Extracellular norepinephrine clearance by the norepinephrine transporter is required for skeletal homeostasis

Abstract

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.

Keywords: ADHD; Bone Remodeling; Gene Knockout; Mouse; Nerve; Neurotransmitter Transport; Norepinephrine Transporter; Osteoblasts; Sympathetic Nervous System.

FIGURE 1.

NET is expressed in differentiated osteoblasts. A and B, RT-PCR analysis of Net, Tyrosine hydroxylase (Th), Dopamine β-hydroxylase (Dbh), Monoamine oxidase (Mao), Catechol-O-methyltransferase (Comt), and Extraneuronal monoamine transporter (Emt). RNA extracts from brain and heart serve as positive controls. Osteocalcin (Ocn) is used as a marker for differentiated osteoblasts. Calcitonin receptor (Ctr) is used as a marker for differentiated osteoclasts. C–F, real-time quantitative PCR analysis of Net expression in undifferentiated (day 0) and differentiated (day 21 or day 33) primary calvarial osteoblasts. Ocn and Sclerostin (Sost) are used as markers for differentiated osteoblasts/osteocytes. Values are given as mean ± S.E., *, p < 0.05 versus day 0, n = 3. G, Western blot analysis of NET expression in undifferentiated (day 0) and differentiated (day 14) mouse calvarial primary osteoblasts. Protein extract from the cerebral cortex serves as a positive control. H, NET immunoreactivity (brown staining) detected by immunohistochemistry in nerves (a) and osteoblasts (b) in neonate tibia sections.

FIGURE 2.

Differentiated osteoblasts transport NE. In vitro uptake assays using: A, non-differentiated osteoblastic cells (CAD: neuronal cell line stably expressing hNET, used as positive control); B, differentiated calvarial POB; and C, differentiated BMSCs. Values are given as mean ± S.E., *, p < 0.05, n = 3.

FIGURE 3.

Chronic reboxetine treatment reduces bone mass. Behavioral tail suspension test in 14-week-old male (A) and female (D) mice treated with vehicle (Veh) or reboxetine (Reb). Micro-CT analyses of femoral (B and E) and vertebral (C and F) trabecular bone in male (B and C) and female (E and F) mice. G, histomorphometric analyses of vertebrae in 14-week-old male mice. ObS/BS, osteoblast surface/bone surface; N.Ob/B.Pm, number of osteoblast per bone perimeter; OcS/BS, osteoclast surface/bone surface; N.Oc/B.Pm, number of osteoclast per bone perimeter; BFR/BS, bone formation rate/bone surface; MAR, mineral apposition rate. Values are given as mean ± S.E., *, p < 0.05 versus vehicle, n = 8–10 per group.

FIGURE 4.

Net^−/− mice are leaner and shorter than WT littermates. A and B, body weight (BW); C and D, femur length. Values are given as mean ± S.E., *, p < 0.05 versus WT, n > 8 per group.

FIGURE 5.

Normal body fat or muscle mass over body weight in 3-month-old Net^−/− mice. A and B, males; C and D, females. Values are given as mean ± S.E., *, p < 0.05 versus WT, n > 8 per group.

FIGURE 6.

Net deficiency causes a low peak bone mass and reduced bone strength. Micro-CT analyses of femoral (A and B) and vertebral (C and D) trabecular bone in males (A and C) and females (B and D) at 1-, 3-, and 6-month-old ages (BV/TV: bone volume/tissue volume). Peak force (E) and stiffness (F) measured by biomechanical three-point bending tests of femurs from 3-month-old males. G, histomorphometric analyses of vertebrae in 6-month-old male mice. ObS/BS (%), osteoblast surface/bone surface; N.Ob/B.Pm (#/mm), number of osteoblast per bone perimeter; OcS/BS (%), osteoclast surface/bone surface; N.Oc/B.Pm (#/mm), number of osteoclast per bone perimeter; BFR/BS (μm/d), bone formation rate/bone surface. Values are given as mean ± S.E., *, p < 0.05 versus WT, n = 8 per group.

FIGURE 7.

Low sympathetic outflow in Net^−/− mice. A and B, brown adipose tissue (BAT) Ucp1 expression measured by quantitative PCR is decreased in Net^−/− mice. C and D, Decreased NE content in Net^−/− bones. Values are given as mean ± S.E., *, p < 0.05 versus WT, n ≥ 8.

FIGURE 8.

NET blockade is required for chronic immobilization stress-induced bone loss. A–D, CIS does not induce bone loss in 3-month-old WT mice. Micro-CT analyses of femoral (A and B) and vertebral (C and D) trabecular bones in males and females. E, CIS induces bone loss in male WT mice only when NE reuptake is inhibited. F, concurrent CIS and NET blockade reduces the number of bone marrow osteoprogenitors (assessed by the number of Cfu-AP colonies, right panel) compared with the control or CIS groups, following 14 days of in vitro differentiation in osteogenic condition. Values are given as mean ± S.E., *, p < 0.05 versus control; #, p < 0.05 versus CIS, n ≥ 8.