Abstract
Mesenchymal stromal cells (MSCs) are multipotent progenitors supporting bone marrow hematopoiesis. MSCs have an efficient DNA damage response (DDR) and are consequently relatively radio-resistant cells. Therefore, MSCs are key to hematopoietic reconstitution following total body irradiation (TBI) and bone marrow transplantation (BMT). The bone marrow niche is hypoxic and via the heterodimeric transcription factor Hypoxia-inducible factor-1 (Hif-1), hypoxia enhances the DDR. Using gene knock-down, we have previously shown that the Hif-1α subunit of Hif-1 is involved in mouse MSC radio-resistance, however its exact mechanism of action remains unknown. In order to dissect the involvement of Hif-1α in the DDR, we used CRISPR/Cas9 technology to generate a stable mutant of the mouse MSC cell line MS5 lacking Hif-1α expression. Herein, we show that it is the whole Hif-1 transcription factor, and not only the Hif-1α subunit, that modulates the DDR of mouse MSCs. This effect is dependent upon the presence of a Hif-1α protein capable of binding to both DNA and its heterodimeric partner Arnt (Hif-1β). Detailed transcriptomic and proteomic analysis of Hif1a KO MS5 cells leads us to conclude that Hif-1α may be acting indirectly on the DNA repair process. These findings have important implications for the modulation of MSC radio-resistance in the context of BMT and cancer.
Highlights
Hematopoiesis is the process whereby through a complex series of commitment and differentiation events, hematopoietic stem cells (HSC) differentiate into all types of blood cells (Brown et al, 2015; Kumar and Evans, 2015)
Since the canonical function of Hypoxia-inducible factor-1 (Hif-1) is to control the expression of a myriad of hypoxiaresponsive genes, we hypothesized that Hif-1α might be involved in mediating an increase in the mRNA expression level of DNA repair genes
Since our group has previously shown that the immortalized mouse Mesenchymal stromal cells (MSCs) cell lines MS5 and ST2 behave very to primary mouse MSCs, in this study the MS5 cell line was chosen for a more detailed molecular investigation of the mechanism behind the effects of hypoxia on the DNA damage response (DDR) of MSCs. mRNA levels of Hif1a, Prkdc, Lig4, and Rad51 were measured by real-time PCR in normoxic and hypoxic MSCs at different timepoints after treatment with 10Gy of IR (Figure 1A)
Summary
Hematopoiesis is the process whereby through a complex series of commitment and differentiation events, hematopoietic stem cells (HSC) differentiate into all types of blood cells (Brown et al, 2015; Kumar and Evans, 2015). Mesenchymal stromal cells (MSCs) are key components of the bone marrow microenvironment, providing support for HSCs and regulating their maintenance and the production and maturation of hematopoietic progenitors (Nagasawa et al, 2011). Hypoxia has been shown to play a key role in both HSC and MSC biology, modulating their proliferation rates, quiescence, differentiation potential, and re-constitutive capacity (Eliasson and Jönsson, 2010; Guitart et al, 2010; Jin et al, 2010; Mohyeldin et al, 2010; Winkler et al, 2010; Tsai et al, 2012a,b; Prado-Lòpez et al, 2014). Hypoxia is involved in angiogenesis, proliferation, metabolism, metastasis, differentiation, and response to radiation therapy, being an important adverse prognostic factor in cancer (Hall and Giaccia, 2006; Bristow and Hill, 2008; Rankin and Giaccia, 2008; Philip et al, 2013)
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