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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 results in embryonic lethality.(A) Summary of genotypes of embryos at different developmental stages and postnatal progenies from interbred Grp94+/− mice. ND, could not be determined by nested PCR. NA, not applicable. (*) denotes deformed embryos. (B) Morphologies of Grp94+/+, +/−, and −/− embryos at E7.5 and E8.5. Embryos were genotyped through PCR. Scale bars represent 200 µm (upper panel), 300 µm (lower panel).
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pone-0010852-g004: Grp94 deficiency results in embryonic lethality.(A) Summary of genotypes of embryos at different developmental stages and postnatal progenies from interbred Grp94+/− mice. ND, could not be determined by nested PCR. NA, not applicable. (*) denotes deformed embryos. (B) Morphologies of Grp94+/+, +/−, and −/− embryos at E7.5 and E8.5. Embryos were genotyped through PCR. Scale bars represent 200 µm (upper panel), 300 µm (lower panel).

Mentions: When the Grp94+/− mice were intercrossed, out of the 80 pups born, the ratio of +/+, +/− and −/− genotype was 25∶55∶0 respectively (Figure 4A). This suggests that Grp94−/− embryos were not viable, whereas Grp94+/− mice were born in the expected Mendelian ratio. Further analysis showed that the Grp94−/− embryos were detectable by nested PCR at E3.5 up to E8.5. By E10.5 and later, no such genotype of embryos was detected and resorbed embryos were evident. Morphological examination of the embryos revealed that at E7.5 and E8.5, the Grp94−/− embryos were much reduced in size with no recognizable structure, compared to normally developing Grp94+/+ and +/− embryos (Figure 4B). The survival of the Grp94−/− embryos past blastocyst stage afforded the opportunity to establish Grp94−/− ESC line for ascertaining GRP94 function in vitro. Two different culture mediums were used in our attempts to derive Grp94−/− ESC lines. The results were summarized in Table 1. Using 44 blastocysts which generated 35 outgrowths, 18 ESC lines were established when they were cultured in the Knockout (KO) DMEM with 15% knockout serum replacement and 5% fetal bovine serum with standard supplements. Surprisingly, none of the 18 lines contained the Grp94−/− genotype. After insulin-transferrin-selenium (ITS) was added to the same medium only a single Grp94−/− ESC line was established among 10 ESC lines derived from 24 outgrowths from 30 blastocysts. This implies that exogenous growth factors are uniquely required for Grp94−/− ESC establishment. Once the lines were established, we observed that ITS was not required for the maintenance of the Grp94−/− ESCs, although upon freezing and thawing, the survival rate of the Grp94−/− ESCs was lower compared to wild-type cells. In order to investigate the differentiation ability of the GRP94 ESCs, paired Grp94+/+ and −/− ESCs were utilized. Our results showed that both genotypes could be induced to differentiate into adipocytes (oil red positive cells), hepatocytes (indocyanine green positive cells) and neurons (Figure 5). The results imply that Grp94−/− ESCs can differentiate into cells of all three germ layers: ectoderm (neurons), mesoderm (adipocytes) and endoderm (hepatocytes). However, the Grp94−/− ESCs are unable to differentiate into cardiomyocyte-like cells in contrast to Grp94+/+ ESCs which can form contracting cells similar to cardiomyocytes (Supplementary Video S1).


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 results in embryonic lethality.(A) Summary of genotypes of embryos at different developmental stages and postnatal progenies from interbred Grp94+/− mice. ND, could not be determined by nested PCR. NA, not applicable. (*) denotes deformed embryos. (B) Morphologies of Grp94+/+, +/−, and −/− embryos at E7.5 and E8.5. Embryos were genotyped through PCR. Scale bars represent 200 µm (upper panel), 300 µm (lower panel).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010852-g004: Grp94 deficiency results in embryonic lethality.(A) Summary of genotypes of embryos at different developmental stages and postnatal progenies from interbred Grp94+/− mice. ND, could not be determined by nested PCR. NA, not applicable. (*) denotes deformed embryos. (B) Morphologies of Grp94+/+, +/−, and −/− embryos at E7.5 and E8.5. Embryos were genotyped through PCR. Scale bars represent 200 µm (upper panel), 300 µm (lower panel).
Mentions: When the Grp94+/− mice were intercrossed, out of the 80 pups born, the ratio of +/+, +/− and −/− genotype was 25∶55∶0 respectively (Figure 4A). This suggests that Grp94−/− embryos were not viable, whereas Grp94+/− mice were born in the expected Mendelian ratio. Further analysis showed that the Grp94−/− embryos were detectable by nested PCR at E3.5 up to E8.5. By E10.5 and later, no such genotype of embryos was detected and resorbed embryos were evident. Morphological examination of the embryos revealed that at E7.5 and E8.5, the Grp94−/− embryos were much reduced in size with no recognizable structure, compared to normally developing Grp94+/+ and +/− embryos (Figure 4B). The survival of the Grp94−/− embryos past blastocyst stage afforded the opportunity to establish Grp94−/− ESC line for ascertaining GRP94 function in vitro. Two different culture mediums were used in our attempts to derive Grp94−/− ESC lines. The results were summarized in Table 1. Using 44 blastocysts which generated 35 outgrowths, 18 ESC lines were established when they were cultured in the Knockout (KO) DMEM with 15% knockout serum replacement and 5% fetal bovine serum with standard supplements. Surprisingly, none of the 18 lines contained the Grp94−/− genotype. After insulin-transferrin-selenium (ITS) was added to the same medium only a single Grp94−/− ESC line was established among 10 ESC lines derived from 24 outgrowths from 30 blastocysts. This implies that exogenous growth factors are uniquely required for Grp94−/− ESC establishment. Once the lines were established, we observed that ITS was not required for the maintenance of the Grp94−/− ESCs, although upon freezing and thawing, the survival rate of the Grp94−/− ESCs was lower compared to wild-type cells. In order to investigate the differentiation ability of the GRP94 ESCs, paired Grp94+/+ and −/− ESCs were utilized. Our results showed that both genotypes could be induced to differentiate into adipocytes (oil red positive cells), hepatocytes (indocyanine green positive cells) and neurons (Figure 5). The results imply that Grp94−/− ESCs can differentiate into cells of all three germ layers: ectoderm (neurons), mesoderm (adipocytes) and endoderm (hepatocytes). However, the Grp94−/− ESCs are unable to differentiate into cardiomyocyte-like cells in contrast to Grp94+/+ ESCs which can form contracting cells similar to cardiomyocytes (Supplementary Video S1).

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