Bethan K. Davies: Extracellular pH is a critical regulator of osteoclast fusion, size and activation

Osteoclast formation is a complex, multi-step process that requires macrophage-colony stimulating factor (M-CSF), receptor activator of nuclear factor κ-B ligand (RANKL) and nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1) for precursor proliferation and commitment, respectively [[1], [2], [3]]. Mononuclear pre-osteoclasts fuse at the bone surface to form multinuclear, mature functional osteoclasts in a process that is not fully understood. Osteoclast fusion is facilitated by many factors including dendritic cell-specific transmembrane protein (DC-STAMP), osteoclast stimulatory transmembrane protein (OC-STAMP), the vacuolar H+-ATPase (vATPase) subunit ATPv60d2, syncytin-B and CD47 [[4], [5], [6], [7], [8]]. To resorb, osteoclasts undergo morphological and ultrastructural changes, principally polarisation, through sealing zone and ruffled border formation. Osteoclast polarisation up-regulates resorption-associated genes (e.g., carbonic anhydrase II (CAII) and vATPase) [9,10]. Proton (H+) and chloride (Cl−) ions are subsequently pumped into the resorption lacunae through the ruffled border to dissolve the hydroxyapatite mineral. The demineralised bone matrix is then degraded by proteases including cathepsin K, matrix metalloproteinases (MMPs) and tartrate resistant acid phosphatase (TRAP).

Systemic acidosis occurs through the dysregulation of the acid-base balance and its profound negative effect on the skeleton is well-characterised [11]. To buffer excess H+ concentrations under acidosis, alkaline material, specifically calcium carbonate, is released from bone [11,12]. Early studies showed that calcium release from live bones was consistent over a physiological pH range (pH 7.03–7.49) but was inhibited at extreme pH (<6.8 or > 7.4) [13]. This, and other work, concluded that acidosis can indirectly stimulate bone resorption by physicochemical dissolution of bone mineral [14]. Other studies, however, showed that parathyroid hormone amplified calcium release, whereas sodium azide (a metabolic inhibitor) inhibited it, implying that mechanisms other than mineral dissolution are involved [15].

The first direct demonstration that osteoclast activity is modulated by protons was by Arnett and Dempster [16]. This and subsequent studies have shown that a step-wise reduction in medium pH between pH 7.4 and pH 6.8 produces a graded increase in resorption area and pit number, depth and width [[16], [17], [18]]. Little or no resorption occurs at a neutral pH (pH 7.4), whereas resorption increases significantly as the pH becomes more acidic. Specifically, osteoclast activity is up to 5-, 9- and 14-fold higher at pH 7.2, 7.0 and 6.8, respectively [[16], [17], [18]]. In fact, resorption pit formation increased 6-fold over pH 7.25 and 7.15, demonstrating that even small changes in pH sensitively modulate osteoclast function [18]. Studies in mouse calvariae cultured in HCl-acidified media similarly report dose-dependent increases in calcium release [19,20]. This H+-induced calcium release was blocked by indomethacin, a prostaglandin inhibitor, and calcitonin, reinforcing the cellular component in bone resorption [19,20]. The acid sensitivity of osteoclast-mediated bone resorption has now been reported in rodent, chick, rabbit, and feline osteoclasts [16,19,[21], [22], [23]].

Acidosis also promotes intracellular and cytoskeletal changes in osteoclasts. For example, acid pH induces TRAP, cathepsin K and CAII mRNA expression for subsequent resorptive function [9]. Similarly, extracellular acidification of osteoclasts to pH 7.0 decreases intracellular pH and Ca2+ concentrations and increases sealing zone formation for attachment to the bone matrix [21]. Conversely, alkalinisation of osteoclasts curtailed sealing zone formation [21,24]. Together, these data indicate that pH regulates the “off/on switch” for osteoclast activity.

The role of extracellular pH on osteoclast formation and fusion, however, is less well-characterised. In a co-culture system of an osteoblast-like cell line and osteoclasts, physiologically low pH (6.8–7.0) was reported to increase the formation of larger, more numerous osteoclasts, whereas pH 7.4 inhibited osteoclast formation [25,26]. However, a preliminary investigation in our laboratory using purified osteoclast-forming mouse marrow cultures on dentine suggested the opposite result [27]. The aim of the present study was, therefore, to characterise in vitro the direct actions of low pH on the formation, fusion, and function of osteoclasts.

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