The Rel family: models for transcriptional regulation and oncogenic transformation

We have previously shown that functional components of the NF-κB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-κB. In this study, we examined the expression of the NF-κB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16⁺ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-κB precursors.

Equine endotoxaemia is an important cause of morbidity and mortality in horses caused by the interaction of bacterial lipopolysaccharide (LPS) with cells such as macrophages and vascular smooth muscle. In this study we isolated equine vascular smooth muscle from a variety of vessels and stimulated it with LPS and human interferon (hIFN)-γ. Using reverse transcriptase polymerase chain reaction (rt-PCR) and Western blot analysis we show that cyclooxygenase-2 (COX-2) is readily expressed in equine vascular smooth muscle. Vascular smooth muscle cells produced prostaglandin E2 in response to LPS and hIFNγ. Using similar approaches we saw very limited expression of inducible nitric oxide synthase (iNOS) in only one vascular smooth muscle preparation. LPS and IFNγ caused translocation of the transcription factor nuclear factor kappa B (NfκB) to the nucleus in equine cells suggesting the limited iNOS production seen in our cells is not due to deficits in this signal transduction pathway. These data suggest that in equine vascular smooth muscle COX-2 and NfκB are likely to play important roles in the pathogenesis of equine endotoxaemia.

The role of nuclear signaling in cancer biology has been studied extensively, from the elucidation of growth factor pathway activation, induction of cell motility and invasive responses, to the inhibition of cell death programs. However, if one is to consider such processes in the context of the complete cellular environment, the influences of cytosolic components should be considered as well. It is becoming more apparent that mitochondria may be the primary players in this putative “cytoplasmic influence” upon tumor cell behavior. In accordance with the endosymbiotic origin proposed for mitochondria and eukaryotic precursors, there exists a large amount of cross talk between mitochondrial- and nuclear-derived signals within the cell. For quite some time the understanding that the coordinated expression of both genomes is required for proper mitochondrial function has been realized, and our realization that such communication has the potential to regulate other critical cellular activities is growing. The mitochondrial genome is more susceptible to mutagenic events than nuclear DNA, suggesting that mtDNA alterations may represent an initial promoting factor for the onset of neoplasia. This effect might be related to changes in cell Ca2+ homeostasis, local redox balances, metabolic pathways, energy production, apoptotic potential or other uncharacterized signals which are controlled at least in part by the mitochondria. Of course, this is a simplistic model. The physiological expression of these putative relationships between mitochondrial signaling and cancer would certainly depend highly upon other variables such as the local cellular environment, level of cellular differentiation and tissue type, all of which are regulated by nuclear signals. The approach to future cancer therapies may include the specific targeting of mitochondria. Drastic changes to this organelle would affect both normal and cancerous tissues, therefore, only more subtle manipulation of mitochondrial functioning would be effective. The alteration of mitochondrial ATP generation, reactive oxygen species production, or sensitization to programmed cell death as an adjunct therapy used along with conventional cancer treatment strategies might enhance chemical or radiation efficacy and reduce the acquired tumor resistance to these agents. As a better understanding of specific mitochondrial signaling is obtained, the usefulness of any such clinical applications can be more appropriately assessed.

Rel/NF-κB proteins are eukaryotic transcription factors that control the expression of genes involved in a large variety of cellular processes. Rel proteins share a highly conserved DNA-binding/dimerization domain called the Rel Homology (RH) domain. We have constructed and characterized a composite cDNA encoding most of the chicken RelB transcription factor. The predicted chicken RelB protein has a high degree of sequence similarity to other vertebrate RelB proteins within the RH domain, but is much less conserved outside this domain. Chicken RelB does not bind DNA as a homodimer, but forms DNA-binding heterodimers with NF-κB p50 or p52. Overexpressed chicken RelB localizes to the nucleus in chicken embryo fibroblasts, and the nonconserved C-terminal sequences of chicken RelB contain a transactivation domain that functions in chicken and mouse fibroblasts. Thus, chicken RelB has functional properties similar to other vertebrate RelB proteins. However, Western blotting of diverse chicken tissues indicates that chicken RelB is more widely expressed than mammalian RelB.

In WEHI-231 cells, anti-immunoglobulin (anti-Ig) treatment leads to both a decrease in the DNA-binding activity of p50/c-Rel/p53 protein complexes and a transient enhancement in the DNA-binding activity of p50 homodimeric complexes. These cells subsequently undergo apoptosis. Because IκB-α plays a pivotal role in the regulation of Rel/NF-κB activity, we have characterized both the nature and kinetics of the expression of IκB-α following anti-Ig-induced apoptosis in WEHI-231 cells. Anti-Ig treatment of WEHI-231 cells decreased the steady-state level of IκB-α mRNA, but enhanced the stability of IκB-α, leading to an accumulation of IκB-α in both the cytosol and nucleus. Concomitant with the increase in IκB-α expression there was a gradual decline in the nuclear expression of c-Rel. Because c-Rel plays an important role in the survival of WEHI-231 cells, these results suggest that post-transcriptional regulation of IκB-α expression might play a role in the anti-Ig-induced apoptosis in WEHI-231 cells.

The initiation of primary immune responses is the key function of specialized antigen presenting cells, the dendritic cells (DC)DC of myeloid origin capture antigens in tissues, migrate to lymphoid organs and stimulate T cell responsesA subset of DC has been described which expresses lymphoid determinants and has potential regulatory functionsConditional transformation of chicken bone marrow progenitors with v-relER, a v-rel estrogen receptor (ER) fusion gene, allows expansion of progenitors that can be induced to differentiate into DC in vitroIn this paper we describe that v-relER cells exhibit both myeloid and lymphoid surface markers, while B cell, T cell and NK (natural killer)-specific surface markers are absentv-relER DC express, however, cytoplasmic CD3 protein and mRNA for CD8α and the lymphoid transcription factor GATA-3These data suggest that v-relER DC might be related to the lymphoid subset of DC described in mammals.