David H. Sherr

Inhibition of NF-kappaB/Rel induces apoptosis of murine B cells.

Apoptosis of the WEHI 231 immature B cell lymphoma line following membrane interaction with an antibody against the surface IgM chains (anti‐IgM) is preceded by dramatic changes in Nuclear Factor‐kappaB (NF‐kappaB)/ Rel binding activities. An early transient increase in NF‐kappaB/Rel binding is followed by a significant decrease in intensity below basal levels. Here we have explored the role of these changes in Rel‐related factors in B cell apoptosis. Treatment of WEH1 231 cells with N‐tosyl‐L‐phenylalanine chloromethyl ketone (TPCK), a protease inhibitor which prevents degradation of the inhibitor of NF‐kappaB (IkappaB)‐alpha, or with low doses of pyrrolidinedithiocarbamate (PDTC) selectively inhibited NF‐kappaB/Rel factor binding and induced apoptosis. Bcl‐XL expression protected WEHI 231 cells from apoptosis induced by these agents. Microinjection of WEHI 231 cells with either IkappaB‐alpha‐GST protein or a c‐Rel affinity‐purified antibody induced apoptosis. Ectopic c‐Rel expression ablated apoptosis induced by TPCK or anti‐IgM. Treatment of BALENLM 17 and A20 B lymphoma cells or normal murine splenic B lymphocytes with either TPCK or PDTC also resulted in apoptosis. These findings indicate that the drop in NF‐kappaB/Rel binding following anti‐IgM treatment activates apoptosis of WEHI 231 cells; furthermore, they implicate the NF‐kappaB/Rel family in control of apoptosis of normal and transformed B cells.

The aryl hydrocarbon receptor interacts with c-Maf to promote the differentiation of type 1 regulatory T cells induced by IL-27

Type 1 regulatory T cells (Tr1 cells ) that produce interleukin 10 (IL-10) are instrumental in the prevention of tissue inflammation, autoimmunity and graft-versus-host disease. The transcription factor c-Maf is essential for the induction of IL-10 by Tr1 cells, but the molecular mechanisms that lead to the development of these cells remain unclear. Here we show that the ligand-activated transcription factor aryl hydrocarbon receptor (AhR), which was induced by IL-27, acted in synergy with c-Maf to promote the development of Tr1 cells. After T cell activation under Tr1-skewing conditions, the AhR bound to c-Maf and promoted transactivation of the Il10 and Il21 promoters, which resulted in the generation of Tr1 cells and the amelioration of experimental autoimmune encephalomyelitis. Manipulating AhR signaling could therefore be beneficial in the resolution of excessive inflammatory responses.

Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals

Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter–reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.

NF-κB and Epithelial to Mesenchymal Transition of Cancer

During progression of an in situ to an invasive cancer, epithelial cells lose expression of proteins that promote cell–cell contact, and acquire mesenchymal markers, which promote cell migration and invasion. These events bear extensive similarities to the process of epithelial to mesenchymal transition (EMT), which has been recognized for several decades as critical feature of embryogenesis. The NF‐κB family of transcription factors plays pivotal roles in both promoting and maintaining an invasive phenotype. After briefly describing the NF‐κB family and its role in cancer, in this review we will first describe studies elucidating the functions of NF‐κB in transcription of master regulator genes that repress an epithelial phenotype. In the second half, we discuss the roles of NF‐κB in control of mesenchymal genes critical for promoting and maintaining an invasive phenotype. Overall, NF‐κB is identified as a key target in prevention and in the treatment of invasive carcinomas. J. Cell. Biochem. 104: 733–744, 2008.

The RelA NF-κB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c- myc promoter in mammary cells

NF-κB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-κB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-κB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-κB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-κB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-κB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells.

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin‐3 gallate (EGCG), which possess anti‐oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen‐induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12‐dimethylbenz(a)anthracene (DMBA) Sprague‐Dawley (S‐D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor‐bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA‐MB‐231 estrogen receptor‐negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA‐transformed D3‐1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27Kip1 cyclin‐dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27Kip1 protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen‐induced mammary tumorigenesis in female S‐D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27Kip1 CKI. J. Cell. Biochem. 82:387–398, 2001.

Inhibition of c-myc expression induces apoptosis of WEHI 231 murine B cells.

Treatment of WEHI 231 immature B-lymphoma cells with an antibody against their surface immunoglobulin (anti-Ig) induces apoptosis and has been studied extensively as a model of B-cell tolerance. Anti-Ig treatment of exponentially growing WEHI 231 cells results in an early transient increase in c-myc expression that is followed by a decline to below basal levels; this decrease in c-myc expression immediately precedes the induction of cell death. Here we have modulated NF-kappaB/Rel factor activity, which regulates the rate of c-myc gene transcription, to determine whether the increase or decrease in c-Myc-levels mediates apoptosis in WEHI 231 cells. Addition of the serine/threonine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), which blocks the normally rapid turnover of the specific inhibitor of NF-kappaB/Rel IkappaBalpha in these cells, caused a drop in Rel-related factor binding. TPCK treatment resulted in decreased c-myc expression, preventing the usual increase seen following anti-Ig treatment. Whereas inhibition of the induction of c-myc expression mediated by anti-Ig failed to block apoptosis, reduction of c-myc expression in exponentially growing WEHI 231 cells induced apoptosis even in the absence of anti-Ig treatment. In WEHI 231 clones ectopically expressing c-Myc, apoptosis induced by treatment with TPCK or anti-Ig was significantly diminished and cells continued to proliferate. Furthermore, apoptosis of WEHI 231 cells ensued following enhanced expression of Mad1, which has been found to reduce functional c-Myc levels. These results indicate that the decline in c-myc expression resulting from the drop in NF-kappaB/Rel binding leads to activation of apoptosis of WEHI 231 B cells.

Mechanisms of environmental influence on human autoimmunity: A national institute of environmental health sciences expert panel workshop

The mechanisms leading to autoimmune diseases remain largely unknown despite numerous lines of experimental inquiry and epidemiological evidence. The growing number of genome-wide association studies and the largely incomplete concordance for autoimmune diseases in monozygotic twins support the role of the environment (including infectious agents and chemicals) in the breakdown of tolerance leading to autoimmunity via numerous mechanisms. The present article reviews the major theories on the mechanisms of the environmental influence on autoimmunity by addressing the different degrees of confidence that characterize our knowledge. The theories discussed herein include (i) the role of innate immunity mediated by toll-like receptors in triggering the autoimmune adaptive response characterizing the observed pathology; (ii) changes in spleen marginal zone B cells in autoantibody production with particular focus on the B10 subpopulation; (iii) Th17 cell differentiation and T regulatory cells in the aryl hydrocarbon receptor model; (iv) self antigen changes induced by chemical and infectious agents which could break tolerance by post-translational modifications and molecular mimicry; and finally (v) epigenetic changes, particularly DNA methylation, that are induced by environmental stimuli and may contribute to autoimmunity initiation. We are convinced that these working hypotheses, in most cases supported by solid evidence, should be viewed in parallel with animal models and epidemiological observations to provide a comprehensive picture of the environmental causes of autoimmune diseases.

Regulation of Constitutive and Inducible AHR Signaling: Complex Interactions Involving the AHR Repressor

The AHR is well known for regulating responses to an array of environmental chemicals. A growing body of evidence supports the hypothesis that the AHR also plays perhaps an even more important role in modulating critical aspects of cell function including cell growth, death, and migration. As these and other important AHR activities continue to be elucidated, it becomes apparent that attention now must be directed towards the mechanisms through which the AHR itself is regulated. Here, we review what is known of and what biological outcomes have been attributed to the AHR repressor (AHRR), an evolutionarily conserved bHLH-PAS protein that inhibits both xenobiotic-induced and constitutively active AHR transcriptional activity in multiple species. We discuss the structure and evolution of the AHRR and the dominant paradigm of a xenobiotic-inducible negative feedback loop comprised of AHR-mediated transcriptional up-regulation of AHRR and the subsequent AHRR-mediated suppression of AHR activity. We highlight the role of the AHRR in limiting AHR activity in the absence of xenobiotic AHR ligands and the important contribution of constitutively repressive AHRR to cancer biology. In this context, we also suggest a new hypothesis proposing that, under some circumstances, constitutively active AHR may repress AHRR transcription, resulting in unbridled AHR activity. We also review the predominant hypotheses on the molecular mechanisms through which AHRR inhibits AHR as well as novel mechanisms through which the AHRR may exert AHR-independent effects. Collectively, this discussion emphasizes the importance of this understudied bHLH-PAS protein in tissue development, normal cell biology, xenobiotic responsiveness, and AHR-regulated malignancy.

Aryl Hydrocarbon Receptor Control of Adaptive Immunity

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that belongs to the family of basic helix-loop-helix transcription factors. Although the AhR was initially recognized as the receptor mediating the pathologic effects of dioxins and other pollutants, the activation of AhR by endogenous and environmental factors has important physiologic effects, including the regulation of the immune response. Thus, the AhR provides a molecular pathway through which environmental factors modulate the immune response in health and disease. In this review, we discuss the role of AhR in the regulation of the immune response, the source and chemical nature of AhR ligands, factors controlling production and degradation of AhR ligands, and the potential to target the AhR for therapeutic immunomodulation.