G-protein-dependent and -independent pathways regulate proteinase-activated receptor-2 mediated p65 NFkappaB serine 536 phosphorylation in human keratinocytes.
The mechanisms underpinning the coupling of GPCRs, such as PAR-2, to the phosphorylation of p65 NFkappaB have not been investigated. In the current study we found that trypsin and the selective PAR-2 activating peptide, 2f-LIGKV-OH, stimulated large and sustained increases in the serine 536 phosphorylation of p65/RelA in a transfected skin epithelial cell line and primary keratinocytes. Parallel experiments showed that in both cell types, p65 NFkappaB phosphorylation is mediated through the selective activation of IKK2. Treatment with PKC inhibitor GF109203X or PKCalpha siRNA reduced phosphorylation at 15 min but not 30 min, whilst rottlerin, a selective PKCdelta inhibitor and PKCdelta siRNA reduced the response at both time points. Pre-treatment of cells with the novel Gq/11 inhibitor YM-254890 and Gq/11 siRNA caused a similar pattern of inhibition and also reduced PAR-2-mediated NFkappaB transcriptional activity. Furthermore, stimulation of cells through a novel PAR-2 mutant PAR-2(34-43), delayed p65 phosphorylation but was without effect on the kinetics of ERK activation. Inhibition of Gi or G12/13 pathways by pertussis toxin pre-treatment or over-expression of the RGS mutant Lsc, also did not effect NFkappaB phosphorylation. Taken together these data indicate dependency for Gq/11 in early phosphorylation of p65 NFkappaB and this subsequently affects initial NFkappaB-dependent gene transcriptional activity, however later regulation of p65 is unaffected. Overall these novel data demonstrate an IKK2-dependent, predominantly G-protein-independent pathway involved in PAR-2 regulation of NFkappaB phosphorylation in keratinocytes.
Goon Goh
Fui
F
Strathclyde Institute of Pharmacy and Biomedical Sciences, Division of Physiology and Pharmacology, University of Strathclyde, 27 Taylor Street, Glasgow, G4 0NR, Scotland, UK.
Sloss
Callum M
CM
Cunningham
Margaret R
MR
Nilsson
Mary
M
Cadalbert
Laurence
L
Plevin
Robin
R
D000098:Acetophenones / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002478:Cells, Cultured; D002999:Clone Cells; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D050505:Mutant Proteins / Q000378:metabolism; D010456:Peptides, Cyclic / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D034622:RNA Interference / Q000187:drug effects; D044464:Receptor, PAR-2 / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism*
D000098:Acetophenones; D001578:Benzopyrans; D020672:I-kappa B Proteins; D007211:Indoles; D008301:Maleimides; D050505:Mutant Proteins; D010456:Peptides, Cyclic; D044464:Receptor, PAR-2; D051996:Transcription Factor RelA; C475455:YM-254890; D010768:Phosphoserine; C085746:rottlerin; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C070515:bisindolylmaleimide I