Tumor necrosis factor alpha induces gamma-glutamyltransferase expression via nuclear factor-kappaB in cooperation with Sp1.
Gamma-glutamyltransferase (GGT) cleaves the gamma-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed. We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFalpha antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-kappaB) isoforms, curcumin, a well characterized natural NF-kappaB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IkappaBalpha), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFalpha-induced stimulation. Over-expression of receptor of TNFalpha-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IkappaBalpha or NF-kappaB p65 further confirmed GGT promoter activation via NF-kappaB. Linker insertion mutagenesis of 536 bp of the proximal GGT promoter revealed NF-kappaB and Sp1 binding sites at -110 and -78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-kappaB site located at -110 additionally inhibited TNFalpha-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFalpha treatment induced in vivo binding of both NF-kappaB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.
Reuter
Simone
S
Laboratoire de Biologie Moléculaire et Cellulaire du Cancer, Fondation de Recherche Cancer et Sang, Hôpital Kirchberg, 9, rue Edward Steichen, L-2540, Luxembourg.
Schnekenburger
Michael
M
Cristofanon
Silvia
S
Buck
Isabelle
I
Teiten
Marie-Hélène
MH
Daubeuf
Sandrine
S
Eifes
Serge
S
Dicato
Mario
M
Aggarwal
Bharat B
BB
Visvikis
Athanase
A
Diederich
Marc
M
D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D006801:Humans; D016296:Mutagenesis; D016328:NF-kappa B / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D017931:DNA Primers; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D014409:Tumor Necrosis Factor-alpha; D005723:gamma-Glutamyltransferase