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AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

Tullai JW, Tacheva S, Owens LJ, Graham JR, Cooper GM - PLoS ONE (2011)

Bottom Line: Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells.Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.

Methodology/principal findings: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.

Conclusions/significance: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

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Effect of c-Jun siRNA on gene induction.A. Knockdown of c-Jun by siRNA. Cells were transfected with either the c-Jun siRNA or a nonspecific construct (NegCtl), or with no construct added (Mock) and then analyzed by c-Jun and β-actin immunoblots. B. Effect of c-Jun knockdown on PDGF induction. T98G cells were transfected with a c-Jun or nonspecific negative control siRNA for 24 hours, and then serum starved for 48 hours. Cells were then stimulated with PDGF for 30 minutes. Expression of the indicated genes was determined by realtime RT-PCR. Data are presented as fold-change as compared to untreated. Data are means for 4 separate experiments ± S.E. * p<0.01. C. Effect of c-Jun knockdown on SB-216763 induction. Transfection and starvation were performed as above, and then cells were treated for 1 hour with SB-216763. Data are presented as fold change as compared to vehicle control (DMSO). Data are means for 4 separate experiments ± S.E., **p<0.05.
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pone-0020150-g005: Effect of c-Jun siRNA on gene induction.A. Knockdown of c-Jun by siRNA. Cells were transfected with either the c-Jun siRNA or a nonspecific construct (NegCtl), or with no construct added (Mock) and then analyzed by c-Jun and β-actin immunoblots. B. Effect of c-Jun knockdown on PDGF induction. T98G cells were transfected with a c-Jun or nonspecific negative control siRNA for 24 hours, and then serum starved for 48 hours. Cells were then stimulated with PDGF for 30 minutes. Expression of the indicated genes was determined by realtime RT-PCR. Data are presented as fold-change as compared to untreated. Data are means for 4 separate experiments ± S.E. * p<0.01. C. Effect of c-Jun knockdown on SB-216763 induction. Transfection and starvation were performed as above, and then cells were treated for 1 hour with SB-216763. Data are presented as fold change as compared to vehicle control (DMSO). Data are means for 4 separate experiments ± S.E., **p<0.05.

Mentions: To determine whether c-Jun is required for the induction of the GSK-3 regulated genes, RNA interference experiments were performed. Transfection of a specific c-Jun siRNA for 24 hours, followed by 48 hours of serum starvation resulted in a knockdown of greater than >90% in T98G cells (Figure 5A). At the end of the 48-hour serum starvation, the effect of the c-Jun knockdown was tested for both the PDGF and SB-216763 induction of the 8 genes for which we demonstrated c-Jun binding. Treatment with c-Jun siRNA decreased the induction of CTGF by PDGF greater than two-fold (p<0.01) (Figure 5B). The induction CYR61 by PDGF was also decreased nearly two-fold in the presence of the c-Jun siRNA, but did not reach statistical significance (p = 0.11) (Figure 5B). RGS2, despite revealing the most dramatic c-Jun recruitment in the ChIP assays upon PDGF stimulation (Figure 2B), does not appear to require c-Jun for its PDGF-mediated induction. RNAi against c-Jun significantly blocked the induction of CTGF, CYR61 and PLAU (p<0.05) resulting from inhibition of GSK-3 with SB-216763 (Figure 5C). These experiments directly demonstrated that c-Jun is required for the induction of these three genes following GSK-3 inhibition.


AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

Tullai JW, Tacheva S, Owens LJ, Graham JR, Cooper GM - PLoS ONE (2011)

Effect of c-Jun siRNA on gene induction.A. Knockdown of c-Jun by siRNA. Cells were transfected with either the c-Jun siRNA or a nonspecific construct (NegCtl), or with no construct added (Mock) and then analyzed by c-Jun and β-actin immunoblots. B. Effect of c-Jun knockdown on PDGF induction. T98G cells were transfected with a c-Jun or nonspecific negative control siRNA for 24 hours, and then serum starved for 48 hours. Cells were then stimulated with PDGF for 30 minutes. Expression of the indicated genes was determined by realtime RT-PCR. Data are presented as fold-change as compared to untreated. Data are means for 4 separate experiments ± S.E. * p<0.01. C. Effect of c-Jun knockdown on SB-216763 induction. Transfection and starvation were performed as above, and then cells were treated for 1 hour with SB-216763. Data are presented as fold change as compared to vehicle control (DMSO). Data are means for 4 separate experiments ± S.E., **p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102068&req=5

pone-0020150-g005: Effect of c-Jun siRNA on gene induction.A. Knockdown of c-Jun by siRNA. Cells were transfected with either the c-Jun siRNA or a nonspecific construct (NegCtl), or with no construct added (Mock) and then analyzed by c-Jun and β-actin immunoblots. B. Effect of c-Jun knockdown on PDGF induction. T98G cells were transfected with a c-Jun or nonspecific negative control siRNA for 24 hours, and then serum starved for 48 hours. Cells were then stimulated with PDGF for 30 minutes. Expression of the indicated genes was determined by realtime RT-PCR. Data are presented as fold-change as compared to untreated. Data are means for 4 separate experiments ± S.E. * p<0.01. C. Effect of c-Jun knockdown on SB-216763 induction. Transfection and starvation were performed as above, and then cells were treated for 1 hour with SB-216763. Data are presented as fold change as compared to vehicle control (DMSO). Data are means for 4 separate experiments ± S.E., **p<0.05.
Mentions: To determine whether c-Jun is required for the induction of the GSK-3 regulated genes, RNA interference experiments were performed. Transfection of a specific c-Jun siRNA for 24 hours, followed by 48 hours of serum starvation resulted in a knockdown of greater than >90% in T98G cells (Figure 5A). At the end of the 48-hour serum starvation, the effect of the c-Jun knockdown was tested for both the PDGF and SB-216763 induction of the 8 genes for which we demonstrated c-Jun binding. Treatment with c-Jun siRNA decreased the induction of CTGF by PDGF greater than two-fold (p<0.01) (Figure 5B). The induction CYR61 by PDGF was also decreased nearly two-fold in the presence of the c-Jun siRNA, but did not reach statistical significance (p = 0.11) (Figure 5B). RGS2, despite revealing the most dramatic c-Jun recruitment in the ChIP assays upon PDGF stimulation (Figure 2B), does not appear to require c-Jun for its PDGF-mediated induction. RNAi against c-Jun significantly blocked the induction of CTGF, CYR61 and PLAU (p<0.05) resulting from inhibition of GSK-3 with SB-216763 (Figure 5C). These experiments directly demonstrated that c-Jun is required for the induction of these three genes following GSK-3 inhibition.

Bottom Line: Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells.Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.

Methodology/principal findings: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.

Conclusions/significance: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

Show MeSH
Related in: MedlinePlus