Sympathetic control of bone mass regulated by osteopontin

Abstract

The sympathetic nervous system suppresses bone mass by mechanisms that remain incompletely elucidated. Using cell-based and murine genetics approaches, we show that this activity of the sympathetic nervous system requires osteopontin (OPN), a cytokine and one of the major members of the noncollagenous extracellular matrix proteins of bone. In this work, we found that the stimulation of the sympathetic tone by isoproterenol increased the level of OPN expression in the plasma and bone and that mice lacking OPN (OPN-KO) suppressed the isoproterenol-induced bone loss by preventing reduced osteoblastic and enhanced osteoclastic activities. In addition, we found that OPN is necessary for changes in the expression of genes related to bone resorption and bone formation that are induced by activation of the sympathetic tone. At the cellular level, we showed that intracellular OPN modulated the capacity of the β2-adrenergic receptor to generate cAMP with a corresponding modulation of cAMP-response element binding (CREB) phosphorylation and associated transcriptional events inside the cell. Our results indicate that OPN plays a critical role in sympathetic tone regulation of bone mass and that this OPN regulation is taking place through modulation of the β2-adrenergic receptor/cAMP signaling system.

Fig. 1.

ISO treatment up-regulates OPN levels in bone and circulation. (A and B) OPN protein expression in circulation for both 129sv (A) and C57B/L6 (B) mice in response to ISO treatment. (n = 3–4 per group, *P < 0.05.) (C) OPN mRNA expression in bone in vivo in response to ISO treatment, normalized to GAPDH mRNA expression. Results are in arbitrary units as mean ± SEM of n = 4 (*P < 0.05).

Fig. 2.

OPN deficiency suppresses ISO-induced bone loss. (A–D) Representative 3D-μCT images of the distal metaphyseal regions of femora in vehicle (A and C) and ISO treatment (B and D) groups of WT (A and B) and OPN-KO (C and D) mice. (E–H) Bone parameters measured from data shown in A–D. (E) BV/TV. (F) Trabecular separation (Tb.Sp). (G) Trabecular number (Tb.N). (H) Trabecular spacing (Tb.Spac). Data represent the mean ± SEM of n = 7–8 mice per group (*P < 0.05). (I–L) Representative 2D-μCT images of vertebrae in mice treated with vehicle (I and K) and ISO treatment (J and L) groups of WT (I and J) and OPN-KO (K and L) mice. (M–P) Bone parameters measured from data shown in I–L. (M) BV/TV. (N) Trabecular separation (Tb.Sp). (O) Trabecular number (Tb.N). (P) Trabecular spacing (Tb.Spac). Data represent the mean ± SEM of n = 7–8 mice per group (*P < 0.05).

Fig. 3.

OPN control of the inhibitory effect of ISO on bone remodeling. (A–D) Secondary trabecular regions of the epiphyses of tibiae were examined for osteoclasts based on TRAP staining in vehicle (A and C) and ISO treatment (B and D) groups of WT (A and B) and OPN-KO (C and D) mice. (E–G) Parameters of osteoclast activity. Number of osteoclasts per bone surface (N.Oc/BS) (E), osteoclast surface per bone surface (Oc.S/BS) (F), and urinary deoxypyridinoline (Dpd) (G) excretion levels are shown. Bars represent the mean ± SEM of n = 5–9 mice per group (*P < 0.05). (H–K) Bone formation activity was evaluated in vivo as described in Materials and Methods. Calcein bands were visualized to obtain dynamic histomorphometric parameters. (L–O) Parameters of osteoblast activity. Levels of mineral apposition rate (MAR) (L), bone formation rate (BFR) (M), osteoblast number (Ob.N/BS) (N), and osteoblast surface (Ob.S/BS) (O) are shown. Bars represent the mean ± SEM of n = 5 mice per group (*P < 0.05).

Fig. 4.

OPN deficiency suppresses ISO-induced osteoclast development and changes in mRNA expression. (A) Bone marrow cells obtained from WT and OPN-KO mice treated with ISO or saline vehicle (Ctrl) were cultured in the presence of vitamin D and dexamethasone to determine osteoclast development (TRAP⁺ multinucleated cells). Bars represent the mean ± SEM of n = 5 mice per group (*P < 0.05). (B–D) Gene expression in bone samples from WT and OPN-KO mice. Expression of TRAP (B), Runx2 (C), and alkaline phosphatase (ALP) (D), all normalized to GAPDH mRNA expression, in bone samples from WT and OPN-KO mice. Results are expressed in arbitrary units as mean ± SEM of n = 5–6 per group (*P < 0.05).

Fig. 5.

Effects of neutralizing antibody against OPN on ISO-induced bone loss. (A–C) μCT analyses of WT proximal tibiae treated with or without 2C5. (D–G) Structural parameter analysis of the tibiae. (D) BV/TV. (E) Trabecular number (Tb.N). (F) Trabecular separation (Tb.Sp). (G) Trabecular spacing (Tb.Spac). (H) In vitro reduction of osteoclast development by anti-OPN antibody, 2C5 (*P < 0.05).

Fig. 6.

OPN control of β2AR signaling. (A) Averaged cAMP responses mediated by ISO. cAMP induction over a 30-min time course was monitored by FRET changes from MC3T3 osteoblastic cells transiently expressing epac-CFP/YFP alone (control, Left) or in combination with si-OPN (Center) or overexpressing OPN (Right). Bars represent the average cAMP responses determined by measuring the area under the curve from 0 to 30 min for cAMP. Data represent the mean ± SEM of n = 15 (ctrl), n = 28 (si-OPN), and n = 34 (OPN). (*P < 0.01, **P < 0.05). (B) Western blot analyses of CREB phosphorylation in control and OPN-depleted MC3T3 cells in response to 10 μM ISO. Bars represent the mean ± SEM of n = 3 (**P < 0.01). (C) CRE-Luc activity (pCRE-Luc) in control cells (control) and cells transfected with si-OPN in response to 10 μM ISO with or without H89, IBMX, or forskolin (Fsk). siRNA for OPN enhanced the ISO-induced increase in the levels of luciferase activity, and H89, a PKA inhibitor, suppressed the enhancement. Bars represent the mean ± SEM of n = 4 (*P < 0.05, **P < 0.01). (D) Coimmunoprecipitation of GαS and OPN. Cells transfected with GαS in combination with Gβ1γ2 (G_S) and empty vector were challenged with carrier or with 10 μM ISO. OPN was immunoprecipitated with Sepharose beads conjugated to anti-C25 monoclonal antibody, and Gα_S was detected with a polyclonal antibody against GFP (n = 4). IP, immunoprecipitation; IB, immunoblot detection.

Fig. 7.

Model of regulation of β2AR signaling by OPN. Our data suggest that intracellular OPN regulates β2AR signaling in osteoblasts by interacting with G_S to reduce the time course of cAMP production and CRE-dependent transcription activity to ultimately reduce bone mass. Extracellular OPN is also involved in regulation of bone cells by activation of CD44/integrin family of receptors.