Characterization of butyrate resistant clones overexpressing p21 waf1Cip1

P21Waf1/Cip1 is a potent inhibitor of cell cycle progression and functions by binding to and inhibiting cyclin-dependent kinases. Millimolar amounts of butyrate can induce cell cycle arrest in mammalian cells through induction of p21Waf1/Cip1 expression. In butyrate resistant variants isolated from HeLa cells, clones 5.1 and 7.5 express high levels of p21Waf1/Cip1, in regular growth medium and in medium containing butyrate. Despite this elevated levels of P21Waf1/Cip1, the cells continue proliferating, albeit at a slower rate than the parental HeLa cells. Western blot analyses showed that other cell cycle proteins were not up regulated to compensate for the elevated expression of this inhibitor. Instead, specific enzymatic assays showed that the cdk2/cyclin E activity in these clones was not inhibited by P21Waf1/Cip1, but remained active to allow the cells to continue to cycle in butyrate

Nrf3-deficient mice are not protected against acute lung and adipose tissue damages induced by butylated hydroxytoluene

AbstractWe found that both wild type and Nrf3 (NF-E2-related factor 3) deficient mice are sensitive to BHT single administration exhibiting respiratory distress and considerably lose body weight following treatment. At time of sacrifice, the BHT-treated Nrf3−/− mice had lost significantly more body weight than their WT counterparts. In the lung, transcript levels of the transcription factors Nrf1, Nrf2 and Nrf3 were differentially regulated by BHT treatment. In addition, genes implicated in adipogenesis were repressed following BHT exposure in the white adipose tissue. Together, our data provide the first evidence that BHT exposure not only affects lung function but also leads to impaired adipogenesis in adipocytes

Functional and Placental Expression Analysis of the Human NRF3 Transcription Factor

International audienceMembers of the Maf protooncogene and cap'n' collar families of basic-leucine zipper transcription factors play important roles in development, differentiation , oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basic-leucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/ MAFG heterodimer recognizes nuclear factor-erythroid 2/Maf recognition element-type DNA-binding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We found that NRF3 transcripts are present in placen-tal chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocar-cinoma cell lines BeWo and JAR, derived from tro-phoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3-specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF-in JAR cells. Our functional studies suggest that human NRF3 is a potent transcrip-tional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development. (Mo-lecular Endocrinology 19: 125-137, 2005

Complexity of CNC Transcription Factors As Revealed by Gene Targeting of the Nrf3 Locus

Cap'n'collar (CNC) family basic leucine zipper transcription factors play crucial roles in the regulation of mammalian gene expression and development. To determine the in vivo function of the CNC protein Nrf3 (NF-E2-related factor 3), we generated mice deficient in this transcription factor. We performed targeted disruption of two Nrf3 exons coding for CNC homology, basic DNA-binding, and leucine zipper dimerization domains. Nrf3 null mice developed normally and revealed no obvious phenotypic differences compared to wild-type animals. Nrf3(−/−) mice were fertile, and gross anatomy as well as behavior appeared normal. The mice showed normal age progression and did not show any apparent additional phenotype during their life span. We observed no differences in various blood parameters and chemistry values. We infected wild-type and Nrf3(−/−) mice with acute lymphocytic choriomeningitis virus and found no differences in these animals with respect to their number of virus-specific CD8 and CD4 T cells as well as their B-lymphocyte response. To determine whether the mild phenotype of Nrf3 null animals is due to functional redundancy, we generated mice deficient in multiple CNC factors. Contrary to our expectations, an absence of Nrf3 does not seem to cause additional lethality in compound Nrf3(−/−)/Nrf2(−/−) and Nrf3(−/−)/p45(−/−) mice. We hypothesize that the role of Nrf3 in vivo may become apparent only after appropriate challenge to the mice