Received for publication February 10, 2005.
Revised March 10, 2005.
Accepted for publication March 11, 2005.

HEME-REVERSIBLE IMPAIRMENT OF CYP2B1/2 INDUCTION IN HEME DEPLETED RAT HEPATOCYTES IN PRIMARY CULTURE : TRANSLATIONAL CONTROL BY A HEPATIC eIF2 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 DDEP (3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine) or NMPP (N-methylprotoporphyrins). 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 -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 heme-sensitive 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.

Key words: CYP2B Induction, Heme regulation, Translational control, eIF2alpha kinase, hepatic heme depletion, transcriptional activation

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