RhoH, a Hematopoietic-Specific Rho Gtpase, Regulates Proliferation, Survival, Migration and Engraftment of Hematopoietic Progenitor Cells.
Authors: Gu Yi , Jasti Aparna C. , Jansen Michael , Siefring Jamie E. , Lee Katherine E. 
Published: April 2020

Abstract
Rho GTPases are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation and survival in hematopoietic cells. Here, we have studied a newly identified hematopoietic-specific Rho GTPase, RhoH. The RhoH gene was first identified as a hypermutable locus in non-Hodgkin’s lymphoma and diffuse large B-cell lymphoma, and alteration of RhoH expression has been implicated in some cases. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and remains in the active, GTP-bound state suggesting that the level of RhoH expression may be a critical determinant of activity. To elucidate the biological role of RhoH in blood cell development and function, we used two complementary approaches, including overexpression via retrovirus-mediated gene transfer and knock-down via RNA interference, to alter levels of RhoH in hematopoietic progenitor cells (HPCs) and examined the effects of altered RhoH expression on hematopoiesis. In cytokine-stimulated HPCs, knock-down of RhoH expression (to ~ 30% of the endogenous level) significantly induces proliferation, survival and stromal cell derived factor-1 α (SDF-1α)-induced migration and cortical filamentous (F)-actin assembly (Table I). Conversely, RhoH overexpression (&gt;2 folds of the endogenous level) in these cells is associated with reduced proliferation and increased apoptosis in response to stem cell factor, and defective F-actin polymerization at cell peripheral membrane and chemotaxis in response to SDF-1 α (Table I). In the irradiated recipient mice, hematopoietic stem/progenitor cells overexpressing RhoH demonstrate defective reconstitution capability in peripheral blood (Table I), bone marrow, spleen and lymph node as compared with control vector-transduced cells. Also, the RhoH-transduced donor cells displayed an increased level of apoptosis and a reduced proliferative response to the 5-fluorouracil treatment when compared with vector-transduced cells. Interestingly, these defective phenotypes associated with the progenitor cells overexpressing RhoH resemble the phenotypes of Rac1-/- ; Rac2-/- cells. Overexpression of RhoH is also associated with reduced Rac activity when stimulated by a Rac agonist. In addition, overexpression of RhoH disrupts the SDF-α-stimulated subcellular redistribution of Rac1 and Rac2 1 to cell peripheral membrane where Rac proteins are activated and induce F-actin polymerization. These results suggest that aberrant expression of RhoH leads to abnormal growth and actin-based function of HPCs. RhoH functions as a negative regulator of proliferation, survival, migration, actin polymerization and engraftment of HPCs possibly through suppression of Rac activation via preventing localization of Rac proteins to cell peripheral membrane.
Table 1. Cellular phenotypes associated with the altered expression levels of RhoH in HPCs.
Proliferation (CPM x 1000) Apoptosis (% Annexin V+ cells) Migration (% migrated cells) F-actin (% cells with cortical F-actin staining) engraftment (%EGFP+ cells in PB) * p &lt; 0.01; ** p &lt; 0.05, HA-RhoH- and siRhoH-transduced vs vector-transduced cells. vector control 16.5 +/− 2.0 3.2 +/− 0.8 22.0 +/− 2.0 75 38.0 +/− 6.0 HA-RhoH 7.0 +/− 0.8 * 6.5 +/− 1.1* 10.5 +/− 1.3 * 22 18.0 +/− 3.5 * siRhoH 23.0 +/− 4.5 ** 1.8 +/− 0.4 ** 32.0 +/− 4.8 * 95 NA