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In the present study, we characterized the gene product of open reading frame 3 encoded at human chromosome 20 (C20orf3), which represents a member of the lactonohydrolase super family. Multiple-tissue Northern blot analysis showed ubiquitous expression of the 2.4 kb transcript coding for 416 amino acids, with highest levels in human liver, placenta and kidney. After recombinant production of protein variants in Escherichia coli and insect cells, antibodies directed against different epitopes within the C20orf3 gene product were generated. Using these immunoreagents, protein expression was demonstrated in the liver, and glomerular and tubular structures of the kidney, as well as in endothelial cells and arterial wall. Positive staining was also observed at the pancreatic islets of Langerhans. Using immunoblotting, we identified three size variants. In line with the results of in silico analysis demonstrating a single transmembrane sequence (amino acids 40–61) at the N-terminus of the full-length protein, FACS cell-surface staining confirmed a mainly extracellular localization of the full-length protein. Sucrose density gradient cell fractionation revealed membrane association of the dominant 50 kDa variant in HepG2 and Rin-5F cells. The finding of a strong arylesterase activity with β-naphthyl acetate and phenyl acetate of the C20orf3 protein-containing fractions suggests potential involvement of this protein in enzymatic processes. C20orf3 promoter-driven reporter assays, which were verified by gene-specific RT-qPCR (real-time quantitative PCR) showed a strong inhibitory effect of human serum on transcription using the HEK-293 human embryonic kidney cell line. In conclusion, we characterized the structure and expression pattern of the C20orf3 gene product. According to a series of analogies with PON (paraoxonase) family members, we speculate that the C20orf3 gene product represents a new member of this important protein family present at the cellular level. [ABSTRACT FROM AUTHOR]

Aiming to identify signalling pathways relevant for ?-cell growth we performed an explorative micro-array analysis comparing the gene expression profiles of three human insulinomas and one normal pancreatic islet preparation. This revealed an insulinoma-associated down-regulation of the transforming growth factor beta 1 (TGF-?1) and its target genes. Comparative quantitative real-time PCR (qRT-PCR) including an expanded sample number of both insulinomas (n=9) and pancreatic islet preparations (n=4) confirmed the decreased TGF-?1 expression and its target molecules (TGFBI, NNMT, RPN2) in insulinomas. Similarly, TGF-?1 immunofluorescense analysis revealed reduced expression in insulinomas when compared to pancreatic islets. In contrast, TGFBR2 (transforming growth factor beta receptor II) was found up-regulated. However, the consistent down-regulation of the TGF-?1 targets TGFBI (transforming growth factor, beta-induced), NNMT (nicotinamide N-methyltransferase), RPN2 (ribophorin II) indicates that the parallel up-regulation of TGFBR2 does not compensate for the only marginal TGF-?1 expression levels in insulinomas. TGFBR2 expression was confirmed at the protein level in insulinomas. SMAD2/3 protein expression was found at higher levels in human pancreatic islets when compared with insulinomas by dual colour confocal microscopy. TGF-?1 signalling is known to be involved in cell replication and is abrogated in ductal pancreatic tumours. The down-regulation of TGF-?1 expression and its target molecules in insulinomas is a new aspect of this cytokine. Our data underline parallels in endocrine and exocrine pancreatic tumour development, which may implicate common progenitor cells. [ABSTRACT FROM AUTHOR]

Aiming to identify signalling pathways relevant for ss-cell growth we performed an explorative micro-array analysis comparing the gene expression profiles of three human insulinomas and one normal pancreatic islet preparation. This revealed an insulinoma-associated down-regulation of the transforming growth factor beta 1 (TGF-beta1) and its target genes. Comparative quantitative real-time PCR (qRT-PCR) including an expanded sample number of both insulinomas (n=9) and pancreatic islet preparations (n=4) confirmed the decreased TGF-beta1 expression and its target molecules (TGFBI, NNMT, RPN2) in insulinomas. Similarly, TGF-beta1 immunofluorescence analysis revealed reduced expression in insulinomas when compared to pancreatic islets. In contrast, TGFBR2 (transforming growth factor beta receptor II) was found up-regulated. However, the consistent down-regulation of the TGF-beta1 targets TGFBI (transforming growth factor, beta-induced), NNMT (nicotinamide N-methyltransferase), RPN2 (ribophorin II) indicates that the parallel up-regulation of TGFBR2 does not compensate for the only marginal TGF-beta1 expression levels in insulinomas. TGFBR2 expression was confirmed at the protein level in insulinomas. SMAD2/3 protein expression was found at higher levels in human pancreatic islets when compared with insulinomas by dual colour confocal microscopy. TGF-beta1 signalling is known to be involved in cell replication and is abrogated in ductal pancreatic tumours. The down-regulation of TGF-beta1 expression and its target molecules in insulinomas is a new aspect of this cytokine. Our data underline parallels in endocrine and exocrine pancreatic tumour development, which may implicate common progenitor cells.