Received for publication April 20, 2007.
Revised August 27, 2007.
Accepted for publication August 28, 2007.

Impaired dexamethasone-mediated induction of tryptophan 2,3-dioxygenase in heme-deficient rat hepatocytes: Translational control by a hepatic eIF2 kinase, the heme-regulated inhibitor (HRI)

Abstract

Tryptophan 2,3-dioxygenase (TDO), a liver specific cytosolic hemoprotein, is the rate-limiting enzyme in L-tryptophan catabolism and thus a key serotonergic determinant. Glucocorticoids transcriptionally activate the TDO gene with marked enzyme induction. TDO is also regulated by heme, its prosthetic moiety, as its expression and function are significantly reduced after acute hepatic heme depletion. We now show in primary rat hepatocytes that this impairment is not due to faulty transcriptional activation of the TDO gene but rather due to its posttranscriptional regulation by heme. Accordingly, in acutely heme-depleted hepatocytes, the de novo synthesis of TDO protein is markedly decreased (>90%) along with that of other hepatic proteins. This global suppression of de novo hepatic protein syntheses in these heme-depleted cells is associated with a significantly enhanced phosphorylation of the -subunit of the eukaryotic initiation factor eIF2 (eIF2), as monitored by the phosphorylated eIF2/total eIF2 ratio. Heme supplementation reversed these effects indicating that heme regulates TDO induction by functional control of an eIF2 kinase. A cDNA was cloned from heme-depleted rat hepatocytes, and DNA sequencing verified its identity to the previously cloned rat brain heme-regulated inhibitor (HRI). Proteomic, biochemical and/or immunoblotting analyses of the purified recombinant protein and the immunoaffinity-captured hepatic protein confirmed its identity as a rat heme-sensitive eIF2 kinase. These findings document not only that a hepatic HRI exists and is physiologically relevant, but also implicate its translational shut-off of key proteins in the pathogenesis and symptomatology of the acute hepatic heme-deficient conditions clinically known as the hepatic porphyrias.

Key words: Hepatic HRI, Rat hepatocytes, Translational control, heme-regulated inhibitor, hepatic eIF2alpha kinase, tryptophan 2,3-dioxygenase

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