SLC4A2-mediated Cl ⁻ /HCO ₃ ⁻ exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts

Abstract

Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H + -ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl − permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2's long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.

Subcellular Elevation of Cytosolic Free Calcium Is Required for Osteoclast Migration

“…However, extended uropodia are observed in other situations, such as blocking myosin II assembly in neutrophils, reflecting the interplay of effectors in cell migration. In this regard, it has recently been shown that osteoclasts lacking the anion exchanger SLC4A2 show reduced spreading and reduced motility, changes attributed to altered pH homeostasis and dysregulation of calpain‐dependent podosome disassembly . This points to the complex orchestration of events underlying cellular retraction, of which Ca 2+ is a key contributor.…”

“…In this regard, it has recently been shown that osteoclasts lacking the anion exchanger SLC4A2 show reduced spreading and reduced motility, changes attributed to altered pH homeostasis and dysregulation of calpain-dependent podosome disassembly. (35) This points to the complex orchestration of events underlying cellular retraction, of which Ca 2þ is a key contributor.…”

Ae4 (Slc4a9) Anion Exchanger Drives Cl− Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells

“…C57Bl/6 and BS/129Svj mice were used as controls when appropriate. Gene targeting and genotyping protocols for Ae2 fl/fl (C57Bl/6 background), Ae4 Ϫ/Ϫ (BS/129Svj background), and Aqp5-Cre (ACID) mice were as described previously (21)(22)(23). Acinusspecific Ae2 Ϫ/Ϫ mice (Ae2 fl/fl -ACID-Cre) were generated using the Cre/LoxP system by crossing Ae2 fl/fl mice with Aqp5-Cre mice.…”

Molecular cloning and functional characterization of zebrafish Slc4a3/Ae3 anion exchanger

“…Mutations in the human SLC4A1 gene cause hereditary spherocytic and stomatocytic anemias and distal renal tubular acidosis [2, 3]. Engineered genetic ablation of the mouse Slc4a2 gene leads to runting and death before weaning [12] with osteopetrosis [10] and reduced gastrointestinal epithelial secretion [13]. A missense polymorphism in the human SLC4A3 gene is associated with increased seizure susceptibility [37].…”