Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorption

doi:10.1016/0006-291X(90)91709-2

Biochemical and Biophysical Research Communications

Volume 168, Issue 1, 16 April 1990, Pages 309-313

https://doi.org/10.1016/0006-291X(90)91709-2 Get rights and content

Abstract

Osteoclasts are the main bone resorbing cells with capacity to acidify their intimate contact area with bone. Recent studies have suggested that osteoclast acid secretion is carried out by an H⁺-ATPase. We demonstrate here, that specific inhibitor of vacuolar type H⁺-ATPase, bafilomycin A₁, inhibits bone resorption in osteoclast cultures as well as blocks proton transport in isolated medullary bone derived microsomes containing a vacuolar type H⁺-ATPase. These results demonstrate an important role of vacuolar H⁺-ATPase in bone resorption.

References (11)

M. Bradford
Anal. Biochem
(1976)
M.D. Fallon
R. Baron et al.
J. Cell Biol
(1985)
G. Vaes
Clin. Orthop
(1988)
H.C. Blair et al.
Science
(1989)

There are more references available in the full text version of this article.

Cited by (161)

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2012, European Journal of Cell Biology
Citation Excerpt :
The V-ATPase is a multiprotein complex which has been found to be localized to the plasma membrane of osteoclasts (Mattsson et al., 1994) and proposed to regulate osteoclast pHi (Nordstrom et al., 1995). Indeed the expression level of both CA2 and subunits of V-ATPase have been shown to be associated with the osteoclastic proteolytic function (Laitala and Vaananen, 1993) and both their expressional or pharmacological (bafilomycin A1) inhibition were found to inhibit bone resorption (Laitala and Vaananen, 1994; Sundquist et al., 1990). Moreover mutations in genes encoding subunits of the V-ATPase have been shown to be associated with severe bone diseases such as osteopetrosis (Kornak et al., 2000; Li et al., 1999; Scimeca et al., 2000).
Invadosomes are actin-rich finger-like cellular structures sensing and interacting with the surrounding extracellular matrix (ECM) and involved in its proteolytic remodeling. Invadosomes are structures distinct from other adhesion complexes, and have been identified in normal cells that have to cross tissue barriers to fulfill their function such as leukocytes, osteoclasts and endothelial cells. They also represent features of highly aggressive cancer cells, allowing them to escape from the primary tumor, to invade surrounding tissues and to reach systemic circulation. They are localized to the ventral membrane of cells grown under 2-dimensional conditions and are supposed to be present all around cells grown in 3-dimensional matrices. Indeed invadosomes are key structures in physiological processes such as inflammation and the immune response, bone remodeling, tissue repair, but also in pathological conditions such as osteopetrosis and the development of metastases. Invadosomes are subdivided into podosomes, found in normal cells, and into invadopodia specific for cancer cells. While these two structures exhibit differences in organization, size, number and half-life, they share similarities in molecular composition, participation in cell-matrix adhesion and promoting matrix degradation. A key determinant in invadosomal function is the recruitment and release of proteases, such as matrix metalloproteinases (MMPs), serine proteases and cysteine cathepsins, together with their activation in a tightly controlled and highly acidic microenvironment. Therefore numerous pH regulators such as V-ATPases and Na⁺/H⁺ exchangers, are found in invadosomes and are directly involved in their constitution as well as their functioning. This review focuses on the participation of pH regulators in invadosome function in physiological and pathological conditions, with a particular emphasis on ECM remodeling by osteoclasts during bone resorption and by cancer cells.
V-ATPases in osteoclasts: Structure, function and potential inhibitors of bone resorption
2012, International Journal of Biochemistry and Cell Biology
The vacuolar-type H⁺-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V₁ and V₀. The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.
Degradation of hydroxyapatite in vivo and in vitro requires osteoclastic sodium-bicarbonate co-transporter NBCn1
2010, Matrix Biology
Dissolution of the inorganic bone matrix releases not only calcium and phosphate ions, but also bicarbonate. Electroneutral sodium-bicarbonate co-transporter (NBCn1) is expressed in inactive osteoclasts, but its physiological role in bone resorption has remained unknown. We show here that NBCn1, encoded by the SLC4A7 gene, is directly involved in bone resorption. NBCn1 protein was specifically found at the bone-facing ruffled border areas, and metabolic acidosis increased NBCn1 expression in rats in vivo. In human hematopoietic stem cell cultures, NBCn1 mRNA expression was observed only after formation of resorbing osteoclasts. To further confirm the critical role of NBCn1 during bone resorption, human hematopoietic stem cells were transduced with SLC4A7 shRNA lentiviral particles. Downregulation of NBCn1 both on mRNA and protein level by lentiviral shRNAs significantly inhibited bone resorption and increased intracellular acidification in osteoclasts. The lentiviral particles did not impair osteoclast survival, or differentiation of the hematopoietic or mesenchymal precursor cells into osteoclasts or osteoblasts in vitro. Inhibition of NBCn1 activity may thus provide a new way to regulate osteoclast activity during pathological bone resorption.
Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7
2009, Biochemical and Biophysical Research Communications
The chloride-proton antiporter ClC-7 has been speculated to be involved in acidification of the lysosomes and the resorption lacunae in osteoclasts; however, neither direct measurements of chloride transport nor acidification have been performed.
Human osteoclasts harboring a dominant negative mutation in ClC-7 (G215R) were isolated, and used these to investigate bone resorption measured by CTX-I, calcium release and pit scoring. The actin cytoskeleton of the osteoclasts was also investigated. ClC-7 enriched membranes from the osteoclasts were isolated, and used to test acidification rates in the presence of a V-ATPase and a chloride channel inhibitor, using a H⁺ and Cl⁻ driven approach. Finally, acidification rates in ClC-7 enriched membranes from ADOII osteoclasts and their corresponding controls were compared.
Resorption by the G215R osteoclasts was reduced by 60% when measured by both CTX-I, calcium release, and pit area when comparing to age and sex matched controls. In addition, the ADOII osteoclasts showed no differences in actin ring formation. Finally, V-ATPase and chloride channel inhibitors completely abrogated the H⁺ and Cl⁻ driven acidification. Finally, the acid influx was reduced by maximally 50% in the ClC-7 deficient membrane fractions when comparing to controls.
These data demonstrate that ClC-7 is essential for bone resorption, via its role in acidification of the lysosomes and resorption lacunae in osteoclasts.
Osteoclast lineage and function
2008, Archives of Biochemistry and Biophysics
Osteoclasts are members of the monocyte/macrophage lineage and are formed by cellular fusions from their mononuclear precursors. Their differentiation is regulated by a number of other cells and their products, especially by RANKL and M-CSF. The resorbing osteoclasts are polarized and show specific plasma membrane domains. Polarization and bone resorption need a continuous membrane trafficking and modulation of the cytoskeleton. The most characteristic feature of osteoclasts is their unique capacity to dissolve crystalline hydroxyapatite by targeted secretion of HCl into the extracellular resorption lacuna. Organic matrix is degraded by enzymes like cathepsin K and the degradation products are transcytosed through the cell for secretion. Dissolution of hydroxyapatite releases large amounts of soluble calcium, phosphate and bicarbonate. Removal of these ions apparently involves the vesicular pathways and direct ion transport via different ion exchangers, channels and pumps. Detailed molecular knowledge of osteoclast differentiation and function has helped us to identify several target molecules and develop specific treatments to inhibit pathological bone resorption in various skeletal diseases.
Visualizing mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells
2008, Bone
Bisphosphonates (BPs) target bone due to their high affinity for calcium ions. During osteoclastic resorption, these drugs are released from the acidified bone surface and taken up by osteoclasts, where they act by inhibiting the prenylation of small GTPases essential for osteoclast function. However, it remains unclear exactly how osteoclasts internalise BPs from bone and whether other cells in the bone microenvironment can also take up BPs from the bone surface. We have investigated this using a novel fluorescently-labelled alendronate analogue (FL-ALN), and by examining changes in protein prenylation following treatment of cells with risedronate (RIS). Confocal microscopic analysis showed that FL-ALN was efficiently internalised from solution or from the surface of dentine by resorbing osteoclasts into intracellular vesicles. Accordingly, unprenylated Rap1A accumulated to the same extent whether osteoclasts were cultured on RIS-coated dentine or with RIS in solution. By contrast, J774 macrophages internalised FL-ALN and RIS from solution, but took up comparatively little from dentine, due to their inability to resorb the mineral. Calvarial osteoblasts and MCF-7 tumour cells internalised even less FL-ALN and RIS, both from solution and from the surface of dentine. Accordingly, the viability of J774 and MCF-7 cells was drastically reduced when cultured with RIS in solution, but not when cultured on dentine pre-coated with RIS. However, when J774 macrophages were co-cultured with rabbit osteoclasts, J774 cells that were adjacent to resorbing osteoclasts frequently internalised more FL-ALN than J774 cells more distant from osteoclasts. This was possibly a result of increased availability of BP to these J774 cells due to transcytosis through osteoclasts, since FL-ALN partially co-localised with trancytosed, resorbed matrix protein within osteoclasts. In addition, J774 cells occupying resorption pits internalised more FL-ALN than those on unresorbed surfaces. These data demonstrate that osteoclasts are able to take up large amounts of BP, due to their ability to release the BP from the dentine surface during resorption. By contrast, non-resorbing cells take up only small amounts of BP that becomes available due to natural desorption from the dentine surface. However, BP uptake by non-resorbing cells can be increased when cultured in the presence of resorbing osteoclasts.

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Inhibition of osteoclast proton transport by bafilomycin A1 abolishes bone resorption

Abstract

Anal. Biochem

J. Cell Biol

Clin. Orthop

Science

Inhibition of osteoclast proton transport by bafilomycin A₁ abolishes bone resorption