1,25-Dihydroxyvitamin D3 Attenuates the Effects of Lipopolysaccharide by Causing ADAM10-Dependent Ectodomain Shedding of Toll-Like Receptor 4

Abstract

Background/Aims: We investigated how 1,25-dihydroxyvitamin D₃ (1,25D₃) inhibits the effects of lipopolysaccharide (LPS) in human aortic endothelial cells. Methods: Cellular signaling was explored by determination of protein abundance with Western blot, measurement of cytosolic Ca²⁺ concentration and immunofluorescence staining for a disintegrin and metalloprotease 10 (ADAM10). Results: LPS stimulated the expression of intercellular adhesion molecule 1 (ICAM-1) through toll-like receptor 4 (TLR4) and subsequent activation of p38 mitogen-activated protein kinase (p38 MAPK). Pretreatment with 1,25D₃ attenuated LPS-induced p38 MAPK activation and ICAM-1 expression by causing ectodomain shedding of TLR4. This effect of 1,25D₃ depended on its ability to induce a rapid extracellular Ca²⁺ influx through L-type calcium channels because the ectodomain shedding was prevented by the absence of extracellular Ca²⁺ or the presence of verapamil. TLR4 ectodomain shedding was also induced by Bay K8644 (L-type calcium channel agonist). Both 1,25D₃ and Bay K8644 caused extracellular Ca²⁺ influx-dependent ADAM10 translocation to the cell surface. Depletion of ADAM10 by siRNA transfection prevented 1,25D₃- and Bay K8644-induced ectodomain shedding of TLR4, and abolished the inhibitory effect of 1,25D₃ on LPS-induced ICAM-1 expression. Conclusion: 1,25D₃ causes ectodomain shedding of TLR4 and thereby decreases the responsiveness of cells to LPS. ADAM10, activated by extracellular Ca²⁺ influx, was implicated in the ectodomain cleavage of TLR4.

Diosgenin, an Activator of 1,25D3-MARRS Receptor/ERp57, Attenuates the Effects of TNF-α by Causing ADAM10-Dependent Ectodomain Shedding of TNF Receptor 1

“…Increase in cytosolic Ca 2+ either by extracellular Ca 2+ influx induced by vitamin D 3 or Bay K8644 or by release of intracellular stored Ca 2+ induced by epigallocatechin-3-gallate, thapsigargin or cyclopiazonic acid was shown to activate ADAM10 in our previous studies [10, 11, 20, 21]. The findings in the present study are consistent with these studies.…”

ADAM17-Mediated Ectodomain Shedding of Toll-Like Receptor 4 as a Negative Feedback Regulation in Lipopolysaccharide-Activated Aortic Endothelial Cells

“…In our previous study [16], LPS stimulated ICAM-1 expression, through TLR4-p38 MAPK axis, with a peak value at 8 h and then a significant decrease at 24 h. HAECs were transfected with control-siRNA or ADAM17-siRNA, and then incubated with LPS for 24 h to determine the delayed effect of LPS. As shown in Fig.…”

“…Ectodomain shedding of TLR4 following LPS stimuli was dependent on ADAM17 as it was completely prevented by specific depletion of ADAM17 by siRNA transfection as well as TAPI-2, a broad spectrum ADAM inhibitor. Recently, we reported that 1,25D 3 induces ectodomain shedding of TLR4 in HAECs [16]. In this case, ADAM10, activated by extracellular Ca 2+ influx, was implicated in ectodomain cleavage of TLR4.…”

“…Endothelial cells also express TLR4 and are known to be activated by LPS through TLR4 [15, 16]. However, LPS signaling in endothelial cells may not be the same with that in monocytes/macrophages.…”

“…Recently, we found that TLR4 is subject to ectodomain shedding by ADAM10 when human aortic endothelial cells (HAECs) are treated with 1,25-dihydroxyvitamin D 3 (1,25D 3 ) or Bay K8644, an L-type calcium channel agonist [16]. Ectodomain shedding of TLR4 resulted in desensitization of the cells to LPS stimuli.…”