Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment

Abstract

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of immunologic disorders. For effective transplant, HSCs must traffic from the peripheral blood to supportive bone marrow niches. We previously showed that HSC trafficking can be enhanced by ex vivo treatment of hematopoietic grafts with 16-16 dimethyl prostaglandin E2 (dmPGE2). While exploring regulatory molecules involved in dmPGE2 enhancement, we found that transiently increasing the transcription factor hypoxia-inducible factor 1-α (HIF1α) is required for dmPGE2-enhanced CXCR4 upregulation and enhanced migration and homing of stem and progenitor cells and that pharmacologic manipulation of HIF1α is also capable of enhancing homing and engraftment. We also now identify the specific hypoxia response element required for CXCR4 upregulation. These data define a precise mechanism through which ex vivo pulse treatment with dmPGE2 enhances the function of hematopoietic stem and progenitor cells; these data also define a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation.

Figure 1

PGE₂ increases HIF1α protein and downstream responsive genes. (A) (Top) Representative blot of HIF1α protein 4 hours after treatment with vehicle, 1 µM of dmPGE₂, or 5 µM of DMOG. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineage^neg BMCs treated with vehicle, dmPGE₂, or DMOG. Data are expressed as X ± standard error of the mean (SEM) percent change in protein levels over vehicle control from 3 separate experiments. (B) Expression of HIF1 responsive genes in mouse lineage^neg BMCs after treatment with dmPGE₂ and DMOG as determined by quantitative reverse-transcription polymerase chain reaction. Data are X ± SEM, N = 3 experiments. *P < .05. (C) (Top) Dose-response blot of HIF1α protein 4 hours after treatment with vehicle or DMOG. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineage^neg BMC. (D) Dose response of CXCR4 on SKL cells treated with vehicle or DMOG. Lineage^neg BMCs were treated with DMOG for 2 hours at 37°C, washed, and incubated in RPMI with 10% heat-inactivated fetal calf serum for 16 hours. CXCR4 expression on LSK-gated cells was analyzed by flow cytometry. (E) In vitro transwell migration of murine SKL cells. One million lineage^neg BMCs were treated with vehicle, dmPGE₂, or DMOG. Cells were assayed for the ability to migrate to 100 ng/mL recombinant murine SDF-1 for 4 hours at 37°C. Data are expressed as X ± SEM, N = 9. (F) BMCs from CD45.1 mice were treated with vehicle, 1 µM of dmPGE₂, or 5 µM of DMOG and 1 × 10⁶ treated lineage^neg cells transplanted into lethally irradiated CD45.2 mice. At 16 hours later, BM was analyzed for homed SKL cells. Data are represented as X ± SEM from 2 separate experiments (N = 4-5 mice/group/experiment, each assayed individually). (G) Similar homing experiment with or without the addition of AMD3100 10 minutes before transplantation. Data are represented as X ± SEM from 2 separate experiments (N = 4-5 mice/group/experiment, each assayed individually) *P < .05. (H) Percent contribution (chimerism) and (I) frequency analysis for DMOG determined by Poisson statistics using LCALC software. Vehicle P₀= 121 319 and DMOG P₀= 53 956. (J) Competitive repopulating units of DMOG and vehicle-treated cells in peripheral blood 6 months after transplant. Data are represented as X ± SEM from 2 pooled experiments (N = 5 mice/group/experiment, each assayed individually). (K) Secondary transplant percent chimerism at 6 months after transplant (N = 6 mice/group each assayed individually). *P < .05.

Figure 2

HIF1α transcriptional activity is necessary for PGE₂-induced CXCR4 upregulation. (A) (Left) CXCR4 cell surface expression (X ± SEM; N = 3 experiments) on HIF1β (−) and HIF1β (+) cells 24 hours after treatment with dmPGE₂. CXCR4 cell surface expression was determined by flow cytometry. Data are expressed as percent change in mean fluorescence intensity of CXCR4 over vehicle. *P < .05. (Right) CXCR4 cell surface expression (X ± SEM; N = 3 experiments) in HIF1β (−) and HIF1β (+) cells 2 hours after treatment with vehicle or dmPGE₂ determined by quantitative reverse-transcription polymerase chain reaction. (B) Schematic of pGL2b luciferase reporter constructs containing various regions of the murine CXCR4 promoter. (C) In vitro Luciferase reporter assay. 293T cells were transfected with either full-length CXCR4 promoter constructs containing all HREs (Full), truncated constructs containing 1 HRE (ΔHRE1) or 2 HREs (ΔHRE2), or a mutated 1.3 kb of HRE (HRE 2 Mut). After 24 hours, cells were split equally and treated with either vehicle or dmPGE₂ for 16 hours at 37°C. Luciferase activity was measured using the Firefly Luciferase assay kit (Promega). Data are represented as X ± SEM from 2 separate experiments (N = 6). *P < .05. (D) (Top) Representative blot of HIF1α protein 4 hours after treatment with vehicle, dmPGE₂, or DMOG, with or without the addition of SNP. (Bottom) Densitometry analysis of HIF1α protein expression in mouse lineage^neg BMCs treated with vehicle, dmPGE₂, or DMOG, with or without the addition of SNP. Data are expressed as X ± standard deviation percent change in protein levels over vehicle control from 2 separate experiments. (E) Expression of HIF1 responsive genes after treatment with vehicle or dmPGE₂ with or without SNP. Data are expressed as X ± SEM, N = 3 experiments. (F) (Left) In vitro transwell migration of murine SKL cells to 100 ng/mL of SDF-1. Lineage^neg cells were treated with vehicle or 1 µM of dmPGE₂ with or without 100 µM of SNP for 2 hours at 37°C. Data are expressed as mean ± SEM, N = 3 experiments. *P < .05. (Right) Representative fluorescence-activated cell sorter histogram showing CXCR4 expression on SKL cells compared with isotype control. (G) In vivo homing of HIF1α KO cells. BMCs from conditional HIF1α KO or floxed control (CD45.2) mice were treated with vehicle or dmPGE₂, and 1 × 10⁶ treated lineage^neg cells were transplanted into lethally irradiated BoyJ (CD45.1) mice. At 16 hours later, BM was analyzed for homed SKL cells. Data are represented as X ± SEM from 1 experiment (N = 4-5 mice/group/experiment, each assayed individually). (H) CXCR4 expression (X ± SEM) on HIF1α KO and HIF1α Floxed SKL cells after treatment with dmPGE₂. N = 3 mice/group, each assayed individually. *P < .05.