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

Abstract

Regulator of G-protein Signaling (RGS) family proteins are known to negatively regulate G-protein-coupled receptor signaling through their GTPase-accelerating activity. In several types of mature hematopoietic cells (e.g., B lymphocytes and megakaryocytes), responses to chemokines are subjected to regulation by R4 subfamily RGS proteins. However, their expression patterns and functional roles in hematopoietic stem and progenitor cells (HSC) are poorly characterized. Here, we showed that human CD34+ HSC derived from cord blood (CB, n = 12) expressed RGS1 , RGS2 , RGS3 , RGS5 , RGS13 , RGS16 and RGS18 at mRNA level, whereas RGS4 , RGS8 and RGS21 were undetectable. Exposure of CB CD34+ cells to the critical homing-related chemokine stromal cell-derived factor-1 (SDF-1) significantly increased RGS1 , RGS2 , RGS13 and RGS16 , and decreased RGS3 and RGS18 expressions (P ≤ 0.040, n = 5). Expressions of RGS1 , RGS13 and RGS16 were significantly higher in bone marrow (BM, n = 14) CD34+ cells when compared to mobilized peripheral blood (MPB, n = 14) CD34+ cells (P ≤ 0.002), while RGS3 and RGS18 expressions were lower in BM CD34+ cells (P ≤ 0.036), suggesting a SDF-1 and niche-dependent regulation of RGS expressions. Lentivirus-mediated overexpression of RGS1 , RGS13 or RGS16 significantly inhibited migration of CD34+ cells to a SDF-1 gradient (P ≤ 0.039, n = 4-5) butdid not alter SDF-1-induced actin polymerization (n = 3). In addition, phosphorylation of signaling molecules downstream of the SDF-1/CXCR4 axis, including Akt, ERK and STAT3 was significantly suppressed in RGS1 - and RGS16 -overexpressing CD34+ cells. In the NOD/SCID mouse xenotransplantation model, overexpression of RGS1 , RGS13 or RGS16 significantly reduced BM engraftment of CD34+ cells by 98% (P = 0.023, n = 7), 89% (P = 0.014, n = 8) and 87% (P = 0.015, n = 8) respectively. Transcriptome profiling (n = 4) of CD34+ cells identified 312 genes with differential expressions upon overexpression of RGS1 , RGS13 or RGS16 , with 32 genes commonly regulated. Gene function enrichment analysis revealed significant downregulation of genes involved in the migration (PROS1 , CCL1 , THBS1 , F2RL2 ), complement activation (C3AR1 , C5AR1 , C5AR2 ), and proteolysis (MMP14 , TIMP3 ) pathways, which were validated by qPCR (n = 8). Taken together, we provided the first evidence that R4 RGS members are expressed and regulated by the SDF-1 in human CD34+ HSC. We also presented data showing that RGS1 , RGS13 or RGS16 negatively regulated in vitro SDF-1-mediated migration and signaling as well as in vivo engraftment of CD34+ cells, suggesting that these RGS members might serve as a feedback mechanism to regulate the SDF-1/CXCR4 axis. Strategies to inhibit specific R4 RGS proteins could potentially enhance efficiency of HSC homing and engraftment, which are particularly important in the setting of CB transplantation. DisclosuresNo relevant conflicts of interest to declare.