The TGFβ1 pathway is required for NFκB dependent gene expression in mouse keratinocytes

The transforming growth factor-beta 1 (TGF beta 1) and NF kappa B pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGF beta 1 induces NF kappa B-luciferase reporter activity that is dependent on both NF kappa B and Smad3. TGF beta 1-induced NF kappa B-luciferase activity was blocked by the I kappa B inhibitor parthenolide, the I kappa B super-repressor, a dominant negative TGF beta 1-activated kinase 1 (TAK1) and genetic deletion of NF kappa B1. Coexpression of NF kappa B p50 or p65 subunits enhanced NF kappa B-luciferase activity. Similarly, inhibition of the TGF beta 1 type! receptor with SB431542 or genetic deletion of Smad3 blocked TGF beta 1 induction of NF kappa B-luciferase. TGF beta 1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic I kappa B levels or nuclear translocation of NF kappa B subunits, although EMSA showed rapid NF kappa B nuclear binding activity that could be blocked by SB431542 treatment. TNF alpha, a well characterized NF kappa B target gene was also induced by TGF beta 1 and this was blocked in NF kappa B+/- and -/- keratinocytes and by the I kappa B super-repressor. To test the effects of the TGF beta 1 pathway on a biologically relevant activator of NF kappa B, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGF beta 1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNF alpha message. Similarly, UVB induction of TNF alpha mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGF beta 1 signaling is required for NF kappa B-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGF beta 1. (C) 2013 Elsevier Ltd. All rights reserved.