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.2008 Nov 4;105(44):16934-9.
doi: 10.1073/pnas.0808763105. Epub 2008 Oct 29.

HCO3-/Cl- anion exchanger SLC4A2 is required for proper osteoclast differentiation and function

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HCO3-/Cl- anion exchanger SLC4A2 is required for proper osteoclast differentiation and function

Jing Wu et al. Proc Natl Acad Sci U S A..

Abstract

As the only cell capable of bone resorption, the osteoclast is a central mediator of skeletal homeostasis and disease. To efficiently degrade mineralized tissue, these multinucleated giant cells secrete acid into a resorption lacuna formed between their apical membrane and the bone surface. For each proton pumped into this extracellular compartment, one bicarbonate ion remains in the cytoplasm. To prevent alkalinization of the cytoplasm, a basolateral bicarbonate/chloride exchanger provides egress for intracellular bicarbonate. However, the identity of this exchanger is unknown. Here, we report that the bicarbonate/chloride exchanger, solute carrier family 4, anion exchanger, member 2 (SLC4A2), is up-regulated during osteoclast differentiation. Suppression of Slc4a2 expression by RNA interference inhibits the ability of RAW cells, a mouse macrophage cell line, to differentiate into osteoclasts and resorb mineralized matrix in vitro. Accordingly, Slc4a2-deficient mice fail to remodel the primary, cartilaginous skeletal anlagen. Abnormal multinucleated giant cells are present in the bone marrow of Slc4a2-deficient mice. Though these cells express the osteoclast markers CD68, cathepsin K, and NFATc1, compared with their wild-type (WT) counterparts they are larger, fail to express tartrate-resistant acid phosphatase (TRAP) activity, and display a propensity to undergo apoptosis. In vitro Slc4a2-deficient osteoclasts are unable to resorb mineralized tissue and cannot form an acidified, extracellular resorption compartment. These data highlight SLC4A2 as a critical mediator of osteoclast differentiation and function in vitro and in vivo.

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Figures

Fig. 1.
Fig. 1.
NFATc1-dependent up-regulation ofSlc4a2 during osteoclastogenesis. (A) Microarray signal intensities forSlc4a2 from two independent mRNA samples isolated from BMOcPs treated for 3 days with MCSF and RANKL. (B) qRT-PCR analysis for the expression ofSlc4a2 isoforms in BMOcPs cultured with MCSF or MCSF and RANKL.ex14 refers to qRT-PCR primers that produce an amplicon within exon 14 ofSlc4a2, an exon present in all five isoforms. (C) qRT-PCR analysis for the expression ofSlc4a2a in BMOcPs treated with MCSF or MCSF and RANKL in the presence of increasing concentrations of CsA (62.5, 125, 250 ng/ml). (D) SLC4A2 immunostaining of WT MCSF-primed BM osteoclast precursors cultured with MCSF or MCSF and RANKL. Two examples of SLC4A2-positive osteoclasts are shown. Green, SLC4A2; blue, Hoechst. (E) Confocal image along thez axis of the osteoclast shown in the rightmost panel of (D).
Fig. 2.
Fig. 2.
Knockdown ofSlc4a2 expression in RAW clone 6 cells prevents osteoclast differentiation and matrix resorption. (A) Western blot analysis for SLC4A2 in RAW clone 6 cells cultured with or without RANKL. (B) qRT-PCR analysis for the expression ofSlc4a2a in RAW clone 6 cells infected with lentiviruses expressing five different shRNA constructs targetingSlc4a2 and cultured with RANKL. The control shown is the average of RAW clone 6 cells infected with three different irrelevant lentiviral shRNA constructs and cultured with RANKL. (C) Supernatant TRAP assay or (D) TRAP stain of RAW clone 6 cells infected with the indicated lentiviral shRNA vectors and treated with or without ([C] only) RANKL. (E) Matrix resorption assay of RAW clone 6 cells infected with the indicated lentiviral shRNAs and cultured with RANKL.
Fig. 3.
Fig. 3.
Slc4a2-deficient mice fail to remodel the primary skeletal anlagen. (A andB) Digital radiographs of (A) mice and (B) femurs. (C andD) Hematoxylin and eosin stain of (C) vertebral bodies and (D) tibias (4× objective). (E) Toluidine blue stain of the tibial metaphysis (10× objective). The radiographs and histology are representative of at least three littermate-matched, 3-week-old mice analyzed per genotype.
Fig. 4.
Fig. 4.
Abnormal osteoclast development and increased osteoclast apoptosis inSlc4a2-deficient mice. (A) TRAP stain of the tibial metaphysis (40× objective). (B) H&E stain showing multinucleated giant cells without (Left) and with (Right) apoptotic features inSlc4a2−/− mice. (C–E) Immunohistochemistry for (C) CD68, (D) cathpesin K, and (E) NFATc1. (F) TUNEL staining at the femoral metaphysis ofSlc4a2−/− mice. Note brown staining in the condensed nuclei of apoptoticSlc4a2−/− osteoclasts (arrows) compared with a nonapoptotic one (arrowhead). (G) Number of cathepsin K-positive, multinucleated cells per microscopic field (40× objective) in the distal femoral metaphysis (P > 0.5,Slc4a2+/+ vs.Slc4a2−/−) (H) Percentage of cathepsin K-positive, multinucleated cells with cytologic features of apoptosis in the distal femoral metaphysis (P < 0.01,Slc4a2+/+ vs.Slc4a2−/−). The images in (B)–(F) were cropped from pictures obtained in the femoral metaphysis with 40× or 60× ([F] only) objectives. Images are representative of at least three littermate-matched, 3-week-old mice analyzed per genotype. Data in (G) and (H) are the average plus SD of three mice per genotype.
Fig. 5.
Fig. 5.
SLC4A2 is required for bone resorption and extracellular acidification by osteoclastsin vitro. (A) Immunostaining for SLC4A2 on MCSF-primed BM cells cultured with MCSF and RANKL. (B) TRAP stain and (C) supernatant TRAP assay of spleen cells cultured with MCSF ([B] only), or MCSF and RANKL. (D) qRT-PCR analysis of spleen cells cultured with MCSF, or MCSF and RANKL. (E) Lectin-TRITC stain of dentin slices cultured with spleen cells and MCSF and RANKL. (F) Acridine orange stain of spleen cells cultured on dentin slices with MCSF and RANKL.
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