Molecular, biochemical, ultrastructural and functional evidence of tight junction formation in normal and neoplastic human neuroendocrine cells

Tight junctions (TJ) provide important features for intestinal epithelial integrity by establishing a paracellular barrier for solute transport and by providing a molecular fence for apico-basolateral polarity of cell membranes. However, little is known about TJ-formation and function of neuroendocrine (NE) cells of the diffuse endocrine system of the gastroenteropancreatic tract and tumors (NET) derived therefrom. We therefore studied TJ-molecules in NE cell lines QGP–1 and BON, normal human intestinal epithelia and NET. We found both TJ-transmembrane proteins (occludin, claudins–1,–3,–4,–7, JAM–1) and TJ-plaque proteins (ZO–1,–2,–3, cingulin, symplekin) expressed in QGP–1 and BON. In both these cell lines biochemical characteristics of homo- and heterooligomeric complex formation of claudins were demonstrated on density gradient centrifugation, native PAGE and coimmunoprecipitation. In confluent QGP–1 a typical circular TJ-staining pattern with subcellular colocalization of TJ-transmembrane and plaque proteins was seen on immunofluoresce microscopy (IFM) while in normal intestinal epithelia we observed continuous apical TJ molecule expression in enterocytes and interspersed NE cells as identified by double staining for TJ proteins (occludin, cingulin) and NE cell markers (synaptophysin, chromogranin A). Electron microscopy revealed typical membrane fusions at the apical junctional complex in QGP–1 and in situ between NE cells and enterocytes of normal human intestinal epithelia. By IFM TJ-proteins (occludin, ZO–1, claudins–1,–3,–4) were also found in NET, often directly related to luminal structures but also in solid areas of the tumor and at the border to the tumor’s stroma. Finally, functional studies using confluent QGP–1 monolayers resulted in a low but significant stable transepithelial electrical resistance of 30–40Ω*cm² while transport studies with variously sized dextrans demonstrated a higher permeability for lower than for higher molecular weight dextrans as is typically seen with leaky epithelia. Here we show molecular and ultrastructural characteristics of TJ in NE cells of normal and neoplastic gastroenteropancreatic tissues. QGP–1 cells provide a useful model of the barrier function for future studies of TJ-function in NE cells and NET.

Key words

barrier - cingulin - claudin - neuroendocrine tumor - occludin - tight junction