Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs–G protein signaling in osteoblasts

Abstract

G protein–coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3-kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein–coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3-kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs.

Gs/Gi Regulation of Bone Cell Differentiation: Review and Insights from Engineered Receptors

“…Finally, bone is increasingly recognized as an endocrine organ that can regulate systemic hormones and metabolism through secreted factors. Since bone loss and increased adiposity appear to be linked clinically, and our prior gene expression studies suggested an increase in metabolism pathways in osteoblastic cells expressing Rs1 [83], we examined how increased osteogenesis might also affect energy metabolism. In addition to the dramatically increased bone formation, we found total body fat was significantly reduced starting at 3 weeks of age [118].…”

“…However, this model also displays an attenuation of ovariectomy-induced bone loss [81] and augmented osteogenic response to mechanical loading [82], which suggests differences in where or how this receptor functions under stress as compared to during homeostasis. Apelin is the ligand for a G i -coupled receptor that is highly expressed in freshly isolated calvarial osteoblasts [83] and can stimulate osteoblast proliferation [77,84]. Global knockout of this ligand may be expected to lead to a reduced bone volume due to reduced osteoblast number; however this is not the case, with the apelin knockout displaying increases in both cortical and trabecular bone associated with increased rates of mineral apposition and bone formation [84].…”

“…Chemogenetic receptors for each of the major G-protein signaling pathways are now available [12,106]. We used this synthetic biology strategy with the engineered RASSL "Rs1" to manipulate G s -GPCR signaling in bone using the Collagen 1a1 2.3 kb promoter fragment to create ColI(2.3) + /Rs1 + mice [83,[107][108][109][110][111][112][113][114]. The Rs1 receptor has strong basal G s signaling activity even in the absence of ligand, similar to the original 5HT4B receptor prior to introduction of the D100A mutation [115].…”

“…Furthermore, activation of the G s signaling pathway in osteoblastic cells of adult mice induced only a slight increase in the initial callus size and increased callus bone formation during fracture healing [114]. In calvarial osteoblasts, gene expression analysis revealed that increased G s -GPCR signaling in osteoblastic cells could lead to activation of cell differentiation, cytokine and growth factor, angiogenesis, coagulation, and energy metabolism pathways [83].…”

Osteoblast-derived FGF9 regulates skeletal homeostasis

“…PTH activates the G s -cyclic AMP signaling pathway by acting on the PTH/PTHrP receptor 1 (PTHR1) in osteoblasts. By assessing the transcriptome of maturing osteoblasts expressing a G s -activating engineered receptor (Rs1) (2,3), we found that fibroblast growth factor 9 (Fgf9) was significantly down-regulated by G s signaling, suggesting a possible role for osteoblast-derived FGF9 in regulating bone homeostasis (4).…”