Assessment of the Osteoblast Transcriptome in a Model of Markedly Enhanced Intramembranous Bone Formation Due to Constitutive Gs-G Protein Signaling in Osteoblasts

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 which were accompanied by an increase in OB lineage cells, especially immature OBs, indicated by an expansion of cells expressing Osterix and Runx2 in the whole femur. In this study, we further evaluated how Gs signaling in OBs affects intramembranous bone formation by examining calvariae of one-and nine-week-old Col1(2.3)/Rs1 mice. Rs1 calvariae displayed a dramatic increase in total volume and trabecular bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space in Rs1 expressing mice while Osteocalcin was expressed predominantly in cells along bone surfaces. These findings resembled that previously seen in Rs1 femoral bones, 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. However, it is still unclear how G protein signaling in mature OBs leads to the observed alterations in bone mass. In this study, we investigated the cellular basis of the skeletal changes by assessing the effect of Rs1 expression in vivo on the transcriptome of mature OBs. We identified the complete set of Gs-GPCRs and other GPCRs that are expressed on OBs which may contribute to the observed skeletal phenotype. Candidate paracrine mediators of the effect of Gs signaling in OBs were determined. Genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. Our results identify novel candidate mediators of the anabolic response to the skeleton to Gs signaling in mature OBs.

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Wattanachanya L, Wang L, Millard SM, Lu WD, O'Carroll D, Hsiao EC, Conklin BR, Nissenson RA