Diphyllin, a Novel and Naturally Potent V-ATPase Inhibitor, Abrogates Acidification of the Osteoclastic Resorption Lacunae and Bone Resorption

Abstract

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 macrophagecolony 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 (IC 50 ‫ס‬ 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC 50 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 (IC 50 ‫ס‬ 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.

Cleistanthin A inhibits the invasion and metastasis of human melanoma cells by inhibiting the expression of matrix metallopeptidase‑2 and ‑9

“…It has also been demonstrated that diphyllin inhibits V-ATPase, and therefore, lysosomal acidification in osteoclasts, which leads to the abrogation of bone resorption (18). The present study suggested that CA inhibits the expression of V-ATPases on A375 cells at concentrations of 0.03, 0.1 or 0.3 µM.…”

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

“…26 In renal principal cells, 4AD interferes with acidification of intracellular vesicles (Figure 4) and leads to inhibition of the PKAdependent phosphorylation of AQP2 at S256, the key trigger for its redistribution to the plasma membrane ( Figure 7B). This effect cannot be explained by global inhibition of PKA because 4AD does not affect total cellular PKA activity ( Figure 6C).…”

“…26,49,50 However, because of the vital role of V-ATPases in normal physiology, adverse effects on blocking of V-ATPase may be expected. Hence, it is unclear whether such candidates reach the clinic.…”

“…Accumulation of AQP2 in the Golgi Compartment 4AD is a selective blocker of vacuolar H + -ATPases(V-ATPases); 26 to compare its effects with another inhibitor of V-ATPases, we used bafilomycin A1 (baf A1). 27,28 Baf A1 also blocked the forskolin-induced AQP2 translocation in IMCD cells ( Figure 4A).…”

Dissociation of Bone Resorption and Bone Formation in Adult Mice with a Non-Functional V-ATPase in Osteoclasts Leads to Increased Bone Strength

“…TRAP activity in cell culture medium was measured as described previously [32]. Briefly, samples were incubated with TRAP reaction buffer, containing p-nitrophenyl phosphate and sodium tartrate, for 1 hour at 37uC in the dark.…”