Helicobacter pylori and toll-like receptor agonists induce syndecan-4 expression in an NF-kappaB-dependent manner.

The syndecans are a family of transmembrane heparan sulfate proteoglycans (HSPG) that have been implicated in a wide variety of biological functions including the regulation of growth factor signaling, adhesion, tumorigenesis, and inflammation. In the current studies, we examined the regulation of syndecan-4 gene expression in gastric epithelial cells and macrophages in response to infection with live Helicobacter pylori and purified toll-like receptor (TLR) agonists. H. pylori, PAM3CSK4 (a TLR2 agonist), and Escherichia coli flagellin (a TLR5 agonist) all induced the rapid expression of syndecan-4 mRNA in MKN45 gastric epithelial cells. Similarly, lipopolysaccharide (LPS) (a TLR4 agonist) also induced the expression of syndecan-4 in macrophages. The H. pylori- and TLR-induced increase in syndecan-4 mRNA was blocked by the proteosome inhibitor MG-132 suggesting a role for nuclear factor kappaB (NF-kappaB) in the regulation of syndecan-4 gene expression. An 895-bp fragment of the human syndecan-4 promoter was cloned upstream of the luciferase reporter. When transfected into MKN45 cells, the activity of this promoter was inducible by H. pylori and TLR agonists. Inducible activity of the syndecan-4 promoter was blocked by cotransfection with a dominant negative IkappaBalpha expression plasmid. Electrophoretic mobility shift assays (EMSA) demonstrated the presence of a highly conserved NF-kappaB-binding site. Mutation of this site within the context of the full-length syndecan-4 promoter resulted in a complete loss of responsiveness to H. pylori and TLR agonists. These results thus demonstrate that the response of the syndecan-4 gene to infectious agents, or their products, is a direct result of NF-kappaB binding to the promoter and induction of de novo transcription.

Role of endogenous IL-10 in LPS-induced STAT3 activation and IL-1 receptor antagonist gene expression.

The regulation of secretory interleukin (IL)-1 receptor antagonist (sIL-1Ra) in response to IL-10 is unique. In contrast to most cytokines, the lipopolysaccharide (LPS)-induced expression of the sIL-1Ra gene is enhanced by concomitant treatment with IL-10. Cotreatment of RAW 264.7 cells with IL-10 + LPS resulted in at least a twofold increase in sIL-1Ra promoter activity and mRNA expression compared with LPS alone; IL-10 alone had no effect on promoter activity or mRNA expression. Examination of sIL-1Ra mRNA expression in bone marrow-derived macrophages (BMDM) resulted in identical results. Transfection of RAW 264.7 cells with the sIL-1Ra/luc reporter and a dominant-negative signal transducer and activator of transcription (STAT)3 (Y705A) expression plasmid inhibited the enhanced response induced by exogenous IL-10 in the presence of LPS. The presence of a functional STAT3-binding site within the proximal sIL-1Ra promoter was demonstrated. As IL-10 is produced by LPS-stimulated macrophages, a role for endogenously produced IL-10 in the response of the sIL-1Ra gene to LPS was suggested. This was confirmed in IL-10-deficient BMDM, which when compared with normal BMDM, had significantly decreased LPS-induced sIL-1Ra mRNA levels that could be restored by exogenously provided IL-10, which induced a fivefold increase of LPS-induced IL-1Ra mRNA in cells from IL-10-/- BMDM. Western blot analysis of phosphorylated STAT3 from wild-type and IL-10-/- BMDM and IL-10 neutralization experiments demonstrated a role for endogenously produced IL-10 in the LPS-induced STAT3 activity. Together, these results demonstrate that endogenously produced IL-10 plays a significant role in LPS-induced sIL-1Ra gene expression via the activation of STAT3.

Gastric cancers overexpress DARPP-32 and a novel isoform, t-DARPP.

Gastric carcinoma is the second most common cause of cancer-related death worldwide. Recently, we have demonstrated that expressed sequence tag AA552509 was frequently amplified and the most consistently overexpressed target at 17q in gastric cancers. Herein, we report that DARPP-32 (dopamine and cAMP-regulated phosphoprotein of M(r) 32,000) is the target gene for overexpression of expressed sequence tag AA552509. In addition, we have identified full-length cDNA of DARPP-32 (GenBank accession number AF464196) with 467 bp of additional untranslated mRNA nucleotides upstream of the previously known translation start site in exon 1. Additionally, we have discovered a novel truncated isoform of DARPP-32 that we named t-DARPP (GenBank accession number AY070271), which is also overexpressed in gastric cancers. Using quantitative real-time reverse transcription-PCR, Western blots, and staining of tumor tissue arrays, the two DARPP mRNA transcripts and proteins were overexpressed in gastric cancer cells and exhibited abundant protein overexpression in neoplastic but not normal gastric epithelial cells. DARPP-32 is the only known protein that acts as a protein phosphatase 1 inhibitor or a protein kinase A inhibitor. The novel truncated isoform, t-DARPP, lacks the phosphorylation site related to protein phosphatase 1 inhibition but maintains the phosphorylation site with the protein kinase A inhibitory effect. Our results reveal for the first time the presence of these signaling molecules in human cancer and suggest that they may be important for gastric tumorigenesis.

Toll-like receptor 2 and 4 (TLR2 and TLR4) agonists differentially regulate secretory interleukin-1 receptor antagonist gene expression in macrophages.

Treatment of macrophages with lipopolysaccharide (LPS) from Gram-negative bacteria or peptidoglycan (PGN) from Gram-positive bacteria activates multiple intracellular signaling pathways and a large, diverse group of nuclear transcription factors. The signaling receptors for PGN and LPS are now known to be the Toll-like receptors 2 and 4 (TLR2 and -4, respectively). While a large body of literature indicates that the members of the TLR family activate nearly identical cytoplasmic signaling programs, several recent reports have suggested that the functional outcomes of signaling via TLR2 or TLR4 are not equivalent. In the current studies, we compared the responses of the secretory IL-1 receptor antagonist (sIL-1Ra) gene to both LPS and PGN. Both LPS and PGN induced IL-1Ra gene expression; however, the combination of both stimuli synergistically increased sIL-1Ra mRNA expression and promoter activity, suggesting that the signals induced by PGN and LPS are not equivalent. While both LPS and PGN utilized the PU.1-binding sites in the proximal sIL-1Ra promoter region to generate a full response, additional distinct promoter elements were utilized by LPS or PGN. Activation of p38 stress-activated protein kinase was required for LPS- or PGN-induced IL-1Ra gene expression, but the p38-responsive promoter elements localized to distinct regions of the sIL-1Ra gene. Additionally, while the LPS-induced, p38-dependent response was dependent upon PU.1 binding, the PGN-induced, p38 response was not. Collectively, these data indicated that while some of the intracellular signaling events by TLR2 and TLR4 agonists are similar, there are clearly distinct differences in the responses elicited by these two bacterial products.