Heme-Reversible Impairment of CYP2B1/2 Induction in Heme-Depleted Rat Hepatocytes in Primary Culture: Translational Control by a Hepatic α-Subunit of the Eukaryotic Initiation Factor Kinase?

Abstract

The role of heme in the phenobarbital-mediated induction of CYP2B1/2 was reexamined in rat hepatocytes in monolayer culture, acutely depleted of heme by treatment with either 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) or N-methylprotoporphyrins (NMPP). The findings revealed that such acute hepatic heme depletion markedly impaired CYP2B1/2 protein induction, an effect that was reversible by heme resupplementation. However, TaqMan analyses of hepatic mRNA isolated from these heme-depleted cells revealed that this impairment was not due to faulty transcriptional activation of either CYP2B1 or CYP2B2 gene expression as previously proposed, thereby confirming literature reports that heme is not a transcriptional regulator of the CYP2B1/2 gene. In contrast, the rate of de novo CYP2B1/2 protein synthesis was found to be dramatically inhibited in both DDEP-and NMPPtreated hepatocytes. Concurrently, a marked (Ͼ80%) suppression of de novo hepatocellular protein synthesis was also observed, along with a significantly enhanced phosphorylation of the ␣-subunit of the eukaryotic initiation factor eIF2 (eIF2␣), as monitored by the phosphorylated eIF2␣/total eIF2␣ ratio in these heme-depleted cells. Indeed, the parallel reversal of all these three effects by heme supplementation suggests that this impaired CYP2B1 induction most likely stems from blocked translational initiation resulting from the activation of a hemesensitive eIF2␣ kinase. Such global suppression of hepatic protein synthesis may disrupt a myriad of vital cellular functions, thereby contributing to the clinical symptoms of acute hepatic heme-deficient states such as the hepatic porphyrias.

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

“…However, our immunoblotting analyses indicate that MG132-induced proteasomal inhibition failed to affect hepatic HRI (Fig. 4), which is otherwise effectively activated in hepatocytes by heme depletion (Han et al, 2005;Liao et al, 2007).…”

“…Lysates from untreated cells obtained at 0 h were diluted to the equivalent volume with the cell culture medium and equivalent aliquots used to monitor the total (100%) cellular AK activity available for release into the media. The assay was performed by the manufacturer's instructions exactly as described previously (Han et al, 2005). In parallel, after treatment with either 0 or 200 M MG132 for 6 h, the medium of some cell cultures was replaced with fresh medium…”

“…Hepatocytes (3 ϫ 10 6 ) were seeded onto 60-mm Permanox culture dishes precoated with type I rat tail collagen. Cells were cultured as described previously (Han et al, 2005) in William's E medium containing insulin-transferrin-selenium G, 0.1 M Dex, 50 U/ml penicillin/streptomycin, 2 mM L-glutamine, and 0.1% BSA. Cells were overlaid with 0.25 mg/ml Matrigel 3 h after plating.…”

“…eIF2␣/eIF2␣P, actin, and Grp78/BiP were subjected to immunoblotting analyses as detailed previously (Han et al, 2005). HRI protein and hepatic cytosolic tryptophan 2,3-dioxygenase (TDO) immunoblotting analyses were carried out with primary rabbit polyclonal antibodies against the corresponding recombinant rat hepatic HRI and TDO proteins as described previously (Liao et al, 2007).…”

Characterization of the Physiological Turnover of Native and Inactivated Cytochromes P450 3A in Cultured Rat Hepatocytes: A Role for the Cytosolic AAA ATPase p97?

“…Cytotoxicity was examined using ToxiLight cytotoxicity assay as per the manufacturer's instructions exactly as described (38). The assay consists of chemiluminescent detection of adenylate kinase (AK) that is released into the medium by damaged hepatocytes using the reconstituted AK detection reagent.…”