Roland M. Schmid

CpG‐DNA‐specific activation of antigen‐presenting cells requires stress kinase activity and is preceded by non‐specific endocytosis and endosomal maturation

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.

Notch mediates TGFα-induced changes in epithelial differentiation during pancreatic tumorigenesis

Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.

SHARP is a novel component of the Notch/RBP-Jκ signalling pathway

Notch proteins are the receptors for an evolutionarily highly conserved signalling pathway that regulates numerous cell fate decisions during development. Signal transduction involves the presenilin‐dependent intracellular processing of Notch and nuclear translocation of the intracellular domain of Notch, Notch‐IC. Notch‐IC associates with the DNA‐binding protein RBP‐Jκ/CBF‐1 to activate transcription of Notch target genes. In the absence of Notch signalling, RBP‐Jκ/CBF‐1 acts as a transcriptional repressor through the recruitment of histone deacetylase (HDAC) corepressor complexes. We identified SHARP as an RBP‐Jκ/CBF‐1‐interacting corepressor in a yeast two‐hybrid screen. In cotransfection experiments, SHARP‐mediated repression was sensitive to the HDAC inhibitor TSA and facilitated by SKIP, a highly conserved SMRT and RBP‐Jκ‐interacting protein. SHARP repressed Hairy/Enhancer of split (HES)‐1 promoter activity, inhibited Notch‐1‐mediated transactivation and rescued Notch‐1‐induced inhibition of primary neurogenesis in Xenopus laevis embryos. Based on our data, we propose a model in which SHARP is a novel component of the HDAC corepressor complex, recruited by RBP‐Jκ to repress transcription of target genes in the absence of activated Notch.

Malignant transformation of duct-like cells originating from acini in transforming growth factor α transgenic mice

BACKGROUND & AIMS In transgenic mice overexpressing transforming growth factor (TGF)-alpha in the exocrine pancreas, progressive pancreatic fibrosis and a transdifferentiation of acinar cells to duct-like cells occurs. The present study was undertaken to analyze this transdifferentiation process. METHODS Pancreatic specimens were characterized using light microscopy and immunohistochemistry. Expression of the epidermal growth factor receptor (EGFR) and TGF-alpha was evaluated with slot blot and Western analysis. To identify other generic events, K-ras mutations were screened with an enriched polymerase chain reaction approach and p53 expression was detected with immunohistochemistry. RESULTS Morphological examination revealed an aggregation of interlobular fibroblasts and a decrease in acinar cell height starting at day 14 after birth. In older animals, these acinar cells change to duct-like cells, which form tubular structures and express ductal markers. Evidence for dysplastic changes was found in 12 of 21 TGF-alpha transgenic mice older than 1 year. We also observed four malignant pancreatic tumors, which were multicentric and originated from dysplastic tubular complexes. They displayed a mixed cystic-papillary phenotype strongly positive for carbonic anhydrase activity. EGFR expression progressively increased in the transition from acinar to duct-like and transformed cells. Activating K-ras mutations could not be detected; however, tubular complexes and tumors displayed increased immunoreactivity for nuclear p53. CONCLUSIONS These data suggest an involvement of the TGF-alpha/EGFR pathway in conjunction with other yet unknown events in pancreatic tumor development. Furthermore, these observations are in favor of an acinar-ductal carcinoma sequence. Thus, these transgenic animals will be useful to define genetic alterations associated with a transition from acinar cells to a neoplastic ductal phenotype.

Mitogenic and antiapoptotic role of constitutive NF‐κB/Rel activity in pancreatic cancer

The transcription factor NF‐κB/Rel was found to be constitutively activated in human pancreatic cancer. RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF‐κB/Rel–binding activity consists of NF‐κB1(p50) and RelA(p65). Constitutive NF‐κB/Rel activity correlates with IκB kinase (IKK) activity and can be blocked by dominant negative mutants of IKKβ and to a lesser extent by IKKα. Constitutive NF‐κB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3 kinase inhibitor LY294002, or dominant negative mutant Akt kinase. Transfection of a dominant negative mutant epidermal growth factor receptor (EGF‐R), EGF‐R kinase inhibitor Tyrphostin and LY 294002 blocked IKK activity and NF‐κB–dependent transcription. Inhibition of constitutive IKK or NF‐κB/Rel activity increased the number of apoptotic cells. Stably expressing a nondegradable form of IκBα inhibited anchorage‐dependent and ‐independent proliferation in MiaPaCa2 and Panc1 cells. Our data demonstrate that an EGF‐R/Ras/PI3 kinase/Akt/IKK‐dependent pathway contributes to constitutive NF‐κB/Rel activity in pancreatic cancer. Inhibition of NF‐κB/Rel activity reveals a mitogenic and antiapoptotic role for NF‐κB/Rel in pancreatic cancer.

bax , but not bcl-2 , influences the prognosis of human pancreatic cancer

Background—bcl-2and bax belong to thebcl-2-related gene family, which marks a new class of genes that influence apoptosis. Thebcl-2 oncogene acts as a broad antiapoptotic factor and extends both normal and tumour cell survival. In contrast, the bax gene is a promoter of apoptosis. Aims—To analyse the expression of bcl-2 andbax in pancreatic cancer and correlate the results with clinical parameters. Patients—Pancreatic cancer tissue samples were obtained from 28 female and 32 male patients (median age 63, range 43–79 years) having surgery for pancreatic cancer. Normal pancreatic tissues obtained from 18 previously healthy organ donors served as controls. Methods—The levels ofbcl-2 and baxmRNA expression were analysed by northern blot and the exact site of mRNA transcription was determined by in situ hybridisation. The presence of the corresponding proteins was determined by immunohistochemistry. Results—Northern blot analysis indicated that, in comparison with the normal pancreas,bcl-2 mRNA was overexpressed in 30% andbax mRNA in 61% of the pancreatic cancer samples. Concomitant overexpression ofbcl-2 and bax was present in 26% of the cancer samples. Pancreatic adenocarcinomas exhibited 3.7-fold and 5.4-fold increases (p<0.001) inbcl-2 and baxmRNA levels respectively. In situ hybridisation showed that bothbcl-2 and baxmRNA were expressed in the cancer cells. Immunohistochemical analysis showed positive Bcl-2 and Bax immunostaining in 28 and 83% of the cancer samples respectively. In multivariate analysis (Cox regression model), bax expression was found to be a strong indicator of survival (p<0.001). Patients whose tumours exhibited Bax immunostaining lived significantly longer (12 months) than those whose tumours were Bax negative (five months) (p<0.039). In contrast, no relation was found between Bcl-2 and survival time. Conclusions—The data indicate that genes that are involved in the regulation of apoptosis are upregulated in human pancreatic cancer cells. Prolonged survival times in patients in whom apoptosis promoting factors are upregulated indicate that apoptotic pathways are of biological significance in pancreatic cancer.

Acute Experimental Pancreatitis and NF-κB/Rel Activation

Acute pancreatitis is a serious disease with a high morbidity and an overall mortality rate of about 10%. However, in its most severe form, which is characterized by pancreatic necrosis, 20–30% of the patients die. Death is often the result of multiorgan dysfunction, including acute respiratory, kidney, and hepatic failure as well as generalized diffuse capillary leak water retention, hypoxia, and acid/base disturbance. The mechanisms by which distant organ systems are involved still remain obscure, but several lines of evidence suggest the participation of cytokines (IL-1, IL-6, and TNF-α) as a response to local tissue damage. A series of studies have now shed new light on the pivotal pathogenic role of the transcription factor NF-ĸB/Rel that binds to the promoter regions of many proinflammatory genes and regulates their transcription.

Inhibition of nuclear factor kappa B and induction of apoptosis in T‐lymphocytes by sulfasalazine

Chronic inflammatory diseases have been shown to be associated with NF‐κB activation and impaired apoptosis of immune cells. The aim of the present study was to investigate if sulfasalazine and its colonic metabolites 5‐aminosalicylic acid (5ASA) and sulfapyridine affect NF‐κB/Rel activation and viability of T‐lymphocytes. Sulfasalazine inhibits NF‐κB/Rel activation in the murine T‐lymphocyte cell line RBL5 using electrophoretic mobility shift assays. In transfection assays sulfasalazine treatment for 4 h inhibits κB‐dependent transcription with an IC50 value of ∼0.625 mM. Higher doses or prolonged treatment result in cell death of T‐lymphocytes in a dose‐ and time‐dependent manner. Cell death is caused by apoptosis as judged by DNA fragmentation, annexin V and Apo 2.7 staining. Induction of apoptosis is a fast event with 50% apoptotic cells after a 4 h incubation with 2.5 mM sulfasalazine. The ED50 value for apoptosis induction after 24 h treatment was ∼0.625 mM. In contrast, 5ASA and sulfapyridine neither inhibit NF‐κB/Rel activation nor induce apoptosis in T‐lymphocytes at doses up to 5.0 mM. These results demonstrate that sulfasalazine, but not 5ASA or sulfapyridine, strongly inhibits NF‐κB activation and potently induces apoptosis in T‐lymphocytes. Inhibition of NF‐κB/Rel activation and subsequent clearance of activated T‐lymphocytes by apoptosis might thus explain the beneficial effects of sulfasalazine in the treatment of chronic inflammatory disorders.

Caerulein-induced NF-κB/Rel activation requires both Ca2+ and protein kinase C as messengers

The eukaryotic transcription factor NF-κB/Rel is activated by a large variety of stimuli. We have recently shown that NF-κB/Rel is induced during the course of caerulein pancreatitis. Here, we show that activation of NF-κB/Rel by caerulein, a CCK analog, requires increasing intracellular Ca2+ levels and protein kinase C activation. Caerulein induces a dose-dependent increase of nuclear NF-κB/Rel binding activity in pancreatic lobules, which is paralleled by degradation of IκBα. IκBβ was only slightly affected by caerulein treatment. Consistent with an involvement of Ca2+, the endoplasmic reticulum-resident Ca2+-ATPase inhibitor thapsigargin activated NF-κB/Rel in pancreatic lobules. The intracellular Ca2+ chelator TMB-8 prevented IκBα degradation and subsequent nuclear translocation of NF-κB/Rel induced by caerulein. BAPTA-AM was less effective. Cyclosporin A, a Ca2+/calmodulin-dependent protein phosphatase (PP2B) inhibitor, decreased caerulein-induced NF-κB/Rel activation and IκBα degradation. The inhibitory effect of bisindolylmaleimide suggests that protein kinase C activity is also required for caerulein-induced NF-κB/Rel activation. These data suggest that Ca2+- as well as protein kinase C-dependent mechanisms are required for caerulein-induced NF-κB/Rel activation.

Helicobacter pylori in the oral cavity: high prevalence and great DNA diversity.

To test the hypothesis that Helicobacter pylori may be transmitted by the oral–oral route, we applied nested PCR and DNA sequencing to detect and analyze H. pylori DNA in the oral cavity of 20 adult patients undergoing endoscopy. Dental plaques of molars, premolars, and incisors and saliva were collected. Additional paraffin-embedded gastric biopsies were analyzed in four patients. Two sets of highly sensitive and specific primers, EHC-U/EHC-L and ET5-U/ET-5L directed to a 860-bp fragment of H. pylori DNA, were used in the nested PCR. Eight patients had an active infection in the stomach determined with the [13C]urea breath test and the other 12 were negative. Nested PCR showed that all 20 subjects (100%) were positive for H. pylori in the oral cavity. DNA sequencing demonstrated that all tested PCR products of the expected size from the oral samples have more than 97% identity with that from H. pylori type strain ATCC 43629. However, sequences differed in oral samples from different subjects as well as between different oral locations and gastric biopsies within the same individuals. In conclusion, the oral cavity may be a permanent reservoir for H. pylori and can harbor multiple H. pylori strains at the same time.