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Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.

Xie W, Pariollaud M, Wixted WE, Chitnis N, Fornwald J, Truong M, Pao C, Liu Y, Ames RS, Callahan J, Solari R, Sanchez Y, Diehl A, Li H - PLoS ONE (2015)

Bottom Line: The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components.In addition, these hits showed NRF2-dependent anti-oxidant gene induction.In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.

ABSTRACT
Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.

No MeSH data available.


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Model for potential crosstalk between PERK and NRF2 signaling pathways.
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pone.0119738.g009: Model for potential crosstalk between PERK and NRF2 signaling pathways.

Mentions: One interesting and yet novel hypothesis that we would like to explore further is the connection between PERK signaling pathway and the NRF2 oxidative stress response signaling pathway. Regulated activation of NRF2 pathway provides cytoprotection against oxidative or electrophilic insults. We examined the effects of our PERK activators on the expression of HO-1 and NQO-1, two key components of the NRF2-dependent defense system. Data demonstrated that these PERK activator compounds induced the expression of both HO-1 and NQO-1 in NHBE cells, the expression of which is NRF2 dependent. This strongly indicates that the compounds are able to modulate the activity of the important transcription factor NRF2 through a potential crosstalk between PERK and NRF2 signaling pathways, as shown in the proposed model (Fig. 9). Indeed, as shown in Figs. 7 and 8, NRF2 siRNA treatment prevented the compound induction on HO-1 and NQO-1, demonstrating that the effect is ultimately NRF2 dependent. These observations are consistent with our initial hypothesis that PERK signaling activation is able to augment NRF2 dependent signaling, with the potential of reducing cell damage and promoting cell growth in COPD lungs. We also noticed that PERK siRNA treatment did not significantly reduce the effect of compounds on HO-1 or NQO-1 expression (Figs. 7 and 8). One possible explanation is that our compounds had some non-PERK dependent activation on HO-1 and NQO-1 expression. This is possible since NRF2 regulation is complexed and related to a number of signaling pathways, such as JNK, PKC and ERK among others. Indeed, both compounds B and C showed some portion of non-PERK dependent activation on eIF2α and ATF-4. Alternatively, PERK siRNA treatment might have generated some stress responses which could stimulate HO-1 and NQO-1 expression. A third possibility is that our compounds activated the very small amount of PERK remained after siRNA treatment, and further upregulated HO-1 and NQO-1 expression, though this is less likely since our data showed a very effective knockdown of PERK protein. In addition, we observed similar results even after combining PERK siRNA treatment and a PERK inhibitor (data not shown). Nevertheless, our data indicates that these PERK activators interacted with NRF2 signaling pathway in our system. Although it is unclear whether the induction of HO-1 and NQO-1 by these compounds is directly PERK dependent, the connection between PERK activation and NRF2 activation is demonstrated. As proposed in Fig. 9, both PERK activation and NRF2 activation can lead to cell survival and provide cytoprotection. Based on this shared outcome, connections between these two pathways can be physiologically meaningful in terms of restoring the oxidant/antioxidant balance in COPD patients. It will be very interesting to further explore these connections. Another interesting observation is the effect of Tg on HO-1 and NQO-1 expression. Our data showed that Tg treatment significantly induced the mRNA level of both HO-1 and NQO-1, but not at the protein level (Figs. 7C and 8C). It is possible that the stability of HO-1 and NQO-1 proteins is regulated through some other cellular processes which may be differentiated from the mRNA regulation.


Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.

Xie W, Pariollaud M, Wixted WE, Chitnis N, Fornwald J, Truong M, Pao C, Liu Y, Ames RS, Callahan J, Solari R, Sanchez Y, Diehl A, Li H - PLoS ONE (2015)

Model for potential crosstalk between PERK and NRF2 signaling pathways.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363567&req=5

pone.0119738.g009: Model for potential crosstalk between PERK and NRF2 signaling pathways.
Mentions: One interesting and yet novel hypothesis that we would like to explore further is the connection between PERK signaling pathway and the NRF2 oxidative stress response signaling pathway. Regulated activation of NRF2 pathway provides cytoprotection against oxidative or electrophilic insults. We examined the effects of our PERK activators on the expression of HO-1 and NQO-1, two key components of the NRF2-dependent defense system. Data demonstrated that these PERK activator compounds induced the expression of both HO-1 and NQO-1 in NHBE cells, the expression of which is NRF2 dependent. This strongly indicates that the compounds are able to modulate the activity of the important transcription factor NRF2 through a potential crosstalk between PERK and NRF2 signaling pathways, as shown in the proposed model (Fig. 9). Indeed, as shown in Figs. 7 and 8, NRF2 siRNA treatment prevented the compound induction on HO-1 and NQO-1, demonstrating that the effect is ultimately NRF2 dependent. These observations are consistent with our initial hypothesis that PERK signaling activation is able to augment NRF2 dependent signaling, with the potential of reducing cell damage and promoting cell growth in COPD lungs. We also noticed that PERK siRNA treatment did not significantly reduce the effect of compounds on HO-1 or NQO-1 expression (Figs. 7 and 8). One possible explanation is that our compounds had some non-PERK dependent activation on HO-1 and NQO-1 expression. This is possible since NRF2 regulation is complexed and related to a number of signaling pathways, such as JNK, PKC and ERK among others. Indeed, both compounds B and C showed some portion of non-PERK dependent activation on eIF2α and ATF-4. Alternatively, PERK siRNA treatment might have generated some stress responses which could stimulate HO-1 and NQO-1 expression. A third possibility is that our compounds activated the very small amount of PERK remained after siRNA treatment, and further upregulated HO-1 and NQO-1 expression, though this is less likely since our data showed a very effective knockdown of PERK protein. In addition, we observed similar results even after combining PERK siRNA treatment and a PERK inhibitor (data not shown). Nevertheless, our data indicates that these PERK activators interacted with NRF2 signaling pathway in our system. Although it is unclear whether the induction of HO-1 and NQO-1 by these compounds is directly PERK dependent, the connection between PERK activation and NRF2 activation is demonstrated. As proposed in Fig. 9, both PERK activation and NRF2 activation can lead to cell survival and provide cytoprotection. Based on this shared outcome, connections between these two pathways can be physiologically meaningful in terms of restoring the oxidant/antioxidant balance in COPD patients. It will be very interesting to further explore these connections. Another interesting observation is the effect of Tg on HO-1 and NQO-1 expression. Our data showed that Tg treatment significantly induced the mRNA level of both HO-1 and NQO-1, but not at the protein level (Figs. 7C and 8C). It is possible that the stability of HO-1 and NQO-1 proteins is regulated through some other cellular processes which may be differentiated from the mRNA regulation.

Bottom Line: The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components.In addition, these hits showed NRF2-dependent anti-oxidant gene induction.In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.

ABSTRACT
Endoplasmic reticulum stress plays a critical role to restore the homeostasis of protein production in eukaryotic cells. This vital process is hence involved in many types of diseases including COPD. PERK, one branch in the ER stress signaling pathways, has been reported to activate NRF2 signaling pathway, a known protective response to COPD. Based on this scientific rationale, we aimed to identify PERK activators as a mechanism to achieve NRF2 activation. In this report, we describe a phenotypic screening assay to identify PERK activators. This assay measures phosphorylation of GFP-tagged eIF2α upon PERK activation via a cell-based LanthaScreen technology. To obtain a robust assay with sufficient signal to background and low variation, multiple parameters were optimized including GFP-tagged eIF2α BacMam concentration, cell density and serum concentration. The assay was validated by a tool compound, Thapsigargin, which induces phosphorylation of eIF2α. In our assay, this compound showed maximal signal window of approximately 2.5-fold with a pEC50 of 8.0, consistent with literature reports. To identify novel PERK activators through phosphorylation of eIF2α, a focused set of 8,400 compounds was screened in this assay at 10 µM. A number of hits were identified and validated. The molecular mechanisms for several selected hits were further characterized in terms of PERK activation and effects on PERK downstream components. Specificity of these compounds in activating PERK was demonstrated with a PERK specific inhibitor and in PERK knockout mouse embryonic fibroblast (MEF) cells. In addition, these hits showed NRF2-dependent anti-oxidant gene induction. In summary, our phenotypic screening assay is demonstrated to be able to identify PERK specific activators. The identified PERK activators could potentially be used as chemical probes to further investigate this pathway as well as the link between PERK activation and NRF2 pathway activation.

No MeSH data available.


Related in: MedlinePlus