASIS, a CREB/ATF-family member, modulates UPR signalling in astrocytes

Endoplasmic reticulum (ER) stress transducers IRE1, PERK and ATF6 are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins are accumulated in the ER. Here, we identified OASIS as a novel ER stress transducer. OASIS is a basic leucine zipper (bZIP) transcription factor of the CREB/ATF family with a transmembrane domain that allows it to associate with the ER. The molecule is cleaved at the membrane in response to ER stress, and its cleaved amino-terminal cytoplasmic domain, which contains the bZIP domain, translocates into the nucleus where it activates the transcription of target genes that are mediated by ER stress-responsive and cyclic AMP-responsive elements. Intriguingly, OASIS was induced at the transcriptional level during ER stress in astrocytes of the central nervous system, but not in other cell types examined. Furthermore, overexpression of OASIS resulted in induction of BiP and suppression of ER-stress-induced cell death, whereas knockdown partially reduced BiP levels and led to ER stress in susceptible astrocytes. Our results reveal pivotal roles for OASIS in modulating the unfolded protein response in astrocytes, and the possibility that cell type-specific UPR signalling also exists in other cells.

Slit and glypican-1 mRNAs are coexpressed in the reactive astrocytes of the injured adult brain

The slit family serves as a repellent for growing axons toward correct targets during neural development. A recent report describes slit mRNAs expressed in various brain regions in adult rats. However, their functions in the adult nervous system remain unknown. In the present study, we investigated whether slit mRNAs were expressed in the cryo‐injured brain, using in situ hybridization. All slit family members were expressed at the lesion. Slit2 mRNA was the most intensely expressed in the cells surrounding the necrotic tissue. A double‐labeling study showed that slit2 mRNA was expressed in the glial fibrillary acidic protein (GFAP)‐positive reactive astrocytes. In addition, glypican‐1, a heparan sulfate proteoglycan that serves as a high‐affinity receptor for Slit protein, was coexpressed with slit2 mRNA in the reactive astrocytes. These findings suggested that slit2 might prevent regenerating axons from entering into the lesion in concert with glypican‐1. GLIA 42:130–138, 2003.

The LIM homeobox gene, L3/Lhx8, is necessary for proper development of basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimers disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene‐disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low‐affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.

Increased syndecan expression by pleiotrophin and FGF receptor-expressing astrocytes in injured brain tissue.

Syndecan‐1, ‐2, ‐3, and ‐4 are heparan sulfate proteoglycans that are differentially expressed during development and wound repair. To determine whether syndecans are also involved in brain injury, we examined the expression of syndecan core proteins genes in cryo‐injured mouse brain, using in situ hybridization. All syndecan mRNA transcripts were similarly expressed in the region surrounding the necrotic tissue, exhibiting peak levels at day 7 after injury. Comparison with cellular markers showed that reactive astrocytes were the primary source of syndecans. Syndecans serve as co‐receptors for fibroblast growth factor (FGF) and as a reservoir for another heparin‐binding growth factor, pleiotrophin (PTN, or heparin‐binding growth‐associated molecule. In our model, FGF receptor1 (FGFR1) and PTN mRNA levels were upregulated in reactive astrocytes. The distribution patterns of FGFR1 and PTN overlapped considerably with those of syndecan‐1 and ‐3 mRNAs, respectively. These results suggest that syndecans are expressed primarily in reactive astrocytes, and may provide a supportive environment for regenerating axons in concert with heparin‐binding growth factors (e.g., FGF and PTN) in the injured brain. GLIA 39:1–9, 2002.

Expression of the novel transcription factor OASIS, which belongs to the CREB/ATF family, in mouse embryo with special reference to bone development

Abstract. The OASIS gene, which encodes a novel CREB/ATF family member, was isolated from long-term cultured astrocytes that were employed as an in vitro gliosis model. In the present study, we examined the expression pattern of the OASIS gene in the developing mouse embryo by in situ hybridization histochemistry and compared it with the expression of osteogenesis markers. OASIS mRNA expression was most strongly detected in preosteoblasts of the outer bony cortex of the ribs. Alveolar bone also showed strong signals for OASIS gene expression. OASIS mRNA was also localized to the preodontoblast of tooth buds. Expression began at embryonic dayxa012 (D12.5), peaked around D14.5–16.5, and continued to D18.5. The pattern of expression was very similar to that of hXBP-1 mRNA, which encodes another CREB/ATF family member. Spatiotemporal patterns of OASIS partly overlapped that of osteopontin, osteonectin, and α1 typexa0I procollagen genes. Among these, the time course of OASIS mRNA expression was most similar to that of osteopontin mRNA expression, suggesting that the OASIS protein is involved in the late phase of osteoblast differentiation, as compared to the Cbfa1 that regulates early phases of osteoblast differentiation.

Comparison of expression patterns between CREB family transcription factor OASIS and proteoglycan core protein genes during murine tooth development.

The transcription factor OASIS gene, which encodes for a CREB/ATF family member, is specifically expressed in the salivary gland, the cartilage and the tooth germs of the mouse embryo. In the present study, the expression patterns were compared between OASIS mRNA and major vertebrate proteoglycans, which might be the downstream genes of OASIS in the tooth germs of mouse first mandibular molars, through in situ hybridization histochemistry. OASIS mRNA expression was observed in the inner enamel epithelium during the cap and bell stages (E14.5-E18.5) in the preodontoblasts during differentiation stage (E18.5-P0) and in the differentiating odontoblasts during the early secretory stage (P2.5-P4.5). Proteoglycans (versican, decorin, biglycan, glypican, syndecan-1, and syndecan-3) were expressed in the tooth germs in various patterns. Decorin, biglycan, syndecan-1 and syndecan-3 showed gene expressions overlapping with OASIS. Especially the expression pattern of decorin and syndecan-3 coincided temporally and spatially exactly with that of OASIS. These results suggest that the OASIS gene might be related to proteoglycan expression and may play an important role in the differentiation of the odontoblast and cells in inner enamel epithelium.

Expression pattern of glypican-1 mRNA after brain injury in mice.

Glypican-1, a heparan sulfate proteoglycan, is expressed in various tissues including developing and postnatal central nervous system. It serves as a receptor for heparin-binding molecules such as fibroblast growth factors (FGFs). We investigated whether glypican-1 was expressed after brain injury in adult mice. In situ hybridization study showed that glypican-1 mRNA was expressed in the region surrounding necrotic tissue, and that the signal intensity peaked 7 days after the cryo-injury. In addition, both FGF-2 and amyloid precursor protein (APP) were concurrently upregulated and colocalized with glypican-1 mRNA. Since FGF-2 and APP can bind to glypican-1 in vitro, the present study suggested that their autocrine/paracrine interactions with glypican-1 may be involved in neuronal regeneration and/or neurite-outgrowth inhibition after brain injury.

Interaction of nucleosome assembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.

Diacylglycerol kinase ζ is associated with chromatin, but dissociates from condensed chromatin during mitotic phase in NIH3T3 cells†

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) to phosphatidic acid, both of which act as second messengers to mediate a variety of cellular mechanisms. Therefore, DGK contributes to the regulation of these messengers in cellular signal transduction. Of DGK isozymes cloned, DGKζ is characterized by a nuclear localization signal that overlaps with a sequence similar to the myristoylated alanine‐rich C‐kinase substrate. Previous studies showed that nuclear DG is differentially regulated from plasma membrane DG and that the nuclear DG levels fluctuate in correlation with cell cycle progression, suggesting the importance of nuclear DG in cell cycle control. In this connection, DGKζ has been shown to localize to the nucleus in fully differentiated cells, such as neurons and lung cells, although it remains elusive how DGK behaves during the cell cycle in proliferating cells. Here we demonstrate that DGKζ localizes to the nucleus during interphase including G1, S, and G2 phases and is associated with chromatin although it dissociates from condensed chromatin during mitotic phase in NIH3T3 cells. Furthermore, this localization pattern is also observed in proliferating spermatogonia in the testis. These results suggest a reversible association of DGKζ with histone or its related proteins in cell cycle, plausibly dependent on their post‐translational modifications. J. Cell. Biochem. 105: 756–765, 2008.

Expression of OASIS, a CREB/ATF family transcription factor, in CNS lesion and its transcriptional activity.

We reported the expression patterns of a novel member of the CREB/ATF family, OASIS, in central nervous system (CNS) lesions and its transcriptional activity. OASIS gene expression was upregulated in the stab-injured spinal cord. Double labeling experiments revealed that the distribution of OASIS mRNA-positive cells overlapped with a population of GFAP-immunoreactive cells. This finding suggested that OASIS might regulate expression of important downstream molecules in certain subset of the reactive astrocytes (e.g. inhibitory substances in injured brain). In gel shift assays, OASIS was able to specifically bind to CRE as CREB family members were. We then examined transcriptional activity of full-length OASIS with GAL4-UAS-luciferase reporter assay in COS7 cells. OASIS protein activated transcription, but did not inhibit basal transcription driven by AdML promoter. To determine critical portion(s) of the OASIS protein in transcriptional activation, we examined the activity of various deletion constructs of OASIS gene. The assay revealed that a strong transcriptional activation domain lay in the N-terminal region where acidic amino acids clustered and a possible repression domain, which had not been reported for other CREB/ATF family members, lay in the more C-terminal region. We therefore proposed that OASIS protein positively regulated gene transcription in a subset of reactive astrocytes, and thereby influenced the reaction of injured CNS tissues.