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Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat IL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, IL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double-stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78. chaperone; cytokine; protein-protein interactions

Menkes disease is a genetic disorder associated with impaired copper (Cu) metabolism. A study of the Cu transport mechanism in different cell lines showed that there is no difference in Cu uptake between normal and Menkes fibroblasts. Treatment with p-chloromercuribenzoic acid had no effect on normal and Menkes fibroblasts. Subsequent treatment with dithiothreitol enhanced Cu uptake in normal fibroblasts but not in Menkes fibroblasts. Menkes fibroblasts also released Cu at rates slower than normal fibroblasts. These results suggest that Cu transport mechanism varies in different cell types.

BeWo cells grown on porous filters have advantages over those grown on plastic dishes in the study of the mechanism of copper (67Cu) transport across human placenta. They show high 67Cu release with normal retention, and are sensitive to p-chloromercuribenzoate. Reverse transcriptase-polymerase chain reaction shows that they also display the Menkes gene transcript. The results indicate a relation between the Menkes gene product and the ability of cells to release copper ions. The expression of the Menkes gene may be controlled by cell polarity.

A study conducted on the transport of copper 67-Cu in the C6 rat glioma cells shows that the influx and efflux of 67-Cu is functional in two varied cellular conditions. Lead and p-Chloromercuribenzoate (p-CMB) suppress the efflux but have no effect on the Michaelis constant for uptake and maximum velocity of influx. The accumulation of 67-Cu and its association with the membrane increases in the presence of p-CMB. The efflux of 67-Cu requires the presence of sulfhydryl agents and a P-type ATPase, whereas the efflux is inhibited by Pb.