Beta 2 Adrenergic Receptor Selective Antagonist Enhances Mechanically Stimulated Bone Anabolism in Aged Mice

Abstract

The anabolic response of aged bone to skeletal loading is typically poor. Efforts to improve mechanotransduction in aged bone have met with limited success. This study investigated whether the bone response to direct skeletal loading is improved by reducing sympathetic suppression of osteoblastic bone formation via β2AR. To test this possibility, we treated aged wild-type C57BL/6 mice with a selective β2AR antagonist, butaxamine (Butax), before each of nine bouts of cantilever bending of the right tibia. Midshaft periosteal bone formation was assessed by dynamic histomorphometry of loaded and contralateral tibias. Butax treatment did not alter osteoblast activity of contralateral tibias. Loading alone induced a modest but significant osteogenic response. However, when loading was combined with Butax pretreatment, the anabolic response was significantly elevated compared with loading preceded by saline injection. Subsequent studies in osteoblastic cultures revealed complex negative interactions between adrenergic and mechanically induced intracellular signaling. Activation of β2AR by treatment with the β1, β2-agonist isoproterenol (ISO) before fluid flow exposure diminished mechanically stimulated ERK1/2 phosphorylation in primary bone cell outgrowth cultures and AKT phosphorylation in MC3T3-E1 pre-osteoblast cultures. Expression of mechanosensitive Fos and Ptgs2 genes was enhanced with ISO treatment and reduced with flow in both MC3T3-E1 and primary cultures. Finally, co-treatment of MC3T3-E1 cells with Butax reversed these ISO effects, confirming a critical role for β2AR in these responses. In combination, these results demonstrate that selective inhibition of β2AR is sufficient to enhance the anabolic response of the aged skeleton to loading, potentially via direct effects upon osteoblasts. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: BONE‐BRAIN‐NERVOUS SYSTEM INTERACTIONS; EXERCISE; OSTEOBLASTS; PRECLINICAL STUDIES; TRANSCRIPTION FACTORS.

Fig. 1

Butax treatment did not alter the decline in body mass in senescent mice. Mice exhibited a decline in body weight over the course of the study (p < 0.0001), but dosage had no effect (p = 0.78) and the interaction between study time course and dosage was not significant (p = 0.51).

Fig. 2

Butax treatment enhanced mechanically induced bone formation across a range of dosages. Periosteal mineral apposition rate (MAR) (A), mineralizing surface (MS) (B), and bone formation rate (BFR) (C) are plotted for loaded (red dots) and contralateral bones (black dots). Representative images (D) of contralateral and loaded tibias from saline and Butax‐treated mice (3 mg/kg dosage), each approximating the mean p.BFR of the specified group. Corresponding group mean values are listed below images. Single‐ and double‐labeled periosteal surfaces are indicated by arrows and arrowheads, respectively. Graphed data are from n = 7–8 mice in the 0, 1, 3, and 10 mg/kg (low, medium, and high) dose groups, respectively. Kruskal–Wallis test comparison across dosages for each limb identified significant dosage effect on p.BFR of loaded limb (p < 0.03). Mann–Whitney test compared each Butax dosage versus saline for this parameter (p < 0.05; ^aButax versus saline, same limb). Wilcoxon's rank‐sum test contrasted outcomes in loaded versus contralateral bones (p < 0.05; ^bloaded versus contralateral limb).

Fig. 3

Isoproterenol (ISO) treatment selectively inhibits flow‐responsive signaling in primary outgrowth bone cells. Cells established from mice were pretreated with 0.1 or 1.0 μM ISO and subjected to fluid flow (FF). (A) Signaling events after flow were analyzed by Western blotting to detect ERK phosphorylation and canonical β‐catenin activation at the time points shown. Values of the normalized quantified signals are aligned below each relevant lane. Three‐way ANOVA was conducted to detect significance of ISO pretreatment (I), flow (F), and time (T) and the combination of these factors (I:F:T) on each signaling event. (B, C) After 1 hour under stationary (gold dots) or flow (blue dots) conditions, cells were analyzed for expression of mechanosensitive genes Fos (B) and Ptgs2 (C) relative to β‐Actin housekeeping gene by qRT‐PCR. Two‐way ANOVA analyses were performed to detect significance of ISO pretreatment (I) and flow (F), alone and in combination (I:F), for the expression of these genes. Western blot results are representative of 3 technical replicates. qRT‐PCR data are pooled from 2 replicate runs (n = 6). Post hoc analyses were used (p < 0.05; ^aversus no ISO; ^bversus no flow).

Fig. 4

Isoproterenol (ISO) selectively inhibits flow‐induced signaling and gene expression in MC3T3‐E1 cells. MC3T3‐E1 cells were pretreated with ISO at the concentrations shown and subjected to fluid flow (FF). (A) Phosphorylation of CREB, AKT, ERK, and canonical β‐catenin activation after flow were analyzed by Western blotting. Quantitative analysis of the blots is shown to the right. (B, C) after 1 hour under stationary (gold dots) or flow (blue dots) conditions, expression of mechanosensitive genes Fos (B) and Ptgs2 (C) was assessed by qRT‐PCR. Two‐way ANOVA analyses were performed to detect significance of ISO pretreatment (I), flow (F), and the combination of these factors (I:F), for the expression of these genes. Western blot results are representative of 3 independent experiments. qRT‐PCR data are pooled from 2 independent experiments (n = 6). Post hoc analyses were used (p < 0.05; ^aversus no ISO; ^bversus no flow).

Fig. 5

Butax co‐treatment reverses isoproterenol (ISO)‐induced effects in MC3T3‐E1 cells. MC3T3‐E1 cells were pretreated with ISO and Butax at the concentrations shown and subjected to fluid flow (FF). (A) Phosphorylation of CREB, AKT, ERK, and canonical β‐catenin activation after flow was analyzed by Western blotting. Quantification of the blots is shown to the right, but the unbalanced study design and small group sizes precluded statistical analysis. (B, C) After 1 hour under stationary (gold dots) or flow (blue dots) conditions, cells were analyzed by qRT‐PCR for expression of mechanosensitive genes Fos (B) and Ptgs2 (C). Three‐way ANOVA analyses were performed to detect significance of ISO (I), flow (F), Butax (B), and the combination of these factors (I:F:B), for the expression of these genes. Post hoc analyses were used (p < 0.05; ^aversus no ISO; ^bversus no flow; ^cversus no Butax). Western blot results are representative of 3 independent experiments. qRT‐PCR data are pooled from 3 independent experiments (n = 3–9).