Claudio Fader Kaiser

Mature erythrocyte results from a finely regulated process called erythropoiesis that produces 2 million RBCs every second in healthy human adults. Maturation from erythroid-committed precursors is called terminal erythropoiesis and occurs in the bone marrow (BM), which consists of a central macrophage surrounded by erythroblasts, and ends in the blood stream where reticulocytes complete their maturation within 1-2 days. During this process, the erythroid precursors undergo a series of physiological changes necessary for the maturation of the erythrocyte. These changes involve membranes remodeling, cell volume decrease, specific protein synthesis such as hemoglobin, membranous organelles clearance by autophagy, and enucleation(1).

A crucial process for the final stage of erythroid differentiation is autophagy, a mechanism by which cytosolic macromolecules and even whole organelles are transported to lysosomes for degradation. Autophagy seems to be active during maturation of orthochromatic erythroblasts and reticulocytes. Studies in both yeast and mammals have allowed the characterization of at least 40 Atg (autophagy-related) genes, which are required for autophagy. One of them is the cytosolic protein LC3/Atg8, which is essential for autophagosome maturation (lipidation) and constitute an autophagosome marker (2).During mitophagy (a specialized autophagic mechanism), mitochondria become trapped in double-membrane vesicles (autophagosomes) and are subsequently degraded and removed by autophagosome-lysosome fusion. This process is related with red blood cells differentiation and maturation, as well as certain mechanisms involved in chemotherapy treatment resistance of particular types of leukemia.

Low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane endocytic receptor involved in many cellular processes, such as cell migration, apoptosis, autophagy and the metabolism of molecules such as alpha-2-macroglobulin (α2m).The ligand binding stimulus triggers clathrin-dependent endocytosis, initiating a signaling cascade that activates different pathways such as PI3K/AKT, ERK1/2 and MAPK (3 and 4). Furthermore, it has been shown that LRP1 is responsible for the elimination of the hemin-hemopexin complex, participating in the regulation of intracellular iron levels (5). Likewise, it has been described that LRP1 levels vary in different cell types, being practically undetectable in mature red blood cells, unlike their more immature progenitors.

Our cellular model isthe K562 cells which are a cell line derived from patients with chronic myeloid leukemia, so they have erythroid characteristics (6). This cell line has been widely used as a model of maturation and differentiation, since depending on the inducing drug they can mature into cells of the megakaryocytic or erythropoietic line (7).

 

Our work group has studied in depth, in this cell type, the molecular machinery involved in intracellular trafficking, required for the maturation of the autophagosome and its relationship during erythropoiesis (8 and 9).We have demonstrated  by biochemical and molecular methos, confocal microscopy an elecron microscopythat hemin, an erythropoiesis inductor, is able to generate an autophagic response (mitophagy) in erythroleukemia cells lines (K562 cells), inducing the expression of LRP1 and some autophagic genes such as LC3, Atg5 and Beclin1. Importantly, we have demonstrated that hemin target LRP1 to autophagosomes and this receptor is in part responsible for hemin autophagy activation. Moreover, hemin induces a traffic modification of LRP1, increasing its localization in later endosomal compartments including lysosomal vesicles (10). In the same way, we have also shown in two different hematopoietic cell lines (K562 and UT7)that alpha 2-macroglobulin (the most studied LRP1 ligand) triggers the autophagic pathway in a LRP1 dependent manner, inducing the autophagy-dependent extracellular vesicles (EVs) secretion.

It has been proposed that autophagy manipulation is a feasible new therapeutic key in erythropoietic disorders as well as in fighting cancer. Taken together, our results suggest that hemin and alpha 2-macroglobulin are two molecules that favoring erythroid maturation by inducing an autophagic response in K562 cells,via LRP1 receptor. This study could provide valuable knowledge regarding the possible role of LRP1 during erythropoiesis, being a possible therapeutic candidate that helps in hematopoietic disorders as well as the chronic myelogenous leukemia (CML) treatment.

 

References

1-Moras M, Lefevre SD, Ostuni MA.2017. From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals.Front Physiol. 19;8:1076.

2- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T. 2000. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 1;19(21):5720–8.

3- Fuentealba RA, Liu Q, Kanekiyo T, Zhang J, Bu G. 2009. Low density lipoprotein receptor-related protein 1 promotes anti-apoptotic signaling in neurons by activating Akt survival pathway. J Biol Chem. 284(49):34045–53.

4- Revuelta-López E, Castellano J, Roura S, Gálvez-Montón C, Nasarre L, Benitez S. 2020.Hypoxia induces metalloproteinase-9 activation and human vascular smooth muscle cell migration through low-density lipoprotein receptor-related protein 1-mediated Pyk2 phosphorylation. Arterioscler Thromb Vasc Biol. 33(12):2877–87.

5-Tsiftsoglou AS, Wong W, Robinson SH, Hensold J.1989. Hemin increases production of β‐like globin RNA transcripts in human erythroleukemia K‐562 cells. Dev Genet. 10(4):311–7.

6- Johnstone RM. Cleavage of the transferrin receptor by human granulocytes: Preferential proteolysis of the exosome-bound TFR.2008. J Cell Physiol. 168(2):333–45.

7- Tsiftsoglou AS, Wong W, Tsamadou AI, Robinson SH. 1991. Cooperative effects of hemin and anthracyclines in promoting terminal erythroid maturation in K562 human erythroleukemia cells. Exp Hematol.19(9):928–33.

8- Fader CM, Sánchez D, Furlán M, Colombo MI. 2008. Induction of autophagy promotes fusion of multivesicular bodies with autophagic vacuoles in K562 cells. Traffic. 9(2):230–50.

9-  Fader CM, Colombo MI. 2009. Autophagy and multivesicular bodies: Two closely related partners. Cell Death and Differentiation.. p. 70–8.

10- Grosso RA, Caldarone PVS, Sánchez MC, Chiabrando GA, Colombo MI, Fader CM. 2019. Hemin induces autophagy in a leukemic erythroblast cell line through the LRP1 receptor. Biosci Rep. ;39(1).