A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells

Figure 6

Treatment of BV2 microglial cells with an amino-terminal fragment of apoE4 leads to the upregulation of inflammatory cytokines. (a) BV2 cells were left untreated (control, green bar) or treated for 5 hours with 25 μg/ml nApoE4_1-151 (blue bar), and RNA was extracted. RT-PCR analysis indicated a 24.5-fold increase in the expression of inflammatory cytokine, TNFα. denotes significant difference, . (b) Secreted TNFα levels are significantly elevated following the treatment of BV2 cells with nApoE4_1-151 (blue bar) as compared to untreated controls (green bar) or an unrelated protein, myoglobin, of similar size and weight to nApoE4_1-151 (red bar, middle). Data are representative of 3 independent denotes significant difference, between control and nApoE4_1-151, while # represents significant difference, between myoglobin and nApoE4_1-151. (c) Secreted IL-1beta levels are significantly elevated following the treatment of BV2 cells with nApoE4_1-151 (blue bar) as compared to untreated controls (green bar) or an unrelated protein, myoglobin, of similar size and weight to nApoE4_1-151 (red bar, middle). Data are representative of 3 independent denotes significant difference, . (d, e) DNA gel mobility shift assay was used to determine a specific interaction of nApoE4_1-151 with the 200 bp promoter region of mouse TNFα. (d) Biotinylated end-labeled DNA corresponding to the mouse TNFα promoter region was incubated alone or in addition of 25 μg/ml of nApoE4_1-151. There was an upward shift in the DNA band indicating a binding of the fragment to DNA. The last lane was performed similarly except that the nApoE4_1-151 fragment was preincubated first with an anti-His antibody that specifically recognizes the His-tagged nApoE4_1-151 fragment. In this case, although shift was still evident, incubation with the anti-His antibody led to a decrease in gel retardation suggesting a specific competition between the DNA and antibody for the nApoE4_1-151 fragment. (e) Identical experiments with the exception of an identical concentration of full-length apoE4 (25 μg/ml) were tested. As denoted in the last lane, in this case, full-length apoE4 had no effect on the DNA shift suggesting that gel retardation of the TNFα DNA promoter region is due to specific binding to the nApoE4_1-151 fragment.

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