Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Abstract

UNLABELLED:

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts.

INTRODUCTION:

Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts.

MATERIALS AND METHODS:

Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1.

RESULTS:

In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic.

CONCLUSIONS:

We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.

• Authors: Mette G Sørensen, Kim Henriksen, Anita V Neutzsky-Wulff, Morten H Dziegiel, Morten A Karsdal
• Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research (J Bone Miner Res) Vol. 22 Issue 10 Pg. 1640-8 (Oct 2007) ISSN: 0884-0431 [Print] United States
• PMID: 17576165 (Publication Type: Journal Article)

Chemical References

• Acids
• Benzodioxoles
• Dioxolanes
• Enzyme Inhibitors
• Lignans
• Vacuolar Proton-Translocating ATPases
• diphyllin

Topics

• Acids (metabolism)
• Adrenal Medulla (drug effects, metabolism)
• Animals
• Benzodioxoles
• Biological Transport
• Bone Resorption (enzymology, pathology)
• Cattle
• Cell Survival (drug effects)
• Cells, Cultured
• Dioxolanes (chemistry, pharmacology)
• Enzyme Inhibitors (chemistry, pharmacology)
• Humans
• Hydrogen-Ion Concentration
• Lignans (chemistry, pharmacology)
• Molecular Structure
• Osteoclasts (cytology, drug effects)
• Vacuolar Proton-Translocating ATPases (antagonists & inhibitors, metabolism)