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RNAi-based screening identifies kinases interfering with dioxin-mediated up-regulation of CYP1A1 activity.

Gilot D, Le Meur N, Giudicelli F, Le Vée M, Lagadic-Gossmann D, Théret N, Fardel O - PLoS ONE (2011)

Bottom Line: Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits.PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway.These data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade.

View Article: PubMed Central - PubMed

Affiliation: EA 4427 Signalisation et Réponse aux Agents Infectieux et Chimiques, Université de Rennes 1, Institut de Recherche Santé, Environnement et Travail, Institut Fédératif de Recherche 140, Rennes, France. david.gilot@univ-rennes1.fr

ABSTRACT

Background: The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose.

Results: We performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKIβ). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway.

Conclusions: These data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

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Hit confirmation and selectivity of siRNA.A, Knock-down of ITPKA, PNCK, CaMKIα and AhR in MCF-7 cells. In this confirmation step, 2 siRNA among 3 of the library were selected for ITPKA and PNCK and effects were compared to those of 2 siRNA targeting CaMKIα and AhR (Positive controls) and to negative control (siNT1). 72 h after transfection, cells were exposed to 5 nM TCDD for 6 h and EROD activity was measured as explained in Materials and Methods. Knock-down efficiencies were evaluated by RT-qPCR assays for ITPKA (B) and PNCK (C). Evaluation of off-target effects of siRNA targeting ITPKA or PNCK through mRNA quantification of ITPKB, ITPKC (D) and CaMKIα (E). mRNA level data are expressed in function of the values of mRNA levels found in siNT1-transfected cells exposed to TCDD, arbitrarily set to 1; they correspond to the means ± S.E.M. of three independent experiments. * p-value<0.05 compared to NT1-transfected cells.
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pone-0018261-g004: Hit confirmation and selectivity of siRNA.A, Knock-down of ITPKA, PNCK, CaMKIα and AhR in MCF-7 cells. In this confirmation step, 2 siRNA among 3 of the library were selected for ITPKA and PNCK and effects were compared to those of 2 siRNA targeting CaMKIα and AhR (Positive controls) and to negative control (siNT1). 72 h after transfection, cells were exposed to 5 nM TCDD for 6 h and EROD activity was measured as explained in Materials and Methods. Knock-down efficiencies were evaluated by RT-qPCR assays for ITPKA (B) and PNCK (C). Evaluation of off-target effects of siRNA targeting ITPKA or PNCK through mRNA quantification of ITPKB, ITPKC (D) and CaMKIα (E). mRNA level data are expressed in function of the values of mRNA levels found in siNT1-transfected cells exposed to TCDD, arbitrarily set to 1; they correspond to the means ± S.E.M. of three independent experiments. * p-value<0.05 compared to NT1-transfected cells.

Mentions: Using two siRNA out of 3 available in our library, we confirmed that knock-down of ITPKA and PNCK expression reduced CYP1A activity in TCDD-treated MCF-7 cells. In addition, to compare the effects of ITPKA and PNCK knock-down on EROD activity to our previous published data with CaMKIα [13], [16], [19], we also used 2 other well-characterized siRNA sequence targeting this kinase [16], [19] (siCaMKIa-1 and a-2, respectively, Fig. 4A). Knock-down consequences were compared to the same controls (siNT1 and siAhR) as those used in the screen.


RNAi-based screening identifies kinases interfering with dioxin-mediated up-regulation of CYP1A1 activity.

Gilot D, Le Meur N, Giudicelli F, Le Vée M, Lagadic-Gossmann D, Théret N, Fardel O - PLoS ONE (2011)

Hit confirmation and selectivity of siRNA.A, Knock-down of ITPKA, PNCK, CaMKIα and AhR in MCF-7 cells. In this confirmation step, 2 siRNA among 3 of the library were selected for ITPKA and PNCK and effects were compared to those of 2 siRNA targeting CaMKIα and AhR (Positive controls) and to negative control (siNT1). 72 h after transfection, cells were exposed to 5 nM TCDD for 6 h and EROD activity was measured as explained in Materials and Methods. Knock-down efficiencies were evaluated by RT-qPCR assays for ITPKA (B) and PNCK (C). Evaluation of off-target effects of siRNA targeting ITPKA or PNCK through mRNA quantification of ITPKB, ITPKC (D) and CaMKIα (E). mRNA level data are expressed in function of the values of mRNA levels found in siNT1-transfected cells exposed to TCDD, arbitrarily set to 1; they correspond to the means ± S.E.M. of three independent experiments. * p-value<0.05 compared to NT1-transfected cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066211&req=5

pone-0018261-g004: Hit confirmation and selectivity of siRNA.A, Knock-down of ITPKA, PNCK, CaMKIα and AhR in MCF-7 cells. In this confirmation step, 2 siRNA among 3 of the library were selected for ITPKA and PNCK and effects were compared to those of 2 siRNA targeting CaMKIα and AhR (Positive controls) and to negative control (siNT1). 72 h after transfection, cells were exposed to 5 nM TCDD for 6 h and EROD activity was measured as explained in Materials and Methods. Knock-down efficiencies were evaluated by RT-qPCR assays for ITPKA (B) and PNCK (C). Evaluation of off-target effects of siRNA targeting ITPKA or PNCK through mRNA quantification of ITPKB, ITPKC (D) and CaMKIα (E). mRNA level data are expressed in function of the values of mRNA levels found in siNT1-transfected cells exposed to TCDD, arbitrarily set to 1; they correspond to the means ± S.E.M. of three independent experiments. * p-value<0.05 compared to NT1-transfected cells.
Mentions: Using two siRNA out of 3 available in our library, we confirmed that knock-down of ITPKA and PNCK expression reduced CYP1A activity in TCDD-treated MCF-7 cells. In addition, to compare the effects of ITPKA and PNCK knock-down on EROD activity to our previous published data with CaMKIα [13], [16], [19], we also used 2 other well-characterized siRNA sequence targeting this kinase [16], [19] (siCaMKIa-1 and a-2, respectively, Fig. 4A). Knock-down consequences were compared to the same controls (siNT1 and siAhR) as those used in the screen.

Bottom Line: Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits.PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway.These data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade.

View Article: PubMed Central - PubMed

Affiliation: EA 4427 Signalisation et Réponse aux Agents Infectieux et Chimiques, Université de Rennes 1, Institut de Recherche Santé, Environnement et Travail, Institut Fédératif de Recherche 140, Rennes, France. david.gilot@univ-rennes1.fr

ABSTRACT

Background: The aryl hydrocarbon receptor (AhR) is a transcription factor activated by several environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and involved in carcinogenesis and various physiological processes, including immune response and endocrine functions. Characterization of kinases-related AhR transduction pathway remains an important purpose.

Results: We performed a kinome-wide siRNA screen in human mammary MCF-7 cells to identify non redundant protein kinases implicated in the up-regulation of cytochrome P-450 (CYP) 1A1 activity, an AhR referent target, in response to TCDD exposure. To this aim, we monitored CYP1A1-related ethoxyresorufin-O-deethylase (EROD) activity and quantified cell density. This normalization was crucial since it allowed us to focus only on siRNA affecting EROD activity and discard siRNA affecting cell density. Analyses of the cell density data allowed us to identify several hits already well-characterized as effectors of the cell cycle and original hits. Collectively, these data fully validated the protocol and the siRNA library. Next, 22 novel candidates were identified as kinases potentially implicated in the up-regulation of CYP1A1 in response to TCDD, without alteration of cell survival or cell proliferation. The siRNA library screen gave a limited number of hits (approximately 3%). Interestingly, four of them are able to bind calmodulin among which the IP3 kinase A (ITPKA) and pregnancy up-regulated non-ubiquitously expressed CaM kinase (PNCK, also named CaMKIβ). Remarkably, for both proteins, their kinase activity depends on the calmodulin binding. Involvement of ITPKA and PNCK in TCDD-mediated CYP1A1 up-regulation was further validated by screening-independent expression knock-down. PNCK was finally shown to regulate activation of CaMKIα, a CaMKI isoform previously reported to interplay with the AhR pathway.

Conclusions: These data fully support a role for both IP3-related kinase and CaMK isoforms in the AhR signaling cascade. More generally, this study also highlights the interest of large scale loss-of-function screens for characterizing the molecular mechanism of action of environmental contaminants.

Show MeSH
Related in: MedlinePlus