Ligand-Mediated Activation of an Engineered Gs G Protein-Coupled Receptor in Osteoblasts Increases Trabecular Bone Formation

Abstract

Age-dependent changes in skeletal growth play important roles in regulating skeletal expansion and in the course of many diseases affecting bone. How G protein-coupled receptor (GPCR) signaling affects these changes is poorly understood. Previously, we described a mouse model expressing Rs1, an engineered receptor with constitutive G(s) activity. Rs1 expression in osteoblasts from gestation induced a dramatic age-dependent increase in trabecular bone with features resembling fibrous dysplasia; however, these changes were greatly minimized if Rs1 expression was delayed until after puberty. To further investigate whether ligand-induced activation of the G(s)-GPCR pathway affects bone formation in adult mice, we activated Rs1 in adult mice with the synthetic ligand RS67333 delivered continuously via an osmotic pump or intermittently by daily injections. We found that osteoblasts from adult animals can be stimulated to form large amounts of bone, indicating that adult mice are sensitive to the dramatic bone- forming actions of G(s) signaling in osteoblasts. In addition, our results show that intermittent and continuous activation of Rs1 led to structurally similar but quantitatively different degrees of trabecular bone formation. These results indicate that activation of a G(s)-coupled receptor in osteoblasts of adult animals by either intermittent or continuous ligand administration can increase trabecular bone formation. In addition, osteoblasts located at the bone epiphyses may be more responsive to G(s) signaling than osteoblasts at the bone diaphysis. This model provides a powerful tool for investigating the effects of ligand-activated G(s)-GPCR signaling on dynamic bone growth and remodeling.

Remote Control of Neuronal Signaling

“…It is noteworthy that unlike Ro1 expression in cardiomyocytes and astrocytes, the timing of Rs1 expression and signaling in osteoblasts is critical for the development of pathologic conditions; Rs1 induction after P28 does not alter trabecular bone mass, which suggests a role for prepubertal bone growth in osteosclerosis . Significantly, induction of Rs1 expression in mice at P28, followed by continuous or intermittent exposure to the Rs1 agonist RS67333 from P70 to P140, greatly increased bone formation (Hsiao et al, 2010b), indicating that adult bone tissue is still sensitive to G s -mediated signaling.…”

Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing

“…These mice showed significant trabecular bone expansion that resembled the fibrous dysplasia-like lesions previously reported in the Col1(2.3) þ /Rs1 þ mice in which Rs1 was expressed from gestation (15) or in Col1/Rs1-late mice treated with 10 weeks of agonist without fractures. (31) In contrast, the contralateral nonfractured limb of mice treated for 3 weeks with agonist showed no major morphologic changes in their structure, likely because of the short duration of ligand exposure.…”

“…(15) However, bone formation can be stimulated in these Col1(2.3) þ /Rs1 þ mice by treatment with RS67333, a synthetic serotonin receptor agonist that strongly activates the Rs1 receptor and further increases G s signaling. (31) These prior results suggested that blockade of G i signaling could produce a bone anabolic effect that shared some features with activation of G s signaling in osteoblasts. (9,14,15) Because G s and G i signaling functions have distinct roles in normal bone formation, we took advantage of our transgenic strategies to delineate the individual effects of the G i and G s pathways by applying fracture models to mice with either inhibited endogenous G i signaling in osteoblasts using PTX or activated G s signaling mediated by the engineered receptor Rs1.…”

“…(15) RS67333 at 12.5 mg/mL in vehicle or vehicle alone was loaded into Durect ALZET miniature osmotic pumps (model 2004 for the 3-week cohort, or model 1002 for up to 2 weeks; Cupertino, CA, USA) to deliver 3 mg/kg/day at a flow rate of 0.25 mL per hour. (31) Pumps were primed overnight in normal saline at 37°C before being implanted into the intraperitoneal space of the treated mice at the time of unstable fracture.…”

“…We previously showed that the engineered receptor Rs1 increases G s signaling in cells (30) and that increased G s activity from osteoblastic Rs1 activated by a synthetic ligand in Col1/Rs1-late mice could dramatically increase bone formation. (31) We examined how G s -GPCR signaling in osteoblastic cells could affect bone healing by using nonstabilized tibial fractures in the Col1/Rs1-late mice and their control littermates. Because we were concerned that G s activation in fractures could shorten the evolution of a fracture callus, we assessed callus formation and the timing of fracture repair at 7, 14, and 21 days (Fig.…”