Lead induces COX-2 expression in glial cells in a NFAT-dependent, AP-1/NFκB-independent manner

Abstract

Epidemiologic studies have provided solid evidence for the neurotoxic effect of lead for decades of years. In view of the fact that children are more vulnerable to the neurotoxicity of lead, lead exposure has been an urgent public health concern. The modes of action of lead neurotoxic effects include disturbance of neurotransmitter storage and release, damage of mitochondria, as well as induction of apoptosis in neurons, cerebrovascular endothelial cells, astroglia and oligodendroglia. Our studies here, from a novel point of view, demonstrates that lead specifically caused induction of COX-2, a well known inflammatory mediator in neurons and glia cells. Furthermore, we revealed that COX-2 was induced by lead in a transcription-dependent manner, which relayed on transcription factor NFAT, rather than AP-1 and NFκB, in glial cells. Considering the important functions of COX-2 in mediation of inflammation reaction and oxidative stress, our studies here provide a mechanistic insight into the understanding of lead-associated inflammatory neurotoxicity effect via activation of pro-inflammatory NFAT3/COX-2 axis.

Keywords: COX-2; Lead; NFAT; Neurotoxicity.

Fig. 1

The effects of lead exposure on COX-2 protein induction in various types of brain cells. BV2 murine microglia cell line(Aand B), C6rat glioma cell line(C and D), primarycortical neurons (E), iPSCs (F) and NSCs (G) were treated with the different doses of lead (25–400 μM) for various time points as indicated. The inductions of COX-2 were determined by Western blotting assay.

Fig. 2

COX-2 transcription was induced by lead in BV2 and C6 cells. The mRNA changes of cox-2 following lead treatment in BV2 cells (A and B) and C6 cells (C and D) were assessed by RT-PCR in both time and dose-dependent responses. (E and F), The transcription of cox-2 was evaluated by using cox-2 promoter-driven luciferase reporter in C6 cells. The symbol (*) indicates a significant difference between medium control and lead treatment (p<0.05). The value was showed as mean ± SD from three independent experiments.

Fig. 3

The effect of lead exposure on potential transcription factors that might involved in regulation of cox-2 transcription. C6 cells were exposed to 40 μM lead for 1h, 3h, 6h, 12h and 24h. The cells were extracted and subjected to Western blotting assay. The induction of COX-2 and activation of AP-1 were detected using anti-COX-2 and anti-p-c-Jun S73 antibodies (A). The lead-associated activation of NFκB pathway was assessed using antibodies recognizing phospho- and total IKKα/β, IkBα, p65 and total p50 (B). Activation of NFAT3 was determined by checking the hypophosphorylated form of NFAT3 using anti-NFAT3 antibody (C).

Fig. 4

NFAT transactivation was crucial for cox-2 transcription following lead exposure. (A), C6 cells were stably transfected with the AP-1, NFκB and NFAT luciferase reporter in combination with the pRL-TK vector (Promega) as an internal control. After the cells were treated with lead at the indicated doses, the luciferase activities were determined using a luminometer to determine transactivation activities of the individual transcription factor. The asterisk (*) indicates a significant increase compared with medium control (p < 0.05). (B), The C6 stable transfectants of NFAT luciferase reporter in combination with the pRL-TK vector were treated with 40 μM lead for 12 h, 24 h and 48 h. The transactivation of NFAT was determined using a luminometer. The asterisk (*) indicates a significant increase compared with medium control (p < 0.05). (C and D), The cox-2-luciferase reporter plasmid containing site-directed mutant of the NFAT binding sequence was used to validate the involvement of NFAT in induction of COX-2 by lead. The asterisk (*) indicates a significant increase compared with medium control (p < 0.05). The heart symbol (♠) indicates a significant decrease compared with that of wild type cox-2 promoter luciferase reporter (p<0.05).