Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II

Abstract

Autosomal dominant osteopetrosis II (ADOII) is a relatively benign disorder caused by a missense mutation in the ClCN7 gene. In this study, we characterize the osteoclasts from patients with ADOII, caused by a G215R mutation, and investigate the effect on osteoclast function in vitro. Osteoclasts from ADOII patients and healthy age- and sex-matched controls, were used to evaluate osteoclastogenesis, cell fusion, acidification, and resorptive activity. ADOII osteoclasts in vivo have increased number and size. However, in vitro we observed no significant changes in the osteoclast formation rate, the morphology, and the expression of markers, such as cathepsin K and tartrate-resistant acid phosphatase. When mature ADOII osteoclasts were investigated on mineralized bone, they degraded the bone material, however only to 10 to 20% of the level in controls. We show by acridine orange, that the reduced chloride transport leads to reduced acidification. We show that the residual activity is sensitive to inhibitors of cathepsins and chloride channels, confirming that resorption is reduced but present. In conclusion, this is the first functional in vitro study of human ADOII osteoclasts. We show normal osteoclastogenesis in ADOII osteoclasts. However, the residual activity of the ClC-7 channel in ADOII osteoclasts does not allow sufficient acidification and thereby resorption.

Figure 1

Fusion is unchanged in ADOII osteoclasts. Peripheral blood was obtained from ADOII patients and age-matched control individuals, and the monocytes were isolated by Ficoll-Paque gradient centrifugation and CD14 magnetic bead isolation. The monocytes were cultured for 3 days in the presence of 25 ng/ml of M-CSF and then lifted and reseeded at a cell density of 100,000 cells/cm² and cultured for 5, 7, 9, or 11 days with M-CSF and 25 ng/ml of RANKL. The cells were then fixed in 3.7% formaldehyde and the number of fused cells was scored. a: The formation of osteoclasts with three or more nuclei. b: The TRAP activity was measured in the conditioned media from the fusion experiments. The results are pooled from six ADOII individuals and six controls, and they represent four replications for each individual.

Figure 2

Expression of TRAP and CKBB appears normal in ADOII osteoclasts. Peripheral blood was obtained from ADOII patients and age-matched control individuals, and the monocytes were isolated by Ficoll-Paque gradient centrifugation and CD14 magnetic bead isolation. The monocytes were cultured for 3 days in the presence of 25 ng/ml of M-CSF and then lifted and reseeded at a cell density of 100,000 cells/cm² and cultured for 5, 7, or 9 days with M-CSF and 25 ng/ml of RANKL. This was followed by lysis of the cells in RIPA+++ buffer at the indicated time points. Thirty μg of total cell lysate was subjected to sodium dodecyl sulfate gel-electrophoresis and immunoblotting with antibodies against creatine kinase BB, TRAP, cathepsin K, and the p38 MAPK as described in the Materials and Methods section. The immunoblots are representative of five different individuals in each group.

Figure 3

The expression and localization of ClC-7 is unchanged in ADOII cells. Peripheral blood was obtained from ADOII patients and age-matched control individuals, and the monocytes were isolated by Ficoll-Paque gradient centrifugation and CD14 magnetic bead isolation. The cells were cultured in the presence of 25 ng/ml of M-CSF and 25 ng/ml of RANKL. a: The lysates for the immunoblots were prepared as described in Figure 2, and then 50 μg of total cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted for ClC-7 and p38 MAPK. b: Mature osteoclasts were cultured as described in Materials and Methods, lifted by trypsin digestion, and reseeded on Osteologic coverslips. The cells were cultured for 48 hours, fixed, and immunostained using the ClC-7 antibody or the corresponding preimmune serum as described in the Materials and Methods section. The asterisks indicate the localization of the resorbed area. The immunoblots are representative of five different individuals in each group.

Figure 4

Acidification in ADOII osteoclasts is more sensitive to bafilomycin A1 treatment. Mature osteoclasts from ADOII patients and healthy controls were cultured as described in Materials and Methods, and then lifted by trypsin digestion and reseeded onto the Osteologic coverslips, where they were cultured for 48 hours. Controls (1 and 3) or ADOII cells (2 and 4) were incubated with acridine orange either in the absence (1 and 2) or presence (3 and 4) of 50 nmol/L of bafilomycin A1 for 45 minutes, and the accumulation of acridine orange was followed using a fluorescence microscope and a digital camera. The white arrowheads indicate multinuclear osteoclasts.

Figure 5

The number and area of resorption events on Osteologic coverslips are reduced in ADOII osteoclasts. Mature osteoclasts from ADOII patients and healthy controls were cultured as described in Materials and Methods, and then lifted by trypsin digestion and reseeded onto the Osteologic coverslips, where they were cultured for 48 hours. The osteoclasts were fixed and the cells and the hydroxyapatite layer were visualized by staining with hematoxylin. a: The area of removed hydroxyapatite (40 osteoclasts with area were measured for each group). b: The number of osteoclasts that formed visible resorption areas relative to the total number of cells (∼1000 cells were scored for each group).

Figure 6

Resorption is severely impaired in ADOII osteoclasts. Mature osteoclasts from ADOII patients and healthy controls were cultured as described in Materials and Methods, lifted by trypsin digestion, and then reseeded on cortical bone slices. a: Resorption of bone measured as CTX fragments released into the culture supernatant by the CrossLaps ELISA. The data are shown as accumulated resorption in percentage of endpoint values in the controls. b: Pit scorings from the endpoint (day 10) of the experiment. Results are shown as pit area in percentage of controls. c: Pictures of hematoxylin-stained pits at the endpoint (day 10) of the culture from ADOII (1) and control (2) osteoclasts. The results are representative of two ADOII and two control individuals, with each condition performed in pentaplicates.

Figure 7

Resorption of bone by ADOII osteoclasts is sensitive to inhibitors of chloride channels and cathepsins. Mature osteoclasts from ADOII patients and healthy controls were cultured as described in Materials and Methods, lifted by trypsin digestion, and then reseeded on mineralized bone. The volume of the resorption pits, measured by the CrossLaps ELISA, is shown in a, and the area of the pits, measured by stereology is shown in b. Controls and ADOII osteoclasts were cultured in the presence of either 30 μmol/L of NS5818 (a1, b1), 40 μmol/L E64 (a2, b2), or 10 μmol/L GM6001 (a3, b3). The data are presented as accumulated CTX release in percentage of endpoint values of the controls, and represent data from two ADOII patients and two matched controls, with each condition performed in pentaplicates. NS5818 significantly inhibited the CTX release in both controls (*) and ADOII cells (^). E64 significantly inhibited the CTX release in both controls (*) and ADOII cells (^). GM6001 did not change the CTX release from either controls or ADOII cells significantly. Endpoint measurements of the pit formation are shown as the percentage of nontreated control osteoclasts. Only NS5818 significantly reduced the area of the pits, when compared to the nontreated conditions.