Erythropoietin Signaling Regulates Heme Biosynthesis

Abstract

Heme plays a fundamental role in a diverse array of cellular processes and is required for the survival of all cells. During erythropoiesis, heme production is drastically upregulated to support the production of oxygen-carrying hemoglobin. This increase in heme production is mediated by transcriptional induction of heme metabolism genes including ferrochelatase (FECH), which is the enzyme that catalyzes the rate-limiting insertion of ferrous iron into protoporphyrin IX in the mitochondria of erythroid cells. However, how heme production is coordinately regulated by extracellular cues is currently unknown. Here, using complementary biochemical and genetics approaches, we show that erythropoietin (EPO) signaling regulates heme biosynthesis via a protein kinase A (PKA)-dependent mechanism. In its inactive state, PKA is a tetrameric complex consisting of two catalytic subunits (C) that are bound to and inhibited by two regulatory subunits (R). The C subunits become activated to phosphorylate downstream target proteins when they dissociate from the R subunits. We demonstrate that EPO-induced JAK2 (janus kinase 2) activity leads to release of the C subunits from the R subunits. We also find that phosphorylated STAT5 (signal transducer and activator of transcription 5) forms a molecular complex with PKA-C. This suggests that phospho-STAT5 can outcompete PKA-R to release PKA-C to directly phosphorylate FECH at a highly conserved threonine residue located in the catalytic site. We examined the importance of FECH phosphorylation in vivo by taking advantage of CRISPR/Cas9-mediated genome editing to knock-in the analogous Thr115Ala substitution into the endogenous Fech gene in murine RBCs. Erythroid cells harboring the homozygous Thr115Ala Fech mutation exhibited a block in hemoglobin production and concomitant intracellular accumulation of upstream protoporphyrin intermediates. Strikingly, this phenotype bears resemblance to erythropoietic protoporphyria (EPP), a human hematologic disorder typically associated with FECHmutations. Together, our results support a model where EPO signaling during erythroid maturation activates PKA by a previously unrecognized JAK2/STAT5-dependent mechanism. Phosphorylation of FECH is required for full activity to support elevated heme biosynthesis and hemoglobin production. Furthermore, our data implicates aberrant EPO/JAK2/PKA signaling in the pathogenesis of human EPP. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Discovering How Heme Controls Genome Function Through Heme-omics

“…Mutation of the murine enhancers abrogates GATA1 occupancy at these sites, decreasing Alas2 expression and heme synthesis, thus causing severe anemia and embryonic lethality (Zhang et al, 2017). Heme synthesis is also controlled by erythropoietin-activated protein kinase A, which phosphorylates and activates the heme synthesis enzyme ferrochelatase (FECH) (Chung et al, 2017).…”

Glutamine via α-ketoglutarate dehydrogenase provides succinyl-CoA for heme synthesis during erythropoiesis

“…6 GATA1-induced erythroid differentiation results in the upregulation of heme synthesis by induction of ALAS2, enhancement of the transcription of all heme pathway enzyme genes, [5][6][7] and phosphorylation of the terminal pathway enzyme, ferrochelatase (FECH). 8 In addition to transcriptional regulation, ALAS2 is also subject to translational regulation by iron via the iron-regulatory protein/iron-responsive element machinery [9][10][11][12] and microRNAs. 13 Posttranslational modification of the protein via the addition of the cofactor pyridoxal phosphate is essential.…”

Early life stress induces dysregulation of the heme pathway in adult mice

“…Similarly, MSEW and control mice did not significantly differ with regard to endogenous concentrations of the free heme scavenger hemopexin in plasma (Figure 2f) or liver (Figure 2g). Erythropoietin (EPO) regulates heme and red blood cell biosynthesis (Chung et al, 2017). Circulating EPO levels showed no significant difference between MSEW and control mice (Figure 2h).…”