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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.


DDIT4 regulates mTOR singling and the expression of pulripotency genes in MSC.(A) MSC were pre-treated with CoCl2 (100 μM), rapamycin (10 nM) or vehicle control for 24 hours, serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. CoCl2 and rapamycin were present at all time during serum deprivation and stimulation period. Data shown is representative of three separate experiments. (B–F) Expression of pluripotency genes Oct4, Nanog and Klf4. (B) MSC were treated with 100 μM of CoCl2 for 1 day (D1) or 4 days (D4) and expression of Oct4 and Klf4 was determined by qRT-PCR (n = 5). (C) MSC were transfected with HIF1α or DDIT4 or control siRNA for 24 h and subsequently treated for a further 24 h with CoCl2 (100 μM) prior to qRT-PCR analysis (n = 5). (D) DDIT4 was transiently overexpressed in MSC and the expression of Oct4, Nanog and Klf4 was determined by qRT-PCR (n = 5). (E) MSC stably transfect with shRNA against DDIT4 or negative control were analysed for mTOR substrate (p-S6K) activation and (F) the expression of pluripotency genes Oct4, Nanog and Klf4. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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f2: DDIT4 regulates mTOR singling and the expression of pulripotency genes in MSC.(A) MSC were pre-treated with CoCl2 (100 μM), rapamycin (10 nM) or vehicle control for 24 hours, serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. CoCl2 and rapamycin were present at all time during serum deprivation and stimulation period. Data shown is representative of three separate experiments. (B–F) Expression of pluripotency genes Oct4, Nanog and Klf4. (B) MSC were treated with 100 μM of CoCl2 for 1 day (D1) or 4 days (D4) and expression of Oct4 and Klf4 was determined by qRT-PCR (n = 5). (C) MSC were transfected with HIF1α or DDIT4 or control siRNA for 24 h and subsequently treated for a further 24 h with CoCl2 (100 μM) prior to qRT-PCR analysis (n = 5). (D) DDIT4 was transiently overexpressed in MSC and the expression of Oct4, Nanog and Klf4 was determined by qRT-PCR (n = 5). (E) MSC stably transfect with shRNA against DDIT4 or negative control were analysed for mTOR substrate (p-S6K) activation and (F) the expression of pluripotency genes Oct4, Nanog and Klf4. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: Given the importance of DDIT4 in the regulation of the mTOR pathway, we next examined the effect of CoCl2-induced DDIT4 expression on activation of mTOR. As shown in Fig. 2A, CoCl2 treatments led to reductions in phosphorylation of the mTOR substrate pS6K and hypo-phosphorylation of 4E-BP1. This is a similar, but less potent, effect to that seen with rapamycin (Fig. 2A).


DDIT4 regulates mesenchymal stem cell fate by mediating between HIF1 α and mTOR signalling
DDIT4 regulates mTOR singling and the expression of pulripotency genes in MSC.(A) MSC were pre-treated with CoCl2 (100 μM), rapamycin (10 nM) or vehicle control for 24 hours, serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. CoCl2 and rapamycin were present at all time during serum deprivation and stimulation period. Data shown is representative of three separate experiments. (B–F) Expression of pluripotency genes Oct4, Nanog and Klf4. (B) MSC were treated with 100 μM of CoCl2 for 1 day (D1) or 4 days (D4) and expression of Oct4 and Klf4 was determined by qRT-PCR (n = 5). (C) MSC were transfected with HIF1α or DDIT4 or control siRNA for 24 h and subsequently treated for a further 24 h with CoCl2 (100 μM) prior to qRT-PCR analysis (n = 5). (D) DDIT4 was transiently overexpressed in MSC and the expression of Oct4, Nanog and Klf4 was determined by qRT-PCR (n = 5). (E) MSC stably transfect with shRNA against DDIT4 or negative control were analysed for mTOR substrate (p-S6K) activation and (F) the expression of pluripotency genes Oct4, Nanog and Klf4. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
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f2: DDIT4 regulates mTOR singling and the expression of pulripotency genes in MSC.(A) MSC were pre-treated with CoCl2 (100 μM), rapamycin (10 nM) or vehicle control for 24 hours, serum starved for 6 hours and phosphorylation of S6K, and 4E-BP1were analyzed in response to stimulation by media containing 10% serum for 20 minutes, 6 and 24 hours using Western blotting. CoCl2 and rapamycin were present at all time during serum deprivation and stimulation period. Data shown is representative of three separate experiments. (B–F) Expression of pluripotency genes Oct4, Nanog and Klf4. (B) MSC were treated with 100 μM of CoCl2 for 1 day (D1) or 4 days (D4) and expression of Oct4 and Klf4 was determined by qRT-PCR (n = 5). (C) MSC were transfected with HIF1α or DDIT4 or control siRNA for 24 h and subsequently treated for a further 24 h with CoCl2 (100 μM) prior to qRT-PCR analysis (n = 5). (D) DDIT4 was transiently overexpressed in MSC and the expression of Oct4, Nanog and Klf4 was determined by qRT-PCR (n = 5). (E) MSC stably transfect with shRNA against DDIT4 or negative control were analysed for mTOR substrate (p-S6K) activation and (F) the expression of pluripotency genes Oct4, Nanog and Klf4. Error bars indicate mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: Given the importance of DDIT4 in the regulation of the mTOR pathway, we next examined the effect of CoCl2-induced DDIT4 expression on activation of mTOR. As shown in Fig. 2A, CoCl2 treatments led to reductions in phosphorylation of the mTOR substrate pS6K and hypo-phosphorylation of 4E-BP1. This is a similar, but less potent, effect to that seen with rapamycin (Fig. 2A).

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.