Oxidation derivative of (-)-epigallocatechin-3-gallate (EGCG) inhibits RANKL-induced osteoclastogenesis by suppressing RANK signaling pathways in RAW 264.7 cells

Biomed Pharmacother. 2019 Oct:118:109237. doi: 10.1016/j.biopha.2019.109237. Epub 2019 Jul 31.

Abstract

Tea consumption has positive effects on the skeletal system and prevents postmenopausal osteoporosis, mainly by inhibiting osteoclastogenesis. In green tea, (-)-epigallocatechin-3-gallate (EGCG) is the most abundant and active compound and has been shown to inhibit RANKL-induced osteoclast formation. Taking into account the highly oxidizable and unstable nature of EGCG, we hypothesized that EGCG oxidation product exhibits greater anti-osteoclastogenesis potential than EGCG. In this study, we successfully isolated and identified an EGCG oxidation derivative, (-)-gallocatechin gallate (compound 2), using a chemical oxidation strategy. We then compared the ability of compound 2 and EGCG to inhibit RANKL-induced osteoclastogenesis in RAW 264.7 cells. The results of TRAP staining and F-actin ring immunofluorescent staining showed that compound 2 exhibits stronger inhibition of RANKL-induced osteoclast differentiation and F-actin ring formation, respectively, than EGCG. Additionally, quantitative real-time PCR (qRT-PCR) and western blotting analyses showed that compound 2 significantly and more strongly inhibited the expression of osteoclastogenesis-related marker genes and proteins, including c-Src, TRAP, cathepsin K, β3-Integrin, and MMP-9, compared with EGCG. Furthermore, compound 2 significantly suppressed RANKL-induced expression of NFATc1 and c-Fos, the master transcriptional regulators of osteoclastogenesis, more strongly than EGCG. Mechanistically, molecular interaction assays showed that compound 2 binds to RANK with high affinity (KD = 189 nM) and blocks RANKL-RANK interactions, thereby suppressing RANKL-induced early RANK signaling pathways including p65, JNK, ERK, and p38 in osteoclast precursors. Taken together, this study demonstrates for the first time that an oxidation derivative of EGCG (compound 2) inhibits RANKL-induced osteoclastogenesis by suppressing RANK signaling pathways in RAW 264.7 cells.

Keywords: EGCG; NFATc1; Osteoclastogenesis; Oxidation derivative; RANK signaling pathways; c-Fos.

MeSH terms

  • Actins / metabolism
  • Animals
  • Carbon-13 Magnetic Resonance Spectroscopy
  • Catechin / analogs & derivatives*
  • Catechin / chemistry
  • Catechin / pharmacology
  • Cell Differentiation / drug effects
  • Gene Expression Regulation / drug effects
  • Mice
  • Mitogen-Activated Protein Kinases / metabolism
  • NFATC Transcription Factors / genetics
  • NFATC Transcription Factors / metabolism
  • Osteoclasts / cytology
  • Osteoclasts / drug effects
  • Osteoclasts / metabolism
  • Osteogenesis / drug effects*
  • Oxidation-Reduction
  • Phosphorylation / drug effects
  • Proto-Oncogene Proteins c-fos / genetics
  • Proto-Oncogene Proteins c-fos / metabolism
  • Proton Magnetic Resonance Spectroscopy
  • RANK Ligand / pharmacology*
  • RAW 264.7 Cells
  • Receptor Activator of Nuclear Factor-kappa B / metabolism*
  • Signal Transduction / drug effects*
  • Transcription Factor RelA / metabolism

Substances

  • Actins
  • NFATC Transcription Factors
  • Proto-Oncogene Proteins c-fos
  • RANK Ligand
  • Receptor Activator of Nuclear Factor-kappa B
  • Transcription Factor RelA
  • gallocatechin gallate
  • Catechin
  • Mitogen-Activated Protein Kinases