Effect Of Environment-responsive Gene JWA On Glutamate Transport

Human diseases are usually the consequences of the co-operation of both environmental and genetic agents. An increasing number of environmental responsive genes related to human diseases have been found during the past years. The research on the mechanism and regularity of the interaction between genetic and environmental agents is important for preventing and treating the injury caused by environmental agents. The aim of this study is to investigate and elucidate the molecular mechanism of environmental agents in induction of diseases. In 1998 Zhou et al cloned a novel environment-responsive gene from human bronchial epithelial (HBE) cells by using the differential display high-throughput technique. The new gene, named JWA, is broadly distributed in most of examined eukaryotic cells and tissues. The intracellular distribution of JWA protein encoded by the gene is very similar to that of cytoskeleton under immunofluorescence microscopy. The gene expressions are modulated by some cell differentiation inducers, e.g., all trans retinoic acid (ATRA), 13-cis-retinoic acid and 12-O-tetradecanoyl-phorbol-13 acetate (PMA). Our recent studies showed that JWA is also a functional protein associated with cell differentiation and apoptosis, and might play an important role in development of acute promyelocytic leukemia (APL). It was reported that glutamate transporterEAAC1-associated protein (GTRAP3-18), expressed by E-18 which is 95% identical to JWA, is a novel protein that interacts with excitatory amino acids transporters 3 (EAAT3) and modulates Na+-dependent glutamate transporter EAAT3-mediated glutamate uptake in rat. In this study, we probe the relationship between the JWA gene and glutamate uptake in HEK293 cell. Further more, we examined the effect of fenvalerate on the expression of JWA gene and glutamate uptake.The aim of this study is to explore the postulated mechanism of JWA, a new microtubule-associated proteins, in the regulation of glutamate transport. JWA transfected HEK293 cell was obtained as a model for enhancing expression of JWA. Antisense oligonucleotide of JWA was used to inhibit the expression of JWA so as to construct a model for down-regulating expression of JWA. The intracellular calcium concentration was measured by laser confocal microscopy. The expressed proteins in the cells were determined by immunofluorescence microscopy and Western blot. The effects of JWA on glutamate uptake in the cells were observed by incorporation of tritium-D, L-glutamate. Construct pEGFP expression vector carrying four different fragments of coding region of JWA. Result shows that glutamate uptake in the FIEK293 cells with enhanced expression of JWA was elevated and down-regulated as a result of JWA blocking by antisense oligonucleotide. PMA (phorbol 12-myristate 13-acetate) contributed to increases of glutamate transport in both of transfected and untransfected cells and independent to the JWA expression levels. PMA also induced a transient boost of intracellular free calcium concentration ([Ca2+]i). PMA increase the expression and alter the localization of JWA in HEK293 cell, transfected with pEGFP-JWA. Fenvalerate can increase the expression of JWA, and glutamate transport in PC 12 cell. In conclusion, JWA enhances the function of glutamate transport in FIEK293 cells, which is not exerted through PKC signal pathway. It is likely to be a new signal molecule associated with malfunction of central nerve system. The increasedglutamate transportation induced by PMA may be unrelated to intracellular JWA expression but associated with the alteration of intercellular calcium level. JWA may be concerned with the effect of fenvalerate to glutamate transportation.