Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

Abstract

Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed.

Keywords: ABCG2; FLVCR1; FLVCR2; HCP1/PCFT; HO-1; hemopexin.

Figure 1

Free heme toxicity. Free heme has potentially toxic properties due to the catalytic active iron atom it coordinates. Here, toxic effects of heme are depicted. Heme causes cellular oxidative damage (1) by promoting ROS formation, lipid peroxidation, DNA and protein damage. Additionally, heme is a source of iron. Therefore, heme overload leads to intracellular accumulation of iron, with further ROS generation. Heme is a hemolytic agent (2), since it intercalates red blood cell membrane, thus favoring cell rupture and further amplifying the hemolytic process. Heme promotes inflammation (3), by stimulating inflammatory cell activation and cytokine production. Finally, heme causes endothelial dysfunction (4) by several mechanisms: increasing adhesion molecule expression and endothelial activation, promoting inflammatory cell recruitment and platelet aggregation, causing nitric oxide (NO) oxidative consumption and vasoconstriction, oxidizing low-density lipoprotein (LDL).

Figure 2

Control steps in heme metabolism. The main mechanisms involved in the control of heme levels outside, inside and across the cell are illustrated. (1) Heme scavenging: Circulating free heme toxicity is avoided thanks to the action of the scavenging proteins Hx and Albumin. (2) Heme Import: Heme might be imported inside the cell via the putative heme importers HCP1/PCFT and FLVCR2. (3) Heme Synthesis: in the mitochondrion and cytosol, the heme biosynthetic enzymes, starting from succinyl-CoA and glycine, give rise to heme. After synthesis, heme is exported out of the mitochondrion to the cytosol by the mitochondrial heme exporter FLVCR1b. (4) Heme Incorporation in Hemoproteins: once released in the cytosol, heme is inserted in apo-hemoproteins to allow the formation of functional hemoproteins. (5) Heme Degradation: in the endoplasmic reticulum, the heme degrading enzyme HO is responsible for heme degradation into iron (Fe), carbon monoxide and biliverdin. (6) Heme Export: the heme exporters FLVCR1a and ABCG2 regulate heme export out of the cell across the plasma membrane. ALAS, aminolevulinic acid synthase; SLC25A38, solute carrier family 25 member 38; ABCB10, ATP-binding cassette sub-family B member 10; ALAD, amino levulinic acid dehydratase; HMBS, hydroxymethylbilane synthase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen decarboxylase; ABCB6, ATP-binding cassette sub-family B member 6; CPOX, coproporphyrinogen oxidase; PPOX, protoporphyrinogen oxidase; FECH, ferrochelatase; MFRN, mitoferrin; FLVCR, feline leukemia virus subgroup C receptor; HCP1/PCFT heme carrier protein 1/proton-coupled folate transporter; ABCG2, ATP-binding cassette sub-family G member 2; HO, heme oxygenase; Hx, Hemopexin.

Figure 3

The loss of the heme exporter FLVCR1a in mice causes embryonic lethality, skeletal malformation and extended hemorrhages. (A) Stereoscopic view of a wild-type (left) and a Flvcr1a^−/− (right) embryo at the embryonic stage of E14,5. At this stage, the wild-type embryo shows normal skeletal structure, with fully formed limbs. The Flvcr1a^−/− embryo shows extended hemorrhages and edema through the body, in particular in the limbs, back and head. Flvcr1a^−/− embryos show skeletal malformations, as suggested by the absence of the lower jaw and properly formed digits. (B) An enlarged view of E15,5 wild-type and Flvcr1a^−/− anterior limbs (marked with a broken line). In the Flvcr1a^−/− embryo, the limbs show severe hemorrhage, leading to impairment in limb and toe formation.

Figure 4

The heme exporter FLVCR1a acts as a new heme detoxifying system. (A) Erythroid progenitors are able to synthesize and handle high amount of heme, in view of their hemoglobin (Hb)-mediated oxygen transport activity. FLVCR1b acts as a mitochondrial heme exporter to allow newly formed heme release from the mitochondrion to the cytosol, where it is incorporated into hemoproteins. FLVCR1a has been described as a system involved in the control of heme levels inside erythroid progenitors. By mediating heme export out of these cells, FLVCR1a regulates intracellular heme amount, thus limiting free heme toxicity and oxidative damage. (B) Hepatocytes have the highest rate of heme synthesis after the erythroid progenitors. Hepatic heme is mostly used for synthesis of P450 enzymes, which metabolize endogenous compounds and xenobiotics. FLVCR1a mediates heme export out of hepatocytes, thus maintaining hepatic heme homeostasis and controlling cell oxidative status. FLVCR1a export function allows the maintenance of a proper cytosolic heme pool that matches cell need for new hemoprotein generation (e.g., cytochrome P450). Block of heme export causes heme pool expansion leading to the inhibition of heme synthesis and the reduction of cytochrome activity. (C) A similar role for FLVCR1a was proposed to occur in endothelial cells. Flvcr1a^−/− embryos show reduced vascular arborization, potentially due to altered endothelial integrity, suggesting that the lack of FLVCR1a leads to intracellular heme overload and oxidative stress. Endothelial cells are highly sensitive to heme overload and, in this context, FLVCR1a function could be of crucial importance to export heme excess, thus maintaining heme homeostasis and controlling heme-induced oxidative stress.