Group A Pak Proteins Are Essential for HSC/P Engraftment.

Abstract 2624

Rho GTPases, including Rac, integrate multiple extracellular signals and play important regulatory roles in critical hematopoietic stem and progenitor (HSC/P) functions such as cell migration, adhesion, proliferation, and survival. Conditional deletion of Rac1 in the hematopoietic cells results in defects in proliferation and engraftment while Rac2 germline deletion leads to enhanced cell death and increased HSC/P mobilization, most likely resulting from decreased adhesion via altered integrin signalling (Gu et al. Science, 2003). Although, Rac1 and Rac2 proteins have been shown to regulate distinct biological processes, simultaneous deletion of both Rac1 and Rac2 reveal overlapping functions in HSC/P survival, mobilization, engraftment, and retention (Cancelas et al. Nat. Med., 2006). Our studies now focus on indentifying potential downstream effector proteins of Rac that are important for normal HSC/P function(s). The best characterized Rac effector proteins are the Pak protein kinases, including Group A (Pak1, Pak2, and Pak3) Paks. Pak proteins not only have an intrinsic kinase activity but also regulate actin-dependent functions. Previous studies have demonstrated inhibition of Rac in HSC/P was associated with reduced phosphorylation of Pak1 (Cancelas et al. Nat. Med., 2006). However, the exact role Pak proteins play in HSC/P function(s) has yet to be elucidated. To further identify the possible role(s) of Pak proteins in hematopoiesis, freshly isolated lineage-, Sca-1+; c-Kit+ (LSK) cells from BoyJ(CD45.1)/C57Bl6J(CD45.2) F1 donor mice were transduced with retroviral vectors containing the Pak Inhibitory Domain (PID), which inhibits Group A Pak protein function, or empty vector control (Mieg3); both constructs co-express GFP. 6.0×10⁴ GFP+ CD45.1/CD45.2 LSK cells were then isolated and co-transplanted with 5.0×10⁵ BoyJ (CD45.1) bone marrow (BM) into lethally irradiated C57Bl/6J (CD45.2) recipients. Percent chimerism was measured from 3-weeks up to 14-weeks post BM transplant. We found that PID transduced LSK cells were incapable of contributing to recipient hematopoietic reconstitution compared to Mieg3 transduced controls at 14 weeks post-transplant (0/9 PID transduced demonstrated >1% engraftment vs 9/9 Mieg3 transduced which had >10% engraftment, p<0.05). To explore the underlying mechanism of this engraftment failure, we cultured transduced LSK in vitro for up to 12 days and compared growth of GFP+ vs GFP- cells. The growth of PID transduced LSK was essentially identical to non-transduced cells for the initial 7 days of in vitro culture. However between day 7 and day 10, the PID LSK cells displayed decreased growth kinetics (Fig. 1). The reduced growth in vitro was correlated with decreased proliferation as measured by BrdU incorporation. There was no change in cell survival, as assessed by AnnexinV staining. Furthermore, LSK cells transduced with a dominant negative Pak construct (DN-Pak), a more general inhibitor of Pak activity, showed defects in cytoskeletal rearrangements after stimulation with the HSC/P chemoattract, SDF-1α. These data suggest that Pak proteins may be critical downstream effectors of Rac GTPases and defective Pak activation may mechanistically link the role of Rac GTPases in regulation of both cytoskeletal and proliferative functions of HSC/P during engraftment.

Fig. 1

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Low-density mononuclear BM cells were depleted of lineage-positive (lin+) cells as described previously (Milsom et al. Blood, (2009). After antibody staining, lin–, Sca-1+, c-Kit+ (LSK) cells were isolated by flow sorting. LSK cells were prestimulated for 48 hours, after which the cells were transduced with a vector expressing PIDK or Mieg3 empty control vector. 48 hours later, % transduction was determined. Cells were then cultured in 10% IMDM + cytokines for an additional 12 days. The percentage of GFP+ cells in culture was determined at days 2, 5, 7, 10, and 12 post-transduction.

Fig. 1

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Low-density mononuclear BM cells were depleted of lineage-positive (lin+) cells as described previously (Milsom et al. Blood, (2009). After antibody staining, lin–, Sca-1+, c-Kit+ (LSK) cells were isolated by flow sorting. LSK cells were prestimulated for 48 hours, after which the cells were transduced with a vector expressing PIDK or Mieg3 empty control vector. 48 hours later, % transduction was determined. Cells were then cultured in 10% IMDM + cytokines for an additional 12 days. The percentage of GFP+ cells in culture was determined at days 2, 5, 7, 10, and 12 post-transduction.

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