Inhibitor Of Heme Biosynthesis Research Articles

Abstract 610: ClpP-dependent and -independent activation of HRI kinase by small molecules

Abstract HRI was initially identified as a kinase essential for maintaining heme-globin balance within red blood cells as well as for controlling the ISR in response to oxidative stress. HRI also responds to a broad range of stresses such as osmotic stress, heat shock, proteasome inhibition. The recently discovered unexpected functions of HRI include innate immunity, translational control of immune evasion in cancer by upregulating PD-L1, proteostasis, mitochondrial stress, inhibition of histone H3 lysine 27 (H3K27) demethylase (KDM6A) and iron deficiency. Importantly, recent evaluation of patient data uncovered high expression of HRI mRNA in a subset of epithelial tumors versus normal tissues. Elevated expression of HRI protein in these tumor cells lead to cell death when BIRC6 ubiquitin complex is inhibited, which mediates degradation of HRI and is required for the survival of the tumors. These further broaden the importance of this member of the eIF2α kinase family as a cancer therapeutic strategy. Dordaviprone (ONC201), an imipridone small molecule, binds to and activates mitochondrial protease ClpP leading to integrated stress response (ISR) and ATF4 transcription factor activation. PG3, a prodigiosin analog, induces ATF4 and pro-apoptotic PUMA. Our data indicate that PG3 activates ATF4 through ISR via eIF2α kinase HRI. ALAS1 (5'-aminolevulinate synthase 1) catalyzes the first rate-limiting step in heme (Iron-protoporphyrin) biosynthesis. ONC201 treatment leads to potent downregulation and inhibition of ALAS1, indicating that ONC201 inhibits heme biosynthesis. It is well known that reduced heme results in activation of the HRI kinase. We show that an inhibitor of heme biosynthesis or knockdown of ALAS1 results in HRI activation, while silencing of HRI or knockout of HRI gene potently inhibit the eIF2α phosphorylation and upregulation of ATF4, CHOP and PUMA by PG3 and imipridones. Knockdown of ClpP rescues ONC201-induced downregulation of ALAS1 which blocks ONC201-induced upregulation of CHOP. Also, silencing of ClpP significantly reduced PARP cleavage in HCT116 p53−/- and MDA-MB-468 cancer cells. Our studies identify a novel link between ClpP activation induced by ONC201 treatment and ATF4 upregulation, via the ClpP/ALAS1/HRI/ATF4 pathway. However, PG3 treatment did not lead to degradation of ALAS1, indicating that PG3 does not activate ClpP. PG3 potently induced cell apoptosis through ISR via HRI/ATF4/PUMA pathway independent of ClpP. We are further investigating the targets of PG3 and the signaling pathway that leads to PG3-induced activation of HRI. Our results suggest that different small molecule inducers of the ISR such as ONC201 and PG3 can achieve an anti-tumor effect through different pathways converging on kinase HRI ultimately leading to ATF4 activation and tumor cell death. Citation Format: Xiaobing Tian, Praveen Srinivasan, Wafik S. El-Deiry. ClpP-dependent and -independent activation of HRI kinase by small molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 610.

Heme Biosynthesis is Crucial for Cell Survival and Mitochondrial OXPHOS after X Irradiation.

Heme is an essential component of the hemoproteins involved in the mitochondrial electron transport chain (ETC). Cancer cells have been reported to display high heme levels and increased activity of heme-containing proteins. Consistently, inhibition of heme biosynthesis by the ALAD inhibitor succinylacetone (SA) has been shown to reduce tumor cell survival. These observations indicate that heme biosynthesis is essential for cancer cell proliferation. X irradiation has been shown to increase mitochondrial mass, membrane potential, oxygen consumption, reactive oxygen species (ROS) production, and ATP synthesis. This finding suggests that radiation activates mitochondrial oxidative phosphorylation (OXPHOS). However, although heme is an essential component of the mitochondrial ETC, whether radiation influences heme biosynthesis remains unclear. In this study, we evaluated heme biosynthesis activity after X irradiation and examined the effects of heme biosynthesis inhibition by SA on cellular radiosensitivity and mitochondrial OXPHOS function. We demonstrated that X irradiation significantly increased ALAS1 mRNA levels and cellular heme content. Inhibition of heme biosynthesis by SA significantly decreased cellular heme content and sensitized cancer cells to radiation. We also showed that SA reduced cellular ATP levels, mitochondrial membrane potential, and mitochondrial ROS production, suggesting mitochondrial OXPHOS dysfunction. SA decreased the expression of mitochondrial heme-related proteins COX2 and cytochrome c but did not influence COX1 and VDAC expression. These results indicate that inhibition of heme biosynthesis decreased mitochondrial ETC protein expression and OXPHOS activity, which triggered cellular ATP depletion and radiosensitization after X irradiation. In summary, heme biosynthesis is upregulated by X irradiation and is essential for mitochondrial OXPHOS and cell survival.

A role of splenic heme biosynthesis pathway in the persistent prophylactic actions of arketamine in lipopolysaccharide-treated mice

Relapse is common in remitted patients with major depressive disorder (MDD). Arketamine, an (R)-enantiomer of ketamine, has persistent prophylactic actions in an inflammatory model of depression. However, the precise mechanisms underlying these prophylactic actions remain unknown. Given the role of the brain–spleen axis in depression, we sought to identify splenic molecular targets that play a role in the prophylactic actions of arketamine. Lipopolysaccharide (LPS) (1.0 mg/kg) was administered 6 days after a single injection of arketamine (10 mg/kg) or saline. RNA-sequencing analysis found altered expression in the heme biosynthesis II pathway. Quantitative RT-PCR revealed that pretreatment with arketamine blocked increased expression of genes involved in the heme biosynthesis II pathway in LPS-treated mice, namely, 5-aminolevulinase synthase 2 (Alas2), ferrochelatase (Fech), hydroxymethylbilane synthase (Hmbs). Interestingly, there were positive correlations between the expression of these genes and spleen weight or plasma levels of pro-inflammatory cytokines. We also found higher expression of ALAS2 and FECH in the spleen from MDD patients. Pretreatment with a key intermediate precursor of heme, 5-aminolaevulinic acid (300 mg/kg/day for 3 days), caused splenomegaly, higher plasma levels of pro-inflammatory cytokines, and depression-like behavior in low-dose LPS (0.1 mg/kg)-treated mice. Interestingly, pretreatment with a heme biosynthesis inhibitor, succinyl acetone (120 mg/kg/day for 3 days), had prophylactic effects in LPS (1.0 mg/kg)-treated mice. These data suggest a novel role for the heme biosynthesis II pathway in the spleen for inflammation-related depression. Therefore, the heme biosynthesis pathway could be a new target for the prevention of relapse in MDD patients.

Abstract 4959: TIC10/ONC201 activates ClpP to degrade ALAS1 while PG3 does not degrade ALAS1 in pathway to HRI, ATF4 and CHOP activation leading to tumor cell death

Abstract TRAIL-inducing compound TIC10/ONC201 binds and activates mitochondrial protease ClpP leading to integrated stress response (ISR) and ATF4 transcription factor activation. Our lab previously reported that ONC201 activates HRI (heme-regulated inhibitor) kinase but does not signal eIF2-alpha phosphorylation through PERK or GCN2. However, the connection between ClpP activation of HRI remains unknown. PG3, a prodigiosin analog that bypasses a defective p53 pathway potently induces ATF4 and pro-apoptotic PUMA. Our data indicate that PG3 activates ATF4 through ISR via HRI as knockdown of HRI but not PKR blocked upregulation of ATF4 and CHOP in several tumor cell lines. We have been exploring how ClpP activation leads to HRI and ISR activation, and the mechanism by which PG3 leads to HRI activation. We find that activation of HRI by PG3 or ONC201 is not through the OMA1/DELE1◊HRI activation pathway. Moreover, ROS and NO are not responsible for ONC201- or PG3-induced HRI activation. ALAS1 (5'-aminolevulinate synthase 1) catalyzes the first rate-limiting step in heme (Iron-protoporphyrin) biosynthesis. ONC201 treatment leads to potent downregulation and inhibition of ALAS1, indicating that ONC201 inhibits heme biosynthesis. It is well known that reduced heme results in activation of the HRI kinase. We show that an inhibitor of heme biosynthesis or knockdown of ALAS1 results in HRI activation, while knockdown of HRI blocks upregulation of ATF4 by ONC201. Knockdown of ClpP rescues ONC201-induced downregulation of ALAS1 which blocks ONC201-induced upregulation of CHOP. Our studies identify a novel link between ClpP activation induced by ONC201 treatment and ATF4 upregulation, via the ClpP/ALAS1◊HRI◊ATF4/CHOP pathway. However, PG3 does not inhibit ALAS1, reduce its expression or therefore signal through ClpP. PG3 treatment did not lead to degradation of ALAS1, indicating that PG3 does not activate ClpP as ALAS1 is a known ClpP direct substrate. We are further investigating the signaling pathway that leads to PG3-induced activation of HRI. Our results suggest that different small molecule inducers of the ISR such as ONC201 and PG3 can achieve an anti-tumor effect through different pathways involving kinase HRI ultimately leading to ATF4/CHOP activation and tumor cell death. Citation Format: Xiaobing Tian, Wafik S. El-Deiry. TIC10/ONC201 activates ClpP to degrade ALAS1 while PG3 does not degrade ALAS1 in pathway to HRI, ATF4 and CHOP activation leading to tumor cell death. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4959.

Comparative hepatotoxicity of a herbicide, epyrifenacil, in humans and rodents by comparing the dynamics and kinetics of its causal metabolite.

A new herbicide, epyrifenacil (S-3100), inhibits protoporphyrinogen oxidase (PPO) in plants. Repeated administration of epyrifenacil in laboratory animals led to some toxicological changes related to PPO inhibition, e.g., hepatotoxicity caused by porphyrin accumulation and anemia caused by the inhibition of heme biosynthesis. In vitro studies revealed that an ester-cleaved metabolite, S-3100-CA, is predominant in mammals, exhibits PPO-inhibitory activity, and thus is the cause of epyrifenacil-induced toxicity. To assess the human risk, the effects of species differences on the dynamics (PPO inhibition) and kinetics (liver uptake) of epyrifenacil were evaluated separately. The results of in vitro assays revealed an approximately tenfold weaker inhibition of PPO by S-3100-CA in humans than in rodents and six- to thirteen-fold less hepatic uptake of S-3100-CA in humans than in mice. Finally, it was suggested that humans are less sensitive to the toxicity of epyrifenacil than are rodents, although further mechanistic research is highly anticipated.

Role of Heme in the Regulation of Transferrin Receptor Expression in Erythroid Cells

Physiologically, iron is transported between sites of absorption, storage, and use by the plasma glycoprotein transferrin (Tf). Most cells acquire iron via the binding of Tf to the Tf receptor (TfR) on the cell membrane. In non-erythroid cells, pivotal players in the regulation of TfR are the iron regulatory proteins, IRP1 and IRP2, which “sense” iron levels in the labile iron pool. IRPs control the synthesis of TfR by binding to iron-responsive elements (IREs) in the 3'UTR region of TfR mRNA. When cellular iron is scarce, IRPs bind to the IREs stabilizing the TfR mRNA, while excess iron prevents the binding of IRPs to IREs resulting in the rapid degradation of TfR mRNA.Developing red blood cells are the largest consumers of iron, which is delivered to them exclusively by Tf via TfR. Erythroid cells have by far the greatest need for iron required for the rapid and efficient synthesis of hemoglobin. Thus, it is not surprising that transcription plays a dominant role over the regulation via the IRE/IRP system in the control of TfR expression (Lok and Ponka (2000) J. Biol. Chem . 275: 24185-24190). The exceptionally high capacity of immature erythroid cells to synthesize heme is primarily a result of their unique control of iron metabolism as well as their distinct heme biosynthetic enzyme, erythroid-specific aminolevulinic acid synthase (ALA-S2). Here we provide evidence that TfR expression and the cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine eryhroleukemia cells with 5-aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone (blocks ALA dehydratase) or N-methylprotoporphyrin (blocks ferrochelatase)), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR expression by ALA is mainly the result of increased mRNA synthesis, since the effect of ALA can be abolished by actinomycin D. Importantly, the TfR promoter is activated in vitro by addition of ALA and hemin to cultured murine erythroleukemia cells differentiated with DMSO. Direct-site specific mutagenesis of the erythroid active element1 in TfR promoter abolished the effects of ALA and hemin. These results suggest that heme may directly or indirectly interact with TfR promoter, consequently activating gene expression. Hence, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels, thus ensuring appropriate iron availability for hemoglobin synthesis. DisclosuresNo relevant conflicts of interest to declare.

GATA/Heme Multi-Omics Reveals a Trace Metal-Dependent Erythrocyte Developmental Mechanism

By functioning as an enzyme cofactor, hemoglobin component and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we used CRISPR/Cas9 to delete intron 1 and 8 GATA motifs in Alas2, encoding the rate-limiting enzyme in heme biosynthesis, in erythroid precursor G1E-ER-GATA-1 cells (Tanimura et al., 2016, EMBO Rep.). These cells stably express a b-estradiol-activated allele encoding the estrogen receptor hormone binding domain fused to GATA-1. The GATA motif mutations abrogated GATA-1-mediated activation of Alas2 and decreased heme ~30 fold. We merged GATA-1/heme-regulated sectors of the proteome and transcriptome. This analysis revealed a GATA-1/heme circuit involving hemoglobin subunits, ubiquitination components, and proteins not implicated in erythrocyte biology, including the zinc exporter Slc30a1.Slc30a1 and the zinc importer Slc39a8 were the most highly expressed zinc transporters in G1E-ER-GATA-1 cells. Both were GATA-1-activated, and Slc30a1, but not Slc39a8, expression declined in heme-deficient Alas2-intronic mutant cells. We tested whether the zinc transporter genes Slc30a1 and Slc39a8 that share GATA-1 regulation and differ in heme regulation are controlled similarly in primary cells. Lineage-depleted hematopoietic precursors from murine fetal livers were cultured for 72 hours, and flow cytometry was used to isolate cells based on erythroid surface markers. Whereas Slc30a1 expression increased 5.7-fold upon differentiation, Slc39a8 increased 4.6-fold during the initial phase, but was downregulated 8.7-fold thereafter. To quantify intracellular zinc levels, we used the fluorescent zinc indicator FluoZin-3. The FluoZin-3 signal increased during early maturation and decreased thereafter, as predicted by the zinc transporter switch, in which expression of the zinc exporter and importer was sustained and decreased, respectively, during terminal differentiation. We demonstrated that the zinc transporter switch occurs in a primary human erythroblast culture system. Intracellular zinc rises during initial erythroid maturation, followed by a steep decline during terminal differentiation. The heme biosynthesis inhibitor succinylacetone reduced SLC30A1 expression 2.8-fold (p < 0.01), as well as other heme-dependent globin genes HBB and HBA1. These primary mouse and human cell studies revealed an evolutionarily conserved mechanism to differentially regulate zinc transporter genes during erythroid differentiation, culminating in a switch from importer and exporter expression to solely exporter expression.To test whether reconfiguring the mechanism governing intracellular zinc impacts differentiation and to elucidate Slc30a1 and Slc39a8 function, we developed multiple shRNAs and used them in loss-of-function studies with lineage-negative hematopoietic precursors from murine fetal livers (E14.5). Slc39a8 downregulation decreased intracellular zinc levels in immature erythroblasts. We also used the zinc chelator TPEN to reduce zinc in immature erythroblasts. TPEN reduced intracellular zinc and considerably decreased the live cell population (p < 0.001). Downregulating Slc30a1 increased intracellular zinc 2.2-fold and, strikingly, accelerated differentiation (p < 0.001). This analysis established a conserved paradigm in which a GATA-1/heme circuit controls trace metal transport machinery and trace metal levels as a mechanism governing cellular differentiation. Ongoing studies involve the development of innovative strategies to leverage this mechanism to enhance or suppress erythrocyte development with primary mouse and human systems. We are conducting multi-disciplinary metallomic analyses to test the hypothesis that the dynamic changes in zinc selectively commission and decommission zinc-binding proteins in erythroblasts. We anticipate that this approach can be readily extended to other sectors of the hematopoietic system and more broadly. DisclosuresNo relevant conflicts of interest to declare.

Oxidative stress, endothelial dysfunction, cytokine imbalance, gonadotropic synergism, or all about tocopherol in the practice of an obstetrician-gynecologist

Generation of reactive oxygen species (ROS) occurs in every human. The condition of oxidative stress develops when the human antioxidant defenses including the enzymes superoxide dismutase, peroxidases etc., and biological antioxidant molecules, are insufficient. Vitamin E is part of the antioxidant family. It plays a key role in neutralizing ROS, and its scope of activity ranges from the inhibition of heme biosynthesis and lipid peroxidation to maintaining the collagen/elastin balance. Vitamin E deficiency may be either primary or secondary; both shift the balance towards the development of oxidative stress, which may affect the normal course of pregnancy. As a result, pregnancy loss or preterm birth may ensue. Moreover, the oxidative stress can have a negative impact on the development of a fetus. In addition, the oxidative stress may play a role in various female reproductive disorders, eventually leading to infertility. In the today’s world, the popular commitment to low-cholesterol and low-fat diets often leads to an increasing number of women with vitamin E deficiency. In order to maintain normal pregnancy, this imbalance requires an adequate correction starting at the stage of preconception care.

5-aminolevulinic acid (ALA) deficiency causes impaired glucose tolerance and insulin resistance coincident with an attenuation of mitochondrial function in aged mice.

In vertebrates, the initial step in heme biosynthesis is the production of 5-aminolevulinic acid (ALA) by ALA synthase (ALAS). ALA formation is believed to be the rate-limiting step for cellular heme production. Recently, several cohort studies have demonstrated the potential of ALA as a treatment for individuals with prediabetes and type-2 diabetes mellitus. These studies imply that a mechanism exists by which ALA or heme can control glucose metabolism. The ALAS1 gene encodes a ubiquitously expressed isozyme. Mice heterozygous null for ALAS1 (A1+/-s) experience impaired glucose tolerance (IGT) and insulin resistance (IR) beyond 20-weeks of age (aged A1+/-s). IGT and IR were remedied in aged A1+/-s by the oral administration of ALA for 1 week. However, the positive effect of ALA proved to be reversible and was lost upon termination of ALA administration. In the skeletal muscle of aged A1+/-s an attenuation of mitochondrial function is observed, coinciding with IGT and IR. Oral administration of ALA for 1-week brought about only a partial improvement in mitochondrial activity however, a 6-week period of ALA treatment was sufficient to remedy mitochondrial function. Studies on differentiated C2C12 myocytes indicate that the impairment of glucose metabolism is a cell autonomous effect and that ALA deficiency ultimately leads to heme depletion. This sequela is evidenced by a reduction of glucose uptake in C2C12 cells following the knockdown of ALAS1 or the inhibition of heme biosynthesis by succinylacetone. Our data provide in vivo proof that ALA deficiency attenuates mitochondrial function, and causes IGT and IR in an age-dependent manner. The data reveals an unexpected metabolic link between heme and glucose that is relevant to the pathogenesis of IGT/IR.

Novel Cell-Killing Mechanisms of Hydroxyurea and the Implication toward Combination Therapy for the Treatment of Fungal Infections.

We have previously reported that an erg11 mutation affecting ergosterol synthesis and a hem13 mutation in the heme synthesis pathway significantly sensitize the fission yeast Schizosaccharomyces pombe to hydroxyurea (HU) (1, 2). Here we show that treatment with inhibitors of Erg11 and heme biosynthesis phenocopies the two mutations in sensitizing wild-type cells to HU. Importantly, HU synergistically interacts with the heme biosynthesis inhibitor sampangine and several Erg11 inhibitors, the antifungal azoles, in causing cell lethality. Since the synergistic drug interactions are also observed in the phylogenetically divergent Saccharomyces cerevisiae and the opportunistic fungal pathogen Candida albicans, the synergism is likely conserved in eukaryotes. Interestingly, our genetic data for S. pombe has also led to the discovery of a robust synergism between sampangine and the azoles in C. albicans Thus, combinations of HU, sampangine, and the azoles can be further studied as a new method for the treatment of fungal infections.

Potential role of heme metabolism in the inducible expression of heme oxygenase-1

BackgroundThe degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite. MethodsWe examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein. ResultsThe blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels. ConclusionsWe demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses. General significanceThe key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.

Potential role of mitochondrial superoxide decreasing ferrochelatase and heme in coronary artery soluble guanylate cyclase depletion by angiotensin II.

Oxidation of the soluble guanylate cyclase (sGC) heme promotes loss of regulation by nitric oxide (NO) and depletion of sGC. We hypothesized that angiotensin II (ANG II) stimulation of mitochondrial superoxide by its type 1 receptor could function as a potential inhibitor of heme biosynthesis by ferrochelatase, and this could decrease vascular responsiveness to NO by depleting sGC. These processes were investigated in a 24-h organoid culture model of bovine coronary arteries (BCA) with 0.1 μM ANG II. Treatment of BCA with ANG II increased mitochondrial superoxide, depleted mitochondrial superoxide dismutase (SOD2), ferrochelatase, and cytochrome oxidase subunit 4, and sGC, associated with impairment of relaxation to NO. These processes were attenuated by organoid culture with 8-bromo-cGMP and/or δ-aminolevulinic acid (a stimulator of sGC by protoporphyrin IX generation and heme biosynthesis). Organoid culture with Mito-TEMPOL, a scavenger of mitochondrial matrix superoxide, also attenuated ANG II-elicited ferrochelatase depletion and loss of relaxation to NO, whereas organoid culture with Tempol, an extramitochondrial scavenger of superoxide, attenuated the loss of relaxation to NO by ANG II, but not ferrochelatase depletion, suggesting cytosolic superoxide could be an initiating factor in the loss of sGC regulation by NO. The depletion of cytochrome oxidase subunit 4 and sGC (but not catalase) suggests that sGC expression may be very sensitive to depletion of heme caused by ANG II disrupting ferrochelatase activity by increasing mitochondrial superoxide. In addition, cGMP-dependent activation of protein kinase G appears to attenuate these ANG II-stimulated processes through both preventing SOD2 depletion and increases in mitochondrial and extramitochondrial superoxide.

Transcriptional Induction of Transferrin Receptors By Heme in Erythroid Cells

The transferrin receptor (TfR) is a membrane glycoprotein whose only clearly defined function is to mediate cellular uptake of iron (Fe) from a plasma glycoprotein, transferrin. Iron uptake from diferric transferrin (Tf) involves the binding of transferrin to the TfR followed by internalization of Tf within an endocytic vesicle by receptor-mediated endocytosis. Iron is then released from transferrin within endosomes by a combination of Fe3+ reduction by Steap3 (likely when transferrin is still bound to TfR) and a decrease in pH (~pH 5.5). Following this, Fe2+ is transported across the endosomal membrane by DMT1. Transferrin receptors are highly expressed on immature erythroid cells, placental tissue, and rapidly dividing cells, both normal and malignant. In proliferating nonerythroid cells the expression of TfR is negatively regulated post-transcriptionally by intracellular iron through iron responsive elements (IREs) in the 3' untranslated region (UTR) of transferrin receptor mRNA. IREs are recognized by specific cytoplasmic proteins (iron regulatory proteins; IRPs) that, in the absence of iron in the labile pool, bind to the IREs of transferrin receptor mRNA, preventing its degradation. On the other hand, the expansion of the labile iron pool leads to a rapid degradation of transferrin receptor mRNA that is not protected, since IRPs are not bound to it. However, some cells and tissues with specific requirements for iron probably evolved mechanisms that can override the IRE/IRP-dependent control of transferrin receptor expression. We previously documented that the TfR gene promoter contains an erythroid active element that stimulates the receptor gene transcription upon induction of hemoglobin synthesis (1).In this study we have demonstrated that incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is primarily a result of increased mRNA synthesis, since the effect of ALA can be abolished by actinomycin D. Moreover, we found that the TfR promoter was activated in vitro by the addition of ALA or hemin to murine erythroleukemia (MEL) cells induced to differentiate using DMSO. Furtehermore, site-directed mutation of erythroid active element (1) in the TfR promoter abolished the effects of ALA or hemin. These results indicate that heme may directly or indirectly interact with the TfR promoter, consequently enhancing the gene expression. Hence, our results show that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. In conclusion, erythroid cells, which are the most avid consumers of iron in the organism, use a transcriptional mechanism to maintain very high transferrin receptor levels.1 Chun-Nam Lok Ponka P. (2000) Identification of an Erythroid Active Element in the Transferrin Receptor Gene. J. Biol. Chem. 275: 24185-24190. DisclosuresNo relevant conflicts of interest to declare.

Delta-Aminolevulinate Preferentially Induces Gamma-Globin Expression in Erythroid Cells through Activation of NRF2 Stress Response

Fetal hemoglobin (HbF,α2γ2) is an important genetic modifier for β-hemoglobinopathies including sickle cell disease (SCD) and β-Thalassemia which occur as a result of mutations in the adult β-globin gene. δ-aminolevulinate (ALA), the first product synthesized in the mammalian heme biosynthesis pathway, has been reported to induce hemoglobin production. In this study, we determined the effects of ALA on γ- and β-globin transcription and HbF production in an erythroid cell line and primary erythroid progenitors. We demonstrated that ALA increased heme levels by 50% with 48 hour treatment in KU812 erythroleukemia cells. By reverse transcription-real time polymerase chain reaction (RT-qPCR), we detected an induction of γ-globin transcription in a time- and dose-dependent manner with 2.4-fold elevation at the 2mM ALA concentration. Moreover, we showed that HbF protein level increased dramatically by 22.9-fold at 48 hour ALA treatment by western blot analysis with HbF specific antibody. By contrast, ALA treatment did not change β-globin transcription level. Subsequent studies in primary erythroid progenitors derived from CD34+ stem cells, we observed γ-globin transcriptional activation up to 6.5-fold by day 22 in culture with 48hour ALA treatment, whereas β-globin transcription was not significantly changed. Studies to determine molecular mechanisms demonstrated that addition of succinylacetone (SA), a specific inhibitor of heme biosynthesis, blocked the ALA-mediated induction of γ-globin transcription and HbF synthesis in KU812 cells detected by RT-qPCR, western blot and enzyme-linked immunosorbent assay. Using 2'-7'-Dichlorodihydrofluorescein diacetate with flow cytometer detection, we observed a 3-fold increase of reactive oxygen species such as hydrogen peroxide with 24-hour ALA treatment in KU812 cells which was inhibited by the addition of SA. In addition, western blot analysis detected increased nuclear translocation of NRF2, a master transcription factor controlling the cellular antioxidant response. Using chromatin immunoprecipitation, we demonstrated that NRF2-associated enrichment of γ-globin proximal promoter chromatin was enhanced 10-fold in the region of the antioxidant responsive element. Furthermore, in KU812 cells transiently transfected with a wild-type and ARE-mutated γ-globin promoter luciferase reporters followed by ALA treatment, we detected activation of the wild-type γ-globin promoter-driven luciferase reporter. As predicted, deletion of the ARE in the γ-globin promoter not only significantly reduced the basal activity by 10-fold but also abolished the ALA-induced promoter activity. These data support a role of ALA in activating heme production and γ-globin expression through an NRF2 activation mechanism. Our results provide evidence that ALA and the heme biosynthesis pathway are novel targets for therapeutic potential in treating β-hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1.

The induction of the gene encoding heme oxygenase 1 (Hmox1, HO-1) by Nrf2 is unique compared with other Nrf2 targets. We previously showed that the Nrf2a-mediated induction of zebrafish hmox1a was liver specific and transient. We screened transcription factors that could repress the induction of hmox1a but not other Nrf2a targets and concluded that Bach1b was a prime candidate. In bach1b-knocked-down larvae, the induction of hmox1a was observed ectopically in nonliver tissues and persisted longer than normal fish, suggesting that Bach1 is the only regulator for both the liver-specific and transient induction of hmox1a. Co-knockdown of bach1b with its co-ortholog bach1a enhanced these effects. To determine why Bach1 could not repress the hmox1a induction in the liver, we analyzed the effects of a heme biosynthesis inhibitor, succinylacetone, and a heme precursor, hemin. Succinylacetone decreased the Nrf2a-mediated hmox1a induction, whereas pre-treatment with hemin caused ectopic induction of hmox1a in nonliver tissues, implying that the high heme levels in the liver may release the repressive activity of Bach1. Our results suggested that Bach1 regulates the liver specificity and transience of the Nrf2a-dependent induction of hmox1a and that heme mediates this regulation through Bach1 inhibition based on its level in each tissue.

Transcriptional regulation of transferrin receptor by heme in erythroid cells

Hemoglobin synthesis consumes most of the iron that is taken up by cells from plasma transferrin, and this process requires very high expression of transferrin receptors (TfR) at the membranes of erythroid cells. In contrast to other cells, developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription (Lok & Ponka, J Biol Chem 275:24185-90, 2000). Our laboratory studies provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of murine erythroleukemia (MEL) cells with 5-aminolevulinic acid (ALA) increased TfR expression as well as iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Recently, IRP2 was proposed to play a role in maintaining TfR mRNA stability in developing RBC (Cooperman et al., Blood 106:108491; 2005; Galy et al., Blood 106: 2580-9, 2005). Importantly, we have demonstrated that ALA added to cultures of erythroid cells derived from IRP2 knock out mice restores the expression of TfR to levels observed in cells obtained from wild type mice. In conclusion, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis.

A Selective Inhibitor of Heme Biosynthesis in Endosymbiotic Bacteria Elicits Antifilarial Activity In Vitro

Lymphatic filariasis and onchocerciasis are severe diseases caused by filarial worms and affect more than 150 million people worldwide. Endosymbiotic α-proteobacteria Wolbachia are essential for these parasites throughout their life cycle. Using a high-throughput chemical screen, we identified a benzimidazole compound, wALADin1, that selectively targets the δ-aminolevulinic acid dehydratase (ALAD) of Wolbachia (wALAD) and exhibits macrofilaricidal effects on Wolbachia-containing filarial worms invitro. wALADin1 is a mixed competitive/noncompetitive inhibitor that interferes with the Mg(2+)-induced activation of wALAD. This mechanism inherently excludes activity against the Zn(2+)-dependent human ortholog and might be translatable to Mg(2+)-responsive orthologs of other bacterial or protozoan pathogens. The specificity profile of wALADin1 derivatives reveals chemical features responsible for inhibitory potency and species selectivity. Our findings validate wALADins as a basis for developing potent leads that meet current requirements for antifilarial drugs.

Sampangine Inhibits Heme Biosynthesis in both Yeast and Human

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.

Transcriptional Regulation of Transferrin Receptor by Heme in Erythroid Cells

AbstractAbstract 2049Erythroid cells are the largest consumers of iron which is delivered to them by tansferrin (Tf) by its cognate receptor (TfR). In contrast to other cells, developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription (Lok & Ponka, J Biol Chem 275:24185-90, 2000). Here we provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression accompanied by increased iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Recently, IRP2 was proposed to play a role in maintaining TfR mRNA stability in developing RBC (Cooperman et al., Blood 106:1084-91; 2005; Galy et al., Blood 106:2580-9, 2005). Importantly, we have demonstrated that ALA added to cultures of erythroid cells derived from IRP2 knock out mice restores the expression of TfR to levels observed in cells obtained from wild type mice. In conclusion, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. Disclosures:No relevant conflicts of interest to declare.

Lack of Hemodynamic Effects After Extended Heme Synthesis Inhibition by Succinylacetone in Rats

Hypertyrosinemia (HT) is a life-threatening condition caused in large part by the buildup of tyrosine metabolites and their derivatives. One such metabolite is succinylacetone (SA), a potent irreversible inhibitor of heme biosynthesis. Heme is a key component of numerous enzymes involved in arterial blood pressure (BP) regulation, including nitric-oxide synthase (NOS) and its downstream mediator soluble guanylyl cyclase (sGC). Because NOS and sGC are important regulators of cardiovascular function, we hypothesized that inhibition of heme supply to these enzymes by SA would result in the induction of a measurable hypertensive response. Male Sprague-Dawley rats were treated with SA (80 mg x kg(-1) x day(-1) i.p.) for 14 days, resulting in a marked increase in urinary SA and delta-aminolevulinic acid (P < 0.001 for both parameters) and decreased heme concentrations in kidney, liver, spleen, and vascular tissues (P < 0.05 for all parameters). After SA treatment, systemic nitrite/nitrate excretion was reduced by 72% (P < 0.001), and renal NOS and sGC activities were decreased by 32 (P < 0.05) and 38% (P < 0.01), respectively. SA administration also compromised the ex vivo sensitivity of aorta to endothelium-dependent and -independent vasodilation. Despite these effects, SA treatment failed to induce any changes in BP, as assessed by radiotelemetry. Moreover, BP profiles in the SA-treated animals were less responsive to altered sodium intake. The present results demonstrate that extended inhibition of heme synthesis with SA affects hemoenzyme function, albeit without consequent effects on BP regulation and sodium excretion.