lipid-a and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Alendronate (ALN) is a nitrogen-containing bisphosphonate (NBP) that inhibits bone resorption. NBPs have inflammatory side effects, and ALN augments bacteria-induced interleukin (IL)-1β production. The present study aimed to examine whether ALN induces pyroptosis, a form of cell death associated with IL-1β release, in macrophage-like J774.1 cells incubated with lipid A, a component of gram-negative bacteria. Pretreatment of J774.1 cells with ALN increased lipid A-induced IL-1β production and cell death, but not IL-6 and TNF-α production. Ac-YVAD-CHO, a caspase-1 inhibitor, inhibited ALN-augmented IL-1β production induced by lipid A, although it did not affect ALN-induced cell death. Moreover, Ac-IETD-CHO, a caspase-8 inhibitor, and Z-VAD-FMK, a pan-caspase inhibitor, did not inhibit ALN-induced cell death, suggesting that the effects of ALN are exerted independently of caspase activation. We also demonstrate that a Smad3 inhibitor (SIS3) suppressed ALN-augmented IL-1β production. Moreover, SIS3 attenuated ALN-augmented release of LDH and caspase-1. These results suggest that ALN augments IL-1β production, cell death, and caspase-1 release in a manner dependent on Smad3. We then investigated whether ALN-augmented IL-1β production and cell death are dependent on apoptosis-associated speck-like protein containing a CARD (ASC) and NOD-like receptor pyrin domain containing-3 (NLRP3), which are associated with Smad3 activation. Both anti-ASC and anti-NLRP3 antibodies suppressed ALN-induced cell death and caspase-1 release, but only anti-ASC antibody inhibited ALN-augmented IL-1β production. Our findings suggest that ALN-augmented IL-1β production and cell death require Smad3 and ASC activation, and that SIS3 and anti-ASC antibodies may serve as palliative agents for necrotizing inflammatory diseases caused by ALN.

Topics: Alendronate; Amino Acid Chloromethyl Ketones; Animals; CARD Signaling Adaptor Proteins; Caspase 1; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Inflammation; Interleukin-1beta; Interleukin-6; Lipid A; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Protein Domains; Smad3 Protein; Tumor Necrosis Factor-alpha