Abstract
We have reported previously that the hepatic heme-regulated inhibitor (HRI)-eukaryotic initiation factor 2α (eIF2α) kinase is activated in acute heme-deficient states, resulting in translational shut-off of global hepatic protein synthesis, including phenobarbital (PB)-mediated induction of CYP2B enzymes in rats. These findings revealed that heme regulates hepatic CYP2B synthesis at the translational level via HRI. As a proof of concept, we have now employed a genetic HRI-knockout (KO) mouse hepatocyte model. In HRI-KO hepatocytes, PB-mediated CYP2B protein induction is no longer regulated by hepatic heme availability and proceeds undeterred even after acute hepatic heme depletion. It is noteworthy that genetic ablation of HRI led to a small albeit significant elevation of basal hepatic endoplasmic reticulum (ER) stress as revealed by the activation of ER stress-inducible RNA-dependent protein kinase-like ER-integral (PERK) eIF2α-kinase, and induction of hepatic protein ubiquitination and ER chaperones Grp78 and Grp94. Such ER stress was further augmented after PB-mediated hepatic protein induction. These findings suggest that HRI normally modulates the basal hepatic ER stress tone. Furthermore, because HRI exists in both human and rat liver in its heme-sensitive form and is inducible by cytochrome P450 inducers such as PB, these findings are clinically relevant to acute heme-deficient states, such as the acute hepatic porphyrias. Activation of this exquisitely sensitive heme sensor would normally protect cells by safeguarding cellular energy and nutrients during acute heme deficiency. However, similar HRI activation in genetically predisposed persons could lead to global translational arrest of physiologically relevant enzymes and proteins, resulting in the severe and often fatal clinical symptoms of the acute hepatic porphyrias.
Footnotes
- Received September 25, 2009.
- Accepted January 12, 2010.
These studies were supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grants DK26506 (to M.A.C.), DK016272, DK078442 (to J.-J.C.)]. We also acknowledge the UCSF Liver Center Core on Cell and Tissue Biology supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant P30-DK26743].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.061259.
ABBREVIATIONS:
- ER
- endoplasmic reticulum
- eIF2α
- α-subunit of the eukaryotic initiation factor eIF2
- eIF2αP
- phosphorylated eIF2α
- PKR
- RNA-dependent protein kinase
- PERK
- PKR-like ER-bound eIF2α-kinase
- GCN2
- general control nonderepressible-2 eIF2α kinase
- PAGE
- polyacrylamide gel electrophoresis
- BSA
- bovine serum albumin
- HRI
- heme-regulated inhibitor
- PB
- phenobarbital
- Dex
- dexamethasone
- BSA
- bovine serum albumin
- DMSO
- dimethyl sulfoxide
- WT
- wild type
- KO
- knockout
- MGB
- mixed genetic background
- TDO
- tryptophan 2,3-dioxygenase
- qRT-PCR
- Quantitative real time polymerase chain reaction
- HRI-P
- 92-kDa phosphorylated HRI species
- MG132
- N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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