RhoA GTPase Is Dispensable for Hematopoietic Stem Cell Maintenance but Essential for Multipotent Progenitor and Lower Hierarchical Hematopoietic Differentiation.

Abstract 2618

Rho family small GTPases are critical regulators of multiple functions of blood cells, including adhesion, migration, proliferation, survival and gene expression. Elucidation of their roles in hematopoiesis could be fundamental for understanding the mechanisms of various blood diseases and improving therapeutic outcomes for hematopoietic abnormalities. The Rho GTPases Rac1, Rac2 and Cdc42 have been found essential in maintaining hematopoietic stem cell (HSC) niche residency and regulating myelopoiesis and lymphopoiesis in previous mouse gene targeting studies. Using a dominant-negative mutant overexpression approach, an earlier study showed that suppression of RhoA activity enhanced HSC proliferation and engraftment potential; however, the bone fide role of RhoA in blood development remains unknown given limitations of such a mutant overexpression approach in specificity, dosage effect, and physiological relevance. Here, we stringently define the function of RhoA in HSC maintenance and hematopoiesis using an interferon inducible RhoA conditional knockout mouse model (Mx-cre⁺; RhoA^loxp/loxp). Systematic deletion of RhoA caused lethality of the mice 7 days post polyIC induction due to hematopoietic failure that was accompanied by drastically decreased bone marrow (BM) cellularity (to ∼1/3 of wild type controls), a loss of splenocytes, and a significant reduction of cell counts of most cell lineages in peripheral blood, suggesting RhoA is required for multiple blood cell lineage differentiation and production. Syngenic transplant experiments yielded similar results, demonstrating that these effects are intrinsic to the hematopoietic compartment. The observed cytopenia resulting from RhoA loss was associated with the exhaustion of BM phenotypic HSPC (Lin⁻Sca1⁺c-kit⁺, LSK) and the hierarchical progenitor cells (Lin⁻c-kit⁺, LK) in number and frequency, a complete loss of colony forming activities, and a total engraftment failure. In addition, BrdU chase labeling and Annexin V/7-AAD staining revealed that RhoA deletion caused a transient increase of proliferation (1.7 fold increase in S phase) and reduction of survival (16.8 fold reduction in Annexin V⁻ 7-AAD⁻) of remaining LSK in the BM. These results indicate that RhoA plays an indispensible, cell autonomous role in HSPC maintenance and hematopoiesis. In a competitive transplantation model where Mx-cre⁺;RhoA^loxp/loxp or Mx-cre⁻;RhoA^loxp/loxp CD45.2⁺ BM cells and WT CD45.1⁺ competitor cells were transplanted at 1:1 ratio into syngenic CD45.1⁺ recipients prior to polyIC induction, RhoA deletion also caused a complete extinction of donor derived (CD45.2⁺) Mac1⁺Gr1⁺ myeloid cells, B220⁺ B cells, as well as CD3⁺ T cells, in the peripheral. Interestingly, distinct from these more differentiated lineages, BM CD45.2⁺ LSK population was only marginally affected (88.9 % of pre-polyIC induction level) while the CD45.2⁺ LK cells and later hierarchical lineages were rapidly eliminated after RhoA deletion in this competitive transplant model. This was associated with increased apoptosis in CD45.2⁺ RhoA^−/− LK, but not LSK, cells, suggesting a specific requirement of RhoA in the myeloid progenitor cell survival. Further, the CD45.2⁺ RhoA^−/− LSK and LSKCD150⁺ cell populations, not differentiated donor-derived progenitors, from the primary competitive transplant BM, were able to be maintained in secondary transplant recipients 5 months post-transplantation, indicating that RhoA serves as a key regulator at an early progenitor differentiation step. Interestingly, RhoA deletion did not affect lin⁻ cell p-MLC and p-cofilin contents and in vitro expansion in response to SCF stimulation, suggesting that RhoA is not required for actomyosin signaling nor SCF induced proliferation. Taken together, our results implicate RhoA as a unique and essential regulator of multipotent progenitor differentiation and survival that controls multi-lineage hematopoiesis.