Symplekin promotes tumorigenicity by up-regulating claudin-2 expression.

Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.

Role of a LIF antagonist in LIF and OSM induced MMP-1, MMP-3, and TIMP-1 expression by primary articular chondrocytes.

Cartilage degradation is mediated by matrix metalloproteinases (MMPs) and their inhibitors, tissue metalloproteinases (TIMPs), which are transcriptionally regulated by a variety of growth factors and cytokines. The levels of various MMPs as well as TIMPs have been shown to increase in response to certain cytokines. These include leukaemia inhibitory factor (LIF) and Oncostatin M (OSM), both of which have been detected in the synovial fluids of patients with rheumatoid arthritis (RA). However, the role of LIF and OSM in the regulation of various MMPs and TIMPs is still incompletely understood. The aims of this study were to examine the effects of LIF and OSM on MMP-1, MMP-3, and TIMP-1 production. In addition, the capacity of the LIF antagonist, MH35-BD, to block LIF and OSM induced MMP expression was examined. Primary chondrocytes, isolated from porcine metacarpophalangeal cartilage, were cultured in the presence and absence of LIF and OSM, with and without a predetermined concentration of the LIF antagonist. We analysed the levels of MMP-1, MMP-3 and TIMP-1 expression using qRT-PCR, Northern blot, and ELISA assays. The results indicate that LIF and OSM increase the expression of MMP-1, MMP-3, and TIMP-1 several fold. Furthermore their expression is reduced to basal levels in the presence of the LIF antagonist MH35-BD.

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function.

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.