Non-Redundant Roles for an Epo/EpoR/STAT5 Regulated Intracellular Serpin, Serpina-3G, during Epo- and Anemia- Induced Erythropoiesis

Interest in advancing a more sophisticated knowledge of EPOR action mechanisms remains intense based on the emergence of novel ESAs;  cytoprotective effects of EPO and ESAs in ischemically injured tissues; and adverse effects of EPO on cancer progression.  By employing primary bone marrow erythroblasts and global transcriptome analyses, we recently have discovered several previously undescribed EPO/EPOR response factors that regulate erythroid progenitor cell development.  These include Cyclin-G2, the pseudokinase Tribbles-3 (TRB3), and a strongly EPO-induced intracellular serpin, Serpina-3G (S3G).  Here we report on EPOR- mediated routes to S3G expression, and on phenotypes resulting from S3G’s reinforced, or disrupted expression.  As determined using early-stage erythroid progenitors with minimal knocked-in EPOR alleles, S3G expression proved to depend upon an EPOR-PY343/STAT5 axis.  At the protein level, S3G was cytoplasmically retained (as was S3G-flag in stably transduced G1E2 cells, and in transgenic Gata1-IE3.9int-S3G mice).  Ectopic expression of S3G further revealed advantaged expansion capacities.  To critically assess S3G’s function, S3G knock-out mice were prepared.  Consistent with a hypothesis that S3G might affect erythropoiesis selectively during anemia, BFUe, CFUe and red cell levels in S3G-null mice at steady-state were not significantly perturbed.  However, S3G’s deletion markedly impaired erythropoiesis during hemolytic anemia, and inhibited EPO-  induced erythropoiesis per se.  In particular, hematocrits during anemia in S3G-null mice fell to 23.0 +/- 2.2% as compared to 38.7 +/- 1.8% for S3G+/+ controls.  In response to EPO, an increase in hematocrit of only 1.75 +/- 0.2% was exhibited for S3G-null mice as compared to 4.9 +/- 0.5% for S3G+/+ controls.  Investigations therefore establish S3G as an important non-redundant mediator of EPO- and anemia- induced erythropoiesis.  They also raise interesting new questions concerning the nature of S3G’s action pathways, and S3G’s candidate pro-erythroblast intrinsic target protease.