RNAi-Based Screening Identifies Kinases Interfering with Dioxin-Mediated Up-Regulation of CYP1A1 Activity

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 CaMKI beta). 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 CaMKI alpha, 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.