Anna Derjuga

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

ABSTRACT Capncollar (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.

Identification of interleukin-1β regulated genes in uterine smooth muscle cells

We analyzed the response of uterine smooth muscle cells to interleukin-1beta (IL-1beta). We first showed that PHM1-31 myometrial cells, our cellular model, are contractile. To determine the molecular mechanisms of uterine smooth muscle cell activation by proinflammatory cytokines, we performed genechip expression array profiling studies of PHM1-31 cells in the absence and the presence of IL-1beta. In total, we identified 198 known genes whose mRNA levels are significantly modulated (> 2.0-fold change) following IL-1beta exposure. We confirmed the expression changes for selected genes by independent mRNA and protein analysis. The group of genes induced by IL-1beta includes transcription factors and inflammatory response genes such as nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFkappaB), pentraxin-related gene (PTX3), and tumor necrosis factor alpha-induced protein 3/A20 (TNFAIP3/A20). We also found up-regulation of chemokines like C-X-C motif ligand 3 (CXCL3) and extracellular matrix remodeling signaling molecules like tenascin C (TNC). Our data suggest that IL-1beta elicits the rapid activation of a cellular network of genes particularly implicated in inflammatory response that may create a cellular environment favorable for myometrial cell contraction. Our results provide novel insights into the mechanisms of uterine smooth muscle cell regulation and possibly infection-induced preterm labor.

Regulation of the MAFF Transcription Factor by Proinflammatory Cytokines in Myometrial Cells

Abstract The MAF (proto-)oncogene family of basic-leucine zipper transcription factors plays crucial roles in the control of mammalian gene expression and development. Here we analyzed the regulation of the human MAFF gene, coding for a small MAF transcription factor, in uterine smooth muscle cells. We found that MAFF transcript levels are induced by proinflammatory cytokines in PHM1–31 myometrial cells. We observed an important induction by interleukin 1 beta (IL1B) and a weaker upregulation by tumor necrosis factor (TNF), whereas interleukin 6 (IL6) treatment had no effect. Time course experiments revealed a rapid induction of MAFF transcripts within 30 min following IL1B treatment. The presence of actinomycin D inhibited the upregulation, suggesting that regulation of MAFF mRNA levels occurs at the transcriptional level. We generated a MAFF-specific antiserum and determined that MAFF protein was also induced by TNF and IL1B in PHM1–31 cells. In contrast, it was particularly interesting that the transcript and protein levels of the highly homologous MAFG and MAFK genes are not modulated by these cytokines. Our results suggest a possible specific role for MAFF in proinflammatory cytokine-mediated control of myometrial gene expression and provide the first link between a small MAF transcription factor and the inflammatory response.

Endoplasmic reticulum association and N-linked glycosylation of the human Nrf3 transcription factor.

We have analysed the molecular and cellular regulation of the basic‐leucine zipper (bZIP) transcription factor Nrf3 (NFE2‐Related Factor 3). Cycloheximide studies revealed a rapid turnover of Nrf3. We showed that the proteasome inhibitor MG‐132 increases Nrf3 protein levels. Furthermore, we demonstrated that Nrf3 is an N‐glycosylated protein associated with the endoplasmic reticulum. Thus, our studies provide the first evidence of a post‐translational modification of Nrf3.

Antagonistic roles of the ERK and p38 MAPK signalling pathways in globin expression, haem biosynthesis and iron uptake.

Late-stage erythroid cells synthesize large quantities of haemoglobin, a process requiring the co-ordinated regulation of globin and haem synthesis as well as iron uptake. In the present study, we investigated the role of the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) signalling pathways in MEL (mouse erythroleukaemia) cell differentiation. We found that treatment of HMBA (hexamethylene bisacetamide)-induced MEL cells with the ERK pathway inhibitor UO126 results in an increase in intracellular haem and haemoglobin levels. The transcript levels of the genes coding for β(major)-globin, the haem biosynthesis enzyme 5-aminolevulinate synthase 2 and the mitochondrial iron transporter mitoferrin 1 are up-regulated. We also showed enhanced expression of globin and transferrin receptor 1 proteins upon UO126 treatment. With respect to iron uptake, we found that ERK inhibitor treatment led to an increase in both haem-bound and total iron. In contrast, treatment of MEL cells with the p38 MAPK pathway inhibitor SB202190 had the opposite effect, resulting in decreased globin expression, haem synthesis and iron uptake. Reporter assays showed that globin promoter and HS2 enhancer-mediated transcription was under the control of MAPKs, as inhibition of the ERK and p38 MAPK pathways led to increased and decreased gene activity respectively. Our present results suggest that the ERK1/2 and p38α/β MAPKs play antagonistic roles in HMBA-induced globin gene expression and erythroid differentiation. These results provide a novel link between MAPK signalling and the regulation of haem biosynthesis and iron uptake in erythroid cells.

Abstract B33: Dissecting the role of Nrf3 (NF-E2-related factor 3) in chemical carcinogenesis using knock-out mice

In this study, we investigated the role of the transcription factor Nrf3 in chemical carcinogenesis induced by benzo[a]pyrene, a common carcinogen found in cigarette smoke, car exhaust and charcoal-grilled meats. Wild type and Nrf3-deficient mice were treated weekly for 4 consecutive weeks with benzo[a]pyrene and then sacrificed 30 weeks after the first administration of carcinogen. Here, we report that mice exposed to benzo[a]pyrene rapidly exhibit labored breathing further accompanied with different signs of morbidity. Kaplan-Meyer survival rate analyses indicate that mice deficient for Nrf3 die earlier that wild type mice following benzo[a]pyrene exposure. Upon necropsy, gross morphology analyses showed the presence of an increased number of lung and thymus lesions in Nrf3-deficient mice compared to wild type mice whereas the number of stomach lesions is identical in both genotypes. Pathology analysis strongly suggests that observed lesions in lung and thymus are from lymphoma origin. Overall, our study reveals that mice deficient for Nrf3 are highly sensitive to chemicalinduced carcinogenesis suggesting a protective role of the transcription factor Nrf3 in this process. This provides new important insights into the molecular mechanisms involved in the development of lymphomas induced by chemical agents. We also propose the Nrf3-deficient mice as a new in vivo model to study tumor formation and response to chemoprotective agents. Citation Information: Cancer Res 2009;69(23 Suppl):B33.