Limits...
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.


Related in: MedlinePlus

DDIT4 regulate MSC differentiation.(A) Expression of vital genes involve in osteogenic and adipogenic differentiation in MSC treated with CoCl2 or vehicle control (n = 4). (B) Osteogenic differentiation of MSC in presence of CoCl2. (Upper left) matrix mineralization visualized and (upper right) quantified following Alizarin Red staining (representative images from four independent experiments) and (lower panel) mRNA expression of the osteogenic markers Runx2, ALP, and Osteopontin (OPN) was analyzed by qRT-PCR (n = 5). (C) Adipogenic differentiation of MSC in presence of CoCl2. (Upper left) Lipid accumulation was visualized and (upper right) quantified following Oil red O staining (×200) and (lower panel) expression of adipogenic markers PPARγ, FAB4, and LPL mRNA was analyzed by qRT-PCR (n = 5). (D) Osteogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were differentiated to osteoblast and (upper) matrix mineralization was visualized and (lower left) quantified by Alizarin Red staining and (lower right) mRNA expression of the osteogenic markers was analyzed by qRT-PCR (n = 4). (E) Adipogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were induced to differentiate to adipocytes and the ability of (upper left) lipid accumulation was visualized (×200) and (lower panel) expression of adipogenic markers was analyzed by qRT-PCR (n = 4). (F) Differentiation following transient overexpression of DDIT4. (Left) Alizarin Red staining of matrix mineralization and (right) Oil Red O staining for lipid droplets following differentiation to osteoblast and adipocyte lineages (×200). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120275&req=5

f3: DDIT4 regulate MSC differentiation.(A) Expression of vital genes involve in osteogenic and adipogenic differentiation in MSC treated with CoCl2 or vehicle control (n = 4). (B) Osteogenic differentiation of MSC in presence of CoCl2. (Upper left) matrix mineralization visualized and (upper right) quantified following Alizarin Red staining (representative images from four independent experiments) and (lower panel) mRNA expression of the osteogenic markers Runx2, ALP, and Osteopontin (OPN) was analyzed by qRT-PCR (n = 5). (C) Adipogenic differentiation of MSC in presence of CoCl2. (Upper left) Lipid accumulation was visualized and (upper right) quantified following Oil red O staining (×200) and (lower panel) expression of adipogenic markers PPARγ, FAB4, and LPL mRNA was analyzed by qRT-PCR (n = 5). (D) Osteogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were differentiated to osteoblast and (upper) matrix mineralization was visualized and (lower left) quantified by Alizarin Red staining and (lower right) mRNA expression of the osteogenic markers was analyzed by qRT-PCR (n = 4). (E) Adipogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were induced to differentiate to adipocytes and the ability of (upper left) lipid accumulation was visualized (×200) and (lower panel) expression of adipogenic markers was analyzed by qRT-PCR (n = 4). (F) Differentiation following transient overexpression of DDIT4. (Left) Alizarin Red staining of matrix mineralization and (right) Oil Red O staining for lipid droplets following differentiation to osteoblast and adipocyte lineages (×200). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: Excessive activation of mTOR signalling is suggested to drive unwanted differentiation and proliferation. mTOR activation has been shown to accelerate differentiation of MSCs and may result in depletion of the stem cell pool. Therefore we next examined the differentiation capacity of MSCs following manipulation of DDIT4 by either CoCl2 activation and gene knock-down or overexpression. Treatment with CoCl2 significantly reduced the expression of genes associated with osteoblast (Runx2 and ALP) and adipocyte (PPARγ) lineages in the absence of any differentiation stimulus (Fig. 3A), suggesting a reduction in spontaneous differentiation by MSC. Similarly when MSC were induced to differentiate in the presence of CoCl2, they showed a significant reduction in differentiation (Fig. 3B,C). Expression of markers of osteogenic (Fig. 3B) and adipogenic (Fig. 3C) differentiation were significantly reduced and the ability of the cells to undergo matrix mineralization (Fig. 3B) or the transformation to lipid containing adipocytes (Fig. 3C) was strongly impaired. We also stably knocked down DDIT4 which resulted in accelerated MSC differentiation toward both lineages, as determined by elevated expression of genes associated with osteoblasts (Fig. 3D) and adipocytes (Fig. 3E), increased matrix mineralization (Fig. 3D) and formation of lipid containing adipocytes (Fig. 3E) in response to stimulation of differentiation. However, no significant effect was observed under basal conditions (Fig. S2G). Interestingly, transient overexpression of DDIT4 in MSC had only a negligible effect on differentiation, which may be due to the temporary nature of expression (Fig. 3F).


DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1 α and mTOR signalling
DDIT4 regulate MSC differentiation.(A) Expression of vital genes involve in osteogenic and adipogenic differentiation in MSC treated with CoCl2 or vehicle control (n = 4). (B) Osteogenic differentiation of MSC in presence of CoCl2. (Upper left) matrix mineralization visualized and (upper right) quantified following Alizarin Red staining (representative images from four independent experiments) and (lower panel) mRNA expression of the osteogenic markers Runx2, ALP, and Osteopontin (OPN) was analyzed by qRT-PCR (n = 5). (C) Adipogenic differentiation of MSC in presence of CoCl2. (Upper left) Lipid accumulation was visualized and (upper right) quantified following Oil red O staining (×200) and (lower panel) expression of adipogenic markers PPARγ, FAB4, and LPL mRNA was analyzed by qRT-PCR (n = 5). (D) Osteogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were differentiated to osteoblast and (upper) matrix mineralization was visualized and (lower left) quantified by Alizarin Red staining and (lower right) mRNA expression of the osteogenic markers was analyzed by qRT-PCR (n = 4). (E) Adipogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were induced to differentiate to adipocytes and the ability of (upper left) lipid accumulation was visualized (×200) and (lower panel) expression of adipogenic markers was analyzed by qRT-PCR (n = 4). (F) Differentiation following transient overexpression of DDIT4. (Left) Alizarin Red staining of matrix mineralization and (right) Oil Red O staining for lipid droplets following differentiation to osteoblast and adipocyte lineages (×200). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120275&req=5

f3: DDIT4 regulate MSC differentiation.(A) Expression of vital genes involve in osteogenic and adipogenic differentiation in MSC treated with CoCl2 or vehicle control (n = 4). (B) Osteogenic differentiation of MSC in presence of CoCl2. (Upper left) matrix mineralization visualized and (upper right) quantified following Alizarin Red staining (representative images from four independent experiments) and (lower panel) mRNA expression of the osteogenic markers Runx2, ALP, and Osteopontin (OPN) was analyzed by qRT-PCR (n = 5). (C) Adipogenic differentiation of MSC in presence of CoCl2. (Upper left) Lipid accumulation was visualized and (upper right) quantified following Oil red O staining (×200) and (lower panel) expression of adipogenic markers PPARγ, FAB4, and LPL mRNA was analyzed by qRT-PCR (n = 5). (D) Osteogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were differentiated to osteoblast and (upper) matrix mineralization was visualized and (lower left) quantified by Alizarin Red staining and (lower right) mRNA expression of the osteogenic markers was analyzed by qRT-PCR (n = 4). (E) Adipogenic differentiation of MSC in DDIT4 depleted cells. MSC stably transfect with shRNA against DDIT4 or negative control were induced to differentiate to adipocytes and the ability of (upper left) lipid accumulation was visualized (×200) and (lower panel) expression of adipogenic markers was analyzed by qRT-PCR (n = 4). (F) Differentiation following transient overexpression of DDIT4. (Left) Alizarin Red staining of matrix mineralization and (right) Oil Red O staining for lipid droplets following differentiation to osteoblast and adipocyte lineages (×200). Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: Excessive activation of mTOR signalling is suggested to drive unwanted differentiation and proliferation. mTOR activation has been shown to accelerate differentiation of MSCs and may result in depletion of the stem cell pool. Therefore we next examined the differentiation capacity of MSCs following manipulation of DDIT4 by either CoCl2 activation and gene knock-down or overexpression. Treatment with CoCl2 significantly reduced the expression of genes associated with osteoblast (Runx2 and ALP) and adipocyte (PPARγ) lineages in the absence of any differentiation stimulus (Fig. 3A), suggesting a reduction in spontaneous differentiation by MSC. Similarly when MSC were induced to differentiate in the presence of CoCl2, they showed a significant reduction in differentiation (Fig. 3B,C). Expression of markers of osteogenic (Fig. 3B) and adipogenic (Fig. 3C) differentiation were significantly reduced and the ability of the cells to undergo matrix mineralization (Fig. 3B) or the transformation to lipid containing adipocytes (Fig. 3C) was strongly impaired. We also stably knocked down DDIT4 which resulted in accelerated MSC differentiation toward both lineages, as determined by elevated expression of genes associated with osteoblasts (Fig. 3D) and adipocytes (Fig. 3E), increased matrix mineralization (Fig. 3D) and formation of lipid containing adipocytes (Fig. 3E) in response to stimulation of differentiation. However, no significant effect was observed under basal conditions (Fig. S2G). Interestingly, transient overexpression of DDIT4 in MSC had only a negligible effect on differentiation, which may be due to the temporary nature of expression (Fig. 3F).

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.


Related in: MedlinePlus