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. Here we 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 α-subunit of the eukaryotic initiation factor eIF2 (eIF2α), as monitored by the phosphorylated eIF2α/total eIF2α ratio. Heme supplementation reversed these effects, indicating that heme regulates TDO induction by functional control of an eIF2α 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 eIF2α kinase. These findings not only document 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.
Footnotes
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↵1 The terms heme and hemin for iron-protoporphyrin IX is used interchangeably throughout the text.
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↵2 It is conceivable that, although cultured hepatocytes are adequately supplied with nutrients and O2, rats with acute hepatic heme depletion being neurologically and/or otherwise physiologically impaired are incapable of maintaining normal hepatocellular gene transcription.
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↵3 An eHRI-like inhibitor was isolated from rat liver perfused free of contaminating blood and erythroid cells (Delaunay et al., 1977), but apparently its identity has remained equivocal.
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↵4 As monitored by 14C-δ-ALA-incorporation into heme (Jover et al., 2000).
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This work was supported by National Institutes of Health (NIH) Grants DK26506 (to M.A.C.) and DK61510 (to J.J.M.). We also acknowledge the UCSF Liver Center Cores on Molecular Analyses (Spectrophotometry and Mass Spectrometry) and on Cell and Tissue Biology supported by NIH NIDDK Center Grant P30DK26743. The UCSF Mass Spectrometry Facility is supported by NIH National Center for Research Resources BRTP 01614, RR019934, and RR015804.
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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.107.124602.
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ABBREVIATIONS: TDO, tryptophan 2,3-dioxygenase; eIF2α, α-subunit of the eukaryotic initiation factor eIF2; eIF2αP, phosphorylated eIF2α; DTT, dithiothreitol; CID, collision-induced dissociation; DDEP, 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine; DEX, dexamethasone; ER, endoplasmic reticulum; eHRI, erythroid HRI; GCN2, general control nonderepressible-2; HRI, heme-regulated inhibitor; IRE-1/ERN1, inositol requiring ER to nucleus signaling 1; mHRI, mouse HRI; NMPP, N-methylprotoporphyrin IX; PERK, PKR-like ER-bound eIF2α-kinase; WME, Williams' Medium E; PKR, RNA-dependent protein kinase; RHL, rat hepatocyte lysate; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; UPR, unfolded protein response; XBP-1, X-box binding protein 1; E-64, N-(trans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide; DMSO, dimethyl sulfoxide; GUS, β-glucuronidase; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; LC, liquid chromatography; MS, mass spectrometry; FT, flow-through; DOC, deoxycholate; δ-ALAS, δ-aminolevulinic acid synthase; HRP, horseradish peroxidase.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received April 18, 2007.
- Accepted August 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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