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Research Article

Inhibition of NF-κB signaling interferes with phorbol ester-induced growth arrest of keratinocytes in a TNFR1-independent manner

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Pages 44-51 | Received 02 Oct 2008, Accepted 10 Dec 2008, Published online: 01 Feb 2009
 

Abstract

A skin-specific block in NF-κB signaling leads to hyperproliferation of the keratinocytes, inflammation, and spontaneous development of squamous cell carcinoma (SCC). Here we show that an inhibition of NF-κB signaling in keratinocytes, via the expression of the super-repressor/ degradation-resistant form of the IκBα protein (IκBαDN), interferes with the growth arrest induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). The IκBαDN cells are able to overcome the TPA-induced cell cycle block. Although SCC development as well as hyperproliferation due to IκBαDN expression in keratinocytes is known to require TNFR1 signaling, the effect of IκBαDN on phorbol ester signaling is downstream/independent of TNFR1. These data thus identify an interaction between IκBαDN and the tumor promoter TPA in the growth regulation of keratinocytes. The proposed mechanism is also likely to be significant in the process of cancer development due to NF-κB inhibition.

Acknowledgments

We are grateful to Pinelopi Vlaches for help with initial experiments and åsa Bergström for technical help. The work was supported by grants from Swedish Cancer Society to IS and RT.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Supplementary Figure 1. Functional characterization of retrovirally transduced keratinocytes: (a) Western blot analysis of IκBαDN expression. N/TERT-1,2G keratinocytes transduced with IκBαDN (lanes 3 and 4) express high levels of the IκBαDN protein compared to the pLNCX2 control cells (lanes 1 and 2). Protein extracts (20 μg) were resolved on 12% SDS-polyacrylamide gel. Subsequently the proteins were transferred to Hybond-P, PVDF transfer membranes(Amersham). The primary antibodies used to detect specific proteins were: rabbit polyclonal anti mouse β-actin antibody (Sigma) and rabbit polyclonal anti IκBαantibody (Santa Cruz). HRP-conjugated secondary antibodies were from GE Healthcare; (b) Confocal imaging of cytoplasmic vs nuclear localization of p65 upon TPA stimulation (100 ng/ml for 1 hr). Greater then 95% of cells transduced with retroviral IκBαDN constructs exhibit a lack of p65 nuclear localization upon TPA stimulation. Green colour denotes p65, nuclei are depicted with blue and torqouise color represents colocalisation/nuclear localization of p65). For immunoflorescence- Cells were fixed with 4% formaldehyde/PBS solution for 10 minutes and were subsequently stained with rabbit polyclonal anti-p65 antibody (Santa Cruz). For secondary antibody, we used Alexa Fluor 488 conjugated goat anti rabbit IgG (Invitrogen). Nuclei were stained with DRAQ5 (Biostatus). The cells were then analysed using the confocal microscope (Zeiss).

Supplementary Figure 1.  Functional characterization of retrovirally transduced keratinocytes: (a) Western blot analysis of IκBαDN expression. N/TERT-1,2G keratinocytes transduced with IκBαDN (lanes 3 and 4) express high levels of the IκBαDN protein compared to the pLNCX2 control cells (lanes 1 and 2). Protein extracts (20 μg) were resolved on 12% SDS-polyacrylamide gel. Subsequently the proteins were transferred to Hybond-P, PVDF transfer membranes(Amersham). The primary antibodies used to detect specific proteins were: rabbit polyclonal anti mouse β-actin antibody (Sigma) and rabbit polyclonal anti IκBαantibody (Santa Cruz). HRP-conjugated secondary antibodies were from GE Healthcare; (b) Confocal imaging of cytoplasmic vs nuclear localization of p65 upon TPA stimulation (100 ng/ml for 1 hr). Greater then 95% of cells transduced with retroviral IκBαDN constructs exhibit a lack of p65 nuclear localization upon TPA stimulation. Green colour denotes p65, nuclei are depicted with blue and torqouise color represents colocalisation/nuclear localization of p65). For immunoflorescence- Cells were fixed with 4% formaldehyde/PBS solution for 10 minutes and were subsequently stained with rabbit polyclonal anti-p65 antibody (Santa Cruz). For secondary antibody, we used Alexa Fluor 488 conjugated goat anti rabbit IgG (Invitrogen). Nuclei were stained with DRAQ5 (Biostatus). The cells were then analysed using the confocal microscope (Zeiss).

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