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Previous Article | Next Article 
Molecular and Cellular Biology, August 2006, p. 5688-5697, Vol. 26, No. 15
0270-7306/06/$08.00+0 doi:10.1128/MCB.00779-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Changhui Mao,
Brenda Lee, and
Amy S. Lee*
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9176
Received 3 May 2006/ Accepted 6 May 2006
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ABSTRACT |
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INTRODUCTION |
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The first hint that GRP78 function is critical for eukaryotic cell function is that its amino acid sequence is highly evolutionarily conserved from yeast to human (45). We reported previously that by the criterion of two-dimensional gel electrophoresis, GRP78 is below the detection limit at the one-cell stage and is weakly detectable from the two-cell stage to the morula stage but is abundant at the blastocyst stage in the developing mouse embryo (20). This suggests a physiological need for the induction of GRP78 during the blastocyst stage. However, the function of GRP78 has not yet been tested directly in vivo. Here, we describe the use of a transgenic (TG) approach to probe the transcriptional activation of the Grp78 promoter during early development and to map the region of the promoter required for its induction. To understand GRP78 function in vivo, we generated heterozygous and knockout (KO) Grp78 mouse models. We discovered that the targeted disruption of one copy of the Grp78 gene results in a 50% reduction in the GRP78 protein level and specific up-regulation of the ER proteins GRP94 and protein disulfide isomerase (PDI) at both the transcript and protein levels. However, these compensations cannot rescue Grp78–/– embryos from lethality at the peri-implantation stage. These findings provide the first evidence that GRP78 is essential for embryonic cell growth and pluripotent cell survival.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of the tri-loxP-targeting vector and Grp78-targeted ES cells.
A 15-kb mouse genomic DNA containing the Grp78 gene (starting from exon 2) and its 3'-adjacent sequences was isolated from a 129/Sv-derived 
FixII genomic library (a gift of Robert Maxson, University of Southern California Keck School of Medicine). A 5.8-kb NotI/HindIII fragment was subcloned into the pBluescript KS vector and used to create the Grp78 tri-loxP-targeting vector, which was constructed by inserting a floxed pgk-neo expression cassette into intron 4, a third loxP site into the intron 7, and a pgk-TK expression cassette at the 3' end of the construct as a negative selection marker (see Fig. 2A). The NotI-linearized DNA was purified by phenol extraction and ethanol precipitation before electroporating the DNA into the 129/Sv-derived embryonic stem (ES) cells. The ES cells were subjected to G418 and ganciclovir selection, and resistant colonies were screened for homologous recombination by PCR and Southern blot analysis.
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Genotyping of E3.5 embryos. The genotype of E3.5 embryos was determined by nested PCR. The embryos were flushed out of the uterus, as described previously (38), with Dulbecco's modified Eagle's medium (containing 10% fetal bovine serum [FBS] and 10 mM HEPES, pH 7.5) and transferred onto another dish containing fresh medium to remove maternal blood cells before each of the embryos was picked to an individual microtube. The embryos were lysed in 20 µl of lysis buffer (50 mM Tris Cl, pH 8.0, 0.5% Triton X-100, and 200 µg/ml of proteinase K) overnight at 50°C. The samples were heated at 95°C for 10 min, which was followed by PCR analysis. The primers for the nested PCR were PF2 and PR2 followed by PF3 and PR3 (WT) and PF2 and PTR followed by PF3 and PR1 (KO). In some cases, E3.5 embryos were cultured for 6 days before they were subjected to PCR genotyping. The PCR primers used for genotyping were PF1 (5'-CTTCGAATCGGCAGCAGCCAGCTTG-3'), PR1 (5'-AATGTATGCTATACGAAGTTATTTAATCG-3'), PF2 (5'-GTTGATATTGGAGGTGGGCAAACCAAG-3'), PR2 (5'-CCCAGGTCAAACACAAGGATGTTCTTC-3'), PF3 (5'-GATTTGAACTCAGGACCTTCGGAAGAGCAG-3'), PR3 (5'-GCAATAGCAGCTGCTGTACTGTGAGGATGA-3'), PTF (5'-TCGTATAGCATACATTATACGAAGTTATACA-3'), and PTR (5'-TTGTTAGGGGTCGTTCACCTAGA-3').
In vitro culture of E3.5 embryos. Blastocysts were flushed from the uterus of Grp78+/– intercrosses at E3.5 and cultured on a gelatin-coated 96-well plate in ES cell medium (15% FBS, 1% penicillin and streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 100 µM ß-mercaptoethanol, and 1000 U/ml leukemia inhibitory factor). For mechanical removal of the zona pellucida (ZP), blastocysts were cultured in vitro for 24 h, and the ZP were removed by teasing with Pasteur pipettes with finely modified tips.
Isolation, culture, and drug treatment of MEFs. Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos. Briefly, the embryo was washed twice in phosphate-buffered saline (PBS) after removal of the head and internal organs, disaggregated with an 18-gauge needle in 0.25% trypsin-EDTA, and incubated at 37°C for 2 to 3 min before being added Dulbecco's modified Eagle's medium with 10% FBS and 1% penicillin and streptomycin. The cells were split after 2 days in culture, and MEFs were used for experiments. For drug treatment, the cells were treated with 300 nM thapsigargin (Tg), 1.5 µg/ml tunicamycin (Tu), or 5 mM azetidine (AzC) for 16 h prior to harvest.
BrdU and TUNEL assays. For bromodeoxyuridine (BrdU) incorporation, the blastocysts were cultured overnight in vitro and treated with 10 mM BrdU for 6 h. After fixing with 70% ethanol for 30 min and permeabilizing for 1 h at room temperature in permeabilization buffer (0.1% Triton X-100, 0.1% NP-40, and 1% bovine serum albumin in PBS), the blastocysts were incubated with anti-BrdU antibody (Amersham Biosciences) at 4°C overnight and washed three times with PBS. The blastocysts were then incubated with fluorescein-conjugated secondary antibody followed by propidium iodide staining. For terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), the blastocysts were cultured for 24 h in vitro, and the TUNEL assay was performed with an in situ cell death detection kit (Roche Applied Sciences) according to the manufacturer's instructions. The blastocysts were subjected to DAPI (4',6'-diamidino-2-phenylindole) staining after TUNEL staining. The embryos were examined and imaged by fluorescent and bright-field microscopy (Olympus IX70) and then subjected to PCR genotyping.
Southern blot. The BamHI-digested DNA (from ES cells or tail biopsy) was run on a 1% agarose gel at 12 V overnight. The DNA was then transferred onto a nylon membrane and immobilized by UV cross-linking. The 5' external probe A was a 1-kb fragment upstream of the Grp78 TATA box amplified by PCR using the following primers: 5'-TACATCTCATGGTGGAAAGTGTCTGTTTGA-3' and 5'-CATCCTCCTTCTTGTCCTCCTCCTCG-3'. The PCR product was gel purified and labeled with radioactive [32P]dCTP or dATP using a Prime It II kit (Stratagene). The 3' probe B used was a 1.2-kb BamHI/HindIII fragment at the Grp78-neighboring Rab9p40 locus. The hybridization was performed at 65°C overnight in PerfectHyb buffer (Sigma) supplemented with 100 µg/ml of salmon sperm DNA. The membrane was then washed under low-stringency (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]-1% sodium dodecyl sulfate [SDS] at 50°C), high-stringency (0.2x SSC-0.2% SDS at 65°C), and ultrastringent (0.1x SSC-0.1% SDS at 65°C) conditions for 30 min each before being exposed to a Kodak film for 24 to 72 h.
Western blot. Whole E10.5 embryos, MEFs, or pieces of liver dissected from 6-week-old mice were homogenized in radioimmunoprecipitation assay buffer (30) with a Dounce homogenizer, followed by centrifugation at 13,000 x g at 4°C for 15 min. The Western blotting was performed as described previously (31). To detect the GRP78, GRP94, and PDI proteins, an anti-KDEL antibody (1:1,000 dilution; Stressgen) was used. To detect the N-terminal fragment of GRP78, an anti-GRP78 (N20) antibody (1:500 dilution; Santa Cruz Biotechnology) was used. The mouse monoclonal anti-ß-actin antibody (Sigma) was used at a 1:5,000 dilution. Both rabbit polyclonal anticalnexin and anticalreticulin antibodies (Stressgen) were used at a dilution of 1:2,000. To detect CHOP, a mouse monoclonal antibody (Santa Cruz Biotechnology) was used at a 1:1,000 dilution. To detect both the unspliced and spliced forms of XBP-1, a rabbit polyclonal antibody (Santa Cruz Biotechnology) was used at a 1:500 dilution. The Western blotting procedure was repeated two to five times.
Northern blot. Total RNAs from E10.5 embryos and MEFs were extracted, and Northern blotting was performed as described previously (34). The probes for detection of Grp78, Grp94, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) have been described previously (11). The probe used for detection of PDI was synthesized by reverse transcription-PCR (RT-PCR) with the following primers: 5'-TCTAGCAGTCAGCGGTCTGTATTCT-3' and 5'-TACTTCTGTAGCCGCAGCAGCCCAT-3'. The probe used for detection of the Rab9- p40 gene was synthesized by RT-PCR with the following primers: 5'-CAAGCCCAGGAAAGCCACATGGTACA-3' and 5'-TTTTGTGCCCGCTGCCACCATCACAT-3'.
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RESULTS |
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Heterozygous Grp78 cells up-regulate GRP94 and PDI. In the Grp78+/– mice, the GRP78 protein level in adult organs such as the liver was about 60% compared with that of the WT siblings (Fig. 3A and B). Interestingly, GRP94 and PDI levels were elevated by 1.7- and 2.1-fold, respectively, in the heterozygous mice (Fig. 3A and B). The up-regulation of GRP94 and PDI appeared to be specific since the levels of calnexin and calreticulin, two other ER chaperones processing glycoproteins, were similar in the Grp78+/– mice and their WT siblings (Fig. 3A and B). Similarly, in E10.5 Grp78+/– embryos, the level of GRP78 was about 50% of that of the WT level, and up-regulation of PDI and GRP94 was also observed (Fig. 3C and D).
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GRP78 is required for embryonic cell proliferation and protecting the inner cell mass from apoptosis. The inability of the Grp78–/– embryos to survive beyond the peri-implantation stage could be due to multiple factors including a defect in cell proliferation and expansion. To test this, the ZP of the E3.5 embryos from the Grp78+/– intercross was mechanically removed. These ZP-free embryos were then cultured in ES cell medium and examined for growth, which was followed by PCR genotyping. As expected, the WT and heterozygous embryos expanded robustly in culture, giving rise to both the ICM and TE cell types. In contrast, the homozygous Grp78 null embryonic cells were unable to expand in culture even after 5 days of culture and appeared degenerative (Fig. 8A). Upon analyzing the BrdU uptake of the E3.5 embryos after 1 day of culture, we discovered that while about 60% of the heterozygous embryonic cells showed BrdU uptake, as previously reported for embryos at this developmental stage (6), only about 7% of the Grp78 null cells showed BrdU uptake (Fig. 8B). TUNEL assays further revealed a dramatic increase in apoptosis of the ICM of the Grp78 null embryos compared to the negligible level of apoptosis in the heterozygous or WT embryos (Fig. 8C and data not shown). Thus, the lack of cell expansion of the homozygous Grp78 null embryos is likely due to a combined effect of a general proliferation defect and an increase in apoptosis of the ICM.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To unveil the role of GRP78 during early embryonic development, we utilized a tri-loxP gene-targeting approach to create Grp78 mutant alleles. Studies on the Grp78 null allele, generated by crossing the tri-loxP Grp78 mice with the EIIA-cre transgenic mice, revealed several novel findings. As commonly observed for most targeted alleles, the GRP78 level is about 50% of the WT level in the Grp78+/– mice and MEFs. Nonetheless, this observation is noteworthy because the Grp78 gene is highly inducible, and it has been shown that mechanisms exist to regulate the level of GRP78 protein within the normal range. For example, CHO cells resisted the overexpression of GRP78 by down-regulating the endogenous Grp78 gene (8). Thus, the 50% expression level of GRP78 in the Grp78+/– embryos and MEFs establishes that both Grp78 alleles contribute to expression and that the compensatory mechanism to restore it to the WT level, if any, is not operational in these cells.
In the Grp78+/– mice, GRP94 and PDI are up-regulated at both the transcript and protein levels. Interestingly, the levels of other ER chaperones such as calreticulin and calnexin are not affected. These novel observations suggest that in mammalian cells, GRP78, GRP94, and PDI may be components of a functional complex or a regulatory network and that a feedback mechanism exists to sense and compensate for the drop in GRP78 levels by specifically up-regulating the other components. This up-regulation is at least in part due to an increase in the transcript level. We further determined that other UPR targets such as CHOP and XBP-1 are not activated in the same cells. However, they are readily inducible under ER stress conditions. Since the UPR appears to be intact in the Grp78+/– MEFs, the specific induction of PDI and GRP94 in the Grp7+/– embryos and MEFs likely occurs through mechanisms independent of the conventional UPR, although this remains to be proven.
Third, under ER stress conditions, both protein and mRNA levels of GRP78 for Grp78+/– MEFs are about 50% of those of the wild-type MEFs, suggesting that the copy number of the Grp78 gene is the rate-limiting factor for optimal induction of GRP78 in these cells. On the other hand, GRP94 and PDI are induced to a higher level in the Grp78+/– MEFs than in the WT MEFs, although the two types of MEFs have the same number of functional Grp94 and PDI genes. Thus, it is tempting to speculate that GRP78 not only monitors the activation of the ER stress response but may also negatively regulate specific pathways leading to the transcriptional activation of Grp94 and PDI. As nuclear localization of GRP78 has been reported previously (35, 37), GRP78 may regulate these pathways as an ER or nuclear protein. Future investigations will be required to address this interesting issue.
Our study also provides the first direct evidence that GRP78 is essential for early embryo development by targeted disruption of the Grp78 gene in the mouse. Heterozygous Grp78+/– mice showed no apparent abnormality in embryos or adults, indicating that one copy of the Grp78 gene is sufficient for normal development. Since the protein levels of PDI and GRP94 are elevated in the heterozygous Grp78+/– mice, it is possible that the apparent normal phenotype of Grp78+/– mice is in part due to these compensations. On the other hand, the lethality of Grp78–/– embryos indicates that GRP78 function is not replaceable by GRP94, PDI, or the existing complements of other cellular chaperones and members of the HSP70 protein family. We did not detect any truncated protein using an antibody derived against the N terminus of GRP78, and in addition, the Grp78+/– embryos showed no obvious defects, suggesting no gain of function for the mutant allele. Thus, it is unlikely that the phenotypes we observed with the Grp78–/– embryos are due to a dominant negative effect of the mutant alleles. Furthermore, the Grp78+/– mice used in the intercrosses had been backcrossed to the C57BL/6J background for three or more generations, further arguing against the possibility that the phenotype observed results from secondary mutations. Interestingly, we noted that the insertion of a neo-resistant gene cassette in the fourth intron of Grp78 is sufficient to inactivate the gene, as Grp78T/T embryos also died at around E3.5, and the GRP78 level was 50% of the WT level, while both GRP94 and PDI were up-regulated (data not shown). This phenomenon of gene inactivation has been reported for other targeted alleles, possibly through the interference of gene splicing (44). Irrespective of the mechanism, our results clearly establish that GRP78 is absolutely required for mouse embryos to grow beyond E3.5.
A likely explanation for why GRP78 is required during early development is that the peri-implantation stage may constitute physiological ER stress in its heightened activity of cell proliferation and secretion, and GRP78 controls one or more of the obligatory steps. If GRP78 is the essential chaperone for the translocation of proteins into the ER lumen as in the case for yeast cells, its elimination will result in a severe deficiency in ER protein synthesis, membrane receptor assembly, and secretion of enzymes and growth factors. Thus, deficiencies in processing and/or secretion of tryptases involved in blastocyst hatching and outgrowth could impede Grp78–/– embryos from hatching and expanding in culture in vitro (39). Likewise, the failure to process cell surface growth factor receptors may also explain the proliferative defect. Our finding that cell replication in the ICM is substantially reduced in homozygous Grp78 null embryos further suggests that GRP78 itself and/or its client proteins are essential for pluripotent cell proliferation. Thus, GRP78 provides a novel model system for the elucidation of interactive protein partners and pathways that may contribute to embryonic stem cell growth and survival, which have important implications for human stem cell therapy (19).
Interestingly, within the environment of the uterus and in the presence of other hatching embryos, most Grp78–/– embryos are apparently able to hatch and implant; however, the embryos quickly degenerated, giving rise to empty deciduas. We demonstrate here that embryos devoid of GRP78 exhibit a substantial increase in apoptosis of the ICM. Since GRP78 is known to suppress ER stress signaling, complete elimination of GRP78 could lead to the inadvertent activation of proapoptotic pathways from the ER (3, 25, 29). GRP78 can also directly interfere with components of the apoptotic pathways (29, 40, 42). It is also possible that the failure to process and secrete blastocyst antiapoptotic factors such as transforming growth factor 
and gonadotropin-releasing hormone I may lead to an increase in apoptosis (5, 18).
The role of GRP78 most likely expands beyond early development. In support of this notion, high levels of GRP78 were detected in the heart, somite, and neural tube of developing mouse embryos at day 9.5 and day 10.5 (2). Elevated levels of GRP78 in the testes also imply that GRP78 might play an important role in spermatogenesis (14). In addition, Grp78 may be crucial for B-cell maturation because of its role in antibody synthesis (10). It was recently reported that genetic disruption of SIL1/BAP, which encodes an adenosine nucleotide exchange factor of GRP78, leads to protein accumulation, ER stress, and subsequent neurodegeneration (47). While this strongly implicates GRP78 in neuroprotection, our finding that a Grp78 deficiency results in early embryonic lethality further reveals that SIL/BAP is not an obligatory cochaperone for GRP78 during early development. Mice homozygous for the floxed Grp78 allele in which partial Cre-mediated recombination resulted in selective excision of the Neo resistance cassette are viable and express GRP78 normally (our unpublished results). These mice will be suitable for future investigations to generate conditional knockout models of GRP78. This will help to reveal the functional role of GRP78 throughout development and in diseases (28).
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ACKNOWLEDGMENTS |
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This work was supported in part by National Cancer Institute grant CA27607 (A.S.L.).
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FOOTNOTES |
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S.L. and C.M. contributed equally to this work.
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), and the pgk-TK expression cassette (TK) are indicated. The location of the primers [
] used in the PCR genotyping, the external 5' probe A and the external 3' probe B used in the Southern blot, and the BamHI restriction sites [B] are also indicated. (B) Southern blot of BamHI-digested wild-type (+/+), heterozygous Grp78+/T, and heterozygous Grp78+/– DNA using the 5' probe A. The size of each band (in kilobases) is indicated. (C) Southern blot of BamHI-digested DNA from wild-type (+/+) and Grp78+/– siblings, using the 3' probe B against the 3'-adjacent Rab9p40 (Rab) gene (GenBank accession no. NM_145522). (D) Whole-cell lysates from livers of wild-type (+/+) adult mice and heterozygous Grp78+/– siblings were subjected to Western blot analysis with anti-GRP78 (N20) antibody. The film was overexposed to detect any truncated form of GRP78, if it exists. The asterisk indicates the 78-kDa full-length GRP78 protein. (E) Total RNA from WT (+/+) or heterozygous (+/–) E10.5 embryos were subjected to Northern blot analysis to analyze the Rab transcript level. The 28S rRNA was used as an RNA loading control.
