ABSTRACT
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.
