Unmethylated CpG motifs in bacterial DNA, plasmid DNA and synthetic oligodeoxynucleotides (CpG ODN) activate dendritic cells (DC) and macrophages in a CD40-CD40 ligand-independent fashion. To understand the molecular mechanisms involved we focused on the cellular uptake of CpG ODN, the need for endosomal maturation and the role of the stress kinase pathway. Here we demonstrate that CpG-DNA induces phosphorylation of Jun N-terminal kinase kinase 1 (JNKK1/SEK/MKK4) and subsequent activation of the stress kinases JNK1/2 and p38 in murine macrophages and dendritic cells. This leads to activation of the transcription factor activating protein-1 (AP-1) via phosphorylation of its constituents c-Jun and ATF2. Moreover, stress kinase activation is essential for CpG-DNA-induced cytokine release of tumor necrosis factor α (TNFα) and interleukin-12 (IL-12), as inhibition of p38 results in severe impairment of this biological response. We further demonstrate that cellular uptake via endocytosis and subsequent endosomal maturation is essential for signalling, since competition by non-CpG-DNA or compounds blocking endosomal maturation such as chloroquine or bafilomycin A prevent all aspects of cellular activation. The data suggest that endosomal maturation is required for translation of intraendosomal CpG ODN sequences into signalling via the stress kinase pathway, where p38 kinase activation represents an essential step in CpG-ODN-triggered activation of antigen-presenting cells.

Transcription factors of the NF-kappaB/Rel family are critical for inducible expression of multiple genes involved in inflammatory responses. Sulfasalazine and its salicylate moiety 5-aminosalicylic acid are among the most effective agents for treating inflammatory bowel disease and rheumatoid arthritis. However, the mode of action of these drugs remains unclear. Here we provide evidence that the transcription factor NF-kappaB is a target of sulfasalazine-mediated immunosuppression. Treatment of SW620 colon cells with sulfasalazine inhibited TNFalpha-, LPS-, or phorbol ester- induced NF-kappaB activation. NF-kappaB-dependent transcription was inhibited by sulfasalazine at micro- to millimolar concentrations. In contrast, 5-aminosalicylic acid or sulfapyridine did not block NF-kappaB activation at all doses tested. TNFalpha-induced nuclear translocation of NF-kappaB was prevented by sulfasalazine through inhibition of IkappaBalpha degradation. When blocking proteasome-mediated degradation of IkappaBalpha, we could demonstrate that sulfasalazine interfered with IkappaBalpha phosphorylation, suggesting a direct effect on an IkappaBalpha kinase or on an upstream signal. Inhibition of NF-kappaB activation seems to be specific since other DNA-binding activities such as AP1 were not affected. These results demonstrate that sulfasalazine is a potent and specific inhibitor of NF-kappaB activation, and thus may explain some of the known biological properties of sulfasalazine.

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NF-B/Rel proteins comprise a family of inducible transcription factors which control the expression of numerous genes involved in the immune, inflammatory, and acute-phase responses (for reviews, see references 3, 5, and 18). There is increasing evidence that members of this family are also involved in the regulation of normal and malignant cellular growth, especially in hematopoietic cells (16). More recently, it was demonstrated that inhibition of NF-B/Rel induces apoptosis in various cell types, suggesting an antiapoptotic potential of NF-B/Rel proteins (6,36,56,60,62).In higher vertebrates, the NF-B/Rel family encompasses five different genes: those encoding NF-B1 (p105/p50), NF-B2 (p100/p52), RelA (p65), and RelB, and the proto-oncogene encoding c-Rel. To a limited extent, these proteins can form homo-and heterodimers with distinct DNA-binding specificity (39,43,49). In most cell types, NF-B/Rel dimers are sequestered in the cytoplasm by a member of the IB family of inhibitory proteins. IB proteins mask the nuclear localization signal of NF-B/Rel, thereby preventing the nuclear translocation of NF-B/Rel. Upon stimulation, IB proteins are phosphorylated on specific serine residues, ubiquitinated, and degraded through proteasome-dependent proteolysis, thereby allowing NF-B/Rel dimers to translocate into the nucleus and bind to their cognant DNA sequences (for reviews, see references 4, 5, 54, and 57).This initial activation of NF-B can occur without de novo protein synthesis. However, it was shown that maintenance of NF-B activity requires ongoing protein synthesis and continuous stimulation, indicating that NF-B/Rel is also regulated at a transcriptional or translational level (23).In a previous study, we demonstrated that NF-B2 is positively autoregulated via two B-responsive elements (34), as shown for other family members, including NF-B1 (53) and IB␣ (28). In addition, mutation of the B elements resulted in a dramatic increase in the basal NF-B2 promoter activity in various cell lines. Therefore, we postulated that there is a negative regulation of NF-B2 transcription mediated by the B elements. A putative repressive DNA binding activity, Rep-B, which interacts with a B motif in the NF-B2 promoter was identified. Rep-B binding activity was partially purified from different cell sources, indicating that its expression is ubiquitous (34).Recombination signal binding protein J (RBP-J), also designated KBF2 or CBF1, was originally purified based on its binding to the recombination signal of the J immunoglobulin gene (38). Subsequently it was demonstrated that RBP-J acts as a transcriptional regulator, via binding to specific DNA motifs, rather than as a recombinase in V(D)J rearrangement (55). RBP-J proteins have been highly conserved during evolution not only in vertebrates but also in invertebrates. Genetic analysis revealed that the gene encoding RBP-J is conserved as the Drosophila melanogaster suppressor of hairless [SuH)], a member of the neurogenic gene family including Notch, Delta, Enhancer of split...

The arterial wail responds to thrombosis or mechanical I jury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. gene: procoUlagen synthesis induced by TGF-131 was greater, and celular proliferation was less prominent. These rmdings show that TGF-8l1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial inJury.

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Pancreatic carcinogenesis is a multistage process that involves numerous genetic alterations, including activating mutations of the Ki-Ras protooncogene by codon 12 mutations. 1 Furthermore, a variety of growth factors and their receptors are found overexpressed and may combine to provide a growth advantage to human pancreatic cancer cells. 2,3 However, downstream targets and in particular nuclear events involved in pancreatic cancer cell growth are poorly understood.Nuclear factor kappa B (NF-B) is a central regulator of the immune response and apoptosis. NF-B/Rel has been implicated in the pathogenesis of certain tumors, especially those of hematopoietic origin. In most cell types, NF-B dimers are sequestered as inactive cytoplasmic complexes by binding to their inhibitors, IBs. A common key event in the activation of NF-B is the inducible phosphorylation of 2 serines at the N-terminus of the inhibitory subunits IB␣, IB␤ or IB⑀. Subsequent IB gets ubiquitinated and degradated by the proteasome. 4 This reaction leaves the active form of NF-B, which is translocated to the nucleus where it binds to regulatory sequences in promoter/enhancer sequences. The mutation or deletion of the 2 serine residues 32 and 36 in IB␣ blocks phosphorylation and degradation of IB␣ protein and results in a superrepressor form of IB␣. 5 Two catalytic subunits, IKK␣ and IKK␤, and a regulatory component, NEMO/IKK␥, have been identified within a large 700 -900 kDa protein complex, which upon stimulation phosphorylates IB␣ at these critical serines. 6 Phosphorylation is followed by an ubiquitination and rapid degradation of IB by a proteasome dependent pathway. 7 This allows the translocation of free active NF-B complexes into the nucleus, where they bind specific DNA motifs in the promoter/enhancer regions of target genes and activate transcription.In this study, we present evidence that the transcription factor NF-B/Rel is constitutively activated in human pancreatic cancer specimen as well as in pancreatic cancer cell lines. NF-B/Rel activity is a result of constitutive IB kinase (IKK) activity and can be inhibited by dominant negative mutants of EGF-R and Ras, the PI3 kinase inhibitor LY294002 or dominant negative mutant Akt kinase. Inhibition of IKK or NF-B/Rel activity induced apoptosis and inhibited anchorage-dependent as well as -independent proliferation of MiaPaCa2 and Panc1 cells. These data demonstrate that NF-B/Rel activity reveals a mitogenic and antiapoptotic role in pancreatic cancer. MATERIAL AND METHODS Tissue samples and immunohistochemistryTissue samples were obtained during surgery on patients with suspected pancreatic cancer and shock frozen in liquid nitrogen. Diagnosis of pancreatic adenocarcinoma was confirmed by an independent pathologist. Sections were fixed with 4% formaldehyde for 15 min and sequentially treated with 0.1% Triton X 100 for 5 min and RNAseA (5 mg/ml) for 30 min at 37°C. Sections were blocked in 2% BSA, 3% goat serum in PBS for 30 min.

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