Abstract
Ectopic delivery of HOXB4 elicits the in vitro and in vivo expansion of hematopoietic stem cells (HSC) although the mechanism is still unknown. We have previously shown that overexpression of HOXB4 attenuates the TNFα signaling pathway at the transcriptional level (Schiedlmeier et al., 2007, PNAS). TNFα is expression is induced at high levels in vivo after irradiation used in bone marrow (BM) transplantation preparative regimens. Since HSC and progenitors (P) derived from Fanconi anemia (FA) knockout mice are hypersensitive to the action of inhibitory cytokines such as TNFα, we chose the Fancc−/− mouse as a model to study the physiologic effects of HOXB4 on TNFα sensitivity of HSC/P and the relationship of these effects to the engraftment defect of FA HSC. Competitive repopulating assays were used to evaluate the effect of HOXB4 overexpression upon engraftment of Fancc−/− BM. Control (CON, expressing eGFP only) transduced Fancc−/− BM demonstrated 80-fold lower engraftment compared with wild type (WT) eGFP+ BM at 26 weeks post-transplant (Table 1). In marked contrast, HOXB4 transduced (HOXB4+) Fancc−/− BM showed a 26-fold higher level of engraftment compared with CON transduced Fancc−/− BM and a 2-fold higher level of engraftment compared with Fancc−/− cells corrected with a FANCC-expressing vector. Fancc−/− BM co-transduced with both vectors (expressing HOXB4 and FANCC) further increased the level of engraftment to that of WT eGFP+ BM suggesting a synergistic correction of the FA HSC engraftment defect. To determine the potential role of TNFα signaling in these effects, we directly assessed the impact of HOXB4 expression on the response of Fancc−/− BM to treatment with TNFα. Fancc−/− BM cells transduced with the CON eGFP vector demonstrated >70% reduction in colony formation upon treatment with 10ng/ml TNFα (Table 2), while Fancc−/− BM overexpressing HOXB4 showed no significant inhibition of CFU at 10ng/ml TNFα compared to either untreated cells or to treated WT BM transduced with eGFP CON vector. Additionally, in vitro treatment of eGFP CON transduced Fancc−/− BM with 100ng/ml TNFα resulted in a 21±5% decrease in lineage-, Sca-1+, c-Kit+ (LSK) cells within 24 hrs. In contrast, similarly treated HOXB4+Fancc−/− demonstrated a 6±9% increase in LSK cells (p<0.01 compared to Fancc−/− eGFP CON). Hence HOXB4 completely protects Fancc−/− HSC/P against the inhibitory effects of TNFα. In order to further define the mechanism through which HOXB4 attenuates TNFα signaling, we determined the level of expression of the TNFα receptors, TNFR1 and TNFR2, on transduced Fancc−/− BM by flow analysis. Fancc−/− LSK BM transduced with eGFP CON or FANCC expressing vectors had equivalent expression of TNFR1 and TNFR2 compared to WT eGFP CON transduced BM (Table 3). In contrast, Fancc−/− LSK cells transduced HOXB4 demonstrated a >25% reduction in the number of cells that stained positive for either TNFR1 or TNFR2. In addition, there was a >35% decrease in the MFI of staining for TNFR1 in HOXB4+Fancc−/− LSK compared to other groups. In summary, ectopic HOXB4 protects Fancc−/− and WT HSC/P from the inhibitory effects of TNFα. Since HOXB4 expression also protects WT cells from TNFα treatment (data not shown), we propose that HOXB4 enhances engraftment by protecting HSC from the elevated TNFα levels, which is a result of conditioning regimens applied to transplant recipients. We suggest that this mechanism reveals a novel target for the pharmacologic manipulation of HSC during engraftment, thus avoiding the potential adverse effects of constitutive HOXB4 overexpression.
Table 1: % peripheral blood chimerism at 26 weeks post-transplant
Fancc−/− + eGFP 0.5±0.2 Fancc−/− + FANCC 7.8±6.5 Fancc−/− + HOXB4 18.1±6.0** Fancc−/− + FANCC + HOXB4 34.1±7.1** WT + eGFP 44.3±5.3**
Table 2: CFU as % non-treated control ± SEM
TNFα dose 1ng/ml 10ng/ml Fancc−/− + eGFP 47±7 20±7 Fancc−/− + FANCC 81±7** 57±7** Fancc−/− + HOXB4 99±5** 92±5** WT + eGFP 90±7** 77±7**
Table 3: % (and MFI) of BM positive for TNFR1 and TNFR2 expression ± SEM
TNFR1 TNFR2 **p<0.01 compared to Fancc-/- + eGFP Fancc−/− + eGFP 75±5 (740±133) 67±8 (676±160) Fancc−/− + FANCC 69±5 (808±133) 64±8 (673±160) Fancc−/− + HOXB4 46±5** (464±133) 37±8** (425±160) WT + eGFP 70±5 (689±133) 67±8 (636±160)
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