Smad3 suppresses Nrf2-mediated expression of GCLC.

3491 Transforming growth factor-beta (TGF-β) regulates cell proliferation and differentiation. TGF-β signaling occurs through transmembrane serine-threonine kinase type II and type I receptors. Smad2 and Smad3 transcription factors activated by type I receptor and common Smad4 translocate to the nucleus to regulate transcription of target genes. In cDNA microarray studies in mouse mammary epithelial NMuMG cells, we identified new target genes suppressed by TGF-β including Gstp2, Gsta4, Gstm1, catalase, and glutamate cysteine ligase. These genes are regulated by the Nuclear Factor-Erythroid 2p45 related transcription factor Nrf2 (Chan and Kwong Biochim. Biophys. Acta 1517: 19-26, 2002; Kwak et al, Mol. Med. 2: 135-145, 2001;Chanas et al., Biochem. J. 356(A2): 405-410, 2002) which binds to antioxidant response elements (AREs) as a heterodimer and participates in the regulation of Phase II gene expression, a component of cancer chemoprevention. (Ramos-Gomez et al. Proc. Natl. Acad. Sci. USA 98:3410-3415, 2001). GCLC, the catalytic subunit of glutamate cysteine ligase (GCL) was used as a molecular surrogate to determine if TGF-β signaling could suppress Nrf2-regulated gene expression. NMuMG cells were transiently co-transfected with either a GCLC Lux or an ARE/GCLC Lux reporter and vectors expressing Nrf2 or Smad3E (a constitutively active Smad3 molecule). Exposure to TGF-β or Smad3E suppressed activity of both Lux reporters and was shown to be dependent upon Nrf2 binding to the ARE as mutation of the GCLC–ARE site abrogated Lux activity. TGF-mediated suppression, however, could be inhibited by forced expression of Nrf2; this could be reversed by co-transfection of Smad3E. GMSAs coupled with immunoprecipitation immunoblotting experiments demonstrated that Smad3 associated with Nrf2 in an off DNA reaction. c-Jun was shown to participate in ARE-dependent expression of GCLC, as demonstrated by the ability of His tagged c-Jun to immunoprecipitate Nrf2, loss of GCLC Lux reporter activity in mouse embryonic fibroblasts containing a targeted disruption of c-Jun, and the ability of forced expression of c-Jun in HepG2 cells to induce ARE-dependent GCLC reporter activity in an ARE-dependent fashion. Similar to the results obtained with Nrf2, induction of GCLC Lux activity by co-transfection of c-Jun was reversed by forced expression of Smad3E. Wild type and Smad3 null dermal mouse fibroblasts were transfected with the GCLC Lux reporter. Loss of Smad3 expression enhanced basal GCLC Lux reporter activity while reconstitution of Smad 3 into the null cells reduced GCLC Lux activity to the level observed in Smad3 +/+ cells. Similar results occurred when cells were exposed to TGFβ. These data suggest that TGF-β suppresses Nrf2-mediated gene expression via Smad signaling.