Small Maf proteins serve as transcriptional cofactors for keratinocyte differentiation in the Keap1-Nrf2 regulatory pathway

Abstract

The small Maf proteins, MafF, MafG, and MafK, possess a leucine zipper (Zip) domain that is required for homodimer or heterodimer complex formation with other bZip transcription factors. In this study we sought to determine the identity of the specific constituent that collaboratively interacts with Nrf2 to bind to the Maf recognition element in vivo. Studies in vitro suggested that Nrf2 forms heterodimers with small Maf proteins and then bind to Maf recognition elements, but the bona fide partner molecules supporting Nrf2 activity in vivo have not been definitively identified. Nrf2 activity is usually suppressed by a cytoplasmic repressor, Keap1, so disruption of the keap1 gene causes constitutive activation of Nrf2. Nrf2 hyperactivity results in hyperproliferation of keratinocytes in the esophagus and forestomach leading to perinatal lethality. However, simultaneous disruption of nrf2 rescued keap1-null mice from the lethality. We exploited this system to investigate whether small Mafs are required for Nrf2 function. We generated keap1 and small maf compound mutant mice and examined whether keratinocyte abnormalities persisted in these animals. The data show that loss of mafG and mafF in the keap1-null mice reversed the lethal keratinocyte dysfunction and rescued the keap1-null mutant mice from perinatal lethality. This rescue phenotype of mafG::mafF::keap1 triple compound mutant mice phenocopies that of the nrf2::keap1 compound mutant mice, indicating that the small Maf proteins MafG and MafF must functionally cooperate with Nrf2 in vivo.

Fig. 1.

Small maf::keap1 compound mutant mice survive beyond weaning. (A) Body weight change for mice bearing single small maf gene disruptions in the keap1-null background. Deletion of either mafK or mafF did not extend the lifespan of keap1-null mutant pups. When mafG alone was disrupted in addition to keap1, pups survived one week longer. (B) Body weight change for mice with compound small maf gene disruptions in the keap1-null mutant background. Simultaneous deletion of mafG and mafF rescued the lethality of keap1-null mutant pups. The body weight of nrf2::keap1 mice was examined as a control. WT indicates wild-type; F0, mafF-/-; K0, mafK-/-; G0, mafG-/-; N0, nrf2-/-; Kp0, keap1-/-.

Fig. 2.

Histological examination of forestomach. Hematoxylin/eosin staining of 10-day-old forestomach thin sections from Kp0 (A), F0Kp0 (B), G0F0Kp0 (C), WT (D), F0 (E), and G0F0 (F) pups are shown. Sections of the forestomach from 4-month-old mice of WT (G), G0F0Kp0 (H), and N0Kp0 (I) genotypes are also presented. Double-ended arrows indicate the cornified layer. (Scale bar, 150 μm.)

Fig. 3.

Expression profiles of Keap1, Nrf2, and small Mafs in the forestomach. (A) LacZ staining of squamous cell epithelia in the forestomach of keap1 heterozygous mutant mice. Blue staining is observed in LacZ-expressing cells. (B and C) Immunohistochemistry with anti-Nrf2 antibody. Brown punctate staining, indicated by arrowheads, is observed within nuclei of keap1-null mutant cells (C), whereas no staining develops in wild-type keratinocytes (B). Nonspecific staining is observed in the cornified epithelial layers. (D–L) LacZ staining of squamous cell epithelia of the forestomach in sections prepared from mafG (D, G, and J), mafK (E, H, and K), and mafF (F, I, and L) heterozygous mutant mice. Samples were prepared from 2-day-old (D–F), 10-day-old (G–I), or 4-month-old (J–L) mice. The green lines indicate the position of basement membranes. (Scale bar, 30 μm throughout.)

Fig. 4.

Quantification of small Maf mRNA abundance in forestomach. cDNA was synthesized from total RNA prepared from WT, Kp0, G0F0, and G0F0Kp0 forestomach at 10 days after birth. MafG, MafK, and MafF mRNA levels were quantified by quantitative real-time PCR using a plasmid containing each cDNA as the abundance standard.

Fig. 5.

Expression of keratin 6 as a marker of Nrf2-mediated transcriptional activity. (A–C) Immunohistochemistry with anti-keratin 6 antibody. Keratin 6 is highly expressed in keap1-null keratinocytes in the forestomach (B), whereas no signals were observed in the wild-type (A) or rescued G0F0Kp0 (C) mice. (Scale bar, 30 μmin A–C.) (D) Keratin 6 expression levels were examined by RNA blot analysis. Total RNA prepared from wild-type (lanes 1 and 2), Kp0 (lanes 3 and 4), F0Kp0 (lanes 5 and 6), G0Kp0 (lanes 7 and 8), G0F0Kp0 (lanes 9 and 10), and N0Kp0 (lanes 11 and 12) mice forestomachs are shown. The arrow and arrowheads indicate keratin 6 mRNA and ribosomal RNAs (18S and 28S), respectively. (E) Relative expression levels of the three small maf genes in the forestomach of wild-type, Kp0, F0Kp0, and G0Kp0 at 10 days after birth (compared with the levels in wild-type mice).