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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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Related in: MedlinePlus

Effects of PMA concentration and treatment duration on ckβ promoter activity.(A) MCF-7 cells were treated with 10, 20, or 30 ng/mL PMA for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 vs. DMSO control; #p<0.05, within the PMA treatment group). (B) MCF-7 cells were treated with 20 ng/mL of PMA for 6 to 72 hr. The values shown are ckβ promoter activities relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (C) Effect of PMA treatment on the ckβ mRNA and protein levels in MCF-7 cells. Cells were treated with 20 ng/mL of PMA or DMSO for 6 to 24 hr. Left panel: The levels of mRNA and proteins are relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. Right panel: The blot shown is representative of three independent experiments that produced similar results. M: SuperSignal Molecular Weight protein ladders (Thermo Scientific, USA), C: control, T: PMA treated, P: purified CKβ protein (45 kDa) as reference.
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pone-0113485-g006: Effects of PMA concentration and treatment duration on ckβ promoter activity.(A) MCF-7 cells were treated with 10, 20, or 30 ng/mL PMA for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 vs. DMSO control; #p<0.05, within the PMA treatment group). (B) MCF-7 cells were treated with 20 ng/mL of PMA for 6 to 72 hr. The values shown are ckβ promoter activities relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (C) Effect of PMA treatment on the ckβ mRNA and protein levels in MCF-7 cells. Cells were treated with 20 ng/mL of PMA or DMSO for 6 to 24 hr. Left panel: The levels of mRNA and proteins are relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. Right panel: The blot shown is representative of three independent experiments that produced similar results. M: SuperSignal Molecular Weight protein ladders (Thermo Scientific, USA), C: control, T: PMA treated, P: purified CKβ protein (45 kDa) as reference.

Mentions: PMA regulates certain Ets and GATA family transcription factors by activating the PKC-mediated mitogen-activated protein kinase (MAPK) pathway [29], [30]. This prompted us to examine the effect of PMA on ckβ promoter activity. The effects of PMA on PKCs are dependent on the PMA concentration and the duration of treatment. Short-term treatment with a low concentration of PMA activates PKCs, while long-term exposure to a high concentration has an inhibitory effect on PKCs [31], [32]. In this study, MCF-7 cells were treated with different concentrations of PMA (10, 20, and 30 ng/mL) for 6 hr after transfection with the wild-type pGL4.10-ckβ(−2000/−1) reporter plasmid. PMA repressed ckβ promoter activity in MCF-7 cells starting at 10 ng/mL and reaching a maximal effect at 20 ng/mL (Fig. 6A). However, a 6 hr exposure to 30 ng/mL PMA attenuated the repression of ckβ promoter activity. The effect of PMA treatment duration on ckβ promoter activity was examined in the next series of experiments, ranging from 6 to 72 hr with a PMA concentration of 20 ng/mL. As shown in Fig. 6B, the maximum repressive effect of PMA occurred at 6 hr after treatment (approximately 38% of the DMSO control). Promoter activity began to increase and was almost equal to that of the DMSO control treatment after 12 hr of PMA treatment. Overall, chronic or extended PMA treatment eliminated the PMA effect on the ckβ promoter activity, suggesting that PMA-sensitive PKC isozymes are involved in the PMA-mediated repression of the ckβ promoter. This result also ruled out the involvement of PKCζ, as this isozyme is known to be resistant to chronic PMA treatment [31], [33].


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)

Effects of PMA concentration and treatment duration on ckβ promoter activity.(A) MCF-7 cells were treated with 10, 20, or 30 ng/mL PMA for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 vs. DMSO control; #p<0.05, within the PMA treatment group). (B) MCF-7 cells were treated with 20 ng/mL of PMA for 6 to 72 hr. The values shown are ckβ promoter activities relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (C) Effect of PMA treatment on the ckβ mRNA and protein levels in MCF-7 cells. Cells were treated with 20 ng/mL of PMA or DMSO for 6 to 24 hr. Left panel: The levels of mRNA and proteins are relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. Right panel: The blot shown is representative of three independent experiments that produced similar results. M: SuperSignal Molecular Weight protein ladders (Thermo Scientific, USA), C: control, T: PMA treated, P: purified CKβ protein (45 kDa) as reference.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4260826&req=5

pone-0113485-g006: Effects of PMA concentration and treatment duration on ckβ promoter activity.(A) MCF-7 cells were treated with 10, 20, or 30 ng/mL PMA for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*p<0.05 vs. DMSO control; #p<0.05, within the PMA treatment group). (B) MCF-7 cells were treated with 20 ng/mL of PMA for 6 to 72 hr. The values shown are ckβ promoter activities relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (C) Effect of PMA treatment on the ckβ mRNA and protein levels in MCF-7 cells. Cells were treated with 20 ng/mL of PMA or DMSO for 6 to 24 hr. Left panel: The levels of mRNA and proteins are relative to their respective controls (control value is set at 1.0, *p<0.05 vs. DMSO control). Each bar represents the mean ±SEM of triplicate samples from three independent experiments. Right panel: The blot shown is representative of three independent experiments that produced similar results. M: SuperSignal Molecular Weight protein ladders (Thermo Scientific, USA), C: control, T: PMA treated, P: purified CKβ protein (45 kDa) as reference.
Mentions: PMA regulates certain Ets and GATA family transcription factors by activating the PKC-mediated mitogen-activated protein kinase (MAPK) pathway [29], [30]. This prompted us to examine the effect of PMA on ckβ promoter activity. The effects of PMA on PKCs are dependent on the PMA concentration and the duration of treatment. Short-term treatment with a low concentration of PMA activates PKCs, while long-term exposure to a high concentration has an inhibitory effect on PKCs [31], [32]. In this study, MCF-7 cells were treated with different concentrations of PMA (10, 20, and 30 ng/mL) for 6 hr after transfection with the wild-type pGL4.10-ckβ(−2000/−1) reporter plasmid. PMA repressed ckβ promoter activity in MCF-7 cells starting at 10 ng/mL and reaching a maximal effect at 20 ng/mL (Fig. 6A). However, a 6 hr exposure to 30 ng/mL PMA attenuated the repression of ckβ promoter activity. The effect of PMA treatment duration on ckβ promoter activity was examined in the next series of experiments, ranging from 6 to 72 hr with a PMA concentration of 20 ng/mL. As shown in Fig. 6B, the maximum repressive effect of PMA occurred at 6 hr after treatment (approximately 38% of the DMSO control). Promoter activity began to increase and was almost equal to that of the DMSO control treatment after 12 hr of PMA treatment. Overall, chronic or extended PMA treatment eliminated the PMA effect on the ckβ promoter activity, suggesting that PMA-sensitive PKC isozymes are involved in the PMA-mediated repression of the ckβ promoter. This result also ruled out the involvement of PKCζ, as this isozyme is known to be resistant to chronic PMA treatment [31], [33].

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