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Targeted mutation of the mouse Grp94 gene disrupts development and perturbs endoplasmic reticulum stress signaling.

Mao C, Wang M, Luo B, Wey S, Dong D, Wesselschmidt R, Rawlings S, Lee AS - PLoS ONE (2010)

Bottom Line: Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response.To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94-/- embryonic stem cells.Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Southern California Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California, USA.

ABSTRACT
Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/- mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94-/- embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function.

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GRP94 deficiency has no effect on some UPR targets.(A) Representative Western blot results on the effect of GRP94 depletion on UPR targets. Grp94+/+ and −/− ESCs were treated with 300 nM Tg for the indicated time (hr). The whole cell lysates were subjected to Western blot to detect the level of EDEM, PERK phosphorylation, total PERK, CHOP and p62. (B) The same as in (A) with Tg treatment for 0, 4 and 16 hr. The level of eIF2α phosphorylation, total eIF2α, ATF4 and HSP70 were measured by Western blot. These experiments were repeated two to four times. (C) The levels of p-PERK and p-eIF2α were quantitated and normalized against total PERK and eIF2α, respectively. The data are presented as mean±s.e.m. The experiments were repeated two to four times.
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pone-0010852-g008: GRP94 deficiency has no effect on some UPR targets.(A) Representative Western blot results on the effect of GRP94 depletion on UPR targets. Grp94+/+ and −/− ESCs were treated with 300 nM Tg for the indicated time (hr). The whole cell lysates were subjected to Western blot to detect the level of EDEM, PERK phosphorylation, total PERK, CHOP and p62. (B) The same as in (A) with Tg treatment for 0, 4 and 16 hr. The level of eIF2α phosphorylation, total eIF2α, ATF4 and HSP70 were measured by Western blot. These experiments were repeated two to four times. (C) The levels of p-PERK and p-eIF2α were quantitated and normalized against total PERK and eIF2α, respectively. The data are presented as mean±s.e.m. The experiments were repeated two to four times.

Mentions: As a downstream target of XBP-1, Tg-induction of EDEM was also reduced in Grp94−/− ESCs (Figure 8A). In contrast, other UPR targets and cellular proteins were only mildly affected or completely unaffected by GRP94 depletion. For example, Tg-induced phosphorylation of PERK was only slightly increased in Grp94−/− ESCs at 16 hr, with no corresponding increase of CHOP (Figure 8A). The level of p62, a cytosolic protein which binds to polyubiquitinated proteins and targets them to the autophagy machinery for degradation [40] was also not affected in the Tg-treated cells of both genotypes (Figure 8A). There were substantial variations on the Tg-induced phosphorylation of eIF2α in the Grp94−/− ESCs however on the average there was no increase (Figure 8B and data not shown). Tg-induction of ATF4 was mildly increased while the level of HSP70, a major heat shock protein related to GRP78, was not affected (Figure 8B). Quantitation of p-PERK and p-eIF2α levels from several independent experiments showed no statistically significant change between the two genotypes (Figure 8C). Thus, the minor differences observed are likely due to experimental variations in the cultured ESC system.


Targeted mutation of the mouse Grp94 gene disrupts development and perturbs endoplasmic reticulum stress signaling.

Mao C, Wang M, Luo B, Wey S, Dong D, Wesselschmidt R, Rawlings S, Lee AS - PLoS ONE (2010)

GRP94 deficiency has no effect on some UPR targets.(A) Representative Western blot results on the effect of GRP94 depletion on UPR targets. Grp94+/+ and −/− ESCs were treated with 300 nM Tg for the indicated time (hr). The whole cell lysates were subjected to Western blot to detect the level of EDEM, PERK phosphorylation, total PERK, CHOP and p62. (B) The same as in (A) with Tg treatment for 0, 4 and 16 hr. The level of eIF2α phosphorylation, total eIF2α, ATF4 and HSP70 were measured by Western blot. These experiments were repeated two to four times. (C) The levels of p-PERK and p-eIF2α were quantitated and normalized against total PERK and eIF2α, respectively. The data are presented as mean±s.e.m. The experiments were repeated two to four times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877114&req=5

pone-0010852-g008: GRP94 deficiency has no effect on some UPR targets.(A) Representative Western blot results on the effect of GRP94 depletion on UPR targets. Grp94+/+ and −/− ESCs were treated with 300 nM Tg for the indicated time (hr). The whole cell lysates were subjected to Western blot to detect the level of EDEM, PERK phosphorylation, total PERK, CHOP and p62. (B) The same as in (A) with Tg treatment for 0, 4 and 16 hr. The level of eIF2α phosphorylation, total eIF2α, ATF4 and HSP70 were measured by Western blot. These experiments were repeated two to four times. (C) The levels of p-PERK and p-eIF2α were quantitated and normalized against total PERK and eIF2α, respectively. The data are presented as mean±s.e.m. The experiments were repeated two to four times.
Mentions: As a downstream target of XBP-1, Tg-induction of EDEM was also reduced in Grp94−/− ESCs (Figure 8A). In contrast, other UPR targets and cellular proteins were only mildly affected or completely unaffected by GRP94 depletion. For example, Tg-induced phosphorylation of PERK was only slightly increased in Grp94−/− ESCs at 16 hr, with no corresponding increase of CHOP (Figure 8A). The level of p62, a cytosolic protein which binds to polyubiquitinated proteins and targets them to the autophagy machinery for degradation [40] was also not affected in the Tg-treated cells of both genotypes (Figure 8A). There were substantial variations on the Tg-induced phosphorylation of eIF2α in the Grp94−/− ESCs however on the average there was no increase (Figure 8B and data not shown). Tg-induction of ATF4 was mildly increased while the level of HSP70, a major heat shock protein related to GRP78, was not affected (Figure 8B). Quantitation of p-PERK and p-eIF2α levels from several independent experiments showed no statistically significant change between the two genotypes (Figure 8C). Thus, the minor differences observed are likely due to experimental variations in the cultured ESC system.

Bottom Line: Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response.To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94-/- embryonic stem cells.Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Southern California Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California, USA.

ABSTRACT
Glucose-regulated protein 94 (GRP94) is one of the most abundant endoplasmic reticulum (ER) resident proteins and is the ER counterpart of the cytoplasmic heat shock protein 90 (HSP90). GRP94, a component of the GRP78 chaperone system in protein processing, has pro-survival properties with implicated function in cancer progression and autoimmune disease. Previous studies on the loss of GRP94 function showed that it is required for embryonic development, regulation of toll-like receptors and innate immunity of macrophages. Here we report the creation of mouse models targeting exon 2 of the Grp94 allele that allows both traditional and conditional knockout (KO) of Grp94. In this study, we utilized the viable Grp94+/+ and +/- mice, as well as primary mouse embryonic fibroblasts generated from them as experimental tools to study its role in ER chaperone balance and ER stress signaling. Our studies reveal that while Grp94 heterozygosity reduces GRP94 level it does not alter ER chaperone levels or the ER stress response. To study the effect of complete loss of GRP94 function, since homozygous GRP94 KO leads to embryonic lethality, we generated Grp94-/- embryonic stem cells. In contrast to Grp94 heterozygosity, complete knockout of GRP94 leads to compensatory upregulation of the ER chaperones GRP78, calnexin and calreticulin but not protein disulphide isomerase. Unexpectedly, loss of GRP94 leads to significant decrease in the level of ER-stress induced spliced form of XBP-1 protein, a downstream target of the IRE1 signaling pathway. Furthermore, from analysis of microarray database and immunohistochemical staining, we present predictions where GRP94 may play an important role in specific adult organ homeostasis and function.

Show MeSH
Related in: MedlinePlus