Hematopoietic stem cell (HSC) transplantation is widely used to treat a number of hematological malignancies. However, to be effective, transplanted HSCs must efficiently “home” to their supportive niches within the bone marrow. Limited HSC number and poor function are complications of transplant in some circumstances, and can lead to impaired immune function and bone marrow failure. Enhancing HSC homing is a strategy to improve stem cell transplantation outcomes. We have previously shown that treatment of mouse or human HSCs with 1μM 16–16 dimethyl PGE2 (dmPGE2) ex vivo increases their bone marrow homing efficiency and engraftment, coordinate with an increase in surface CXCR4 expression. The transcription factor Hypoxia Inducible Factor 1α (HIF1α) has been shown to up-regulate CXCR4 in certain cancer models. In addition, PGE2 has been shown to stabilize HIF1α under normoxic conditions. We hypothesized that the effects of PGE2 on CXCR4 expression and homing might result from stabilization of HIF1α and its subsequent transcriptional activity in the transplanted HSCs. Treatment of lineageneg bone marrow cells with 1μM dmPGE2 resulted in a 33 ± 8.6% increase in HIF1α protein levels over vehicle (p<0.05) measured by Western Blot analysis. In addition, treatment of HSC with the hypoxia mimetic dimethyloxallyl glycine (DMOG) also resulted in an increase in CXCR4 cell surface expression in mouse LinnegSca-1posc-kitpos (SKL) cells (11.3 ± 3.9% MFI over vehicle, p<0.05) and human CD34+ umbilical cord blood cells (20.1 ± 3.6% MFI over vehicle), which was comparable to treatment with dmPGE2. This increase in CXCR4 expression translated to a functional increase in SKL migration to SDF-1 (46.7 ± 1.5% migration compared to 35 ± 3.1% migration in vehicle, p<0.05). To determine whether the effects of PGE2 on CXCR4 were due effects on HIF1 transcriptional activity, we employed two mouse hepatoma cell lines (ARNT- and ARNT+). These cells express detectable levels of CXCR4 as well as all four of the PGE2 G-protein coupled receptors (EP1-4). However the ARNT- cell line lacks the HIF nuclear translocator, and thus lacks HIF1 transcriptional activity. While ARNT+ cells showed the characteristic increase in CXCR4 cell surface protein (91.3 ± 28.7%) and mRNA (5.69-fold increase) after treatment with 1μM dmPGE2, ARNT- cells failed to show a similar up-regulation of CXCR4 protein (15 ± 6.1% increase) and mRNA (0.44-fold increase) after PGE2 treatment. These data suggest that HIF1 transcriptional activity is necessary for PGE2-mediated CXCR4 regulation. To determine whether HIF1α stabilization has similar effects on HSC function after transplant, in vivo homing studies were performed. Pulse-treatment of mouse SKL cells with 5μM DMOG ex vivo for 1 hour resulted in a 2.9-fold increase in homing (p<0.005), compared to a 2.5-fold increase for cells treated with dmPGE2. Treatment of donor cells with the CXCR4 antagonist AMD3100 resulted in a statistically significant reduction in SKL homing (74.8 ± 9%, p<0.05), indicating that DMOG's positive effects on homing are mainly due to CXCR4 up-regulation and are similar to effects seen after dmPGE2 treatment. These data suggest the effects of PGE2 on CXCR4 expression are at least partially due to the stabilization of HIF1α. Due to the importance of HIF1α and its involvement in HSC regulation, PGE2 treatment could also have effects on other pathways associated with the hypoxic response mediated through HIF1α that could be targeted to modulate HSC function. Further studies could potentially identify more specific targets to improve the efficacy of HSCs after transplant.
Hoggatt:Fate Therapeutics: Consultancy. Pelus:Fate Therapeutics: Consultancy.
Asterisk with author names denotes non-ASH members.