Calcium signaling pathway involving calcineurin regulates interleukin-8 gene expression through activation of NF-kappaB in human osteoblast-like cells.
Involvement of aberrant IL-8 production by osteoblasts was demonstrated in pathogenesis of inflammatory joint diseases. We thus investigated intracellular signaling pathways leading to IL-8 expression in human osteoblast-like HOS-TE85 cells. It was demonstrated that Ca2+ signaling pathway involving calcineurin regulates IL-8 gene expression through activation of a transcription factor, NF-kappaB.


BACKGROUND
Involvement of aberrant interleukin (IL)-8 production by osteoblasts was demonstrated in pathogenesis of inflammatory joint diseases. However, intracellular signaling pathways leading to IL-8 expression in osteoblasts have been poorly explored. Because a variety of external stimuli was shown to increase intracellular Ca2+ in osteoblasts, we investigated effects of Ca(2+)-ionophore and phorbol-myristate-acetate (Ion/PMA) on IL-8 expression in human osteoblast-like HOS-TE85 cells and compared the effects with those elicited by TNF-alpha.


METHODS
HOS-TE85 cells were treated with Ion/PMA or TNF-alpha in the presence and absence of calcineurin inhibitors (CnI), cyclosporin A, and FK506. IL-8 mRNA levels and its promoter activities were examined by Northern blot and luciferase reporter analyses, respectively. Electrophoretic mobility shift assay (EMSA) was used to evaluate DNA binding activities of transcription factors such as NF-kappaB. Degradation of IkappaB, a cytoplasmic NF-kappaB-inhibitory protein, was examined by Western blot analysis.


RESULTS
Ion/PMA and TNF-alpha induced IL-8 mRNA expression. Interestingly, CnI attenuated the induction by Ion/PMA, but not that by TNF-alpha. Promoter activity was also increased by both stimuli, and only the Ion/PMA-dependent increase was suppressed by CnI. Introduction of mutations in the promoter demonstrated that one NF-kappaB site was responsible for the suppression by CnI. EMSA revealed that this site binds with NF-kappaB containing p65 that was activated by Ion/PMA and TNF-alpha and that CnI inhibited only Ion/PMA-dependent NF-kappaB activation. Accordingly, CnI blocked only Ion/PMA-dependent degradation of IkappaB-alpha. In addition, the basal and Ion/PMA-dependent IL-8 promoter activities were enhanced by co-transfection of constitutively active calcineurin.


CONCLUSIONS
These results show that the Ca2+ signaling pathway involving calcineurin regulates IL-8 gene expression through activation of NF-kappaB in human osteoblast-like cells.
Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan.
Hirohito
H
Mitsuyama
Fukushi
F
Kambe
Ryuichiro
R
Murakami
Xia
X
Cao
Naoki
N
Ishiguro
Hisao
H
Seo
D000970:Antineoplastic Agents / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D019703:Calcineurin / Q000096:biosynthesis*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics*; D002460:Cell Line; D016572:Cyclosporine / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D007476:Ionophores / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D016559:Tacrolimus / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000970:Antineoplastic Agents; D007166:Immunosuppressive Agents; D016209:Interleukin-8; D007476:Ionophores; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D000001:Calcimycin; D016572:Cyclosporine; D019703:Calcineurin; D013755:Tetradecanoylphorbol Acetate; D016559:Tacrolimus