Impaired dexamethasone-mediated induction of tryptophan 2,3-dioxygenase in heme-deficient rat hepatocytes: translational control by a hepatic eIF2alpha kinase, the heme-regulated inhibitor.
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 alpha-subunit of the eukaryotic initiation factor eIF2 (eIF2alpha), as monitored by the phosphorylated eIF2alpha/total eIF2alpha ratio. Heme supplementation reversed these effects, indicating that heme regulates TDO induction by functional control of an eIF2alpha 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 eIF2alpha 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.
Liao
Mingxiang
M
Dept. of Cellular and Molecular Pharmacology, Mission Bay Campus, Genentech Hall, 600 16th Street, N572F/Box 2280, University of California, San Francisco, CA 94158, USA.
Pabarcus
Michael K
MK
Wang
YongQiang
Y
Hefner
Colleen
C
Maltby
David A
DA
Medzihradszky
Katalin F
KF
Salas-Castillo
Saida Patricia
SP
Yan
James
J
Maher
Jacquelyn J
JJ
Correia
Maria Almira
MA
D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003907:Dexamethasone / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005938:Glucocorticoids / Q000494:pharmacology*; D006418:Heme / Q000172:deficiency* / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D014176:Protein Biosynthesis; D011524:Protoporphyrins / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics; D053719:Tandem Mass Spectrometry; D014366:Tryptophan Oxygenase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D019892:eIF-2 Kinase / Q000235:genetics / Q000502:physiology*
D005938:Glucocorticoids; D011524:Protoporphyrins; D011994:Recombinant Proteins; D006418:Heme; C028842:N-methylprotoporphyrin IX; D003907:Dexamethasone; D014366:Tryptophan Oxygenase; D019892:eIF-2 Kinase