R4 Rgs Subfamily Proteins Suppress Engraftment and Regulate SDF-1/CXCR4 Signaling in Human CD34+ Hematopoietic Stem/Progenitor Cells

Members of the Regulators of G-protein Signaling (RGS) are GTPase-accelerating proteins and have been implicated in SDF-1-directed trafficking of mature hematopoietic cells. However, their roles in hematopoietic stem and progenitor cells (HSPC) remain largely unknown. In this study, we investigated the expression, functions and mechanism of R4 RGS subfamily members on migration and engraftment of human HSPC. Our results demonstrated that cord blood (CB), bone marrow (BM) and mobilized peripheral blood (MPB) CD34⁺ cells expressed specific RGS mRNAs, of which RGS1, RGS2, RGS13 and RGS16 were significantly upregulated by SDF-1 (1.6-1.9 fold, n=5, P<0.05). In the presence of AMD3100, a CXCR4 inhibitor, the stimulating effects of SDF-1 on RGS expression were completely abolished (n=6). SDF-1-directed functions (chemotaxis, trans-matrigel migration and calcium flux) and signaling (phosphorylation of Akt, ERK and Stat3) were significantly inhibited in RGS1, RGS13 and RGS16-overexpressing CD34⁺ cells (n=4-6, P<0.05) but not in RGS2-overexpressing cells, whereas actin polymerization, adhesion and colony formation were unaffected by these RGS members. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1, RGS13 or RGS16 in CD34⁺ cells did not impact their short-term homing but substantially compromised their long-term engraftment efficiency in bone marrow and spleens of recipient mice by 91.4%, 83.7% and 71.2%, respectively (n= 8-9; P<0.05). Genome-wide expression microarray and qPCR validation identified 32 common differentially expressed genes (1 upregulated and 31 downregulated) in RGS1, RGS13 or RGS16-overexpressing CD34⁺ cells. Network analysis revealed the potential mechanisms of RGS1, RGS13 and RGS16 downstream of SDF1/CXCR4 and Gαi protein, leading to compromised Akt, ERK and Stat3 phosphorylation and negative regulation of stem cell functions (CCNA1, SPP1, LPAR5, IL1RL1, HPSE), complement activation (C3AR1, C5AR2, C5AR1), proteolysis (TIMP3, MMP14) and cell migration (THBS1, F2RL2, PROS1, CCL1). Our results highlight the unprecedented functions of R4 RGS proteins in HSPC migration and engraftment, and provide the foundation of future design of RGS-targeting strategies to enhance the efficiency of clinical HSPC transplantation.