Osteocytes Acidify Their Microenvironment in Response to PTHrP In Vitro and in Lactating Mice In Vivo

Abstract

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG-SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno-canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)-tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP-fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research.

Keywords: CELLS OF BONE; OSTEOCLASTS; OSTEOCYTES.

Fig. 1

Increased expression of bone resorption related genes in osteocytes. IDG-SW3 cells were differentiated for 29 d. (A) CTSK (CT=23-21), (B) ACP5 (CT=29-28), (C) CA1 (CT=34-29), (D) CA2 (CT=32-29), (E) ATP6V0B (CT=24-23), and (F) ATP6V0D2 (CT=40-32) were significantly increased with osteocyte-differentiation. Graphs show individual data points, mean and SEM for fold change in gene expression compared to day 1 (4 experiments, representative experiment shown with n=3). Statistical analysis: one-way ANOVA and Dunnett posthoc test compared to day 1.

Fig. 2

The expression of PTH1R (A) increases during osteocyte differentiation in IDG-SW3 cells (CT=25-21). PTHrP induced a further increase in ATP6V0D2 expression (CT=32-27) with a long-term treatment of 7 d (B). Graphs show individual data points, mean and SEM for fold change in gene expression compared to day 1 (4 experiments, representative experiment shown with n=3). Statistical analysis: one-way ANOVA and (A) Dunnett posthoc test compared to day 1, and (B) Tukey posthoc test comparing all groups.

Fig. 3

Osteocytes acidify their microenvironment in vitro in response to PTHrP and this is mediated by the vacuolar ATPase. Cells were treated for 20 min with PTHrP in the presence of SNARF; (A) NIH3T3 fibroblasts (8 experiments, n=3 each) did not respond to PTHrP, while (B) SaOs2 osteosarcoma cells (5 experiments, n=3-4 each) and (C) primary osteocytes (4 experiments, n=2-3 each) demonstrated significant increase in SNARF fluorescence upon PTHrP treatment (mean and SEM, unpaired two-tailed t-test compared to vehicle). (D) PTHrP-induced acidification upon osteocyte differentiation in IDG-SW3 cells (mean and SEM, 3 experiments, n=3 each, one-way; ANOVA and Bonferroni posthoc test compared to vehicle group on each day). (E) Bafilomycin blocked PTHrP-induced acidification in primary osteocytes (5 experiments, n=2-3 each, mean and SEM, one-way ANOVA and Tukey posthoc test comparing all groups).

Fig. 4

Osteocytes remain viable at low pH. Three cell types were cultured in media with adjusted pH, viability was assessed by trypan blue assay (ratio live cells / dead cells with mean and SEM). (A) MLO-Y4 cells maintained higher viability in pH 5 media at 24 h than L292 and MC3T3 (3 experiments, n=3 each, two-way ANOVA and Tukey posthoc test comparing all groups). (B) MLO-Y4 remain more viable than NIH3T3 and MLO-A5 during 48 h in media with pH≤6.5. (6 experiments, n=2-4 each, two-way ANOVA and Tukey posthoc test comparing all groups). (C) Osteocyte enriched primary calvarial cells outgrown from pre-digested bone chips showed the highest cell viability after 24 h at pH 5 compared to DsRed-negative cells and DsRed-positive cells (3 experiments, n=3 each, two-way ANOVA and Tukey posthoc test comparing all groups).

Fig. 5

Lower pH in osteocyte lacunae of lactating mice. (A) Representative BSEM images of cortical (upper surface = periosteal, lower surface = endocortical) and trabecular bone from femora of virgin and lactating CD-1 mice. (B) Increased osteocyte lacunar area in cortical (100-300 lacunae) and trabecular (25-150 lacunae) bone in lactation group (mean and SEM, n=4 animals/group, unpaired two-tailed t-test compared to virgin). (C) Representative confocal images of acridine orange fluorescence in osteocyte lacunae in tibial cortex of virgin and lactating mice. In the left images the scale bar represents 50 μm, and in the right images, which are higher magnifications of the left the scale bar represents 10 μm. (D) Ratio of acidic to neutral AO fluorescence is significantly higher in lactating mice (mean and SEM, n=4 animals/group, 30-60 lacunae each, unpaired two-tailed t-test compared to virgin).

Fig. 6

Decreased GFP fluorescence in bones of lactating GFP-collagen mice. (A) Representative BSEM images of cortical (upper surface = periosteal, lower surface = endocortical) and trabecular bone from femora of virgin and lactating GFP-collagen mice. (B) Lactation led to increased osteocyte lacunar area in cortical (125- 235 lacunae) and trabecular (25-100 lacunae) bone (mean and SEM, n=8 animals/group, unpaired two-tailed t-test comparing to virgin). (C) Representative confocal images of GFP fluorescence in the femoral cortex of virgin and lactating mice (GFP fluorescence in green, autofluorescence in yellow; scale bar = 50 μm for each image). (D) GFP fluorescence is significantly lower in cortical bone of the proximal femur of lactating mice (mean and SEM, n=8 animals/group, 4 images w/ 8 randomly assigned areas, unpaired two-tailed t-test compared to virgin).

Fig. 7

Illustration of theoretical acidification by osteocytes and visualization of the osteocyte environment. (A) Schematic image showing osteocyte with dendrites (blue) in bone (light grey) that is bathed in lacuno-canalicular fluid filled volume (yellow) and surrounded by perilacunar matrix (dark grey). (B) Representative BSEM images of acid etched murine bone specimens containing osteocyte lacuna. With short acid etching (left image) the perilacunar matrix is etched away but the removal of the neighboring and deeper bone matrix requires longer acid etching (right image).