Migration of hemopoietic stem and progenitor cells (HSPC) is required for homing to bone marrow following transplantation. Therefore, it is critical to understand signals underlying directional movement of HSPC. Stromal cell-derived factor-1 (SDF-1)/CXCL12 is a potent chemoattractant for HSPC. In this study, we demonstrate that the serine-threonine protein phosphatase (PP)2A plays an important role in regulation of optimal level and duration of Akt/protein kinase B activation (a molecule important for efficient chemotaxis), in response to SDF-1. Inhibition of PP2A, using various pharmacological inhibitors of PP2A including okadaic acid (OA) as well as using genetic approaches including dominant-negative PP2A-catalytic subunit (PP2A-C) or PP2A-C small interfering RNA, in primary CD34+ cord blood (CB) cells led to reduced chemotaxis. This was associated with impairment in polarization and slower speed of movement in response to SDF-1. Concomitantly, SDF-1-induced Akt phosphorylation was robust and prolonged. Following SDF-1 stimulation, Akt and PP2A-C translocate to plasma membrane with enhanced association of PP2A-C with Akt observed at the plasma membrane. Inhibition of PI3K by low-dose LY294002 partially recovered chemotactic activity of cells pretreated with OA. In addition to chemotaxis, adhesion of CD34+ cells to fibronectin was impaired by OA pretreatment. Our study demonstrates PP2A plays an important role in chemotaxis and adhesion of CD34+ CB cells in response to SDF-1. CD34+ CB cells pretreated with OA showed impaired ability to repopulate NOD-SCID mice in vivo, suggesting physiological relevance of these observations.
Although cord blood contains significant numbers of hematopoietic stem and progenitor cells (HSPC), its applicability has remained largely in children (1Broxmeyer and Smith, 2004). Homing of hematopoietic stem progenitor cells (HSPC) to bone marrow is a critical determinant for success of transplantation. Enhancement of homing of HSPC from cord blood has the potential to increase the applicability of cord blood transplantation in adults. Therefore, it is important to understand the molecules that underlie directional movement of HSPC. Stromal derived factor-1 (SDF-1)/CXCL12 is the most potent chemoattractant for both mouse and human HSPC and has been shown to play an important role in the homing and retention of HSPC in bone marrow. Earlier studies have shown that PI-3 kinase plays a critical role in chemotaxis by attracting various pleckstin homology (PH) containing domain proteins at the leading edge and spatial localization of PI3Kinase and PTEN is critical for maintaining the leading edge. Controlled activation of Akt/PKB, one of the various PH containg domains, has been described to be required for efficient chemotaxis. In this study we have demonstrated that protein phosphatase 2A (PP2A), a serine-threonine phosphatase, plays an important role in chemotaxis of cord blood CD34+ cells towards SDF-1, primarily by modulating Akt activity. Inhibition of PP2A by okadaic acid (OA) or siRNA impairs chemotaxis; this involved impairment in the ability of CD34+ cells to polarize and reduced speed of movement. This was associated with robust and prolonged Akt phosphorylation. Indeed, over expression of constitutive active Akt in CD34+ cells impaired SDF-1 directed chemotaxis. Co-immunoprecipitation experiments demonstrated increased association of Akt with PP2A following SDF-1 stimulation and this increased association of Akt and PP2A-catalytic subunit was observed at the plasma membrane of SDF-1 stimulated CD34+ cells by confocal microscopy. The importance of PP2A in maintaining a critical level and duration of activated Akt was also supported by our finding that inhibition of PI-3kinase by low dose of LY294002, partially recovered chemotactic activity in CD34+ cells pretreated with OA. Interestingly, glycogen synthase kinase-3 (GSK-3) associated with Akt following SDF-1 stimulation, although it was found to be constitutively associated with PP2A-C. Inhibition of GSK-3 using GSK-3 IX inhibitor impaired chemotaxis, raising the possibility that GSK-3 is a downstream target for Akt in SDF-1 directed chemotaxis. The physiological relevance of our in vitro findings is established from the observation that OA pretreatment impaired engraftment potential of CD34+ cells in NOD-SCID mice. Our findings contribute to the growing understanding of molecules that affect directional movement and may have the potential implications for homing and engraftment of HSPC.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.