DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1α and mTOR signalling

Stem cell fate decisions to remain quiescent, self-renew or differentiate are largely governed by the interplay between extracellular signals from the niche and the cell intrinsic signal cascades and transcriptional programs. Here we demonstrate that DNA Damage Inducible Transcript 4 (DDIT4) acts as a link between HIF1α and mTOR signalling and regulation of adult stem cell fate. Global gene expression analysis of mesenchymal stem cells (MSC) derived from single clones and live RNA cell sorting showed a direct correlation between DDIT4 and differentiation potentials of MSC. Loss and gain of function analysis demonstrated that DDIT4 activity is directly linked to regulation of mTOR signalling, expression of pluripotency genes and differentiation. Further we demonstrated that DDIT4 exert these effects down-stream to HIF1α. Our findings provide an insight in regulation of adult stem cells homeostasis by two major pathways with opposing functions to coordinate between states of self-renewal and differentiation.


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or low differentiation potentials (12,14,15). (E&F) Global gene expression was carried out using the Illumina microarray platform and differences in gene expression were compared by the Differential Expression Algorithm using the Illumina custom error model of GenomeStudio software (Illumina, Inc.). Genes with a Diffscore of ±13, equivalent to p<0.05, were considered as being significantly upregulated; a DiffScore of ±20, ±30 or ±40 was equivalent to a p-value of <0.01, <0.001, or <0.0001, respectively. Cluster of genes highly expressed in (E) osteogenic and (F) adipogenic clones compare with clones with low differentiation capacity. (G) QRT-PCR analysis of DDIT4 expression in clones with high osteogenic (4, 7, 19), high adipogenic (1, 8, 18), high multi (2, 6, 9) or low differentiation potentials (12,14,15). (H) Combined data showing DDIT4 expression in clones with high or low differentiation potentials.

Cell culture
Primary human MSCs from young male and female adults (aged 21-25) were purchased from Lonza (Slough, UK) and characterized as shown previously (Gharibi and Hughes, 2012) quantified following staining with Oil red O dye as described previously (Gharibi et al., 2011).

Production of single cell clones
To achieve single cell clones from subpopulations of MSCs, cell were diluted to 1 cell per 100µl per well of 96 well plates. The presence of single cell in each well was evaluated using a phase-contrast microscope, and wells with more than one cell were excluded from the study. MSC single clones were subsequently expanded and assessed for differentiation capability and subdivided into 3 categories; clones with high osteogenic potential, high adipogenic potential, or with low differentiation potential. Subsequently three clones from each category were chosen to be analysed for gene expression by microarray.

Microarray analysis
Total RNA was extracted using TRI reagent (Life Technologies) and Phase Lock Gel

Colony Forming Unit (CFU) assay
MSCs were seeded at a density of 400cell/well in 6 well plates and cultured for up to14 days with media replenished every 3-4 days. Cells were washed with PBS, fixed for 15 min with 4% formaldehyde in PBS, stained for 30 min with 0.5% Crystal Violet and washed with PBS.

Cell proliferation assay
Cell number was counted at each passage using a haemocytometer and subcultured for further expansion. Cell proliferation (DNA synthesis) was assessed by measuring 5ethynyl-2′-deoxyuridine (EdU) DNA incorporation using the Click-iT EdU Alexa Fluor 647 cell proliferation assay kit (Life Technologies). Briefly, cells were treated with EdU at 10 µg/ml for 48 hours, harvested by trypsinization, washed in PBS/1% BSA, and fixed with Click-iT fixative. The cells were then permeabilized using saponin-based permeabilization reagent, treated with the Click-iT EdU reaction cocktail in the dark, and washed with saponin-based permeabilization reagent. The number of EdUpositive cells was determined using a FACS-Canto II flow cytometer, and data analysis was performed using DIVA software (Becton Dickinson Biosciences).

Immunostaining
For immunohistochemistry femurs of 5 weeks old CD-1 wild type mice were dissected and cleaned of surrounding tissue. The bones were then fixed in 4% formaldehyde in PBS for 3 days and decalcified in 10% EDTA for further 7 days. Samples were dehydrated and embedded in paraffin and sectioned. Paraffin sections were stained with goat-anti-LepR-biotin (R&D system, Abingdon, UK) and rabbit anti-DDIT4