The osteocytic actions of glucocorticoids on bone mass, mechanical properties, or perilacunar remodeling outcomes are not rescued by PTH(1-34)

Abstract

Glucocorticoids (GC) and parathyroid hormone (PTH) are widely used therapeutic endocrine hormones where their effects on bone and joint arise from actions on multiple skeletal cell types. In osteocytes, GC and PTH exert opposing effects on perilacunar canalicular remodeling (PLR). Suppressed PLR can impair bone quality and joint homeostasis, including in GC-induced osteonecrosis. However, combined effects of GC and PTH on PLR are unknown. Given the untapped potential to target osteocytes to improve skeletal health, this study sought to test the feasibility of therapeutically mitigating PLR suppression. Focusing on subchondral bone and joint homeostasis, we hypothesize that PTH(1-34), a PLR agonist, could rescue GC-suppressed PLR. The skeletal effects of GC and PTH(1-34), alone or combined, were examined in male and female mice by micro-computed tomography, mechanical testing, histology, and gene expression analysis. For each outcome, females were more responsive to GC and PTH(1-34) than males. GC and PTH(1-34) exerted regional differences, with GC increasing trabecular bone volume but reducing cortical bone thickness, stiffness, and ultimate force. Despite PTH(1-34)'s anabolic effects on trabecular bone, it did not rescue GC's catabolic effects on cortical bone. Likewise, cartilage integrity and subchondral bone apoptosis, tartrate-resistant acid phosphatase (TRAP) activity, and osteocyte lacunocanalicular networks showed no evidence that PTH(1-34) could offset GC-dependent effects. Rather, GC and PTH(1-34) each increased cortical bone gene expression implicated in bone resorption by osteoclasts and osteocytes, including Acp5, Mmp13, Atp6v0d2, Ctsk, differences maintained when GC and PTH(1-34) were combined. Since PTH(1-34) is insufficient to rescue GC's effects on young female mouse bone, future studies are needed to determine if osteocyte PLR suppression, due to GC, aging, or other factors, can be offset by a PLR agonist.

Keywords: PTH (1-34); bone; glucocorticoids; osteocyte; osteocytic osteolysis; parathyroid hormone (PTH); perilacunar canalicular remodeling; prednisolone.

Figure 1

GC and PTH(1-34) effects on bone quantity and quality are sexually dimorphic. Femora of 16-week-old control and GC and/or PTH(1-34) treated male (n=4-8/group) and female (n=6-7/group) mice were analyzed using µCT for trabecular (Tb.) (A–D) and cortical (Ct.) parameters (E–H) on distal femur and mid-femur respectively. Results reveal trabecular bone/volume fraction (Tb. BV/TV, A), trabecular number (Tb. N, B), trabecular thickness (Tb. Th, C), trabecular separation (Tb. Sp, D), cortical bone volume fraction (Ct. BV/TV, E), cortical thickness (Ct. Th, F), cortical bone mineral density (Ct. BMD, G), and cortical tissue mineral density (Ct. TMD, H). Representative µCT reconstructions display sexual dimorphism (scale bar = 500µm) (I). Three-point bending on male (n=4-8/group) and female (n=6-8/group) left femora show outcomes of flexural strength (J–L). In each graph, male data is displayed as blue dots, with female data displayed as red dots. Data are presented as mean ± SD. Statistically significant differences (*p≤0.05) were determined by two-way ANOVA with post-hoc Holm Sidak within sex.

Figure 2

GC and PTH(1-34) effects on skeletal gene expression in female cortical bone. Volcano plots of 96 skeletal-associated mouse genes from Nanostring analysis shows significantly up- and down-regulated genes associated with bone remodeling (red dots) in treated (GC, PTH(1-34), and GC+PTH(1-34)) female mice (n=4) compared to controls (A–C). Statistically expressed genes (gray dots) are above the horizontal p-value threshold (dotted gray line) and up-regulated or down-regulated genes fall to either to the right or left sides, respectively. Highly significantly gene expression fold changes was determined by unpaired t-test between experimental groups, normalized to 7 housekeeping genes (Gapdh, Rpl19, Gilz (Tsc22d3), bone sialoprotein (Ibsp), beta-2 microglobulin (B2m), beta actin (Actb), Serpine2). (D–F) show statistically up- or down-regulated genes in each condition, with red bars indicating genes that are regulated in the same manner as combined GC+PTH(1-34) treatment, and blue bars indicating genes that are opposingly regulated between GC and PTH(1-34) treatment.

Figure 3

Osteocyte-intrinsic suppression of Mmp13 by GC is not rescued by PTH(1-34). Real-time qPCR analysis on MLO-Y4 cells treated with low (0.1µM) or high (1µM) dose of Dexamethasone (DEX) causes induction of Atrogin1 (A), Murf1 (B) and dose-dependent down-regulation of Mmp13 (C) mRNA (n=3 replicates/group and 2 independent experiments) normalized to GAPDH. PTH(1-34) did not mitigate effects of GC treatment on Mmp13 (C). Data is displayed as mean ± SD and statistically significant differences (*p≤0.05) were determined using one-way ANOVA.

Figure 4

Joint and osteoarthritis assessment of GC and PTH(1-34) treated females. Safranin O/Fast Green stain of right knee joints from 16-week-old control and GC and/or PTH(1-34) treated females (n=4/group) show no changes in cartilage (red) and subchondral bone (counterstain blue/green) knee joint phenotypes in representative images (20X, scale bar = 200µm) (A), supported by quantified total OARSI (B) and total Modified Mankin Score (C). Data are presented as mean ± SEM and statistically significant differences were determined by two-way ANOVA with post-hoc Holm Sidak between experimental groups.

Figure 5

Effects of GC and PTH(1-34) on TRAP activity. TRAP staining on subchondral knee sections of control and treated (GC, PTH(1-34), or GC+PTH(1-34)) 16-week-old female mice (n=4-5/group). Representative images from each condition (A 20X, scalebar = 200 µm) provide visualization of TRAP+ stained cells (red), counterstained in methyl green. Quantification of Osteoclast Surface per Bone Surface (Oc.S/BS %) and Number of Osteoclasts per Tissue Volume (N.Oc/TV mm^-2) were analyzed in each joint compartment (femur, tibia, medial, lateral) and displayed as total (B, E), lateral (C, F), and medial (D, G). Data are presented as mean ± SD and statistically significant differences (*p≤0.05) were determined by two-way ANOVA with post-hoc Tukey was performed between experimental groups.

Figure 6

Subchondral bone assessment of GC and/or PTH(1-34) treated female mouse knees. Representative high-resolution images (100X, scale bar = 50 µm) of the right knee joints of control and treated (GC, PTH(1-34), or GC+PTH(1-34)) females at 16-week-old (n=4/group) stained with Ploton silver nitrate stain and counterstained with Cresyl Violet show the subchondral bone lacunocanalicular network (LCN) (A). Quantitative analysis of the number (#) of lacunae (B) and average lacunae size (C) shows treatment effects on the LCN in each joint compartments (femur, tibia, medial, lateral). Data are presented as mean ± SD, and statistically significant differences (*p≤0.05) were determined by unpaired t-test between experimental groups.