Abstract
Calnexin is a type I integral endoplasmic reticulum (ER) membrane chaperone involved in folding of newly synthesized (glycol)proteins. In this study, we used β-galactosidase reporter gene knock-in and reverse transcriptase polymerase chain reaction (RT-PCR) to investigate activation of the calnexin gene during embryonic development. We showed that the calnexin gene was activated in neuronal tissue at the early stages of embryonic development but remained low in the heart, intestine, and smooth muscle. At early stages of embryonic development, large quantities of calnexin messenger RNA (mRNA) were also found in neuronal tissue and liver. There was no detectable calnexin mRNA in the heart, lung, and intestine. The absence of calnexin had no significant effect on ER stress response (unfolded protein response, UPR) at the tissue level as tested by IRE1-dependent splicing of Xbp1 mRNA. In contrast, non-stimulated calnexin-deficient cells showed increased activation of IRE1, as measured by RT-PCR and luciferase reporter gene analysis of splicing of Xbp1 mRNA and activation of the BiP promoter. This indicates that cnx −/− cells have increased constitutively active UPR. Importantly, cnx −/− cells have significantly increased proteasomal activity, which may play a role in the adaptive mechanisms addressing the acute ER stress observed in the absence of calnexin.
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Abbreviations
- ER:
-
endoplasmic reticulum
- UPR:
-
unfolded protein response
- IRE1:
-
inositol-requiring enzyme 1
- Xbp1:
-
X-box binding protein 1
- ERAD:
-
ER-associated degradation
- PDI:
-
protein disulfide isomerase
- PBS:
-
phosphate buffered saline
- OCT:
-
optimal cutting temperature compound
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Acknowledgment
We thank Dr. R. J. Kaufman for his generous gifts of the luciferase reporter plasmids and anti-IRE1 antibodies. We thank S. Aldred and P. Gajda for superb technical assistance. This work was supported by a grant from the Canadian Institutes of Health Research. H. Coe and J. Groenendyk are supported by the Canadian Institutes of Health Research (MOP-15291) and Heart and Stroke Foundation of Canada Membrane Protein and Cardiovascular Disease Training Program. H. Coe and J. Jung are recipients of an Alberta Heritage Foundation for Medical Research Studentship.
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Coe, H., Bedard, K., Groenendyk, J. et al. Endoplasmic reticulum stress in the absence of calnexin. Cell Stress and Chaperones 13, 497–507 (2008). https://doi.org/10.1007/s12192-008-0049-x
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DOI: https://doi.org/10.1007/s12192-008-0049-x