NF-κB is a pleiotropic transcription factor with key functions in the intestinal immune system. NF-κB family members control transcriptional activity of various promoters of proinflammatory cytokines, cell surface receptors, transcription factors, and adhesion molecules that are involved in intestinal inflammation. The perpetuated activation of NF-κB in patients with active inflammatory bowel disease suggests that regulation of NF-κB activity is a very attractive target for therapeutic intervention. Such strategies include antioxidants, proteasome inhibitors, inhibition of NF-κB by adenoviral IκBα expression vectors, and antisense DNA targeting of NF-κB. These approaches will hopefully permit the design of new treatment strategies for chronic intestinal inflammation.

Immunosuppressive therapy with methotrexate (MTX) has been established as effective treatment for patients with rheumatoid arthritis. To analyse the therapeutic potential and mechanisms of action of MTX, we determined serum cytokine levels and cytokine production by splenic T cells and macrophages in untreated and MTX-treated mice. Furthermore, we assessed the role of MTX in a murine model of experimental arthritis induced by collagen type II (CIA). MTX reduced spontaneous and IL-15-induced tumour necrosis factor (TNF) production by splenic T cells but not by macrophages from healthy mice in vitro in a dose-dependent manner. In contrast, interferon-gamma (IFN-gamma) production was less strikingly reduced and IL-4 production was virtually unaffected. In addition, treatment of healthy mice with MTX in vivo led to reduced TNF serum levels and diminished TNF production by splenic T cells and macrophages. Intraperitoneal administration of MTX prior to the onset of arthritis completely prevented clinical and pathological signs of CIA. This was associated with a striking reduction of TNF production by spleen cells from MTX-treated mice. The role of TNF in MTX-mediated effects on cytokine production was further underlined by the finding that MTX effects on IFN-gamma production were augmented in TNF-transgenic mice but abrogated in mice in which the TNF-alpha gene had been inactivated by homologous recombination. Thus, MTX specifically modulates spontaneous and IL-15-induced TNF-alpha production in mice and prevents experimental murine CIA. These data suggest that TNF production by T cells is an important target of MTX and may serve as a basis to understand and further analyse MTX-mediated mechanisms of immunosuppression in patients with RA.

The Pem homeobox transcription factor is expressed under androgen control in the testis and epididymis. It is also transcribed in the ovary, muscle, and placenta. The mouse Pem gene promoter was cloned and sequenced. It was analyzed in transactivation tests using CV-1 and PC-3 cells expressing the AR and found to be strongly stimulated by androgens. EMSAs and mutational analysis of the Pem promoter allowed the identification of two functional androgen response elements named ARE-1 and ARE-2. They both differed from the consensus semipalindromic steroid response element and exhibited characteristics of direct repeats of the TGTTCT half-site. Unlike the steroid response element, both Pem androgen response elements were selectively responsive to androgen stimulation. Specific mutations in the left half-site of Pem ARE-1 and ARE-2, but not of the steroid response element, were still compatible with AR binding in the EMSA. In addition, Pem ARE-1, but not ARE-2 or the steroid response element, showed some flexibility with regard to spacing between half-sites. These results strongly suggest that the AR interacts differently with direct repeats than with inverted repeats, potentially leading to cis element-driven selective properties. Thus, the existence of several classes of DNA response elements might be an essential feature of differential androgen regulation.

In addition to the steroid response elements (SREs), which are recognized by several steroid receptors, a second class of DNA elements exhibiting selectivity for the androgen receptor (AR) and named androgen response elements (AREs) has been identified. Here we provide evidence for the differential role of these element classes in modulating AR function. AR complexes attached to response elements representative of each class were purified. Limited protease digests of ARE- or SRE-bound AR complexes led to the generation of different patterns, in line with differential accessibilities. In transactivation assays, mutations in the AR dimerization interface of the DNA-binding domain had various effects, depending on the response elements tested. The R598D mutant displayed much enhanced activity on SREs, whereas far less effect was seen on the selective AREs. The A596T mutant had reduced activity on AREs but not on SREs. Ectopic expression of the coactivators transcriptional intermediary factor 2 (TIF2) and ARA55 stimulated AR activity to different extents, depending on the response element. When using cysteine-rich secretory protein 1 (CRISP-1) SRE as reference, the most significant difference was observed with Pem ARE-2. A differential response of each element class was furthermore observed in the presence of two enzymes involved in the sumoylation pathway. Ubiquitin-conjugating enzyme 9 (Ubc9) overexpression enhanced AR action conveyed by SREs, whereas little effect was seen on Pem ARE-1 and repression on Pem ARE-2. Protein inhibitor of activated STAT (PIAS)xalpha overexpression had little influence on SRE-mediated AR activity but was repressive when using AREs. Altogether, these results demonstrate that DNA response elements play an important modulatory role in transmitting AR action and may be determinative for specificity of gene expression in cell or tissue types.