ROCK1 Functions As a Critical Regulator of Stress Erythropoiesis and Survival by Regulating p53

Abstract 916

Erythropoiesis is a dynamic, multistep process in which hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Under normal physiologic condition, erythropoiesis takes place primarily in the bone marrow of humans and mice. Efficient stress erythropoiesis is crucial to survival and recovery from various pathophysiologic conditions including blood loss, anemia, and therapeutic procedures used in the treatment of hematological malignancies such as chemotherapy and stem cell transplantation. Impaired stress erythropoiesis can be fatal under these conditions. In order to develop improved strategies to stimulate stress erythropoiesis in patients with such conditions, it is critical to identify the molecular mechanisms that regulate this process. While many of the downstream signaling molecules and pathways have been elucidated in steady state erythroid cell development, the major regulators under stress conditions remain to be defined. The small families of Rho GTPases and their downstream effectors, Rho kinases have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells and inflammatory cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases, and their physiologic role in erythropoiesis is not known. Utilizing mice deficient in the expression of ROCK1, we demonstrate no significant difference with respect to erythroid parameters in peripheral blood under steady-state conditions (n=19 mice for each genotype). The frequency of Ter119+, CD71+ or double-positive erythroid cells in the bone marrow and spleen of ROCK1-deficient mice was comparable to wild type (WT) controls at basal levels (n=7). In response to myelotoxic stress, ROCK1 deficient Ter119 positive cells demonstrated enhanced survival and recovery compared to WT controls (n=3–6, *p< 0.05). Further, using a phenylhydrazine (PHZ)-induced murine model of hemolytic anemia, we demonstrate that ROCK1-deficient mice exhibit increased red blood cells and enhanced hematocrits relative to WT mice (n=6, *p< 0.05). In addition, ROCK1−/− mice support efficient erythro-splenomegaly, and exhibit a threefold increase in splenic weight after PHZ injection compared to WT controls (n=6, *p< 0.05). Flow cytometric analysis revealed increased frequency of Ter119/CD71 double positive erythroid progenitor pool in ROCK1−/− spleens after PHZ treatment compared to WT (n=6, *p< 0.05). Furthermore, histopathological analysis of WT and ROCK1−/− spleens following PHZ treatment revealed a defined population of white pulp and red pulp in control spleens but enhanced extramedullary erythropoiesis in ROCK1−/− spleens (n=3, *p< 0.05). In vitro colony forming assays showed that ROCK1-deficient splenocytes generated more erythroid colony forming units (CFU-Es) and eventually generated more erythroid-burst forming units (BFU-Es) compared to WT splenocytes in response to different concentrations of EPO and combination of EPO and SCF (n=3, *p< 0.05). Deficiency of ROCK1 also resulted in enhanced survival of mice treated with PHZ compared to controls (n=17 mice per group, *p< 0.05). The enhanced survival of ROCK1-deficient mice in response to PHZ was associated with reduced reactive oxygen species (ROS) levels compared to WT (n=6 mice per group, *p< 0.05). Bone marrow transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous (n=6 mice per genotype, *p< 0.05). Remarkably, the red cell phenotype observed in ROCK1−/− mice is similar to that reported in mice deficient in p53. We show that ROCK1 binds p53 directly and regulates its stability and expression (n=3). In absence of ROCK1, p53 phosphorylation and expression is reduced in ROCK1−/− erythroblasts (n=3). Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.