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 NMPP-treated hepatocytes. Concurrently, a marked (>80%) suppression of de novo hepatocellular protein synthesis was also observed, along with a significantly enhanced phosphorylation of the α-subunit of the eukaryotic initiation factor eIF2 (eIF2α), as monitored by the phosphorylated eIF2α/total eIF2α 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 heme-sensitive eIF2α 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.
Footnotes
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↵1 Heme and hemin for iron-protoporphyrin IX are used interchangeably throughout the text.
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These studies were supported by National Institutes of Health Grants DK26506 (to M.A.C.) and DK61510 (to J.J.M.). We also acknowledge the University of California San Francisco Liver Core Center Facilities on Molecular Analyses (Spectrophotometry) and on Cell and Tissue Biology supported by P30DK26743.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.084699.
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ABBREVIATIONS: ER, endoplasmic reticulum; P450, cytochrome P450; TDO, tryptophan 2,3-dioxygenase; PB, phenobarbital; PBGD, porphobilinogen deaminase; ALA, δ-aminolevulinic acid; DDEP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine; NMPP, N-methylprotoporphyrins; eIF2, α-subunit of the eukaryotic initiation factor; WME, Williams' medium E; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; E-64, l-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane; PBS, phosphate-buffered saline; PROD, 7-pentoxyresorufin O-dealkylase; PCR, polymerase chain reaction; GUS, β-glucuronidase; bp, base pair(s); AK, adenylate kinase; eEF2, eukaryotic elongation factor 2; eEF2P, phosphorylated eEF; Grp78, 78-kDa glucose-regulated protein; HRP, horseradish peroxidase; eIF2αP, phosphorylated α-subunit of the eukaryotic initiation factor; HRI, heme-regulated inhibitor; PERK, PKR-like ER kinase.
- Received February 10, 2005.
- Accepted March 11, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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