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. 2015 Mar 26;11(3):e1005057.
doi: 10.1371/journal.pgen.1005057. eCollection 2015 Mar.

Osteopetrorickets due to Snx10 deficiency in mice results from both failed osteoclast activity and loss of gastric acid-dependent calcium absorption

Liang Ye  1 , Leslie R Morse  2 , Li Zhang  3 , Hajime Sasaki  4 , Jason C Mills  5 , Paul R Odgren  6 , Greg Sibbel  5 , James R L Stanley  7 , Gee Wong  7 , Ariane Zamarioli  8 , Ricardo A Battaglino  1
Affiliations

Osteopetrorickets due to Snx10 deficiency in mice results from both failed osteoclast activity and loss of gastric acid-dependent calcium absorption

Liang Ye et al. PLoS Genet. .

Abstract

Mutations in sorting nexin 10 (Snx10) have recently been found to account for roughly 4% of all human malignant osteopetrosis, some of them fatal. To study the disease pathogenesis, we investigated the expression of Snx10 and created mouse models in which Snx10 was knocked down globally or knocked out in osteoclasts. Endocytosis is severely defective in Snx10-deficient osteoclasts, as is extracellular acidification, ruffled border formation, and bone resorption. We also discovered that Snx10 is highly expressed in stomach epithelium, with mutations leading to high stomach pH and low calcium solubilization. Global Snx10-deficiency in mice results in a combined phenotype: osteopetrosis (due to osteoclast defect) and rickets (due to high stomach pH and low calcium availability, resulting in impaired bone mineralization). Osteopetrorickets, the paradoxical association of insufficient mineralization in the context of a positive total body calcium balance, is thought to occur due to the inability of the osteoclasts to maintain normal calcium-phosphorus homeostasis. However, osteoclast-specific Snx10 knockout had no effect on calcium balance, and therefore led to severe osteopetrosis without rickets. Moreover, supplementation with calcium gluconate rescued mice from the rachitic phenotype and dramatically extended life span in global Snx10-deficient mice, suggesting that this may be a life-saving component of the clinical approach to Snx10-dependent human osteopetrosis that has previously gone unrecognized. We conclude that tissue-specific effects of Snx10 mutation need to be considered in clinical approaches to this disease entity. Reliance solely on hematopoietic stem cell transplantation can leave hypocalcemia uncorrected with sometimes fatal consequences. These studies established an essential role for Snx10 in bone homeostasis and underscore the importance of gastric acidification in calcium uptake.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Morphological and radiographic examination of Snx10 KD mice.
A) 2-week-old Snx10 KD mice are growth retarded. 2-week-old Snx10 KD mice (C and E) have a tooth eruption defect compared to their WT littermates (B and D). The white arrows (D and E) demonstrate the presence of tooth buds that failed to erupt in the KD mice. Radiographic images demonstrate that 3-week-old Snx10 KD mice (G and I) have bones that lack marrow cavities. Compare the femur (white arrow) and pelvis (black arrow) with WT (F and H) mice. The metaphyseal cupping in the Snx10 KD femur and tibia (I) are consistent with rickets.
Fig 2
Fig 2. Micro CT analysis of Snx10 KD mice.
(A, top) Surface images of the femur demonstrate a deficiency of mineralized cortical bone. Longitudinal mid-plane images of the femur (A, bottom) show a marrow cavity filled with unresorbed bone in Snx10 KD mice. Other examined bones: head, (B) and mandible (C) also were deficient in cortical bone, producing a “moth-eaten” appearance. Cross sectional micro CT images of femur (D, E, F and G) reveal the presence of an inner ring of cortex-like (i.e., denser) bone in Snx10 KD bones (E and G).
Fig 3
Fig 3. Histology of bone from WT and Snx10 KD mice.
Low magnification of Von Kossa/van Gieson staining (A and B) of femur longitudinal sections from 3 week-old mice demonstrate endosteum filled with unresorbed cartilage (purple stained trabecular cores) and thin cortical bone (black arrowheads) in Snx10 KD mice (B). Higher magnification (C and D) reveals non-mineralized osteoid (pink-stained surface, indicated with asterisks), covering the mineralized trabeculae (black) from Snx10 KD bones (D) Bar = 0.05 mm
Fig 4
Fig 4. Snx10 is expressed in the stomach and is required for gastric acid production and calcium homeostasis.
A) Representative stomachs from 3-week old Snx10 KD mice (n = 6) and WT littermates (n = 6). Snx10 KD mice have abnormal stomachs. B) mRNA expression of Snx10 (left panel) is mainly observed in stomach and bone. Within the stomach (right panel) expression of Snx10 mRNA is mainly observed in the body/corpus of the stomach ("Body"), where levels are comparable to the femur; expression is lower in the antrum and the non-glandular forestomach. C) Immunofluorescence images from stomach sections stained for pepsinogen (a zymogenic cell marker), Snx10 and GSII (a mucous neck cell marker) shows Snx10-specific staining (green) in zymogenic cells. D) Sections of the body of the stomach stained for the parietal cell marker VEGF-B (red), show that Snx10-deficient parietal cells are smaller, have abnormal nuclear morphology, increased nuclear to cytoplasmic ratio, and an abnormal cellular distribution of VEGF-B. E) Gastric pH is significantly elevated in Snx10 KD mice indicating that Snx10 is required for gastric acid production (* P<0.05). F) Serum calcium is reduced in Snx10 KD mice, indicating that Snx10 is required for calcium homeostasis (*P<0.05). G) Serum parathyroid hormone (PTH) is significantly elevated in Snx10 KD (* P = 0.001). H) 1,25-dihydroxyvitamin D levels are reduced in Snx10 KD mice (* P = 0.01).
Fig 5
Fig 5. Calcium supplementation can normalize calcium homeostasis and correct the rickets phenotype in Snx10 KD mice.
Serum calcium (A, left), GpTh (A, center) and OV/BV, (%;A, right) are normalized by calcium supplementation of Snx10 KD mice (Snx10 KD + Ca) (*, P<0.05, **, P<0.01). BV/TV(%; A, bottom right) is not normalized by calcium supplementation. B) Radiographic images of femora and tibiae show mineralization of femoral condyles (see inset) (C) Micro CT analysis of femora confirms partial re-mineralization of condyles and prevention of the “moth-eaten” cortical bone phenotype, as a result of calcium supplementation in Snx10 KD mice.
Fig 6
Fig 6. Snx10 OC KO mice are osteopetrotic: Expression, morphological and radiographic analysis.
A) qPCR analysis of RNA from femur shows a 95% reduction in Snx10 expression in bones from Snx10 OC KO mice. B) Snx10 expression in the stomach is not affected. C) 3-week-old Snx10 OC KO mice (right) are growth retarded. E and G) 3-week-old Snx10 OC KO mice have a tooth eruption defect compared to their WT littermates (D and F). Radiographs (I and L) show that 3-week-old Snx10 OC KO mice have bones without marrow spaces and with a higher radio-density than WT counterparts (H and K) or Snx10 KD (J and M). The metaphyseal fraying and cupping seen in the Snx10 KD femur and tibia are not observed in the Snx10 OC KO mice (I and L), consistent with osteopetrosis with no rickets.
Fig 7
Fig 7. Histology and Micro CT analysis of Snx10 OC KO mice.
A) Surface images of the femur (top), longitudinal mid-plane sections (middle) and skull images (bottom) show that osteoclast-specific Snx10 deficiency leads to severely osteopetrotic bone, but unlike the global deficiency it does not result in a lack of cortical bone. The white line in the longitudinal mid-plane sections (A, middle) indicates the location of the transverse micro-CT images were taken. The transverse images show that, in contrast to the Snx10 KD mouse, Snx10 OC KO mice have cortical bone. B) Snx10 OC KO mice are normo-calcemic, suggesting that that osteoclast-specific Snx10 deficiency does not have a major impact in calcium homeostasis. C) (top panel) H&E staining of longitudinal sections of the proximal femur show abundant unresorbed cartilage in Snx10 OC KO and Snx10 KD mice, typical of osteopetrosis. The growth plate (GP) is thicker in Snx10 KD mice. C) (FEMUR) Von Kossa/van Gieson staining of femur transverse sections close to the growth plate (cut at the depth of the white line shown in the upper panels) show numerous trabeculae in both Snx10 OC KO and Snx10 KD tibiae, confirming the osteopetrotic phenotype. However, while the trabeculae of Snx10 KD mice are not fully mineralized as evidenced by the pink-stained osteoid (right panels), the trabeculae of osteoclast-specific Snx10 KO mice are fully mineralized (bottom panels). Similar results were observed for vertebra (C. VERTEBRA panel) and bones of the skull base/floor (C. SKULL panel)
Fig 8
Fig 8. Snx10-deficient osteoclasts are unable to acidify have impaired endosomal pathways, and have defective ruffled borders.
Acridine orange indicates acidification by osteoclasts derived from WT splenocytes (A), whereas none is present in Snx10 KD osteoclasts (B)(Nuclei labeled green). (C) WT osteoclasts can internalize fluorescent dextran (green) but Snx10 KD osteoclasts (D) cannot. E) TEM images of bone sections show a well-developed ruffled border in WT osteoclasts (black arrowheads). F) Snx10-deficent osteoclasts form a severely defective ruffled border. BM: bone matrix. (HV = 80.0 kV).
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