Effects of Bafilomycin A1: An inhibitor of vacuolar H (+)‐ATPases on endocytosis and apoptosis in RAW cells and RAW cell‐derived osteoclasts

Abstract

Bafilomycin A1, a specific inhibitor of V-ATPases, is a potent inhibitor of bone resorption, but the underlying mechanisms of its action remain unclear. In this study, we investigated the effect of Bafilomycin A1 on endocytosis and apoptosis in RAW cells and RAW cell-derived osteoclasts. Quantitative analysis by flow cytometry showed that Bafilomycin A1 increased total transferrin levels when RAW cells were exposed to labeled transferrin and decreased the total uptake of Dextran-rhodamine B, both in a dose- and time-dependent fashion, indicating that Bafilomycin influences receptor-mediated and fluid phase endocytosis in these cells. Furthermore, Bafilomycin A1 induced apoptosis of RAW cells in a dose dependent manner as evidenced by Annexin V flow cytometry. The action of Bafilomycin A1 on endocytotic events appeared to be more sensitive and occurred earlier than on its apoptosis inducing effects, suggesting that interrupting of endocytosis might be an early sign of Bafilomycin-mediated osteoclast inhibition. Semi-quantitative RT-PCR analysis showed that the gene transcripts of putative Bafilomycin A1 binding subunit, V-ATPase-subunit a3, were expressed in the preosteoclastic RAW cell line, and up-regulated during RANKL-induced osteoclastogenesis. Osteoclasts treated with Bafilomycin A1 exhibited apoptosis as well as altered cellular localization of Transferrin Alexa 647. Given that endocytosis and apoptosis are important processes during osteoclastic bone resorption, the potent effect of Bafilomycin A1 on endocytosis and apoptosis of osteoclasts and their precursor cells may in part account for Bafilomycin A1 inhibited bone resorption.

Selective upregulation of TNFα expression in classically-activated human monocyte-derived macrophages (M1) through pharmacological interference with V-ATPase

“…Previous studies showed that macrophages from different origins and species express V-ATPase and respond to VATPase inhibitors in different ways [13,31,[34][35][36][37][38][39]. We first confirmed V-ATPase expression in human monocyte-derived macrophages [40] by Western blot (not shown) and by using immunofluorescence microscopy where no obvious differences regarding V-ATPase subcellular localization between the macrophage phenotypes were immediately apparent (Fig.…”

“…Pharmacological manipulation of V-ATPase in macrophages by respective inhibitors such as bafilomycin A1 was reported before [13,31,35,37,38,48,50]. Many of these studies focused on osteoclasts [51], macrophage cell lines [31] and macrophages from rodents [37,39,48] and from other animals [52], but only few studies addressed V-ATPase functions in human primary macrophages [40,50], and to the best of our knowledge there are no reports that studied the role of V-ATPase on cytokine/chemokine secretion of different macrophage phenotypes, such as M1 and M2.…”

“…Many of these studies focused on osteoclasts [51], macrophage cell lines [31] and macrophages from rodents [37,39,48] and from other animals [52], but only few studies addressed V-ATPase functions in human primary macrophages [40,50], and to the best of our knowledge there are no reports that studied the role of V-ATPase on cytokine/chemokine secretion of different macrophage phenotypes, such as M1 and M2. Therefore, one significant novelty of our study is the consideration of distinct human macrophage subtypes, that is, uncommitted M(0), classically-activated M1-like and so-called "resolving" M2-like macrophages that significantly differ in their bioactions [53].…”

Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity

“…Acidic pH ex benefits tumor cells because it promotes invasiveness, whereas an alkaline pH cyt gives them a competitive advantage over normal cells for growth. Furthermore, V-ATPase is anti-apoptotic in tumor cells (49,59).…”

Bergapten prevents lipopolysaccharide mediated osteoclast formation, bone resorption and osteoclast survival

“…Hence, we supposed that apoptosis of osteoclasts and their precursors may contribute to the abovedescribed inhibition. New evidence demonstrates that triggering osteoclast apoptosis occurs via a variety of extracellular stimuli, such as estrogens [18][19][20], bisphosphonates [21] and bafilomycin A1 [22]. The consequences are diminished bone loss and reduced bone turnover.…”