No abstract

In a transgenic model of multi-stage squamous carcinogenesis induced by human papillomavirus (HPV) oncogenes, infiltrating CD4+ T cells can be detected in both premalignant and malignant lesions. The lymph nodes that drain sites of epidermal neoplasia contain activated CD4+ T cells predominantly reactive toward Staphylococcal bacterial antigens. HPV16 mice deficient in CD4+ T cells were found to have delayed neoplastic progression and a lower incidence of tumors. This delay in carcinogenesis is marked by decreased infiltration of neutrophils, and reduced activity of matrix metalloproteinase-9, an important cofactor for tumor progression in this model. The data reveal an unexpected capability of CD4 T cells, whereby, proinflammatory CD4+ T cells, apparently responding to bacterial infection of dysplastic skin lesions, can inadvertently enhance neoplastic progression to invasive cancer.

/MMP2 -/-mice (four mice/experimental group) in response to mustard oil (black bars), as compared with mineral oil (white bars). Data reflect mean ± S.E.M. *P<0.005.

No abstract

Migration of cells requires interactions with the extracellular matrix mediated, in part, by integrins, proteases, and their receptors. Previous studies have shown that ␤ 3 -integrin interacts with the urokinase-type plasminogen activator receptor (u-PAR) at the cell surface. Since integrins mediate signaling into the cell, the current study was undertaken to determine if in addition ␤ 3 -integrin regulates u-PAR expression. Overexpression of ␤ 3 -integrin in CHO cells, which are avid expressers of the receptor, downregulated u-PAR protein and mRNA expression. The u-PAR promoter (؊1,469 bp) that is normally constitutively active in CHO cells was downregulated by induced ␤ 3 -integrin expression. A region between ؊398 and ؊197 bp of the u-PAR promoter was critical for ␤ 3 -integrin-induced downregulation of u-PAR promoter activity. Deletion of the PEA3/ets motif at ؊248 bp substantially impaired the ability of ␤ 3 -integrin to downregulate the u-PAR promoter, suggesting that the PEA3/ets site acts as a silencing element. An expression vector encoding the transcription factor PEA3 caused inhibition of the wild-type but not the PEA3/ets-deleted u-PAR promoter. The PEA3/ets site bound nuclear factors from CHO cells specifically, but binding was enhanced when ␤ 3 -integrin was overexpressed. A PEA3 antibody inhibited DNA-protein complex formation, indicating the presence of PEA3. Downregulation of the u-PAR promoter was achieved by the ␤ 3 A-integrin isoform but not by other ␤ 3 -integrin isoforms and required the cytoplasmic membrane NITY 759 motif. Moreover, overexpression of the short but not the long isoform of the ␤ 3 -integrin adapter protein ␤ 3 -endonexin blocked u-PAR promoter activity through the PEA3/ets binding site. Thus, besides the physical interaction of ␤ 3 -integrin and u-PAR at the cell surface, ␤ 3 signaling is implicated in the regulation of u-PAR gene transcription, suggesting a mutual regulation of adhesion and proteolysis receptors.

We studied the expression of interleukin-1 (IL-1) receptors and the effect of IL-1β on the function of highly enriched (>97%) rat parietal cells. RT-PCR of parietal cell poly(A)+RNA with primers specific for the rat IL-1 receptor revealed a single 547-kb PCR product highly homologous to the published sequence of the IL-1 receptor. Northern blot analysis of poly(A)+RNA of rat parietal cells and brain revealed a single RNA species of 5.7 kb. Cytochemistry of parietal cell IL-1 receptor was performed with biotinylated recombinant human IL-1β, visualized by avidin-coupled fluorescein. Corresponding to the high degree of parietal cell enrichment, 95% of the cells stained positive. Basal H+production ([14C]aminopyrine accumulation) was not changed by IL-1β (0.25–100 pg/ml) nor was the response to histamine or carbachol when added simultaneously with the cytokine. However, when parietal cells were preincubated with IL-1β (0.5–5 pg/ml) for 10 min before the addition of histamine or carbachol, the response to these secretagogues was reduced by 35 and 67%, respectively. Inhibition by IL-1β was fully reversed by the human recombinant IL-1 receptor antagonist. Preincubation of parietal cells with IL-1β failed to alter histamine-stimulated cAMP production but markedly inhibited carbachol-induced formation ofd- myo-inositol 1,4,5-trisphosphate. In fura 2-loaded, purified parietal cells, 10 min preincubation with IL-1β dramatically reduced the initial transient peak elevation of intracellular Ca2+concentration in response to carbachol. We conclude that rat parietal cells express IL-1 receptors mediating inhibition of H+production. The antisecretory effect of IL-1β may contribute to hypoacidity secondary to acute Helicobacter pylori infection or during chronic colonization by H. pylori preferring the fundic mucosa.

We have previously shown that in highly enriched rat gastric parietal cells the intestinal peptide hormones oxyntomodulin and glucagon-like peptide-2 (GLP-2) compete for receptor-binding with glucagon-like peptide-1 (GLP-1), a potent cAMP-dependent stimulus of H⁺ production in vitro. It is, however, unknown whether oxyntomodulin and GLP-2 elicit a biological response by interacting with the GLP-1 receptor. Therefore, we used enriched rat parietal cells to investigate the effects of both hormones on the production of cAMP and H⁺ ([¹⁴C]aminopyrine accumulation). Both parameters were stimulated by oxyntomodulin in a concentration-dependent manner. EC50 values were 6.2-10^-8 and 2.5·10^-7M oxyntomodulin for stimulation of H⁺ and cAMP production, respectively. The maximally effective concentrations for stimulation of [¹⁴C]aminopyrine accumulation and cAMP production were l·10^-6 and 1 · 10^-5M oxyntomodulin, respectively. At these concentrations oxyntomodulin was nearly as effective as 10^-4Mhistamine and equally effective as 10^-8 MGLP-1 (7-36)NH₂. In the enriched parietal cell preparation there was no immunocytochemical evidence of contaminating D cells. Accordingly, the responses to oxyntomodulin and GLP-1 (7-36)NH2 were not augmented by incubating the cells in the presence of a polyclonal anti-somatostatin antibody. [¹⁴C]Aminopyrine accumulation in response to oxyntomodulin was inhibited by the GLP-1 (7-36)NH₂ receptor antagonist, exendin (9-39)NH₂, but not by the H₂-receptor antagonist, ranitidine. Oxyntomodulin and carbachol acted additively to stimulate [¹⁴C]aminopyrine accumulation. GLP-2 (10^-7 to 10^-5M) was without effect on basal H⁺ and cAMP production; however, at 10^-5M GLP-2 markedly inhibited oxyntomodulin-stimulated [¹⁴C]aminopy-rine accumulation. It is concluded that, by interacting with parietal cell receptors for GLP-1 (7-36)NH₂, oxyntomodulin, but not GLP-2, directly stimulates H⁺ production by activating the adenylate cyclase.

The intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36) amide is a potent stimulus of H+ production in isolated rat parietal cells, suggesting the presence of specific GLP-1-receptors on this cell type. Our aim was to characterize these receptors. Enzymatically isolated rat gastric mucosal cells (F0) were fractionated by counterflow elutriation, resulting in five fractions (F1-F5) according to increasing cell diameter and parietal cell content (3, 5, 4, 27, 81%). Additional density gradient centrifugation of F4 yielded enriched chief cells (74%; parietal cells: 1%; F6), whereas density gradient centrifugation of F5 almost purified parietal cells (97%; chief cells: 1%; F7). Northern blot of total cellular RNA from F0-F7 with a probe specific for the GLP-1-(7-36) amide receptor revealed two RNA species of 2.7 and 3.6 kb. These messages were present to some extent in small cells (F1, F2), much more pronounced in F5, abundant in F7, barely detectable in F3 and F4, and absent from F6. Cross-linking of 125I-labeled GLP-1-(7-36) amide to parietal cell membranes revealed a single 59-kDa band that was abolished by unlabeled GLP-1-(7-36) amide. Throughout fractions F1-F7 specific binding of 125I-GLP-1-(7-36) amide was correlated with parietal cell content (r = 0.99; P < 0.01) and H+ production ([14C]aminopyrine accumulation) in response to GLP-1-(7-36) amide or histamine (r = 0.96; P < 0.01). Binding was maximal in purified parietal cells (F7), whereas almost no binding was detectable in enriched chief cells (F6). In F7, Scatchard analysis revealed a single class of high-affinity binding sites (KD = 2.8 +/- 0.6 x 10(-10) M, Bmax = 6.8 +/- 1.4 fmol/10(6) cells, 4,096 +/- 793 receptors/parietal cells). The following half-maximal inhibition values were found for GLP-1-(7-36) amide and (1-37) and (1-36) amide: 6.6 +/- 0.9 x 10(-10), 1.4 +/- 0.7 x 10(-7), and 2.6 +/- 0.4 x 10(-7) M, respectively. Pancreatic glucagon, GLP-2, and oxyntomodulin, products of the proglucagon gene, were 3-4 log units less potent displacers while gastric inhibitory peptide, vasoactive intestinal peptide, and secretin were ineffective. We conclude that rat parietal cells are equipped with specific high-affinity receptors for GLP-1-(7-36) amide, which, in addition, are present in as yet unidentified small cells (F1, F2) but not in chief cells.