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Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML) cells results in the downregulation of Hsp70.

Banerjee Mustafi S, Chakraborty PK, Raha S - PLoS ONE (2010)

Bottom Line: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity.Blocking ERK1/2 activation resulted in induction of Hsp70.Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

View Article: PubMed Central - PubMed

Affiliation: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

ABSTRACT

Background: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1.

Methodology/principal findings: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

Conclusion/significance: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.

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Schematic representation of the signaling pathways operative in Hsp70 regulation in Chronic Myelogenous Leukemia.Akt and ERK1/2 both phosphorylate HSF1 on serines (ERK1/2 on serine 307 and GSK3β on serine 303 to negatively influence HSF1 activity). In fact, the phosphorylation by ERK1/2 at Serine 307 is the priming event and it facilitates the phosphorylation by GSK3β at serine 303 which has vital regulatory influence on HSF1 activity. Resveratrol inhibits Akt but stimulates the activity of ERK1/2. Therefore, Resveratrol is able to influence both the priming and the subsequent inhibitory phosphorylation. 17-AAG inhibits Akt and as a consequence decreases the phosphorylation of GSK3β and makes GSK3β active. However, 17AAG also decreases the activation of ERK1/2. Therefore, the active GSK3β is not able to negatively influence HSF1 activity. 17-AAG binds Hsp90 and releases Hsp90 bound HSF1. Free HSF1 in the absence of negative influence of phosphorylations enters the nucleus and triggers the transcription of Hsp70.
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pone-0008719-g006: Schematic representation of the signaling pathways operative in Hsp70 regulation in Chronic Myelogenous Leukemia.Akt and ERK1/2 both phosphorylate HSF1 on serines (ERK1/2 on serine 307 and GSK3β on serine 303 to negatively influence HSF1 activity). In fact, the phosphorylation by ERK1/2 at Serine 307 is the priming event and it facilitates the phosphorylation by GSK3β at serine 303 which has vital regulatory influence on HSF1 activity. Resveratrol inhibits Akt but stimulates the activity of ERK1/2. Therefore, Resveratrol is able to influence both the priming and the subsequent inhibitory phosphorylation. 17-AAG inhibits Akt and as a consequence decreases the phosphorylation of GSK3β and makes GSK3β active. However, 17AAG also decreases the activation of ERK1/2. Therefore, the active GSK3β is not able to negatively influence HSF1 activity. 17-AAG binds Hsp90 and releases Hsp90 bound HSF1. Free HSF1 in the absence of negative influence of phosphorylations enters the nucleus and triggers the transcription of Hsp70.

Mentions: We also checked the effect of Resveratrol on phospho ERK1/2 as this kinase is required for the primary priming of HSF1 at ser 307 so that GSK3β can perform the secondary phosphorylation on HSF1 at ser 303 resulting in a cytoplasmic –inactive form of HSF1 [33]. In presence of Resveratrol there was a considerable increase in the phosphorylated ERK1/2 signal and ERK1/2 activity was also enhanced (Fig 3A–C). The command of phospho ERK1/2 on the suppression of Hsp70 expression in K562 cells was shown by inhibiting ERK1/2 kinase activity with U0126 which resulted in an elevated expression of Hsp72 (Fig. 3E). From these aforesaid results it can be concluded that Resveratrol influences the two kinases namely GSK3β and ERK1/2 (Fig. 6).


Modulation of Akt and ERK1/2 pathways by resveratrol in chronic myelogenous leukemia (CML) cells results in the downregulation of Hsp70.

Banerjee Mustafi S, Chakraborty PK, Raha S - PLoS ONE (2010)

Schematic representation of the signaling pathways operative in Hsp70 regulation in Chronic Myelogenous Leukemia.Akt and ERK1/2 both phosphorylate HSF1 on serines (ERK1/2 on serine 307 and GSK3β on serine 303 to negatively influence HSF1 activity). In fact, the phosphorylation by ERK1/2 at Serine 307 is the priming event and it facilitates the phosphorylation by GSK3β at serine 303 which has vital regulatory influence on HSF1 activity. Resveratrol inhibits Akt but stimulates the activity of ERK1/2. Therefore, Resveratrol is able to influence both the priming and the subsequent inhibitory phosphorylation. 17-AAG inhibits Akt and as a consequence decreases the phosphorylation of GSK3β and makes GSK3β active. However, 17AAG also decreases the activation of ERK1/2. Therefore, the active GSK3β is not able to negatively influence HSF1 activity. 17-AAG binds Hsp90 and releases Hsp90 bound HSF1. Free HSF1 in the absence of negative influence of phosphorylations enters the nucleus and triggers the transcription of Hsp70.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806839&req=5

pone-0008719-g006: Schematic representation of the signaling pathways operative in Hsp70 regulation in Chronic Myelogenous Leukemia.Akt and ERK1/2 both phosphorylate HSF1 on serines (ERK1/2 on serine 307 and GSK3β on serine 303 to negatively influence HSF1 activity). In fact, the phosphorylation by ERK1/2 at Serine 307 is the priming event and it facilitates the phosphorylation by GSK3β at serine 303 which has vital regulatory influence on HSF1 activity. Resveratrol inhibits Akt but stimulates the activity of ERK1/2. Therefore, Resveratrol is able to influence both the priming and the subsequent inhibitory phosphorylation. 17-AAG inhibits Akt and as a consequence decreases the phosphorylation of GSK3β and makes GSK3β active. However, 17AAG also decreases the activation of ERK1/2. Therefore, the active GSK3β is not able to negatively influence HSF1 activity. 17-AAG binds Hsp90 and releases Hsp90 bound HSF1. Free HSF1 in the absence of negative influence of phosphorylations enters the nucleus and triggers the transcription of Hsp70.
Mentions: We also checked the effect of Resveratrol on phospho ERK1/2 as this kinase is required for the primary priming of HSF1 at ser 307 so that GSK3β can perform the secondary phosphorylation on HSF1 at ser 303 resulting in a cytoplasmic –inactive form of HSF1 [33]. In presence of Resveratrol there was a considerable increase in the phosphorylated ERK1/2 signal and ERK1/2 activity was also enhanced (Fig 3A–C). The command of phospho ERK1/2 on the suppression of Hsp70 expression in K562 cells was shown by inhibiting ERK1/2 kinase activity with U0126 which resulted in an elevated expression of Hsp72 (Fig. 3E). From these aforesaid results it can be concluded that Resveratrol influences the two kinases namely GSK3β and ERK1/2 (Fig. 6).

Bottom Line: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity.Blocking ERK1/2 activation resulted in induction of Hsp70.Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

View Article: PubMed Central - PubMed

Affiliation: Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.

ABSTRACT

Background: Resveratrol is known to downregulate the high endogenous level of Heat shock protein 70 (Hsp70) in Chronic Myelogenous Leukemia (CML) K562 cells and induce apoptosis. Since Heat Shock Factor 1 (HSF1) controls transcription of Hsp70, we wanted to probe the signaling pathways responsible for transcriptional activation of HSF1.

Methodology/principal findings: Cells exposed to 40microM Resveratrol rapidly abolished serine473 phosphorylation of Akt and significantly reduced its kinase activity. Inactivation of Akt pathway by Resveratrol subsequently blocked serine9 phosphorylation of Gsk3beta. Active non-phosphorylated Gsk3beta rendered HSF1 transcriptionally inactive and reduced Hsp70 production. Blocking PI3K/Akt activity also demonstrated similar effects on Hsp70 comparable to Resveratrol. Inactivation of Gsk3beta activity by inhibitors SB261763 or LiCl upregulated Hsp70. Resveratrol significantly modulated ERK1/2 activity as evident from hyper phosphorylation at T302/Y304 residues and simultaneous upregulation in kinase activity. Blocking ERK1/2 activation resulted in induction of Hsp70. Therefore, increase in ERK1/2 activity by Resveratrol provided another negative influence on Hsp70 levels through negative regulation of HSF1 activity. 17-allylamino-17-demethoxygeldanamycin (17AAG), a drug that inhibits Hsp90 chaperone and degrades its client protein Akt concomitantly elevated Hsp70 levels by promoting nuclear translocation of HSF1 from the cytosol. This effect is predominantly due to inhibition of both Akt and ERK1/2 activation by 17AAG. Simultaneously treating K562 with Resveratrol and 17AAG maintained phosho-ERK1/2 levels close to untreated controls demonstrating their opposite effects on ERK1/2 pathway. Resveratrol was found not to interfere with Bcr-Abl activation in K562 cells.

Conclusion/significance: Thus our study comprehensively illustrates that Resveratrol acts downstream of Bcr-Abl and inhibits Akt activity but stimulates ERK1/2 activity. This brings down the transcriptional activity of HSF1 and Hsp70 production in K562 cells. Additionally, Resveratrol can be used in combination with chemotherapeutic agents such as 17AAG, an Hsp90 inhibitor reported to induce Hsp70 and hence compromise its chemotherapeutic potential.

Show MeSH
Related in: MedlinePlus