LL-37-induced human osteoblast cytotoxicity and permeability occurs independently of cellular LL-37 uptake through clathrin-mediated endocytosis

The host defense peptide LL-37 is cytotoxic for bacteria but it has also been reported to reduce host cell viability through an intracellular mechanism. LL-37-evoked cytotoxicity may be involved in the loss of bone tissue in periodontitis which is an inflammatory disease characterized by high concentrations of LL-37 observed locally in the periodontal tissue at the inflammation process. Here, we showed that LL-37 reduced human osteoblast-like MG63 cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and increased plasma membrane permeability determined by measuring intracellular Ca²⁺ levels and lactate dehydrogenase (LDH) release. Treatment with chlorpromazine, a well-recognized inhibitor of clathrin-mediated endocytosis, reduced cellular uptake of synthesized LL-37 b y about 30% assessed by Western blotting and ELISA, while filipin, an inhibitor of caveolin-mediated endocytosis, had no effect. The chlorpromazine-induced attenuation of LL-37 uptake was not associated with modulation of LL-37-induced cytotoxicity and LL-37-evoked plasma membrane permeability. Clathrin heavy chain 2 is a major protein of the polyhedral coat of coated pits and vesicles encoded by clathrin heavy chain like 1 gene. Down-regulation of clathrin heavy chain like 1 gene activity by siRNA reduced uptake of LL-37 but did not affect LL-37-induced cytotoxicity and permeability. Thus, we show, using both a pharmacological approach and knockdown of clathrin heavy chain like 1 expression, that LL-37-induced MG63 cell cytotoxicity and permeability occurs independently of LL-37 uptake via clathrin-mediated endocytosis.