Characterization of Caco-2 cells stably expressing the protein-based zinc probe eCalwy-5 as a model system for investigating intestinal zinc transport.

Intestinal zinc resorption, in particular its regulation and mechanisms, are not yet fully understood. Suitable intestinal cell models are needed to investigate zinc uptake kinetics and the role of labile zinc in enterocytes in vitro. Therefore, a Caco-2 cell clone was produced, stably expressing the genetically encoded zinc biosensor eCalwy-5. The aim of the present study was to reassure the presence of characteristic enterocyte-specific properties in the Caco-2-eCalwy clone. Comparison of Caco-2-WT and Caco-2-eCalwy cells revealed only slight differences regarding subcellular localization of the tight junction protein occludin and alkaline phosphatase activity, which did not affect basic integrity of the intestinal barrier or the characteristic brush border membrane morphology. Furthermore, introduction of the additional zinc-binding protein in Caco-2 cells did not alter mRNA expression of the major intestinal zinc transporters (zip4, zip5, znt-1 and znt-5), but increased metallothionein 1a-expression and cellular resistance to higher zinc concentrations. Moreover, this study examines the effect of sensor expression level on its saturation with zinc. Fluorescence cell imaging indicated considerable intercellular heterogeneity in biosensor-expression. However, FRET-measurements confirmed that these differences in expression levels have no effect on fractional zinc-saturation of the probe.

Mitochondrial hexokinase II (HKII) and phosphoprotein enriched in astrocytes (PEA15) form a molecular switch governing cellular fate depending on the metabolic state.

The metabolic state of a cell is a key determinant in the decision to live and proliferate or to die. Consequently, balanced energy metabolism and the regulation of apoptosis are critical for the development and maintenance of differentiated organisms. Hypoxia occurs physiologically during development or exercise and pathologically in vascular disease, tumorigenesis, and inflammation, interfering with homeostatic metabolism. Here, we show that the hypoxia-inducible factor (HIF)-1-regulated glycolytic enzyme hexokinase II (HKII) acts as a molecular switch that determines cellular fate by regulating both cytoprotection and induction of apoptosis based on the metabolic state. We provide evidence for a direct molecular interactor of HKII and show that, together with phosphoprotein enriched in astrocytes (PEA15), HKII inhibits apoptosis after hypoxia. In contrast, HKII accelerates apoptosis in the absence of PEA15 and under glucose deprivation. HKII both protects cells from death during hypoxia and functions as a sensor of glucose availability during normoxia, inducing apoptosis in response to glucose depletion. Thus, HKII-mediated apoptosis may represent an evolutionarily conserved altruistic mechanism to eliminate cells during metabolic stress to the advantage of a multicellular organism.

Role of the G-protein-coupled receptor GPR12 as high-affinity receptor for sphingosylphosphorylcholine and its expression and function in brain development.

Lysophospholipids are bioactive molecules influencing numerous cellular processes such as proliferation, differentiation, and motility. As extracellular ligands, they interact with specific members of the G-protein-coupled receptor family. We show in this paper that the lysophospholipid sphingosylphosphorylcholine is a high-affinity ligand for the orphan G-protein-coupled receptor GPR12. Heterologous expression of GPR12 in Chinese hamster ovary cells and in frog oocytes revealed a high-affinity interaction with sphingosylphosphorylcholine in the nanomolar range. Blockade of its action by pertussis toxin was taken as evidence that GPR12 is coupled to an inhibitory G-protein. In the adult mouse brain, GPR12 was expressed in the limbic system. During mouse embryonal development, GPR12 transcripts were detected in the CNS, especially in areas where neuronal differentiation occurs. Consistent with this we found that cultures of embryonal cerebral cortical neurons responded to sphingosylphosphorylcholine with an increase in synaptic contacts. The GPR12-expressing hippocampal cell line HT22 reacted to sphingosylphophorylcholine with an increase in cell proliferation and cell clustering. Other receptors known to interact at nanomolar concentrations with sphingosylphosphorycholine were expressed neither in the developing cerebral cortex nor in the HT22 cell line. We therefore hypothesize that sphingosylphosphorylcholine, most likely by interaction with GPR12, has positive effects on the differentiation and maturation of postmitotic neurons and that it may also influence the proliferation of neuronal precursor cells.

Short-term serum supplementation improves glucose-oxygen deprivation in primary cortical cultures grown under serum-free conditions.

Brain ischemia can be studied in vitro by depriving primary neurons of oxygen and glucose by replacing oxygen with argon and glucose with its antimetabolite 2-deoxy-D-glucose. In this contribution, we explain how to construct a reliably functioning ischemia chamber and use it to study neuronal cell death in neuron-enriched fetal primary cortical cultures grown under serum-free conditions. We observed that these cultures exhibited a significant cell death even during exposure to oxygenated balanced salt solution used as control for oxygen-glucose deprivation. We show that addition of only 2% fetal calf serum 24 h prior, during, and after treatment almost abolished this undesirable cell loss and proportionally increased cell death induced by oxygen-glucose deprivation. Western blots and immunocytochemistry showed that these effects were mainly due to an increase in neuronal viability under control conditions accompanied by a limited glial proliferation independent of the treatment condition. Under these modified conditions, the cultures could also still be effectively preconditioned by a short-term oxygen-glucose deprivation. In summary, this modified protocol combines the advantages of serum-free neuronal culture, where potentially toxic antimitotic substances can be omitted, with a serum-mediated protection of neurons against unspecific factors and concomitant sensitization for oxygen-glucose deprivation.