Clioquinol increases the expression of interleukin-8 by suppression of GATA-2 and GATA-3

[Speaker] Masato Katsuyama:1
[Co-author] Chihiro Yabe-Nishimura:2
1:Radioisotope Center, Kyoto Prefectural University of Medicine, Japan, 2:Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan

Clioquinol was used extensively as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have yet to be elucidated. We performed a global analysis on SH-SY5Y cells and found that clioquinol increased the expression of interleukin-8 (IL-8) mRNA.
Levels of IL-8 mRNA and secreted IL-8 were measured by quantitative PCR and enzyme-linked immunosorbent assay, respectively. Promoter analyses were performed on the human IL-8 gene. Electrophoresis mobility shift assays (EMSA) were performed using a probe corresponding to -159/-113 of the human IL-8 gene. Genome editing was performed using a lentiviral system.
Clioquinol significantly increased the level of IL-8 mRNA and the secretion of IL-8 into culture media. A region responsive to clioquinol existed between -152 and -144 of the IL-8 gene, which contains a consensus GATA-binding site. Introducing mutations at this site or the activator protein (AP)-1 site at -126/-120 significantly reduced clioquinol-induced transcriptional activation of the IL-8 gene. Among the GATA transcription factors expressed in SH-SY5Y cells, protein levels of GATA-2 and GATA-3 were significantly decreased by the addition of clioquinol. EMSA revealed two major shifted bands, one of which was increased and the other was decreased by clioquinol. The introduction of mutations showed that the former corresponded to binding to the AP-1 site, and the latter to binding to the GATA site. Supershift analyses revealed that the binding of c-Jun and c-Fos to the AP-1 site was increased, whereas that of GATA-3 to the GATA site was decreased by clioquinol. Genome editing against GATA-2 or GATA-3 significantly enhanced clioquinol-induced IL-8 mRNA expression.
These results suggest that the clioquinol-induced suppression of GATA-2 and GATA-3 expression mediates the up-regulation of IL-8.

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