Analysis of ATF6 Activation in Site-2 Protease-deficient Chinese Hamster Ovary Cells

Nadanaka, Satomi; Yoshida, Hiderou; Sato, Ryuichiro; Mori, Kazutoshi

doi:10.1247/csf.06015

Cited by 26 publications

(20 citation statements)

References 30 publications

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“…This is similar to a recent study showing that basal GRP78 levels are decreased in muscle tissue from ATF6␣ Ϫ/Ϫ mice (44). In addition, Chinese hamster ovary cells deficient in Site-2 protease have reduced ER function, suggesting that activation of ATF6␣ is important for the homeostasis of the ER in unstressed as well as ER stressed cells (26). In pancreatic ␤-cells an additional effect of maintaining some active ATF6␣ expression is that it can contribute to keeping insulin gene expression turned off under basal (low glucose) conditions.…”

Section: Present In Certain Cells Such As ␤-Cells Under Control Non-esupporting

confidence: 91%

Pancreatic β-cells depend on basal expression of active ATF6α-p50 for cell survival even under nonstress conditions

Teodoro

Odisho

Sidorova

et al. 2012

American Journal of Physiology-Cell Physiology

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Activating transcription factor 6 (ATF6) is one of three principle endoplasmic reticulum (ER) stress response proteins and becomes activated when ER homeostasis is perturbed. ATF6 functions to increase ER capacity by stimulating transcription of ER-resident chaperone genes such as GRP78. Using an antibody that recognizes active ATF6α-p50, we found that active ATF6α was detected in insulinoma cells and rodent islets even under basal conditions and the levels were further increased by ER stress. To examine the function of ATF6α-p50, we depleted endogenous ATF6α-p50 levels using small interfering RNA in insulinoma cells. Knockdown of endogenous ATF6α-p50 levels by ∼60% resulted in a reduction in the steady-state levels of GRP78 mRNA and protein levels in nonstressed cells. Furthermore, ATF6α knockdown resulted in an apoptotic phenotype. We hypothesized that removal of the ATF6α branch of the unfolded protein response (UPR) would result in ER stress. However, neither the PKR-like endoplasmic reticulum kinase (PERK), nor the inositol requiring enzyme 1 (IRE1) pathways of the UPR were significantly activated in ATF6α knockdown cells, although these cells were more sensitive to ER stress-inducing compounds. Interestingly, phosphorylation of JNK, p38, and c-Jun were elevated in ATF6α knockdown cells and inhibition of JNK or p38 kinases prevented apoptosis. These results suggest that ATF6α may have a role in maintaining β-cell survival even in the absence of ER stress.

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Section: Present In Certain Cells Such As ␤-Cells Under Control Non-esupporting

confidence: 91%

Pancreatic β-cells depend on basal expression of active ATF6α-p50 for cell survival even under nonstress conditions

Teodoro

Odisho

Sidorova

et al. 2012

American Journal of Physiology-Cell Physiology

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“…We showed in a preceding paper (Nadanaka et al, 2006) that the ERSE-mediated transcriptional enhancement in response to ER stress observed in WT cells was abolished in M19 cells, and here reproduced this result in vector-transfected WT and M19 cells (Fig. 2B).…”

Section: Defective Induction Of Xbp1 and Its Target In M19 Cellssupporting

confidence: 88%

“…In the meantime, the plausibility of this notion can at least be tested using mammalian cells unable to activate ATF6. We showed in a preceding paper (Nadanaka et al, 2006) that, as expected, the M19 cells, a CHO cell mutant deficient in Site-2 protease, are unable to activate ATF6. This cell line therefore afforded us the opportunity to determine the consequences of a lack of ATF6 activation.…”

Section: Discussionsupporting

confidence: 78%

“…Importantly, comparable levels of pXBP1(S) were produced after the addition of tunicamycin in WT and M19 cells overexpressing XBP1 unspliced mRNA (lanes 4 and 8, respectively), showing that the introduction of a large amount of XBP1 unspliced mRNA into M19 cells restored the ability of these cells to produce pXBP1(S) to a level comparable to that in WT cells. We showed in a preceding paper (Nadanaka et al, 2006) that IRE1 was constitutively activated in M19 cells as judged from almost complete splicing of XBP1 mRNA in the absence of ER stress. This notion was supported by the detection of a small amount of pXBP1(S) in unstressed M19 cells (Fig.…”

Section: Defective Induction Of Xbp1 and Its Target In M19 Cellsmentioning

confidence: 85%

“…Nonetheless, marked difference in the level of pXBP1(S) detected between unstressed and tunicamycintreated M19 cells after overexpression of XBP1 unspliced mRNA ( Fig. 2A, compare lane 7 with lane 8) indicated that IRE1 was activated very weakly in unstressed M19 cells, similarly to PERK perhaps, only a small portion of which was shown to be phosphorylated and thus activated in unstressed M19 cells (Nadanaka et al, 2006). This extent of activation was enough for splicing of all XBP1 mRNA expressed at a low level but far below for that overexpressed by transfection.…”

Section: Defective Induction Of Xbp1 and Its Target In M19 Cellsmentioning

confidence: 94%

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XBP1 is Critical to Protect Cells from Endoplasmic Reticulum Stress: Evidence from Site-2 Protease-deficient Chinese Hamster Ovary Cells

Yoshida

Nadanaka

Sato

et al. 2006

Cell Struct. Funct.

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ABSTRACT. XBP1 is a transcription factor downstream of IRE1, a transmembrane protein in the endoplasmic reticulum (ER) which functions as a sensor and transducer of ER stress. XBP1 mRNA is constitutively expressed at a low level as an intron-containing precursor mRNA (unspliced mRNA), which is subject to IRE1-mediated splicing reaction upon ER stress to produce the active form of XBP1, pXBP1(S). Because the XBP1 promoter carries a perfect ER stress-response element, namely, the cis-acting element responsible for the induction of ER chaperones, and XBP1 mRNA is induced in response to ER stress with a time course similar to that of ER chaperone mRNAs, it is conjectured that transcription factor ATF6, activated immediately upon ER stress, induces the transcription of not only ER chaperone genes but also of XBP1 gene, such that pXBP1(S) produced by the splicing of an increased level of XBP1 mRNA escapes from proteasome-mediated degradation. Here, we examined this notion by determining the induction of XBP1 mRNA and pXBP1(S) in mutant Chinese hamster ovary (M19) cells deficient in Site-2 protease, which executes the last step of ER stress-induced activation of ATF6. We found that the induction of XBP1 mRNA and pXBP1(S) was greatly reduced in M19 cells as compared with wildtype cells, leading to a marked reduction in the extent of induction of XBP1-target gene. M19 cells were much more sensitive to ER stress than wild-type cells. Importantly, overexpression of XBP1 unspliced mRNA in M19 cells reversed all of these phenotypes. We concluded that ATF6-mediated induction of XBP1 mRNA is important to the production of pXBP1(S), activation of XBP1-target genes, and protection of cells from ER stress.

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Regulation of Golgi signaling and trafficking by the KDEL receptor

Cancino

Jung

Luini

2013

Histochem Cell Biol

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Intracellular membrane transport involves the well-coordinated engagement of a series of organelles and molecular machineries that ensure that proteins are delivered to their correct cellular locations according to their function. To maintain the homeostasis of the secretory system, the fluxes of membranes and protein across the transport compartments must be precisely balanced. This control should rely on a mechanism that senses the movement of the traffic and generates the required homeostatic response. Due to its central position in the secretory pathway and to the large amounts of signaling molecules associated with it, the Golgi complex represents the ideal candidate for this regulation. The generation of autonomous signaling by the Golgi complex in response to the arrival of cargo from the endoplasmic reticulum (ER) has been experimentally addressed only in recent years. These studies have revealed that cargo moving from the ER to the Golgi activates a series of signaling pathways, the functional significance of which appears to be to maintain the homeostasis of the Golgi complex and to activate Golgi trafficking according to internal demand. We have termed this regulatory mechanism the Golgi control system. A key player in this Golgi control system is the KDEL receptor, which has previously been shown to retrieve chaperones back to the endoplasmic reticulum and more recently to behave as a signaling receptor. Here, we discuss the particular role of KDEL receptor signaling in the regulation of important pathways involved in the maintenance of the homeostasis of the transport apparatus, and in particular, of the Golgi complex.

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Analysis of ATF6 Activation in Site-2 Protease-deficient Chinese Hamster Ovary Cells

Cited by 26 publications

References 30 publications

Pancreatic β-cells depend on basal expression of active ATF6α-p50 for cell survival even under nonstress conditions

Pancreatic β-cells depend on basal expression of active ATF6α-p50 for cell survival even under nonstress conditions

XBP1 is Critical to Protect Cells from Endoplasmic Reticulum Stress: Evidence from Site-2 Protease-deficient Chinese Hamster Ovary Cells

Regulation of Golgi signaling and trafficking by the KDEL receptor

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