Ligation of erythropoietin (EPO) receptor (EPOR) JAK2 kinase complexes propagates signals within erythroid progenitor cells (EPCs) that are essential for red blood cell production. To reveal hypothesized novel EPOR/JAK2 targets, a phosphotyrosine (PY) phosphoproteomics approach was applied. Beyond known signal transduction factors, 32 new targets of EPO-modulated tyrosine phosphorylation were defined. Molecular adaptors comprised one major set including growth factor receptor-bound protein 2 (GRB2)–associated binding proteins 1–3 (GAB1-3), insulin receptor substrate 2 (IRS2), docking protein 1 (DOK1), Src homology 2 domain containing transforming protein 1 (SHC1), and sprouty homologue 1 (SPRY1) as validating targets, and SPRY2, SH2 domain containing 2A (SH2D2A), and signal transducing adaptor molecule 2 (STAM2) as novel candidate adaptors together with an ORF factor designated as regulator of human erythroid cell expansion (RHEX). RHEX is well conserved in Homo sapiens and primates but absent from mouse, rat, and lower vertebrate genomes. Among tissues and lineages, RHEX was elevated in EPCs, occurred as a plasma membrane protein, was rapidly PY-phosphorylated >20-fold upon EPO exposure, and coimmunoprecipitated with the EPOR. In UT7epo cells, knockdown of RHEX inhibited EPO-dependent growth. This was associated with extracellular signal-regulated kinase 1,2 (ERK1,2) modulation, and RHEX coupling to GRB2. In primary human EPCs, shRNA knockdown studies confirmed RHEX regulation of erythroid progenitor expansion and further revealed roles in promoting the formation of hemoglobinizing erythroblasts. RHEX therefore comprises a new EPO/EPOR target and regulator of human erythroid cell expansion that additionally acts to support late-stage erythroblast development.

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for red blood cell production and exert important cytoprotective effects on select vascular, immune, and cancer cells. To discover novel EPO action modes, we profiled the transcriptome of primary erythroid progenitors. We report Serpina3g /Spi2A as a major new EPO/EPOR target for the survival of erythroid progenitors. In knockout mice, loss of Spi2A worsened anemia caused by hemolysis, radiation, or transplantation. EPO-induced erythropoiesis also was compromised. In particular, maturing erythroblasts required Spi2A for cytoprotection, with iron and reactive oxygen species as cytotoxic agents. Spi2A defects were ameliorated by cathepsin-B/L inhibition, and by genetic co-deletion of lysosomal cathepsin B. Pharmacological inhibition of cathepsin B/L enhanced EPO-induced red cell formation in normal mice. Overall, we define an unexpected EPO action mode via an EPOR–Spi2A serpin–cathepsin axis in maturing erythroblasts, with lysosomal cathepsins as novel therapeutic targets.