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Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway.

Kuan CS, Yee YH, See Too WC, Few LL - PLoS ONE (2014)

Bottom Line: In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements.PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated.These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

View Article: PubMed Central - PubMed

Affiliation: School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

ABSTRACT

Background: Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.

Methodology/principal findings: In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.

Conclusion/significance: These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

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Effect of PKC inhibitors (PKC412 and Go 6983) on the PMA-mediated repression of ckβ promoter activity.(A) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 1 mM PKC412 and 0.1 mM Go 6983 for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control). (B) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 10 µM PKCε inhibitor peptide and 10 µM PKCη pseudo-substrate inhibitor for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control, #p<0.05 between the two indicated treatments).
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pone-0113485-g007: Effect of PKC inhibitors (PKC412 and Go 6983) on the PMA-mediated repression of ckβ promoter activity.(A) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 1 mM PKC412 and 0.1 mM Go 6983 for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control). (B) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 10 µM PKCε inhibitor peptide and 10 µM PKCη pseudo-substrate inhibitor for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control, #p<0.05 between the two indicated treatments).

Mentions: PKC412 (midostaurin, or CGP 41251), an inhibitor of PKCα, −β, −γ, −δ, −ε, −η, and −ζ [34], completely abolished the PMA-induced down-regulation of the ckβ promoter, whereas Go 6983, an inhibitor of PKCα, −β, −γ, −δ, and −ζ [32], [35], did not show the same effect (Fig. 7A). The ckβ promoter activity was also increased by the individual treatment with PKC412 but not Go 6983. These findings, along with the resistance of PKCζ to chronic PMA treatment, suggest that PKCε or PKCη is most likely the PKC isozyme that mediates the repressive effect of PMA in this system. Subsequently, PKCε and PKCη specific inhibitors were used to identify the isozyme involved. The results in Fig. 7B show that PKCε inhibition abolished the PMA repression of ckβ promoter. PKCη inhibition did not affect the down-regulation of ckβ promoter by PMA treatment. The promoter activity of treatment with PMA and PKCη inhibitor was also significantly lower than treatment with PKCη alone. This further excludes the involvement of PKCη isozyme in the PMA repression of ckβ promoter.


Ets and GATA transcription factors play a critical role in PMA-mediated repression of the ckβ promoter via the protein kinase C signaling pathway.

Kuan CS, Yee YH, See Too WC, Few LL - PLoS ONE (2014)

Effect of PKC inhibitors (PKC412 and Go 6983) on the PMA-mediated repression of ckβ promoter activity.(A) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 1 mM PKC412 and 0.1 mM Go 6983 for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control). (B) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 10 µM PKCε inhibitor peptide and 10 µM PKCη pseudo-substrate inhibitor for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control, #p<0.05 between the two indicated treatments).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260826&req=5

pone-0113485-g007: Effect of PKC inhibitors (PKC412 and Go 6983) on the PMA-mediated repression of ckβ promoter activity.(A) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 1 mM PKC412 and 0.1 mM Go 6983 for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control). (B) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 10 µM PKCε inhibitor peptide and 10 µM PKCη pseudo-substrate inhibitor for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 and **p<0.01 vs. DMSO control, #p<0.05 between the two indicated treatments).
Mentions: PKC412 (midostaurin, or CGP 41251), an inhibitor of PKCα, −β, −γ, −δ, −ε, −η, and −ζ [34], completely abolished the PMA-induced down-regulation of the ckβ promoter, whereas Go 6983, an inhibitor of PKCα, −β, −γ, −δ, and −ζ [32], [35], did not show the same effect (Fig. 7A). The ckβ promoter activity was also increased by the individual treatment with PKC412 but not Go 6983. These findings, along with the resistance of PKCζ to chronic PMA treatment, suggest that PKCε or PKCη is most likely the PKC isozyme that mediates the repressive effect of PMA in this system. Subsequently, PKCε and PKCη specific inhibitors were used to identify the isozyme involved. The results in Fig. 7B show that PKCε inhibition abolished the PMA repression of ckβ promoter. PKCη inhibition did not affect the down-regulation of ckβ promoter by PMA treatment. The promoter activity of treatment with PMA and PKCη inhibitor was also significantly lower than treatment with PKCη alone. This further excludes the involvement of PKCη isozyme in the PMA repression of ckβ promoter.

Bottom Line: In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements.PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated.These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

View Article: PubMed Central - PubMed

Affiliation: School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

ABSTRACT

Background: Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.

Methodology/principal findings: In this report, the distal promoter region of the ckβ gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.

Conclusion/significance: These results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.

Show MeSH
Related in: MedlinePlus