Interleukin-6/Soluble IL-6 Receptor-Induced Secretion of Cathepsin B and L from Human Gingival Fibroblasts is Regulated by Caveolin-1 and ERK1/2 Pathways

Ayaka Goto Kazuhiro Omori Tomoko Yamaguchi-Tomikawa Hiroya Kobayashi Yuki Shinoda-Ito Kimito Hirai Atsushi Ikeda Shogo Takashiba ^*

• Okayama University, Okayama, Japan

Aims: Cathepsins are essential lysosomal enzymes that maintain organismal homeostasis by degrading extracellular substrates. The inflammatory cytokine interleukin-6 (IL-6) increases the production of cathepsins through the caveolin-1 (Cav-1) and c-Jun N-terminal kinase (JNK) signaling pathways, which have been implicated in the destruction of periodontal tissue. This study investigated the effect of the IL-6/soluble IL-6 receptor (sIL-6R) complex on the extracellular secretion of cathepsins in human gingival fibroblasts (HGFs) and examined the function of extracellularly secreted cathepsins B and L under acidic culture conditions in vitro.Methods: HGFs were isolated from healthy volunteer donors. The expression of Cav-1 was suppressed via transfection with small interfering RNA (siRNA) targeting Cav-1. The expression levels of cathepsins B and L induced by extracellular IL-6/sIL-6R were measured using western blotting and enzyme-linked immunosorbent assay.Extracellular cathepsin activity following IL-6/sIL-6R stimulation was assessed using a methylcoumarylamide substrate in a fluorescence-based assay. IL-6/sIL-6R-induced expression of cathepsins B and L in HGFs was quantified under inhibitory conditions for extracellular signal-regulated kinase (ERK) 1/2 and/or JNK signaling, both of which are transduction pathways activated by IL-6/sIL-6R. This quantification was also performed in HGFs with suppressed Cav-1 expression using western blotting.Results: Cathepsins B and L were secreted in their precursor forms from HGFs, with significantly elevated protein levels observed at 24, 48, and 72 h post-IL-6/sIL-6R stimulation. Under acidic culture conditions, cathepsin B activity increased at 48 and 72 h. Cav-1 suppression inhibited the secretion of cathepsin B regardless of IL-6/sIL-6R stimulation, whereas the secretion of cathepsin L was reduced only after 48 h of IL-6/sIL-6R stimulation. Inhibition of ERK1/2 and JNK pathways decreased the secretion of cathepsin B after 48 h of IL-6/sIL-6R stimulation, and JNK inhibition reduced the secretion of cathepsin L under similar conditions.IL-6/sIL-6R stimulation increased the extracellular secretion of cathepsin B and L precursors in HGFs, and these precursors became activated under acidic conditions. Cav-1 and ERK1/2 are involved in regulating the secretion of cathepsin B precursors.