RETRACTION: Hepatic Heme-Regulated Inhibitor (HRI) Eukaryotic Initiation Factor 2α Kinase: A Protagonist of Heme-Mediated Translational Control of CYP2B Enzymes and a Modulator of Basal Endoplasmic Reticulum Stress Tone

Abstract

We have reported previously that the hepatic heme-regulated inhibitor (HRI)-eukaryotic initiation factor 2␣ (eIF2␣) 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 ERintegral (PERK) eIF2␣-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.Suppression of global protein synthesis through translational control is an effective way to preserve cellular energy and nutrients after various forms of cellular stress and injury. Through a rapid and reversible control of gene expression, it critically regulates various vital cellular processes, including growth stimulation, cell cycle progression, differentiation, hypoxia, ER stress, and heme deficiency

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases

“…Lipofectamine 2000 was obtained from Invitrogen. Common cell culture media, supplements, culture plasticware, and commercial sources of protease inhibitors and dexamethasone (Dex) have been reported previously (52,53). Goat polyclonal IgGs were raised commercially against purified recombinant rat hepatic CYP3A23 and purified by Hi-Trap protein A-Sepharose affinity chromatography.…”

Liver Cytochrome P450 3A Endoplasmic Reticulum-associated Degradation

“…Materials-The sources of common cell culture media, supplements, and culture plastic ware and the commercial sources of protease inhibitors, dexamethasone (Dex), ␤-glucuronidase/arylsulfatase mixture, 7-hydroxy-4-trifluoromethylcoumarin (HFC), and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) have been reported previously (35,66,67). Goat or rabbit polyclonal IgGs were raised commercially against purified recombinant rat hepatic CYP3A23 and purified by HiTrap Protein A-Sepharose affinity chromatography.…”

“…Rat Hepatocyte Viral Infection and Culture-Hepatocytes were isolated from rats (4 -6 weeks old) by in situ liver perfusion with collagenase (liver digest medium) and cultured as described (14,66,67,69). Cells were maintained for 2 days with a daily change of medium to enable recovery and hepatic function restoration (70).…”

“…Densitometric Quantification-Direct quantification of the immunoblots was performed by ImageJ (National Institutes of Health) analyses as described (67). Densitometrically derived arbitrary units of individual immunoblots were normalized against corresponding values of the actin loading controls and then expressed as a percentage of the control/basal values.…”

“…qRT-PCR Analyses-Total RNA was extracted and reverse transcribed to cDNA exactly as described (67,69). Universal PCR master mix (catalog no.…”