Forest bathing has beneficial effects on human health via showering of forest aerosols as well as... more Forest bathing has beneficial effects on human health via showering of forest aerosols as well as physical relaxation. Terpenes that consist of multiple isoprene units are the largest class of organic compounds produced by various plants, and one of the major components of forest aerosols. Traditionally, terpene-containing plant oil has been used to treat various diseases without knowing the exact functions or the mechanisms of action of the individual bioactive compounds. This review categorizes various terpenes easily obtained from forests according to their anti-inflammatory, anti-tumorigenic, or neuroprotective activities. Moreover, potential action mechanisms of the individual terpenes and their effects on such processes, which are described in various in vivo and in vitro systems, are discussed. In conclusion, the studies that show the biological effectiveness of terpenes support the benefits of forest bathing and propose a potential use of terpenes as chemotherapeutic agents ...
Plasma cells (PCs) are exposed to intense endoplasmic reticulum (ER) stress imposed by enormous r... more Plasma cells (PCs) are exposed to intense endoplasmic reticulum (ER) stress imposed by enormous rates of immunoglobulin (Ig) synthesis and secretion. Therefore, protein homeostasis is crucial for the survival of PCs, but its molecular mechanism remains largely unknown. Here, we found marked overexpression of FK506-binding protein 13 (FKBP13) in long-lived PCs from autoimmune mice and investigated its function using a plasmacytoma cell line secreting IgA. FKBP13 expression was induced largely in the lumen of ER in response to treatment with an ER stressor tunicamycin or overexpression of an adaptive unfolded protein response (UPR) protein X-box binding protein 1 (XBP1). Silencing of FKBP13 expression led to induction of molecules involved in the terminal UPR and ER stress-associated apoptosis. FKBP13 interacted with Ig, facilitated its ubiquitination, and lowered the extent of ER stress. FKBP13 overexpression caused a significant reduction in secreted IgA in plasmacytoma cells, and F...
The American journal of Chinese medicine, Jan 25, 2016
Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widesp... more Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widespread pathology that is characterized by the accumulation of amyloid [Formula: see text]-peptide (A[Formula: see text]) in the brain and various cellular abnormalities, including increased oxidative damage, an amplified inflammatory response, and altered mitogen-activated protein kinase signaling. Based on the complex etiology of AD, traditional medicinal plants with multiple effective components are alternative treatments for patients with AD. In the present study, we investigated the neuroprotective effects of an ethanol extract of Coriandrum sativum (C. sativum) leaves on A[Formula: see text] cytotoxicity and examined the molecular mechanisms underlying the beneficial effects. Although recent studies have shown the benefits of the inhalation of C. sativum oil in an animal model of AD, the detailed molecular mechanisms by which C. sativum exerts its neuroprotective effects are unclear. ...
Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles i... more Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles in inflammation in early-stage rheumatoid arthritis (RA). This study investigated whether glucosamine hydrochloride (GlcN), a nonsteroidal anti-inflammatory agent (NSAID) widely used to treat arthritis, affects the expression of inflammatory cytokines via the unfolded protein response (UPR) in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells). Pretreatment with GlcN reduced the expression of inflammatory cytokines and inhibited cell differentiation. Moreover, GlcN treatment increased the expression of CHOP and BiP/Grp78, the UPR target genes, in the presence or absence of LPS. Indeed, knockdown of CHOP using siRNAs prevented the GlcN-mediated reduction of inflammatory cytokines in LPS-stimulated RAW264.7 cells. Finally, we found that GlcN-mediated induction of CHOP reduced the phosphorylation of JNK and NF-?B in LPS-stimulated RAW264.7 cells. Combined, these results suggest that the GlcN-mediated induction of CHOP negatively regulates the inflammatory response by modulating JNK and NF-?B in LPS-stimulated RAW264.7 cells.
The endoplasmic reticulum (ER) serves many specialized functions in the cell including calcium st... more The endoplasmic reticulum (ER) serves many specialized functions in the cell including calcium storage and gated release, biosynthesis of membrane and secretory proteins, and production of lipids and sterols. Therefore, the ER integrates many internal and external signals to coordinate downstream responses, although the mechanism(s) that maintain homeostasis are largely unknown. When misfolded or unfolded proteins accumulate in the ER, an intracellular signaling pathway termed the unfolded protein response (UPR) is activated. Identification of IRE1 in the yeast Saccharomyces cerevisiae as a proximal sensor in the UPR pathway was a milestone in understanding how the ER responds to the accumulation of unfolded protein and signals transcriptional activation through regulated nonconventional splicing of its substrate mRNA encoding the transcription factor Hac1p. Subsequent studies identified IRE1 and HAC1 homologues in mammalian cells. Here, we summarize various approaches to study the ...
The endoplasmic reticulum (ER) is a reticular membranous network that extends through the cytopla... more The endoplasmic reticulum (ER) is a reticular membranous network that extends through the cytoplasm of the cell and is contiguous with the nuclear envelope. It can therefore sense and transmit signals that originate in any cellular subcompartment. To carry out the folding of intra-organellar, secretory and transmembrane proteins in the ER, the ER has evolved as a specialized protein-folding machine that promotes productive folding and prevents aggregation. Perturbations that alter ER homeostasis therefore disrupt folding, and lead to the accumulation of unfolded proteins and protein aggregates, which are detrimental to cell survival. These perturbations include disturbances in calcium homeostasis or redox status, an increased demand for protein folding due to elevated synthesis of secretory proteins, the expression of mutant or misfolded proteins and nutrient/glucose deprivation. As a consequence, the cell has evolved an adaptive coordinated response to limit further accumulation of unfolded proteins in the ER. This signalling pathway is termed the unfolded protein response (UPR), and the original description came from the demonstration that the UPR is induced when the SV40 T antigen is mistargeted to the ER 1. The UPR is also induced by high-level expression of wild-type secretory proteins 2. On a cellular level, the accumulation of unfolded proteins in the ER lumen induces the transcription of a large set of genes, many of which encode functions to increase the volume and capacity for protein folding, or to increase the degradation of misfolded proteins 3. For example, transcription of the ER CHAPERONE KAR2/BiP/GRP78 (binding Ig protein/glucose regulated protein of molecular weight 78 kDa) is a classical marker for UPR activation in yeast and mammalian cells 4. In addition, translation is attenuated to decrease the protein-folding load on the ER 5. This complex network of physiological responses to ER stress is regulated by only a few ER transmembrane proteins: 'inositol requiring 1' (IRE1), PKR-like endoplasmic reticulum kinase/pancreatic eIF2α kinase (PERK/PEK) and activating transcription factor 6 (ATF6) (REF. 6). In multicellular organisms, if these adaptive responses are not sufficient to relieve the unfolded protein load ('ER stress'), the cell enters one of the cell-death pathways-APOPTOSIS or NECROSIS. Recent evidence supports the hypothesis that the UPR not only promotes cell survival in response to ER stress, but that
An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic... more An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress–associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1β, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimulated by thapsigargin or proinflammatory cytokines. Down-regulation of Grp78 transcripts increased the apoptosis of RA synoviocytes while abolishing TNF- or TGF-β–induced synoviocyte proliferation and cyclin D1 up-regulation. Conversely, overexpression of the Grp78 gene prevented synoviocyte apoptosis. Moreover, Grp78 small interfering RNA inhibited VEGF165-induced angiogenesis in vitro and also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants engrafted into...
HAC1 encodes a transcription factor that mediates the unfolded protein response (UPR) in Saccharo... more HAC1 encodes a transcription factor that mediates the unfolded protein response (UPR) in Saccharomyces cerevisiae. We characterized hac1⌬ mutants in the sporulation-proficient SK1 genetic background and found a novel function for HAC1 in haploid tolerance. hac1⌬ spore clones contain a diploid DNA content as determined by fluorescence-activated cell sorting and genetic analyses. Autodiploidization of hac1 spore clones occurred after germination; hac1 spores were born haploid, but efficiently generated diploid progeny during the subsequent mitotic division. Once the hac1 mutant acquired a diploid DNA content, no further ploidy increase was observed. Interestingly, the increase in genome content following meiosis was not a general property associated with hac1 spore clones; instead, it was restricted to an inability to tolerate the haploid state. Genetic analyses involving the UPR target gene KAR2 and the UPR regulator IRE1 revealed that autodiploidization associated with hac1 mutants is a consequence of its role in the UPR pathway. Inhibition of the UPR pathway induces autodiploidization, and constitutive activation of UPR target genes suppresses this response.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular si... more Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signal transduction program termed the unfolded protein response (UPR). In mammalian cells, the UPR is signaled in part through dimerization of ER membrane-localized IRE1␣ to activate its protein kinase and endoribonuclease activities. Activated IRE1␣ cleaves XBP1 mRNA at two sites to initiate an unconventional splicing reaction. The 5 and 3 fragments are subsequently joined by an RNA ligase activity, thereby removing a 26-base intron. This splicing reaction creates a translational frameshift to produce a functional XBP1 transcription factor. However, the cellular location and physiological processes required for splicing of XBP1 mRNA are not well characterized. To study these processes, XBP1 mRNAs were engineered in which translation of enhanced green fluorescence protein or luciferase required splicing of the XBP1 intron. Using cell lines that continuously or transiently express these reporter constructs, we show that cytoplasmic unspliced XBP1 mRNA is efficiently spliced by activated IRE1␣ and requires ongoing cellular transcription but not active translation. The XBP1 intron was effectively removed from RNA substrates transcribed from T7 RNA polymerase or delivered directly to the cytoplasm by RNA transfection, thus indicating that the splicing reaction does not require nuclear processing of the RNA substrate. Analysis of nuclear and cytoplasmic RNA fractions demonstrated that XBP1 mRNA splicing occurs in the cytoplasm. Moreover, an artificial F v-IRE1␣⌬N was engineered that was able to splice XBP1 mRNA upon chemical-induced dimerization. These findings demonstrate that IRE1␣ dimerization is sufficient to activate XBP1 mRNA splicing in the absence of the UPR. We propose that XBP1 mRNA cytoplasmic splicing provides a novel mechanism to rapidly induce translation of a transcription factor in response to a specific stimulus.
All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulu... more All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed the unfolded protein response (UPR). In yeast, a type-I ER transmembrane protein kinase, Ire1p, is the proximal sensor of unfolded proteins in the ER lumen that initiates an unconventional splicing reaction onHAC1mRNA. Hac1p is a transcription factor required for induction of UPR genes. In higher eukaryotic cells, the UPR also induces site-2 protease (S2P)-mediated cleavage of ER-localized ATF6 to generate an N-terminal fragment that activates transcription of UPR genes. To elucidate the requirements for IRE1α and ATF6 for signaling the mammalian UPR, we identified a UPR reporter gene that was defective for induction inIRE1α-null mouse embryonic fibroblasts and S2P-deficient Chinese hamster ovary (CHO) cells. We show that the endoribonuclease activity of IRE1α is required to spliceXBP1(X-boxbindingprotein) mRNA to generate a new C terminus, ...
The unfolded protein response (UPR) is a signal transduction pathway that is activated by the acc... more The unfolded protein response (UPR) is a signal transduction pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In Saccharomyces cerevisiae the ER transmembrane receptor, Ire1p, transmits the signal to the nucleus culminating in the transcriptional activation of genes encoding an adaptive response. Yeast Ire1p requires both protein kinase and site-specific endoribonuclease (RNase) activities to signal the UPR. In mammalian cells, two homologs, Ire1α and Ire1β, are implicated in signaling the UPR. To elucidate the RNase requirement for mammalian Ire1 function, we have identified five amino acid residues within IRE1α that are essential for RNase activity but not kinase activity. These mutants were used to demonstrate that the RNase activity is required for UPR activation by IRE1α and IRE1β. In addition, the data support that IRE1 RNase is activated by dimerization-induced trans-autophosphorylation and requires a homodimer of catalytic...
Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis... more Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.
A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UP... more A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6α (ATF6α), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6...
Forest bathing has beneficial effects on human health via showering of forest aerosols as well as... more Forest bathing has beneficial effects on human health via showering of forest aerosols as well as physical relaxation. Terpenes that consist of multiple isoprene units are the largest class of organic compounds produced by various plants, and one of the major components of forest aerosols. Traditionally, terpene-containing plant oil has been used to treat various diseases without knowing the exact functions or the mechanisms of action of the individual bioactive compounds. This review categorizes various terpenes easily obtained from forests according to their anti-inflammatory, anti-tumorigenic, or neuroprotective activities. Moreover, potential action mechanisms of the individual terpenes and their effects on such processes, which are described in various in vivo and in vitro systems, are discussed. In conclusion, the studies that show the biological effectiveness of terpenes support the benefits of forest bathing and propose a potential use of terpenes as chemotherapeutic agents ...
Plasma cells (PCs) are exposed to intense endoplasmic reticulum (ER) stress imposed by enormous r... more Plasma cells (PCs) are exposed to intense endoplasmic reticulum (ER) stress imposed by enormous rates of immunoglobulin (Ig) synthesis and secretion. Therefore, protein homeostasis is crucial for the survival of PCs, but its molecular mechanism remains largely unknown. Here, we found marked overexpression of FK506-binding protein 13 (FKBP13) in long-lived PCs from autoimmune mice and investigated its function using a plasmacytoma cell line secreting IgA. FKBP13 expression was induced largely in the lumen of ER in response to treatment with an ER stressor tunicamycin or overexpression of an adaptive unfolded protein response (UPR) protein X-box binding protein 1 (XBP1). Silencing of FKBP13 expression led to induction of molecules involved in the terminal UPR and ER stress-associated apoptosis. FKBP13 interacted with Ig, facilitated its ubiquitination, and lowered the extent of ER stress. FKBP13 overexpression caused a significant reduction in secreted IgA in plasmacytoma cells, and F...
The American journal of Chinese medicine, Jan 25, 2016
Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widesp... more Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widespread pathology that is characterized by the accumulation of amyloid [Formula: see text]-peptide (A[Formula: see text]) in the brain and various cellular abnormalities, including increased oxidative damage, an amplified inflammatory response, and altered mitogen-activated protein kinase signaling. Based on the complex etiology of AD, traditional medicinal plants with multiple effective components are alternative treatments for patients with AD. In the present study, we investigated the neuroprotective effects of an ethanol extract of Coriandrum sativum (C. sativum) leaves on A[Formula: see text] cytotoxicity and examined the molecular mechanisms underlying the beneficial effects. Although recent studies have shown the benefits of the inhalation of C. sativum oil in an animal model of AD, the detailed molecular mechanisms by which C. sativum exerts its neuroprotective effects are unclear. ...
Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles i... more Macrophages secrete inflammatory cytokines and mono-nitrogen oxide (NO), and play crucial roles in inflammation in early-stage rheumatoid arthritis (RA). This study investigated whether glucosamine hydrochloride (GlcN), a nonsteroidal anti-inflammatory agent (NSAID) widely used to treat arthritis, affects the expression of inflammatory cytokines via the unfolded protein response (UPR) in lipopolysaccharide (LPS)-stimulated mouse macrophages (RAW264.7 cells). Pretreatment with GlcN reduced the expression of inflammatory cytokines and inhibited cell differentiation. Moreover, GlcN treatment increased the expression of CHOP and BiP/Grp78, the UPR target genes, in the presence or absence of LPS. Indeed, knockdown of CHOP using siRNAs prevented the GlcN-mediated reduction of inflammatory cytokines in LPS-stimulated RAW264.7 cells. Finally, we found that GlcN-mediated induction of CHOP reduced the phosphorylation of JNK and NF-?B in LPS-stimulated RAW264.7 cells. Combined, these results suggest that the GlcN-mediated induction of CHOP negatively regulates the inflammatory response by modulating JNK and NF-?B in LPS-stimulated RAW264.7 cells.
The endoplasmic reticulum (ER) serves many specialized functions in the cell including calcium st... more The endoplasmic reticulum (ER) serves many specialized functions in the cell including calcium storage and gated release, biosynthesis of membrane and secretory proteins, and production of lipids and sterols. Therefore, the ER integrates many internal and external signals to coordinate downstream responses, although the mechanism(s) that maintain homeostasis are largely unknown. When misfolded or unfolded proteins accumulate in the ER, an intracellular signaling pathway termed the unfolded protein response (UPR) is activated. Identification of IRE1 in the yeast Saccharomyces cerevisiae as a proximal sensor in the UPR pathway was a milestone in understanding how the ER responds to the accumulation of unfolded protein and signals transcriptional activation through regulated nonconventional splicing of its substrate mRNA encoding the transcription factor Hac1p. Subsequent studies identified IRE1 and HAC1 homologues in mammalian cells. Here, we summarize various approaches to study the ...
The endoplasmic reticulum (ER) is a reticular membranous network that extends through the cytopla... more The endoplasmic reticulum (ER) is a reticular membranous network that extends through the cytoplasm of the cell and is contiguous with the nuclear envelope. It can therefore sense and transmit signals that originate in any cellular subcompartment. To carry out the folding of intra-organellar, secretory and transmembrane proteins in the ER, the ER has evolved as a specialized protein-folding machine that promotes productive folding and prevents aggregation. Perturbations that alter ER homeostasis therefore disrupt folding, and lead to the accumulation of unfolded proteins and protein aggregates, which are detrimental to cell survival. These perturbations include disturbances in calcium homeostasis or redox status, an increased demand for protein folding due to elevated synthesis of secretory proteins, the expression of mutant or misfolded proteins and nutrient/glucose deprivation. As a consequence, the cell has evolved an adaptive coordinated response to limit further accumulation of unfolded proteins in the ER. This signalling pathway is termed the unfolded protein response (UPR), and the original description came from the demonstration that the UPR is induced when the SV40 T antigen is mistargeted to the ER 1. The UPR is also induced by high-level expression of wild-type secretory proteins 2. On a cellular level, the accumulation of unfolded proteins in the ER lumen induces the transcription of a large set of genes, many of which encode functions to increase the volume and capacity for protein folding, or to increase the degradation of misfolded proteins 3. For example, transcription of the ER CHAPERONE KAR2/BiP/GRP78 (binding Ig protein/glucose regulated protein of molecular weight 78 kDa) is a classical marker for UPR activation in yeast and mammalian cells 4. In addition, translation is attenuated to decrease the protein-folding load on the ER 5. This complex network of physiological responses to ER stress is regulated by only a few ER transmembrane proteins: 'inositol requiring 1' (IRE1), PKR-like endoplasmic reticulum kinase/pancreatic eIF2α kinase (PERK/PEK) and activating transcription factor 6 (ATF6) (REF. 6). In multicellular organisms, if these adaptive responses are not sufficient to relieve the unfolded protein load ('ER stress'), the cell enters one of the cell-death pathways-APOPTOSIS or NECROSIS. Recent evidence supports the hypothesis that the UPR not only promotes cell survival in response to ER stress, but that
An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic... more An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress–associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1β, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimulated by thapsigargin or proinflammatory cytokines. Down-regulation of Grp78 transcripts increased the apoptosis of RA synoviocytes while abolishing TNF- or TGF-β–induced synoviocyte proliferation and cyclin D1 up-regulation. Conversely, overexpression of the Grp78 gene prevented synoviocyte apoptosis. Moreover, Grp78 small interfering RNA inhibited VEGF165-induced angiogenesis in vitro and also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants engrafted into...
HAC1 encodes a transcription factor that mediates the unfolded protein response (UPR) in Saccharo... more HAC1 encodes a transcription factor that mediates the unfolded protein response (UPR) in Saccharomyces cerevisiae. We characterized hac1⌬ mutants in the sporulation-proficient SK1 genetic background and found a novel function for HAC1 in haploid tolerance. hac1⌬ spore clones contain a diploid DNA content as determined by fluorescence-activated cell sorting and genetic analyses. Autodiploidization of hac1 spore clones occurred after germination; hac1 spores were born haploid, but efficiently generated diploid progeny during the subsequent mitotic division. Once the hac1 mutant acquired a diploid DNA content, no further ploidy increase was observed. Interestingly, the increase in genome content following meiosis was not a general property associated with hac1 spore clones; instead, it was restricted to an inability to tolerate the haploid state. Genetic analyses involving the UPR target gene KAR2 and the UPR regulator IRE1 revealed that autodiploidization associated with hac1 mutants is a consequence of its role in the UPR pathway. Inhibition of the UPR pathway induces autodiploidization, and constitutive activation of UPR target genes suppresses this response.
Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular si... more Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signal transduction program termed the unfolded protein response (UPR). In mammalian cells, the UPR is signaled in part through dimerization of ER membrane-localized IRE1␣ to activate its protein kinase and endoribonuclease activities. Activated IRE1␣ cleaves XBP1 mRNA at two sites to initiate an unconventional splicing reaction. The 5 and 3 fragments are subsequently joined by an RNA ligase activity, thereby removing a 26-base intron. This splicing reaction creates a translational frameshift to produce a functional XBP1 transcription factor. However, the cellular location and physiological processes required for splicing of XBP1 mRNA are not well characterized. To study these processes, XBP1 mRNAs were engineered in which translation of enhanced green fluorescence protein or luciferase required splicing of the XBP1 intron. Using cell lines that continuously or transiently express these reporter constructs, we show that cytoplasmic unspliced XBP1 mRNA is efficiently spliced by activated IRE1␣ and requires ongoing cellular transcription but not active translation. The XBP1 intron was effectively removed from RNA substrates transcribed from T7 RNA polymerase or delivered directly to the cytoplasm by RNA transfection, thus indicating that the splicing reaction does not require nuclear processing of the RNA substrate. Analysis of nuclear and cytoplasmic RNA fractions demonstrated that XBP1 mRNA splicing occurs in the cytoplasm. Moreover, an artificial F v-IRE1␣⌬N was engineered that was able to splice XBP1 mRNA upon chemical-induced dimerization. These findings demonstrate that IRE1␣ dimerization is sufficient to activate XBP1 mRNA splicing in the absence of the UPR. We propose that XBP1 mRNA cytoplasmic splicing provides a novel mechanism to rapidly induce translation of a transcription factor in response to a specific stimulus.
All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulu... more All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed the unfolded protein response (UPR). In yeast, a type-I ER transmembrane protein kinase, Ire1p, is the proximal sensor of unfolded proteins in the ER lumen that initiates an unconventional splicing reaction onHAC1mRNA. Hac1p is a transcription factor required for induction of UPR genes. In higher eukaryotic cells, the UPR also induces site-2 protease (S2P)-mediated cleavage of ER-localized ATF6 to generate an N-terminal fragment that activates transcription of UPR genes. To elucidate the requirements for IRE1α and ATF6 for signaling the mammalian UPR, we identified a UPR reporter gene that was defective for induction inIRE1α-null mouse embryonic fibroblasts and S2P-deficient Chinese hamster ovary (CHO) cells. We show that the endoribonuclease activity of IRE1α is required to spliceXBP1(X-boxbindingprotein) mRNA to generate a new C terminus, ...
The unfolded protein response (UPR) is a signal transduction pathway that is activated by the acc... more The unfolded protein response (UPR) is a signal transduction pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In Saccharomyces cerevisiae the ER transmembrane receptor, Ire1p, transmits the signal to the nucleus culminating in the transcriptional activation of genes encoding an adaptive response. Yeast Ire1p requires both protein kinase and site-specific endoribonuclease (RNase) activities to signal the UPR. In mammalian cells, two homologs, Ire1α and Ire1β, are implicated in signaling the UPR. To elucidate the RNase requirement for mammalian Ire1 function, we have identified five amino acid residues within IRE1α that are essential for RNase activity but not kinase activity. These mutants were used to demonstrate that the RNase activity is required for UPR activation by IRE1α and IRE1β. In addition, the data support that IRE1 RNase is activated by dimerization-induced trans-autophosphorylation and requires a homodimer of catalytic...
Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis... more Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.
A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UP... more A link exists between endoplasmic reticulum (ER) biogenesis and the unfolded protein response (UPR), a complex set of signaling mechanisms triggered by increased demands on the protein folding capacity of the ER. The UPR transcriptional activator X-box binding protein 1 (XBP1) regulates the expression of proteins that function throughout the secretory pathway and is necessary for development of an expansive ER network. We previously demonstrated that overexpression of XBP1(S), the active form of XBP1 generated by UPR-mediated splicing of Xbp1 mRNA, augments the activity of the cytidine diphosphocholine (CDP-choline) pathway for biosynthesis of phosphatidylcholine (PtdCho) and induces ER biogenesis. Another UPR transcriptional activator, activating transcription factor 6α (ATF6α), primarily regulates expression of ER resident proteins involved in the maturation and degradation of ER client proteins. Here, we demonstrate that enforced expression of a constitutively active form of ATF6...
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Papers by Kyungho Lee