Abstract
Rho family GTPases are key regulators of many different biological processes including cell motility, growth, and differentiation. Cdc42, Rac and Rho are the most extensively studied members of this family. Although the role of GTPases is becoming increasingly clear in blood cells, virtually nothing is known about the role of downstream effectors of Rho GTPases in hematopoiesis. Rho activates the serine/threonine protein kinases ROCKI and ROCKII. To determine the role of ROCK kinases in hematopoiesis, we generated mice deficient in the expression of ROCKI. Under steady state conditions, deficiency of ROCKI did not demonstrate any significant changes in peripheral blood white cell counts nor defects in the production of red cells compared to controls were observed. Since deficiency of ROCKI alone did not result in perturbed steady state erythropoiesis, we examined the role of ROCKII in this process. For these studies, we used an erythroid progenitor cell line derived from GATA-1 (G1E) null mice. These cells proliferate continuously in culture as developmentally arrested erythroid precursors and upon restoration of GATA-1 activity, undergo cell cycle arrest and terminal maturation in a fashion that largely recapitulates normal erythropoiesis. Using these cells, we generated a conditionally regulated system to study the consequences of ROCKII inactivation on erythropoiesis. We utilized a retroviral vector encoding the activated and kinase dead version of ROCKII by swapping the COOH-terminal negative regulatory portion of ROCKII with the estrogen receptor hormone binding domain. Specifically, we utilized a fused active and kinase dead ROCKII mutant to EGFP and to the estrogen receptor hormone binding domain, which can be stimulated by the estrogen analogs tamoxifen or 4-hydroxytamoxifen (4-HT). In contrast to a lack of a positive role for ROCKI in erythropoiesis, inactivation of ROCKII in erythroid progenitors in the absence of GATA-1 activation resulted in complete repression of cytokine mediated growth (n=4, p<0.001). Consistently, a dose dependent repression in the growth of these cells was also observed in the presence of two distinct ROCK kinase inhibitors (n=3, p<0.001). Importantly, when these cells were induced to differentiate by conditionally activating GATA-1, cells expressing the empty vector or the dominant negative version of ROCKII under went cell cycle arrest and terminal maturation as expected. In contrast, cells expressing the activated version of ROCKII, also under went terminal differentiation, however these cells continued to proliferate throughout the course of differentiation (n=3, p<0.005). To determine the mechanism(s) of growth repression in the absence of GATA-1 activation, we conducted survival and cell cycle analysis. Surprisingly, expression of dominant negative (DN) ROCKII in these cells did not induce apoptosis, but significantly impaired cell cycle entry, with majority of cells falling in the G1/G0 phase of cell cycle (n=5, p<0.005). The extensive arrest in cell cycle progression due to ROCKII inactivation in these cells was associated with complete loss of cyclinD1 expression. Our results reveal that ROCKI and ROCKII play distinct roles in erythropoiesis. Furthermore, in addition to its well established function as an activator of erythroid genes, GATA-1 also participates in a genetic program that inhibits cell proliferation by repressing the activation of ROCKII kinase. In summary, we demonstrate a novel aspect of cross talk between GATA-1 and Rho kinase ROCKII in erythropoietic development.
Dual role of hematopoietic progenitor kinase 1 (HPK1) as a positive regulator of 1α,25-dihydroxyvitamin D-induced differentiation and cell cycle arrest of AML cells and as a mediator of vitamin D resistance