Smad3-ATF3 signaling mediates TGF-beta suppression of genes encoding Phase II detoxifying proteins.
This study provides evidence that in mammary epithelial cells the pluripotent cytokine TGF-beta1 repressed expression of multiple genes involved in Phase II detoxification. GCLC, the gene that encodes the catalytic subunit of the enzyme glutamate cysteine ligase, the rate-limiting enzyme in the biosynthesis of glutathione, was used as a molecular surrogate for investigating the mechanisms by which TGF-beta suppressed Phase II gene expression. TGF-beta was found to suppress luciferase reporter activity mediated by the human GCLC proximal promoter, as well as reporter activity mediated by the GCLC antioxidant response element, ARE4. TGF-beta downregulated expression of endogenous GCLC mRNA and GCLC protein. TGF-beta suppression of the Phase II genes correlated with a decrease in cellular glutathione and an increase in cellular reactive oxygen species. Ectopic expression of constitutively active Smad3E was sufficient to inhibit both reporters in the absence of TGF-beta, whereas dominant negative Smad3A blocked TGF-beta suppression. Smad3E suppressed Nrf2-mediated activation of the GCLC reporter. We demonstrate that TGF-beta increased ATF3 protein levels, as did transient overexpression of Smad3E. Ectopic expression of ATF3 was sufficient to suppress the GCLC reporter activity, as well as endogenous GCLC expression. These results demonstrate that Smad3-ATF3 signaling mediates TGF-beta repression of ARE-dependent Phase II gene expression and potentially provide critical insight into mechanisms underlying TGF-beta1 function in carcinogenesis, tissue repair, and fibrosis.
Bakin
Andrei V
AV
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263, USA. andrei.bakin@roswellpark.org
Stourman
Nina V
NV
Sekhar
Konjeti R
KR
Rinehart
Cammie
C
Yan
Xuexian
X
Meredith
Michael J
MJ
Arteaga
Carlos L
CL
Freeman
Michael L
ML
D051700:Activating Transcription Factor 3; D000818:Animals; D002374:Catalase / Q000187:drug effects / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D005721:Glutamate-Cysteine Ligase / Q000187:drug effects / Q000235:genetics*; D005978:Glutathione / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D005982:Glutathione Transferase / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D008658:Inactivation, Metabolic / Q000235:genetics; D051379:Mice; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D051900:Smad3 Protein; D015534:Trans-Activators / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*
D051700:Activating Transcription Factor 3; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D002374:Catalase; D005982:Glutathione Transferase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione