Impaired Dexamethasone-Mediated Induction of Tryptophan 2,3-Dioxygenase in Heme-Deficient Rat Hepatocytes: Translational Control by a Hepatic eIF2α Kinase, the Heme-Regulated Inhibitor

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 (Ͼ90%) 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 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.Tryptophan 2,3-dioxygenase (TDO) is a liver-specific cytosolic enzyme highly specific for L-tryptophan (L-Trp) as the substrate (Greengard and Feigelson, 1961;Schimke et al., 1965;Knox, 1966). As the rate-limiting enzyme in L-Trp oxidative breakdown to kynurenine, its activity critically determines the relative L-Trp flux into serotonergic (5-HT) and kynurenine [NAD, NADP, poly(ADP-ribose)] pathways (Greengard and Feigelson, 1961;Schimke et al., 1965;Knox, 1966). TDO is a 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

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

“…However, our immunoblotting analyses indicate that MG132-induced proteasomal inhibition failed to affect hepatic HRI (Fig. 4), which is otherwise effectively activated in hepatocytes by heme depletion (Han et al, 2005;Liao et al, 2007).…”

“…Concentration-dependent effects of MG132 on hepatic TDO content. Aliquots of lysates (20 g of protein) from hepatocytes treated with 0, 10, 20, 50, 100, 200, and 300 M MG132 along with a purified recombinant rat liver TDO (1 pg of protein) as a standard (STD) were also subjected to immunoblotting analyses with rabbit polyclonal anti-TDO IgGs as detailed (Liao et al, 2007). Corresponding aliquots were used for actin immunoblotting analyses as loading controls.…”

“…eIF2␣/eIF2␣P, actin, and Grp78/BiP were subjected to immunoblotting analyses as detailed previously (Han et al, 2005). HRI protein and hepatic cytosolic tryptophan 2,3-dioxygenase (TDO) immunoblotting analyses were carried out with primary rabbit polyclonal antibodies against the corresponding recombinant rat hepatic HRI and TDO proteins as described previously (Liao et al, 2007). PERK.…”

RETRACTION: Hepatic Heme-Regulated Inhibitor (HRI) Eukaryotic Initiation Factor 2α Kinase: A Protagonist of Heme-Mediated Translational Control of CYP2B Enzymes and a Modulator of Basal Endoplasmic Reticulum Stress Tone

“…at ASPET Journals on May 12, 2018 molpharm.aspetjournals.org in cultured rat hepatocytes (Liao et al, 2007). Through a combination of approaches including cloning, heterologous expression, isolation, purification, immunoblotting, immunoaffinity capture, and proteomic analyses we have identified it as a bona fide HRI that is activated via autophosphorylation by acute hepatic heme depletion, resulting in global protein translational shutoff, and functionally inhibited by heme repletion (Han et al, 2005b;Liao et al, 2007).…”

“…Through a combination of approaches including cloning, heterologous expression, isolation, purification, immunoblotting, immunoaffinity capture, and proteomic analyses we have identified it as a bona fide HRI that is activated via autophosphorylation by acute hepatic heme depletion, resulting in global protein translational shutoff, and functionally inhibited by heme repletion (Han et al, 2005b;Liao et al, 2007). Accordingly, in hemedepleted rat hepatocytes, the de novo syntheses (determined by [ 35 S]Met/Cys-incorporation into immunoprecipitable protein) of the phenobarbital (PB)-inducible CYP2B enzymes (Han et al, 2005b) and dexamethasone (Dex)-inducible tryptophan 2,3-dioxygenase (TDO; Liao et al, 2007) were greatly impaired in a heme-reversible manner. To directly verify the role of hepatic HRI in the translational shutoff of PB-inducible CYP2B enzymes, we have used a HRI gene knockout mouse model [HRI(Ϫ/Ϫ)] .…”