The TGFβ1 pathway is required for NFκB dependent gene expression in mouse keratinocytes.
The transforming growth factor-beta 1 (TGFβ1) and NFκB 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, TGFβ1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFβ1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFβ1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFβ1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFβ1 induction of NFκB-luciferase. TGFβ1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFβ1 and this was blocked in NFκB+/- and -/- keratinocytes and by the IκB super-repressor. To test the effects of the TGFβ1 pathway on a biologically relevant activator of NFκB, 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 TGFβ1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGFβ1 signaling is required for NFκB-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 TGFβ1.
Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, USA. Electronic address: kahogan@umich.edu.
Kelly A
KA
Hogan
Anand
A
Ravindran
Michael A
MA
Podolsky
Adam B
AB
Glick
D000818:Animals; D000831:Animals, Newborn; D001549:Benzamides / Q000494:pharmacology; D002478:Cells, Cultured; D004149:Dioxoles / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D015151:Immunoblotting; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D016328:NF-kappa B; D018125:Receptors, Transforming Growth Factor beta; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D053773:Transforming Growth Factor beta1; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases
ALK5; EMSA; I kappa Kinaseα; I kappaβ; I kappaβ super repressor; IKKα; IKβ; IKβsr; JNK; Keratinocyte; NFkappaB; SBE; Smad binding elements; TAK1; TGFbeta1; TGFβ1; TGFβ1 activated kinase 1; TNFalpha; TNFα; UVB; activin receptor-like kinase 5; cJun N-terminal kinases; electrophoretic mobility shift assay; nuclear factor kappaB; transforming growth factor-beta 1; tumor necrosis factor alpha