Anemia as associated with end-stage renal disease, chemo- and radiation therapies, inflammation, myelodysplasia, iron dysregulation and hemoglobinopathies leads to chronic bouts of accelerated, yet stressed and often compromised erythropoiesis. A specific understanding of key regulators of such stress erythropoiesis is of basic importance for insight into new anemia therapies. rhEPO per se can be therapeutic, but has substantial hypertensive side-effects, evokes thrombolytic events, is a high-cost recombinant glycoprotein, and may worsen certain cancers. Via investigations of EPO's effects on primary erythroid progenitors, we have uncovered and initially characterized two important new factors (and associated pathways) that selectively regulate stress erythropoiesis. First, our studies of a novel EPO-induced intracellular Spi2A serpin (via a new KO model) reveal that under select stress conditions, erythroblasts become highly sensitive to lysosome damage to the extent of leaching lysosomal executioner cathepsins- which are inhibited by Spi2A. If unchecked, cathepsin-associated damage and erythroblast death escalate, with ROS and iron as cytotoxic co-factors. We also demonstrate that a Cathepsin B/L inhibitor can phenocopy Spi2A's cytoprotective effects. A second EPO co-induced erythropoietic regulator we have discovered (and are beginning to understand) is the molecular adaptor and pseudokinase, Trib3. By developing a Trib3-KO mouse model, we reveal non-redundant novel stress erythropoiesis specific roles for Trib3 during both early, and late- stage erythroid cell formation We will now advance insight into these two new stress erythropoiesis factors, and coupled pathways, via the following SPECIFIC AIMS: SA#1 will first define how Spi2A and linked pathways impact on stress erythropoiesis due to ?-thalassemia and iron imbalance (#1.1). At a mechanistic level, consequences of Spi2A- deficiency on erythroblast autophagy will be determined and molecular damage incurred by erythroblast lysosomes during oxidative stress plus Spi2A-deficiency will be defined (#1.2). Via LOF studies, SA#2 first will define effects exerted by Trib3 pseudokinase on early- and late-stage erythropoiesis during ?-thalassemia, and iron imbalance (#2.1).
Aim 2. 2 will determine Trib3's roles in unfolded protein response pathways together with Trib3's regulatory molecular action mechanisms in early- and late- stage erythroid cells. SA#3 will advance studies to primary human erythroid progenitors (including thalassemia), and will first determine effects of lysosomal compromise, and cathepsin inhibition on erythroblast cytoprotection (#3.1).
Aim 3. 2 will define effects of Trib3 LOF and GOF during erythropoietic stress. Substantial insight will be gained concerning new regulators of red cell formation and novel target pathways within compromised erythroid progenitors for anti- anemia agents.

Public Health Relevance

During anemia, integrated sets of stress response pathways engage to escalate RBC formation. Chronic stress erythropoiesis, however, can dysregulate response circuits, and compromise the erythron. This includes the anemia of chronic kidney disease (CKD), myelodysplastic syndromes (MDS), cancer therapies, inflammation, iron imbalance, and hemoglobinopathies. For CKD, MDS and chemotherapy, the RBC-inducing hormone EPO is often therapeutic but has major hypertensive side-effects, can be ineffective, and may worsen certain cancers. For hemoglobinopathies (e.g., thalassemia, sickle cell disease) present therapies also are limited (transfusions, BMT, HU). R01 DK89439 will investigate two important new erythroid cell intrinsic stress erythropoiesis regulators as Spi2A and Trib3, together with their action pathways and potential as new targets for anti-anemia agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK089439-04A1
Application #
8817637
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
2010-08-01
Project End
2018-05-31
Budget Start
2014-09-09
Budget End
2015-05-31
Support Year
4
Fiscal Year
2014
Total Cost
$360,110
Indirect Cost
$121,145
Name
Maine Medical Center
Department
Type
DUNS #
071732663
City
Portland
State
ME
Country
United States
Zip Code
04102
Verma, Rakesh; Su, Su; McCrann, Donald J et al. (2014) RHEX, a novel regulator of human erythroid progenitor cell expansion and erythroblast development. J Exp Med 211:1715-22
Li, Lei; Byrne, Susan M; Rainville, Nicole et al. (2014) Brief report: serpin Spi2A as a novel modulator of hematopoietic progenitor cell formation. Stem Cells 32:2550-6
Dev, Arvind; Byrne, Susan M; Verma, Rakesh et al. (2013) Erythropoietin-directed erythropoiesis depends on serpin inhibition of erythroblast lysosomal cathepsins. J Exp Med 210:225-32
Sathyanarayana, Pradeep; Dev, Arvind; Pradeep, Anamika et al. (2012) Spry1 as a novel regulator of erythropoiesis, EPO/EPOR target, and suppressor of JAK2. Blood 119:5522-31
Dev, Arvind; Fang, Jing; Sathyanarayana, Pradeep et al. (2010) During EPO or anemia challenge, erythroid progenitor cells transit through a selectively expandable proerythroblast pool. Blood 116:5334-46