Deficiency of ATP6V1H Causes Bone Loss by Inhibiting Bone Resorption and Bone Formation through the TGF-β1 Pathway

Abstract

Vacuolar-type H ⁺-ATPase (V-ATPase) is a highly conserved, ancient enzyme that couples the energy of ATP hydrolysis to proton transport across vesicular and plasma membranes of eukaryotic cells. Previously reported mutations of various V-ATPase subunits are associated with increased bone density. We now show that haploinsufficiency for the H subunit of the V₁ domain (ATP6V1H) is associated with osteoporosis in humans and mice. A genome-wide SNP array analysis of 1625 Han Chinese found that 4 of 15 tag SNPs (26.7%) within ATP6V1H were significantly associated with low spine bone mineral density. Atp6v1h^+/- knockout mice generated by the CRISPR/Cas9 technique had decreased bone remodeling and a net bone matrix loss. Atp6v1h^+/- osteoclasts showed impaired bone formation and increased bone resorption. The increased intracellular pH of Atp6v1h^+/- osteoclasts downregulated TGF-β1 activation, thereby reducing induction of osteoblast formation but the bone mineralization was not altered. However, bone formation was reduced more than bone resorption. Our data provide evidence that partial loss of ATP6V1H function results in osteoporosis/osteopenia. We propose that defective osteoclast formation triggers impaired bone formation by altering bone remodeling. In the future, ATP6V1H might, therefore, serve as a target for the therapy of osteoporosis.

Keywords: ATP6V1H; CRISPR/Cas9; OPG.; RANKL; TGF-β1; V-ATPase; osteoclasts; osteoporosis; pH.

Figure 1

Increased bone resorption in Atp6v1h^+/- mice. A. Atp6v1h^+/- mouse with a 5bp (CGAGG) deletion and one base replacement (T>G) which caused an early stop codon. B. Right: sABC immunohistochemical (IHC) staining for ATP6V1H in wild-type osteoclasts, parallel to TRAP-positive staining. Bars=20μm. Left: Strong IHC staining for ATP6V1H in wild-type osteoclasts (red arrow). Bars=10μm. C. TRAP staining of the distal femurs from 3-month-old Atp6v1h^+/- mice and control littermates. Bars = 50μm. The number of TRAP-positive osteoclasts (N. Oc/BS,) and osteoclast surface area (OC.S/BS) were not altered in Atp6v1h^+/- mice. BS. Bone surface. n=8. D. Three-dimensional micro-CT images of calvarial bones. Right: Whole appearance. Left: Middle-sagittal view of a 3D image. Atp6v1h^+/- mouse showed decreased bone density (white arrow). Bars=1mm. E. Micro CT images of the distal region of femurs from3-month-old Atp6v1h^+/- mice showed decreased bone mass. Bars=0.5mm. BV/TV, Tb.Th, and Ct.Th were decreased and BS/TV was increased in Atp6v1h^+/- mice compared with wild type. n=8. F. Modified Ponceau red staining of non-mineralized sections from the 4-week old wild-type and Atp6v1h^+/- mice. The Atp6v1h^+/- mouse showed less bone mass and decreased BV/TV and Tb.Th. Bars = 100μm. n=8. G. TEM images of Atp6v1h^+/- osteoclasts showed a normal ruffled border. Bars=1μm (large image), 5 μm (small image). H and I. ELISA results of TRAP and CTX-I in sera. There was no significant difference between wild-type and Atp6v1h^+/- mice. n=9. J and K. The serum level of calcium was higher and that of phosphate was lower in Atp6v1h^+/- mice than in wild-type. n=8. All data represent mean ±s.d. ** P<0.01, *P<0.05. NS: not significant difference.

Figure 2

Decreased bone formation in Atp6v1h^+/- mice. A.H&E staining of femurs from 3-month-old Atp6v1h^+/- mice and control littermates. Bars = 200μm. B. Modified Ponceau red staining of femurs from 3-month-old male Atp6v1h^+/- mice and control littermates. The two images on the right are magnified presentations of the white outline in the left two images. Bars= 200μm (left two images), 50μm (right two images). C. Dynamic analysis of femurs from 4-week-old Atp6v1h^+/- mice and control littermates. C₁: Images of laser scanning confocal microscope. Stars represent growth plate. Bars=50 μm. C₂~C₄: Analysis results. The rate of mineral apposition (MAR, mcm/d) and bone formation rate (BFR, mm³/mm²/d) were decreased in Atp6v1h^+/- mice, while the mineralizing surface (MS, %) was not altered. n=8. D. ALP staining of femurs from 3-month-old Atp6v1h^+/- mice and control littermates. D₁: ALP staining was reduced and unevenly distributed in Atp6v1h^+/- mice. Bars = 50μm. D₂, D₃: The number of ALP+ osteoblasts (N. Ob/BS, /mm) and the osteoblast surface (Ob. S/BS, %) were significantly reduced in Atp6v1h^+/- mice. n=9. D₄: The average intensity of ALP-positive staining was reduced in Atp6v1h^+/- mice by Image J software analysis. n=9. E. The serum ALP level of Atp6v1h^+/- mice was higher than for controls. n=9. F. There was no significant difference in serum osteocalcin levels between wild type and Atp6v1h^+/- mice. n=9. All data represent mean ±s.d. ** P<0.01, *P<0.05. NS: not significant difference.

Figure 3

Reduced formations and function of Atp6v1h^+/- osteoclasts. A. Fewer TRAP⁺ Atp6v1h^+/- osteoclasts with normal size were induced in a general 96 well plate. Bars=100μm. n≥100. B. When osteoclasts were induced in osteoassay plate, the number of osteoclasts per well, average size of osteoclasts, resorptive area (%), and resorptive area (%)/N.Oc (number of osteoclasts) were all decreased in Atp6v1h^+/- group. n=3. C. TRAP staining (left) and toluidine blue staining (right) of bone slices. The decreased number of pits matched the reduced number of TRAP+ osteoclasts. The diameter of Atp6v1h^+/- pits was smaller than for wild-type. n ≥100. D. Merged images for TRAP and F-actin staining. Both Atp6v1h^+/- and wild-type cells could form actin rings. Bars=50μm. F. Western blot analysis showed the decreased ATP6V1H protein in Atp6v1h^+/- cells induced by RANKL and M-CSF 1~ 4 day. F. Atp6v1h siRNA reduced the mRNA level of Atp6v1h in wild type osteoclasts and the formation of TRAP+ osteoclasts. n=3. G. Atp6v1h^+/- osteoclasts showed a decreased V-ATPase activity which was inhibited by 10nM Bafilomycin A1. n=3. H. The intracellular pH of Atp6v1h^+/- osteoclasts was increased by staining with lysosensor DND-160. n≥50. All data represent mean ±s.d. ** P<0.01, *P<0.05. NS: not significant difference.

Figure 4

Decreased interactions between osteoblasts and osteoclasts in Atp6v1h^+/- mice. A. Comparison of osteogenic markers in newborn calvarial bone and its derived osteoblasts with Q-PCR analysis. Primary cultured Atp6v1h ^+/- osteoblasts with osteogenic induction had decreased Col1a1, Alp, and Bsp. n≥3. B. Atp6v1h siRNA decreased the mRNA level of Atp6v1h more than 50 percent in primary cultured osteoblasts (OB) and Mc3T3-E1 cells (M), and the mRNA level of Ocn and Opn were changed. n≥3. C. Alizarin red S and ALP staining showed that calvarial osteoblasts from wild-type and Atp6v1h^+/- mice formed mineralized nodules but no difference existed between them. n≥3. D. Western results showed the decreased TGF-β1 in Atp6v1h^+/- osteoclasts and unchanged TGF-β1 in Atp6v1h^+/- osteoblasts, respectively. n=3. E. ELISA detection revealed the decreased active TGF-β1 in sera of Atp6v1h^+/- mice. n≥8. F. The active TGF-β1 level in the supernatant of Atp6v1h^+/- osteoclasts cultured on the bone slices was also decreased. Control: control medium. n≥3. G. Atp6v1h^+/- osteoblasts with osteogenic induction secreted more active TGF-β1 by ELISA detection. No difference existed between wild-type and Atp6v1h^+/- osteoblasts under a non-osteogenic induction condition. n≥3. H. Western blot results showed the decreased RANKL and unchanged OPG in Atp6v1h^+/- osteoblasts. n≥3. I. ELISA results revealed the less soluble RANKL (sRANKL) in sera and the supernatant of Atp6v1h^+/- osteoblasts with osteogenic induction. n≥3. J. OPG level was increased in sera of Atp6v1h^+/- mice and the supernatant of Atp6v1h^+/- osteoblasts with osteogenic induction. n≥3. K. Comparison of the number of osteoclasts in the co-culture system of osteoblasts and osteoclasts. When co-cultured with wild-type osteoblasts, wild-type and Atp6v1h^+/- monocytes formed the same amount TRAP+ osteoclasts. When co-cultured with Atp6v1h^+/- osteoblasts, fewer TRAP+ osteoclasts were formed in Atp6v1h^+/- group. n≥3. All data represent mean ±s.d. ** P<0.01, *P<0.05. NS: not significant difference.

Figure 5

Schematic map of haploinsufficiency of Atp6v1h and bone loss. TGF-β1 stimulates the proliferation of mesenchymal stem cells (MSC) and induces MSC to differentiate into osteoblasts (OB); TGF-β1 also promotes the maturation of osteoclasts (OC). The increased intracellular pH resulted in less secretion of TGF-β1 from Atp6v1h^+/- osteoclasts. Decreased TGF-β1 negatively regulates osteoblasts differentiation, results in the fewer and smaller osteoblasts and finally causes the decreased bone formation in Atp6v1h^+/-mice. The low level of TGF-β1 is associated with reduced RANKL and increased OPG signal, sending decreased signals to induce the osteoclasts formation. The decreased bone formation is more than decreased osteoclast function. Thus, the final in vivo net effect is bone loss.