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DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1 α and mTOR signalling

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Endogenous DDIT4 level governs MSC fate and function.SmartFlare™ live cell DDIT4 RNA sorting and functional analysis of DDIT4-low and DDIT4-high sub-populations. (A) Isolation of MSC subsets based on DDIT4 expression using SmartFlare RNA detection probes and fluorescence activated cell sorting. MSC were isolated into DDIT4-low and high populations by sorting the lowest and highest 15% of the population based on APC fluorescence. (B) MSC were serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1 were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. Data shown is representative of four separate experiments. (lower panel) Quantitative densitometry of pS6K normalised to GAPDH. (C) The frequency of colony formation by each population was assessed by colony forming unit assay and crystal violet staining (representative images from five independent experiments). (D) Differences in rate of cell cycle were determined by Click-iT EdU assay following 48 hours of incubation using flow cytometry (n = 4). (E) Cumulative cell doubling per passage was calculated by cell counting (n = 5). (F) Osteogenic differentiation capacity was determined by Alizarin Red staining of mineralized matrix and (I) mRNA expression of the osteogenic markers Runx2 and ALP was assessed by qRT-PCR (n = 5). (J) Adipogenic differentiation was analyzed following Oil Red O staining for lipid droplets (×200) and (K) expression of adipogenic markers PPARγ and LPL by qRT-PCR (n = 5). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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f5: Endogenous DDIT4 level governs MSC fate and function.SmartFlare™ live cell DDIT4 RNA sorting and functional analysis of DDIT4-low and DDIT4-high sub-populations. (A) Isolation of MSC subsets based on DDIT4 expression using SmartFlare RNA detection probes and fluorescence activated cell sorting. MSC were isolated into DDIT4-low and high populations by sorting the lowest and highest 15% of the population based on APC fluorescence. (B) MSC were serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1 were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. Data shown is representative of four separate experiments. (lower panel) Quantitative densitometry of pS6K normalised to GAPDH. (C) The frequency of colony formation by each population was assessed by colony forming unit assay and crystal violet staining (representative images from five independent experiments). (D) Differences in rate of cell cycle were determined by Click-iT EdU assay following 48 hours of incubation using flow cytometry (n = 4). (E) Cumulative cell doubling per passage was calculated by cell counting (n = 5). (F) Osteogenic differentiation capacity was determined by Alizarin Red staining of mineralized matrix and (I) mRNA expression of the osteogenic markers Runx2 and ALP was assessed by qRT-PCR (n = 5). (J) Adipogenic differentiation was analyzed following Oil Red O staining for lipid droplets (×200) and (K) expression of adipogenic markers PPARγ and LPL by qRT-PCR (n = 5). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: MSCs were sorted into two subpopulations with high and low DDIT4 mRNA expression levels using SmartFlare mRNA probes and fluorescence activated cell sorting (FACS) (Fig. 5A). The DDIT4-low and -high populations displayed contrasting levels of mTOR signalling following stimulation (Fig. 5B), as measured by levels of phosphorylation of substrates. In particular S6K phosphorylation was considerably higher in DDIT4-low compared with DDIT4-high cells. In line with studies that have reported an inverse association between mTOR activity and self-renewal and longevity, the DDIT4-high population showed greater stem cell properties with high clonogenic capacity (Fig. 5C), proliferation rate (Fig. 5D) and increased osteogenic and adipogenic differentiation following transfer of cells to differentiation media (Fig. 5F–I). The DDIT4-high population showed increased expression of markers of differentiation for both osteoblast (Runx2 and ALP) (Fig. 5G) and adipocyte (PPARγ and LPL) (Fig. 5I) lineages and produced higher matrix mineralization (Fig. 5F) and lipid containing adipocytes (Fig. 5H) when cells were induced to differentiate. These observations are in agreement with data obtained with the single clone analysis reported at Fig. 1A,B. The DDIT-high population also showed a greater number of cell doublings with long-term continuous culture (Fig. 5E). The data presented here are in line with previous studies in which long term inhibition of mTOR and the excessive differentiation prevent aging and maintained stemness of number of stem cell types69101112.


DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1 α and mTOR signalling
Endogenous DDIT4 level governs MSC fate and function.SmartFlare™ live cell DDIT4 RNA sorting and functional analysis of DDIT4-low and DDIT4-high sub-populations. (A) Isolation of MSC subsets based on DDIT4 expression using SmartFlare RNA detection probes and fluorescence activated cell sorting. MSC were isolated into DDIT4-low and high populations by sorting the lowest and highest 15% of the population based on APC fluorescence. (B) MSC were serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1 were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. Data shown is representative of four separate experiments. (lower panel) Quantitative densitometry of pS6K normalised to GAPDH. (C) The frequency of colony formation by each population was assessed by colony forming unit assay and crystal violet staining (representative images from five independent experiments). (D) Differences in rate of cell cycle were determined by Click-iT EdU assay following 48 hours of incubation using flow cytometry (n = 4). (E) Cumulative cell doubling per passage was calculated by cell counting (n = 5). (F) Osteogenic differentiation capacity was determined by Alizarin Red staining of mineralized matrix and (I) mRNA expression of the osteogenic markers Runx2 and ALP was assessed by qRT-PCR (n = 5). (J) Adipogenic differentiation was analyzed following Oil Red O staining for lipid droplets (×200) and (K) expression of adipogenic markers PPARγ and LPL by qRT-PCR (n = 5). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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f5: Endogenous DDIT4 level governs MSC fate and function.SmartFlare™ live cell DDIT4 RNA sorting and functional analysis of DDIT4-low and DDIT4-high sub-populations. (A) Isolation of MSC subsets based on DDIT4 expression using SmartFlare RNA detection probes and fluorescence activated cell sorting. MSC were isolated into DDIT4-low and high populations by sorting the lowest and highest 15% of the population based on APC fluorescence. (B) MSC were serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1 were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. Data shown is representative of four separate experiments. (lower panel) Quantitative densitometry of pS6K normalised to GAPDH. (C) The frequency of colony formation by each population was assessed by colony forming unit assay and crystal violet staining (representative images from five independent experiments). (D) Differences in rate of cell cycle were determined by Click-iT EdU assay following 48 hours of incubation using flow cytometry (n = 4). (E) Cumulative cell doubling per passage was calculated by cell counting (n = 5). (F) Osteogenic differentiation capacity was determined by Alizarin Red staining of mineralized matrix and (I) mRNA expression of the osteogenic markers Runx2 and ALP was assessed by qRT-PCR (n = 5). (J) Adipogenic differentiation was analyzed following Oil Red O staining for lipid droplets (×200) and (K) expression of adipogenic markers PPARγ and LPL by qRT-PCR (n = 5). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: MSCs were sorted into two subpopulations with high and low DDIT4 mRNA expression levels using SmartFlare mRNA probes and fluorescence activated cell sorting (FACS) (Fig. 5A). The DDIT4-low and -high populations displayed contrasting levels of mTOR signalling following stimulation (Fig. 5B), as measured by levels of phosphorylation of substrates. In particular S6K phosphorylation was considerably higher in DDIT4-low compared with DDIT4-high cells. In line with studies that have reported an inverse association between mTOR activity and self-renewal and longevity, the DDIT4-high population showed greater stem cell properties with high clonogenic capacity (Fig. 5C), proliferation rate (Fig. 5D) and increased osteogenic and adipogenic differentiation following transfer of cells to differentiation media (Fig. 5F–I). The DDIT4-high population showed increased expression of markers of differentiation for both osteoblast (Runx2 and ALP) (Fig. 5G) and adipocyte (PPARγ and LPL) (Fig. 5I) lineages and produced higher matrix mineralization (Fig. 5F) and lipid containing adipocytes (Fig. 5H) when cells were induced to differentiate. These observations are in agreement with data obtained with the single clone analysis reported at Fig. 1A,B. The DDIT-high population also showed a greater number of cell doublings with long-term continuous culture (Fig. 5E). The data presented here are in line with previous studies in which long term inhibition of mTOR and the excessive differentiation prevent aging and maintained stemness of number of stem cell types69101112.

View Article: PubMed Central - PubMed

ABSTRACT

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 HIF1&alpha; 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 HIF1&alpha;. 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.

No MeSH data available.