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FOXO target gene CTDSP2 regulates cell cycle progression through Ras and p21(Cip1/Waf1).

Kloet DE, Polderman PE, Eijkelenboom A, Smits LM, van Triest MH, van den Berg MC, Groot Koerkamp MJ, van Leenen D, Lijnzaad P, Holstege FC, Burgering BM - Biochem. J. (2015)

Bottom Line: Our data suggest that CTDSP2 induces p21(Cip1/Waf1) through increasing the activity of Ras.As has been described previously, Ras induces p21(Cip1/Waf1) through p53-dependent and p53-independent pathways and indeed both p53 and MEK inhibition can mitigate the CTDSP2-induced p21(Cip1/Waf1) mRNA up-regulation.In support of Ras activation by CTDSP2, depletion of endogenous CTDSP2 results in reduced Ras activity and thus CTDSP2 seems to be part of a larger set of genes regulated by FOXO proteins, which increase growth factor signalling upon FOXO activation.

View Article: PubMed Central - PubMed

Affiliation: University Medical Centre Utrecht, Universiteitsweg 100 STR3.217, 3584CG Utrecht, The Netherlands.

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CTDSP2 expression is regulated by FOXO proteins(A) CTDSP2 mRNA was measured by qPCR in DL23 cells after FOXO3 was activated by 4OHT for the indicated times or (B) for 8 h in the presence of cycloheximide (CHX). (C) U2OS cells were treated for 16 h with LY294002 or Akti to inhibit PI3K and PKB/Akt and thereby activate endogenous FOXO proteins. CTDSP2 mRNA was measured by qPCR. Either treatment induces CTDSP2 expression significantly. (D) ChIP–qPCR-determined percentage input of the CTDSP2 promoter precipitated by endogenous FOXO3 (black bars) or non-specific IgG (white bars). Nuclear localization of endogenous FOXO3 was induced using LY294002 and precipitated using anti-FOXO3 antibody. (E) U2OS cells were transfected as indicated in the Materials and methods section. FOXO3.A3 induces luciferase expression driven by a 479 bp fragment overlapping the 5′-UTR of human CTDSP2 (white bars), which is decreased if the FOXO-binding sites are mutated (black bars). WT, wild-type. Results are means±S.D. of three biological replicates or representative examples in the case of ChIP. Student's t tests: *P<0.05, **P<0.005; indication absent, P>0.05. NT, not treated.
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Figure 2: CTDSP2 expression is regulated by FOXO proteins(A) CTDSP2 mRNA was measured by qPCR in DL23 cells after FOXO3 was activated by 4OHT for the indicated times or (B) for 8 h in the presence of cycloheximide (CHX). (C) U2OS cells were treated for 16 h with LY294002 or Akti to inhibit PI3K and PKB/Akt and thereby activate endogenous FOXO proteins. CTDSP2 mRNA was measured by qPCR. Either treatment induces CTDSP2 expression significantly. (D) ChIP–qPCR-determined percentage input of the CTDSP2 promoter precipitated by endogenous FOXO3 (black bars) or non-specific IgG (white bars). Nuclear localization of endogenous FOXO3 was induced using LY294002 and precipitated using anti-FOXO3 antibody. (E) U2OS cells were transfected as indicated in the Materials and methods section. FOXO3.A3 induces luciferase expression driven by a 479 bp fragment overlapping the 5′-UTR of human CTDSP2 (white bars), which is decreased if the FOXO-binding sites are mutated (black bars). WT, wild-type. Results are means±S.D. of three biological replicates or representative examples in the case of ChIP. Student's t tests: *P<0.05, **P<0.005; indication absent, P>0.05. NT, not treated.

Mentions: To establish the kinetics of CTDSP2 regulation, we measured CTDSP2 mRNA levels in response to activation of FOXO3.A3-ER in DL23 cells. We observed a rapid increase in CTDSP2 mRNA levels after addition of 4OHT (Figure 2A), even in the presence of the protein translation inhibitor cycloheximide (Figure 2B), both strong indications that FOXO3 can directly control CTDSP2 expression. Furthermore, in DLD1, U2OS, RPE and NIH/3T3 cells, expression of CTDSP2 mRNA is increased in response to inhibitors of PI3K or PKB/Akt (Figure 2C and Supplementary Figure S1A), which are known to control the activity of endogenous FOXO proteins [31]. Interestingly, CTDSP2 regulation is not restricted to FOXO3, as ectopic expression of GFP–FOXO1, GFP–FOXO3 or GFP–FOXO4 also elevates CTDSP2 mRNA levels in U2OS cells (Supplementary Figure S1B). Lastly, shRNA-mediated knockdown of FOXO1 and FOXO3 in U2OS cells decreases the basal and PKB/Akt-inhibition-induced increase in CTDSP2 mRNA (Supplementary Figure S1C), showing that endogenous FOXO1 and FOXO3 are involved in the control of CTDSP2 expression levels.


FOXO target gene CTDSP2 regulates cell cycle progression through Ras and p21(Cip1/Waf1).

Kloet DE, Polderman PE, Eijkelenboom A, Smits LM, van Triest MH, van den Berg MC, Groot Koerkamp MJ, van Leenen D, Lijnzaad P, Holstege FC, Burgering BM - Biochem. J. (2015)

CTDSP2 expression is regulated by FOXO proteins(A) CTDSP2 mRNA was measured by qPCR in DL23 cells after FOXO3 was activated by 4OHT for the indicated times or (B) for 8 h in the presence of cycloheximide (CHX). (C) U2OS cells were treated for 16 h with LY294002 or Akti to inhibit PI3K and PKB/Akt and thereby activate endogenous FOXO proteins. CTDSP2 mRNA was measured by qPCR. Either treatment induces CTDSP2 expression significantly. (D) ChIP–qPCR-determined percentage input of the CTDSP2 promoter precipitated by endogenous FOXO3 (black bars) or non-specific IgG (white bars). Nuclear localization of endogenous FOXO3 was induced using LY294002 and precipitated using anti-FOXO3 antibody. (E) U2OS cells were transfected as indicated in the Materials and methods section. FOXO3.A3 induces luciferase expression driven by a 479 bp fragment overlapping the 5′-UTR of human CTDSP2 (white bars), which is decreased if the FOXO-binding sites are mutated (black bars). WT, wild-type. Results are means±S.D. of three biological replicates or representative examples in the case of ChIP. Student's t tests: *P<0.05, **P<0.005; indication absent, P>0.05. NT, not treated.
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Figure 2: CTDSP2 expression is regulated by FOXO proteins(A) CTDSP2 mRNA was measured by qPCR in DL23 cells after FOXO3 was activated by 4OHT for the indicated times or (B) for 8 h in the presence of cycloheximide (CHX). (C) U2OS cells were treated for 16 h with LY294002 or Akti to inhibit PI3K and PKB/Akt and thereby activate endogenous FOXO proteins. CTDSP2 mRNA was measured by qPCR. Either treatment induces CTDSP2 expression significantly. (D) ChIP–qPCR-determined percentage input of the CTDSP2 promoter precipitated by endogenous FOXO3 (black bars) or non-specific IgG (white bars). Nuclear localization of endogenous FOXO3 was induced using LY294002 and precipitated using anti-FOXO3 antibody. (E) U2OS cells were transfected as indicated in the Materials and methods section. FOXO3.A3 induces luciferase expression driven by a 479 bp fragment overlapping the 5′-UTR of human CTDSP2 (white bars), which is decreased if the FOXO-binding sites are mutated (black bars). WT, wild-type. Results are means±S.D. of three biological replicates or representative examples in the case of ChIP. Student's t tests: *P<0.05, **P<0.005; indication absent, P>0.05. NT, not treated.
Mentions: To establish the kinetics of CTDSP2 regulation, we measured CTDSP2 mRNA levels in response to activation of FOXO3.A3-ER in DL23 cells. We observed a rapid increase in CTDSP2 mRNA levels after addition of 4OHT (Figure 2A), even in the presence of the protein translation inhibitor cycloheximide (Figure 2B), both strong indications that FOXO3 can directly control CTDSP2 expression. Furthermore, in DLD1, U2OS, RPE and NIH/3T3 cells, expression of CTDSP2 mRNA is increased in response to inhibitors of PI3K or PKB/Akt (Figure 2C and Supplementary Figure S1A), which are known to control the activity of endogenous FOXO proteins [31]. Interestingly, CTDSP2 regulation is not restricted to FOXO3, as ectopic expression of GFP–FOXO1, GFP–FOXO3 or GFP–FOXO4 also elevates CTDSP2 mRNA levels in U2OS cells (Supplementary Figure S1B). Lastly, shRNA-mediated knockdown of FOXO1 and FOXO3 in U2OS cells decreases the basal and PKB/Akt-inhibition-induced increase in CTDSP2 mRNA (Supplementary Figure S1C), showing that endogenous FOXO1 and FOXO3 are involved in the control of CTDSP2 expression levels.

Bottom Line: Our data suggest that CTDSP2 induces p21(Cip1/Waf1) through increasing the activity of Ras.As has been described previously, Ras induces p21(Cip1/Waf1) through p53-dependent and p53-independent pathways and indeed both p53 and MEK inhibition can mitigate the CTDSP2-induced p21(Cip1/Waf1) mRNA up-regulation.In support of Ras activation by CTDSP2, depletion of endogenous CTDSP2 results in reduced Ras activity and thus CTDSP2 seems to be part of a larger set of genes regulated by FOXO proteins, which increase growth factor signalling upon FOXO activation.

View Article: PubMed Central - PubMed

Affiliation: University Medical Centre Utrecht, Universiteitsweg 100 STR3.217, 3584CG Utrecht, The Netherlands.

Show MeSH
Related in: MedlinePlus