Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

<p>Abstract</p> <p>Background</p> <p>During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis.</p> <p>Results</p> <p>Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic cell lineage and extracellular matrix deposition markers in three dimensions. Samples from human prenatal livers ranging from 7 weeks + 2 days to 15½ weeks post conception as well as adult normal and acetaminophen intoxicated liver were used. The markers included cytokeratins (CK) 7 and 19, the epithelial cell adhesion molecule (EpCAM), hepatocyte paraffin 1 (HepPar1), sex determining region Y (SRY)-box 9 (SOX9), laminin, nestin, and aquaporin 1 (AQP1).</p> <p>Digital three-dimensional reconstructions using CK19 as a single marker protein disclosed a fine network of CK19 positive cells in the biliary tree in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte tubulogenesis.</p> <p>Conclusions</p> <p>The developed protocols provide a novel and sophisticated three-dimensional visualization of vessels and protein expression in human liver during development and disease.</p

The level of claudin-7 is reduced as an early event in colorectal carcinogenesis

<p>Abstract</p> <p>Background</p> <p>Compromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both <it>in vivo </it>and <it>in vitro</it>. We found in an <it>in-silico </it>search tight co-regulation between <it>matriptase </it>and <it>claudin-7 </it>expression. We have previously shown that the <it>matriptase </it>expression level decreases during colorectal carcinogenesis. In the present study we investigated whether <it>claudin-7 </it>expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue.</p> <p>Methods</p> <p>The mRNA level of <it>claudin-7 </it>(CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings.</p> <p>Results</p> <p>A 2.7-fold reduction in the <it>claudin-7 </it>mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the <it>claudin-7 </it>mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.</p> <p>Conclusions</p> <p>Our results show that the <it>claudin-7 </it>mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.</p

Expression of prostasin and its inhibitors during colorectal cancer carcinogenesis

<p>Abstract</p> <p>Background</p> <p>Clinical trials where cancer patients were treated with protease inhibitors have suggested that the serine protease, prostasin, may act as a tumour suppressor. Prostasin is proteolytically activated by the serine protease, matriptase, which has a very high oncogenic potential. Prostasin is inhibited by protease nexin-1 (PN-1) and the two isoforms encoded by the mRNA splice variants of <it>hepatocyte growth factor activator inhibitor-1 </it>(<it>HAI-1</it>), <it>HAI-1A</it>, and <it>HAI-1B</it>.</p> <p>Methods</p> <p>Using quantitative RT-PCR, we have determined the mRNA levels for <it>prostasin </it>and <it>PN-1 </it>in colorectal cancer tissue (n = 116), severe dysplasia (n = 13), mild/moderate dysplasia (n = 93), and in normal tissue from the same individuals. In addition, corresponding tissues were examined from healthy volunteers (n = 23). A part of the cohort was further analysed for the mRNA levels of the two variants of HAI-1, here denoted <it>HAI-1A </it>and <it>HAI-1B</it>. mRNA levels were normalised to <it>β-actin</it>. Immunohistochemical analysis of prostasin and HAI-1 was performed on normal and cancer tissue.</p> <p>Results</p> <p>The mRNA level of prostasin was slightly but significantly decreased in both mild/moderate dysplasia (p < 0.001) and severe dysplasia (p < 0.01) and in carcinomas (p < 0.05) compared to normal tissue from the same individual. The mRNA level of <it>PN-1 </it>was more that two-fold elevated in colorectal cancer tissue as compared to healthy individuals (p < 0.001) and elevated in both mild/moderate dysplasia (p < 0.01), severe dysplasia (p < 0.05) and in colorectal cancer tissue (p < 0.001) as compared to normal tissue from the same individual. The mRNA levels of <it>HAI-1A </it>and <it>HAI-1B </it>mRNAs showed the same patterns of expression. Immunohistochemistry showed that prostasin is located mainly on the apical plasma membrane in normal colorectal tissue. A large variation was found in the degree of polarization of prostasin in colorectal cancer tissue.</p> <p>Conclusion</p> <p>These results show that the mRNA level of <it>PN-1 </it>is significantly elevated in colorectal cancer tissue. Future studies are required to clarify whether down-regulation of prostasin activity via up regulation of PN-1 is causing the malignant progression or if it is a consequence of it.</p

UPF2 is a critical regulator of liver development, function and regeneration

BACKGROUND: Nonsense-mediated mRNA decay (NMD) is a post-transcriptional RNA surveillance process that facilitates the recognition and destruction of mRNAs bearing premature terminations codons (PTCs). Such PTC-containing (PTC+) mRNAs may arise from different processes, including erroneous processing and expression of pseudogenes, but also from more regulated events such as alternative splicing coupled NMD (AS-NMD). Thus, the NMD pathway serves both as a silencer of genomic noise and a regulator of gene expression. Given the early embryonic lethality in NMD deficient mice, uncovering the full regulatory potential of the NMD pathway in mammals will require the functional assessment of NMD in different tissues. METHODOLOGY/PRINCIPAL FINDINGS: Here we use mouse genetics to address the role of UPF2, a core NMD component, in the development, function and regeneration of the liver. We find that loss of NMD during fetal liver development is incompatible with postnatal life due to failure of terminal differentiation. Moreover, deletion of Upf2 in the adult liver results in hepatosteatosis and disruption of liver homeostasis. Finally, NMD was found to be absolutely required for liver regeneration. CONCLUSION/SIGNIFICANCE: Collectively, our data demonstrate the critical role of the NMD pathway in liver development, function and regeneration and highlights the importance of NMD for mammalian biology