Synergism and Antagonism of Two Distinct, but Confused, Nrf1 Factors in Integral Regulation of the Nuclear-to-Mitochondrial Respiratory and Antioxidant Transcription Networks

Figure 6

Identification of pitx2 as an upstream regulator of Nfe2l1, besides α-pal^NRF1. (a) HepG2 cells were cotransfected for 8 h with mNfe2l1-luc (A1) or empty pGL3-Basic (A2), together with the pRL-TK control, plus a Pitx2 expression construct or an empty pcDNA3.1, and then allowed for a 24 h recovery before the luciferase activity was measured. The data are shown as () with significant increases ($, ) or NS (not significance). (b) Putative cis-regulatory binding sites for Pitx2, α-Pal^NRF1, and AREs within the human Nfe2l1 promoter region were indicated. Various lengths of hNfe2l1-luc were cloned into the pGL3-Basic vector as shown schematically. (c) HepG2 cells were cotransfected for 8 h (i) with hNfe2l1-luc and pRL-TK and also treated for 24 h with 50 μmol/L tBHQ or the DMSO vehicle (C1); (ii) with hNfe2l1-luc (C2), hNfe2l1-luc1 (C3), or hNfe2l1-luc2 (C4), along with the pRL-TK control, plus a Pitx2 expression construct or an empty pcDNA3.1, before being allowed for additional 24 h recovery. The samples were then subjected to dual luciferase assays. The results are shown as () with significant increases ($, ). (d) HepG2 cells were cotransfected for 8 h with each of PitxRE-luc reporters or their mutants, together with pRL-TK plus a Pitx2 expression construct or an empty pcDNA3.1, and then allowed for 24 h recovery. Thereafter, the reporter activity was detected and calculated as () with significant increases ($, ). (e) Distinct mRNA levels of Pitx2, Nfe2l1, and HO-1 were detected by real-time qPCR of HepG2 cells that had been transfected with an expression construct for Pitx2 or an empty pcDNA3.1 vector. (f) Pitx2-expressing HepG2 cells were subjected to Western blotting of Pitx2, Nfe2l1, Nfe2l2, HO-1, GCLM, α-Pal^NRF1, TFAM, and SOD1. (g–n) Distinct mRNA levels of Pitx2 (g), α-Pal^NRF1 (h), TFAM (i), Nfe2l1 (j), HO-1 (k), GCLM (l), COX5a (m), and SOD1 (n) were determined by real-time qPCR analysis of HepG2 cells that had been transfected with three different siRNAs against Pitx2. The results are shown as () with significant decreases (, ). (o) A model is proposed for effects of Pitx2 on Nfe2l1, Nfe2l2, α-Pal^NRF, TFAM, and other genes in HepG2 cells.

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