G protein-coupled receptor 84 gene expression is regulated by the ER stress response in the liver.

G protein-coupled receptor 84 (Gpr84) is reportedly activated by medium-chain fatty acids and is involved in the pathology of liver fibrosis. Inflammatory stimulants, such as lipopolysaccharide and tumor necrosis factor-α, upregulate Gpr84 expression. However, the detailed molecular mechanism by which Gpr84 is induced remains unknown. Inflammatory stimulation also evokes endoplasmic reticulum (ER) stress, but there has been no direct evidence to link Gpr84 expression and the ER stress response. Administration of tunicamycin (Tm) provokes ER stress and acute steatosis in the liver tissue of mice. Here, in situ hybridization analysis revealed that induction of Gpr84 expression occurred in parenchymal cells in the liver tissue following Tm administration. Gene expression analysis using a reporter assay showed that the intron 1 region of Gpr84 was involved in induction of the gene under ER stress conditions. Furthermore, Tm-dependent upregulation of Gpr84 was blocked by the small chemical compound AEBSF, an inhibitor of ER stress transducers, in vitro and in vivo. In conclusion, the current study marks the discovery that the ER stress agent Tm induces the expression of Gpr84.

Neuronal activity-dependent local activation of dendritic unfolded protein response promotes expression of brain-derived neurotrophic factor in cell soma.

Unfolded protein response (UPR) has roles not only in resolving the accumulation of unfolded proteins owing to endoplasmic reticulum (ER) stress, but also in regulation of cellular physiological functions. ER stress transducers providing the branches of UPR signaling are known to localize in distal dendritic ER of neurons. These reports suggest that local activation of UPR branches may produce integrated outputs for distant communication, and allow regulation of local events in highly polarized neurons. Here, we demonstrated that synaptic activity- and brain-derived neurotrophic factor (BDNF)-dependent local activation of UPR signaling could be associated with dendritic functions through retrograde signal propagation by using murine neuroblastoma cell line, Neuro-2A and primary cultured hippocampal neurons derived from postnatal day 0 litter C57BL/6 mice. ER stress transducer, inositol-requiring kinase 1 (IRE1), was activated at postsynapses in response to excitatory synaptic activation. Activated dendritic IRE1 accelerated accumulation of the downstream transcription factor, x-box-binding protein 1 (XBP1), in the nucleus. Interestingly, excitatory synaptic activation-dependent up-regulation of XBP1 directly facilitated transcriptional activation of BDNF. BDNF in turn drove its own expression via IRE1-XBP1 pathway in a protein kinase A-dependent manner. Exogenous treatment with BDNF promoted extension and branching of dendrites through the protein kinase A-IRE1-XBP1 cascade. Taken together, our findings indicate novel mechanisms for communication between soma and distal sites of polarized neurons that are coordinated by local activation of IRE1-XBP1 signaling. Synaptic activity- and BDNF-dependent distinct activation of dendritic IRE1-XBP1 cascade drives BDNF expression in cell soma and may be involved in dendritic extension. Cover Image for this issue: doi. 10.1111/jnc.14159.

ER Stress and Disease: Toward Prevention and Treatment.

Secretory and membrane proteins are synthesized in ribosomes, then mature in the endoplasmic reticulum (ER), but if ER function is impaired, immature defective proteins accumulate in the ER. This situation is called ER stress: in response, a defensive mechanism called the unfolded protein response (UPR) is activated in cells to reduce the defective proteins. During the UPR, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and RNA-dependent protein kinase (PKR)-like ER kinase (PERK) are activated, stress signals are transduced to the outside of the ER, and various cell responses, including gene induction, occur. In ER-associated degradation (ERAD), one type of UPR, defective proteins are eventually expelled from the ER and degraded in the cytoplasm through the ubiquitin proteasome system. Since ER stress has been reported to have relationships with neurodegenerative diseases, diabetes, metabolic syndromes, and cancer, it is the focus of increased attention from the perspectives of elucidating pathogenic mechanisms, and in the development of therapeutics.

The androgen-induced protein AIbZIP facilitates proliferation of prostate cancer cells through downregulation of p21 expression.

Androgen-Induced bZIP (AIbZIP) is structurally a bZIP transmembrane transcription factor belonging to the CREB/ATF family. This molecule is highly expressed in androgen-sensitive prostate cancer cells and is transcriptionally upregulated by androgen treatment. Here, we investigated molecular mechanism of androgen-dependent expression of AIbZIP and its physiological function in prostate cancer cells. Our data showed that SAM pointed domain-containing ETS transcription factor (SPDEF), which is upregulated by androgen treatment, directly activates transcription of AIbZIP. Knockdown of AIbZIP caused a significant reduction in the proliferation of androgen-sensitive prostate cancer cells with robust expression of p21. Mechanistically, we demonstrated that AIbZIP interacts with old astrocyte specifically induced substance (OASIS), which is a CREB/ATF family transcription factor, and prevents OASIS from promoting transcription of its target gene p21. These findings showed that AIbZIP induced by the androgen receptor (AR) axis plays a crucial role in the proliferation of androgen-sensitive prostate cancer cells, and could be a novel target of therapy for prostate cancer.

OASIS modulates hypoxia pathway activity to regulate bone angiogenesis.

OASIS/CREB3L1, an endoplasmic reticulum (ER)-resident transcription factor, plays important roles in osteoblast differentiation. In this study, we identified new crosstalk between OASIS and the hypoxia signaling pathway, which regulates vascularization during bone development. RT-PCR and real-time PCR analyses revealed significant decreases in the expression levels of hypoxia-inducible factor-1α (HIF-1α) target genes such as vascular endothelial growth factor A (VEGFA) in OASIS-deficient (Oasis(-/-)) mouse embryonic fibroblasts. In coimmunoprecipitation experiments, the N-terminal fragment of OASIS (OASIS-N; activated form of OASIS) bound to HIF-1α through the bZIP domain. Luciferase assays showed that OASIS-N promoted the transcription activities of a reporter gene via a hypoxia-response element (HRE). Furthermore, the expression levels of an angiogenic factor Vegfa was decreased in Oasis(-/-) osteoblasts. Immunostaining and metatarsal angiogenesis assay showed retarded vascularization in bone tissue of Oasis(-/-) mice. These results suggest that OASIS affects the expression of HIF-1α target genes through the protein interaction with HIF-1α, and that OASIS-HIF-1α complexes may play essential roles in angiogenesis during bone development.

IRE1α-XBP1 is a novel branch in the transcriptional regulation of Ucp1 in brown adipocytes.

The unfolded protein response (UPR) not only resolves endoplasmic reticulum (ER) stress, but also regulates cellular physiological functions. In this study, we first linked the UPR to the physiological roles of brown adipose tissue (BAT). BAT is one of the tissues that control energy homeostasis in the body. Brown adipocytes are able to dissipate energy in the form of heat owing to their mitochondrial protein, uncoupling protein 1 (UCP1). We found that one of the UPR branches, the IRE1α-XBP1 pathway, was activated during the transcriptional induction of Ucp1. Inhibiting the IRE1α-XBP1 pathway reduced the induction of Ucp1 expression. However, the activation of the IRE1α-XBP1 pathway by ER stress never upregulated Ucp1. On the other hand, the activation of protein kinase A (PKA) induced Ucp1 transcription through the activation of IRE1α-XBP1. The inhibition of PKA abrogated the activation of IRE1α-XBP1 pathway, while the inhibition of a p38 mitogen activated protein kinase (p38 MAPK), which is one of the downstream molecules of PKA, never suppressed the activation of IRE1α-XBP1 pathway. These data indicate that PKA-dependent IRE1α-XBP1 activation is crucial for the transcriptional induction of Ucp1 in brown adipocytes, and they demonstrate a novel, ER stress -independent role of the UPR during thermogenesis.

Luman is involved in osteoclastogenesis through the regulation of DC-STAMP expression, stability and localization.

Luman (also known as CREB3) is a type-II transmembrane transcription factor belonging to the OASIS family that localizes to the endoplasmic reticulum (ER) membrane under normal conditions. In response to ER stress, OASIS-family members are subjected to regulated intramembrane proteolysis (RIP), following which the cleaved N-terminal fragments translocate to the nucleus. In this study, we show that treatment of bone marrow macrophages (BMMs) with cytokines - macrophage colony-stimulating factor (M-CSF) and RANKL (also known as TNFSF11) - causes a time-dependent increase in Luman expression, and that Luman undergoes RIP and becomes activated during osteoclast differentiation. Small hairpin (sh)RNA-mediated knockdown of Luman in BMMs prevented the formation of multinucleated osteoclasts, concomitant with the suppression of DC-STAMP, a protein that is essential for cell-cell fusion in osteoclastogenesis. The N-terminus of Luman facilitates promoter activity of DC-STAMP, resulting in upregulation of DC-STAMP expression. Furthermore, Luman interacts with DC-STAMP, and controls its stability and localization. These results suggest that Luman regulates the multinucleation of osteoclasts by promoting cell fusion of mononuclear osteoclasts through DC-STAMP induction and intracellular distribution during osteoclastogenesis.

Promotion of Cancer Cell Proliferation by Cleaved and Secreted Luminal Domains of ER Stress Transducer BBF2H7.

BBF2H7 is an endoplasmic reticulum (ER)-resident transmembrane basic leucine zipper (bZIP) transcription factor that is cleaved at the transmembrane domain by regulated intramembrane proteolysis in response to ER stress. The cleaved cytoplasmic N-terminus containing transcription activation and bZIP domains translocates into the nucleus to promote the expression of target genes. In chondrocytes, the cleaved luminal C-terminus is extracellularly secreted and facilitates proliferation of neighboring cells through activation of Hedgehog signaling. In the present study, we found that Bbf2h7 expression levels significantly increased by 1.070-2.567-fold in several tumor types including glioblastoma compared with those in respective normal tissues, using the ONCOMINE Cancer Profiling Database. In some Hedgehog ligand-dependent cancer cell lines including glioblastoma U251MG cells, the BBF2H7 C-terminus was secreted from cells into the culture media and promoted cancer cell proliferation through activation of Hedgehog signaling. Knockdown of Bbf2h7 expression suppressed the proliferation of U251MG cells by downregulating Hedgehog signaling. The impaired cell proliferation and Hedgehog signaling were recovered by addition of BBF2H7 C-terminus to the culture medium of Bbf2h7-knockdown U251MG cells. These data suggest that the secreted luminal BBF2H7 C-terminus is involved in Hedgehog ligand-dependent cancer cell proliferation through activation of Hedgehog signaling. Thus, the BBF2H7 C-terminus may be a novel target for the development of anticancer drugs.

Transcriptional regulation of VEGFA by the endoplasmic reticulum stress transducer OASIS in ARPE-19 cells.

BACKGROUND: Vascular endothelial growth factor-A (VEGFA) is the main mediator of angiogenesis. Angiogenesis plays important roles not only in many physiological processes, but also in the pathophysiology of many diseases. VEGFA is one of the therapeutic targets of treatment for ocular diseases with neovascularization. Therefore, elucidation of the regulatory mechanisms for VEGFA expression is important for the development of pharmaceutical drugs. Recent studies have demonstrated that the unfolded protein response is involved in the transcriptional regulation of VEGFA. However, the precise regulation of VEGFA in the human retina is not fully understood. PRINCIPAL FINDINGS: When human retinal pigment epithelial cells, ARPE-19, were exposed to endoplasmic reticulum stressors, VEGFA mRNA was significantly upregulated. The unfolded protein response-related transcription factors XBP1, ATF4, ATF6, and OASIS were expressed in ARPE-19 cells. To determine which transcription factors preferentially contribute to the induction of VEGFA expression after endoplasmic reticulum stress, we carried out reporter assays using an approximately 6-kbp 5'-upstream region of the human VEGFA gene. Among these transcription factors, OASIS acted most effectively on the VEGFA promoter in ARPE-19 cells. Based on data obtained for certain deleted and mutated reporter constructs, we determined that OASIS promoted VEGFA expression by acting on a cyclic AMP-responsive element-like site located at around -500 bp relative to the VEGFA transcription start site. Furthermore, we confirmed that OASIS directly bound to the promoter region containing this site by chromatin immunoprecipitation assays. CONCLUSIONS AND SIGNIFICANCE: We have demonstrated a novel regulatory mechanism for VEGFA transcription by OASIS in human retinal pigment epithelial cells. Chemical compounds that regulate the binding of OASIS to the promoter region of the VEGFA gene may have potential as therapeutic agents for ocular diseases with neovascularization.

The endoplasmic reticulum stress transducer BBF2H7 suppresses apoptosis by activating the ATF5-MCL1 pathway in growth plate cartilage.

BBF2H7 (box B-binding factor 2 human homolog on chromosome 7) is a basic leucine zipper transmembrane transcription factor that belongs to the cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) family. This novel endoplasmic reticulum (ER) stress transducer is localized in the ER and is cleaved in its transmembrane region in response to ER stress. BBF2H7 has been shown to be expressed in proliferating chondrocytes in cartilage during the development of long bones. The target of BBF2H7 is Sec23a, one of the coat protein complex II components. Bbf2h7-deficient (Bbf2h7(-/-)) mice exhibit severe chondrodysplasia, with expansion of the rough ER in proliferating chondrocytes caused by impaired secretion of extracellular matrix (ECM) proteins. We observed a decrease in the number of proliferating chondrocytes in the cartilage of Bbf2h7(-/-) mice. TUNEL staining of the cartilage showed that apoptosis was promoted in Bbf2h7(-/-) chondrocytes. Atf5 (activating transcription factor 5), another member of the CREB/ATF family and an antiapoptotic factor, was also found to be a target of BBF2H7 in chondrocytes. ATF5 activated the transcription of Mcl1 (myeloid cell leukemia sequence 1), which belongs to the antiapoptotic B-cell leukemia/lymphoma 2 family, to suppress apoptosis. Finally, we found that the BBF2H7-ATF5-MCL1 pathway specifically suppressed ER stress-induced apoptosis in chondrocytes. Taken together, our findings indicate that BBF2H7 is activated in response to ER stress caused by synthesis of abundant ECM proteins and plays crucial roles as a bifunctional regulator to accelerate ECM protein secretion and suppress ER stress-induced apoptosis by activating the ATF5-MCL1 pathway during chondrogenesis.

The endoplasmic reticulum stress transducer OASIS is involved in the terminal differentiation of goblet cells in the large intestine.

OASIS is a basic leucine zipper transmembrane transcription factor localized in the endoplasmic reticulum (ER) that is cleaved in its transmembrane region in response to ER stress. This novel ER stress transducer has been demonstrated to express in osteoblasts and astrocytes and promote terminal maturation of these cells. Additionally, OASIS is highly expressed in goblet cells of the large intestine. In this study, we investigated the roles of OASIS in goblet cell differentiation in the large intestine. To analyze the functions of OASIS in goblet cells, we examined morphological changes and the expression of goblet cell differentiation markers in the large intestine of Oasis(-/-) mice. By disrupting the Oasis gene, the number of goblet cells and production of mucus were decreased in the large intestine. Oasis(-/-) goblet cells showed abnormal morphology of mucous vesicles and rough ER. The expression levels of mature goblet cell markers were lower, and conversely those of early goblet cell markers were higher in Oasis(-/-) mice, indicating that differentiation from early to mature goblet cells is impaired in Oasis(-/-) mice. To determine the association of OASIS with other factors involved in goblet cell differentiation, in vitro experiments using a cell culture model were performed. We found that OASIS was activated in response to mild ER stress that is induced in differentiating goblet cells. Knockdown of the Oasis transcript perturbed goblet cell terminal differentiation. Together, our data indicate that OASIS plays crucial roles in promoting the differentiation of early goblet cells to mature goblet cells in the large intestine.

TMP21 transmembrane domain regulates gamma-secretase cleavage.

TMP21 has been shown to be associated with the gamma-secretase complex and can specifically regulate gamma-cleavage without affecting epsilon-mediated proteolysis. To explore the basis of this activity, TMP21 modulation of gamma-secretase activity was investigated independent of epsilon-cleavage using an amyloid-beta precursor proteinepsilon (APPepsilon) construct which lacks the amyloid intracellular domain domain. The APPepsilon construct behaves similarly to the full-length precursor protein with respect to alpha- and beta-cleavages and is able to undergo normal gamma-processing. Co-expression of APPepsilon and TMP21 resulted in the accumulation of membrane-embedded higher molecular weight Abeta-positive fragments, consistent with an inhibition of gamma-secretase cleavage. The APPepsilon system was used to examine the functional domains of TMP21 through the investigation of a series of TMP21-p24a chimera proteins. It was found that chimeras containing the transmembrane domain bound to the gamma-secretase complex and could decrease gamma-secretase proteolytic processing. This was confirmed though investigation of a synthetic peptide corresponding to the TMP21 transmembrane helix. The isolated TMP21 TM peptide but not the homologous p24a domain was able to reduce Abeta production in a dose-dependent fashion. These observations suggest that the TMP21 transmembrane domain promotes its association with the presenilin complex that results in decreased gamma-cleavage activity.

Endoplasmic reticulum stress in myotonic dystrophy type 1 muscle.

In myotonic dystrophy type 1 (DM1), alternative splicing of ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) genes has been reported. These proteins are essential for maintaining intracellular Ca2+ in skeletal muscle. To clarify involvement of endoplasmic reticulum (ER) stress in DM1 muscles, we examined the activation of ER stress-related proteins by immunohistochemistry, western blot analysis and RT-PCR. In four of five DM1 muscle biopsies, except for a muscle biopsy from a patient with the shortest CTG expansion and no myotonia, increased expression of GRP78 and calnexin, and phosphorylation of PERK and eIF-2 alpha were revealed in fibers with sarcoplasmic masses and in highly atrophic fibers with pyknotic nuclear clumps. Caspase-3 and -7 were also expressed in these fibers. Increased expression of GRP78 in these DM1 muscles was confirmed by western blot analysis. GRP78 mRNA and spliced isoform of XBP1 mRNA were also increased in DM1 muscle biopsies. Furthermore, we demonstrated increased expression of GRP78 in highly atrophic fibers with pyknotic nuclear clumps in all three muscle biopsies from neurogenic muscular atrophies. However, five muscle biopsies from central core disease presumably with disturbed intracellular Ca2+ homeostasis and a muscle biopsy from paramyotonia congenita with myotonia showed no activation of these proteins. Taken together, ER stress is involved in muscle wasting in DM1. However, it seems to be evoked not only by disrupted intracellular Ca2+ homeostasis.

BBF2H7, a novel transmembrane bZIP transcription factor, is a new type of endoplasmic reticulum stress transducer.

Endoplasmic reticulum (ER) stress transducers IRE1 (inositol requiring 1), PERK (PKR-like endoplasmic reticulum kinase), and ATF6 (activating transcription factor 6) are well known to transduce signals from the ER to the cytoplasm and nucleus when unfolded proteins accumulate in the ER. Recently, we identified OASIS (old astrocyte specifically induced substance) as a novel ER stress transducer expressed in astrocytes. We report here that BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident transmembrane protein with the bZIP domain in the cytoplasmic portion and structurally homologous to OASIS, is cleaved at the membrane in response to ER stress. The cleaved fragments of BBF2H7 translocate into the nucleus and can bind directly to cyclic AMP-responsive element sites to activate transcription of target genes. Interestingly, although BBF2H7 protein is not expressed under normal conditions, it is markedly induced at the translational level during ER stress, suggesting that BBF2H7 might contribute to only the late phase of unfolded protein response signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently induced in neurons in the peri-infarction region. Furthermore, in a neuroblastoma cell line, BBF2H7 overexpression suppresses ER stress-induced cell death, while small interfering RNA knockdown of BBF2H7 promotes ER stress-induced cell death. Taken together, our results suggest that BBF2H7 is a novel ER stress transducer and could play important roles in preventing accumulation of unfolded proteins in damaged neurons.

Autophagy is activated for cell survival after endoplasmic reticulum stress.

Eukaryotic cells deal with accumulation of unfolded proteins in the endoplasmic reticulum (ER) by the unfolded protein response, involving the induction of molecular chaperones, translational attenuation, and ER-associated degradation, to prevent cell death. Here, we found that the autophagy system is activated as a novel signaling pathway in response to ER stress. Treatment of SK-N-SH neuroblastoma cells with ER stressors markedly induced the formation of autophagosomes, which were recognized at the ultrastructural level. The formation of green fluorescent protein (GFP)-LC3-labeled structures (GFP-LC3 "dots"), representing autophagosomes, was extensively induced in cells exposed to ER stress with conversion from LC3-I to LC3-II. In IRE1-deficient cells or cells treated with c-Jun N-terminal kinase (JNK) inhibitor, the autophagy induced by ER stress was inhibited, indicating that the IRE1-JNK pathway is required for autophagy activation after ER stress. In contrast, PERK-deficient cells and ATF6 knockdown cells showed that autophagy was induced after ER stress in a manner similar to the wild-type cells. Disturbance of autophagy rendered cells vulnerable to ER stress, suggesting that autophagy plays important roles in cell survival after ER stress.

Cleavage of the membrane-bound transcription factor OASIS in response to endoplasmic reticulum stress.

When unfolded or misfolded proteins accumulate in the endoplasmic reticulum (ER), unfolded protein response (UPR) signals are transmitted from the ER to the nucleus and cytoplasm to facilitate protein folding. OASIS (old astrocyte specifically induced substance) is an ER stress transducer in astrocytes, a membrane-bound transcription factor that activates genes in the ER stress response. When unfolded proteins accumulate in the ER, OASIS is cleaved at the membrane to release its cytoplasmic domain, which then enters the nucleus and activates target genes. Here, we showed that OASIS is processed by Site-1 and -2 proteases (S1P and S2P), enzymes that reside at the Golgi apparatus and process activating transcription factor 6 (ATF6), in response to ER stress. We also showed that the cleavage of OASIS is triggered by its translocation to the Golgi apparatus. All deletion mutants for luminal domain of OASIS showed that proteolytic processing and translocation to the Golgi apparatus remained intact, indicating that OASIS does not have significant sequences for Golgi localization signals, different from the case of ATF6, and that there could be other systems for translocation of OASIS to the Golgi apparatus in response to ER stress.

Candidates for tumor-specific alternative splicing.

Gene expression can be regulated not only by transcription and post-transcriptional modifications, but also by splicing regulation. Recent genome-wide analyses have indicated that up to 70% of human genes may have alternatively spliced forms, suggesting that splicing regulation affects a wide range of gene expression. Tumor tissues show significantly altered protein expressions, and this is also thought to be affected by alternative splicing. Although some alternative splicing events have been reported to be cancer specific and others have been predicted from database analyses, the process of alternative splicing and its regulatory machinery are hardly understood. We searched for and detected alternative splicing events that alter protein splicing in all or a subset of tumor tissues. The results revealed tissue-specific alterations of splicing regulation by tumorigenesis, and regulatory cis-element analyses further suggested that multiple splicing regulatory machineries were affected by this process.

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.

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions.

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.