Keap1 regulates the constitutive expression of GST A1 during differentiation of Caco-2 cells.
Kelch-like ECH-associated protein 1 (Keap1), a BTB-Kelch substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex, regulates the induction of the phase 2 enzymes, such as glutathione S-transferase (GST), by repressing the transcription factor Nrf2. It is known that, in the human gastrointestinal tract, both GST A1 and P1 are constitutively expressed as the major GST isozymes. In the present study, using the Keap1-overexpressing derivatives of Caco-2 cells, human carcinoma cell line of colonic origin, by stable transfection of wild type Keap1, we investigated the molecular mechanism underlying the constitutive expression of these GST isozymes during differentiation. It was revealed that the overexpression of Keap1 completely repressed the constitutive expression of GST A1, but not GST P1. In Keap1-overexpressed cells, dome formation disappeared, and the formation of the intact actin cytoskeletal organization at cell-cell contact sites and the recruitment of E-cadherin and beta-catenin to adherens junctions were inhibited. The constitutive GST A1 expression in Caco-2 cells was repressed by disruption of E-cadherin-mediated cell-cell adhesion, suggesting the correlation between epithelial cell polarization and induction of the basal GST A1 expressions during Caco-2 differentiation. Keap1 overexpression indeed inhibited the activation of the small guanosine triphosphatase Rac1 on the formation of E-cadherin-mediated cell-cell adhesion. The transfection of V12Rac1, the constitutively active Rac1 mutant, into Keap1-overexpressed cells promoted the basal GST A1 expression, suggesting that Keap1 regulated the basal GST A1 expression during Caco-2 differentiation via Rac1 activation on the formation of E-cadherin-mediated cell-cell adhesion. The results of this study suggest the involvement of a novel Keap1-dependent signaling pathway for the induction of the constitutive GST A1 expression during epithelial cell differentiation.
Kusano
Yuri
Y
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Horie
Shunsuke
S
Shibata
Takahiro
T
Satsu
Hideo
H
Shimizu
Makoto
M
Hitomi
Eri
E
Nishida
Motohiro
M
Kurose
Hitoshi
H
Itoh
Ken
K
Kobayashi
Akira
A
Yamamoto
Masayuki
M
Uchida
Koji
K
D018938:Caco-2 Cells; D002454:Cell Differentiation / Q000502:physiology*; D002458:Cell Fractionation; D003001:Cloning, Molecular; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D051549:Glutathione S-Transferase pi / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism*; D000072019:Kelch-Like ECH-Associated Protein 1; D016296:Mutagenesis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; C484555:GSTA1 protein, human; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; D005982:Glutathione Transferase