Project description: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. We utilized a microarray approach to examine Rs1-induced alterations in the OB transcriptome. The approach used to identify an approximate snapshot of the OB transcriptome at the time of sacrifice by isolating the OB population that expresses Rs1 by GFP labeling, without the use of cell culture. We compared gene expression between control OBs and OBs-expressing Rs1 in triplicates.

Project description: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.

Project description:Background Skeletal morbidity in cancer patients has a major impact on quality of life and preserving bone health while improving outcomes is an important goal of modern antitumor treatment strategies. Despite their widespread use in early disease stages, the effects of immune-checkpoint inhibitors (ICI) on the skeleton are still poorly defined. Here, we initiated a comprehensive investigation of the impact of ICI on bone turnover by longitudinal assessment of bone turnover markers (BTM) in cancer patients and by validation in a novel bioengineered 3D model of bone remodeling. Methods Serum markers of bone resorption (C-terminal telopeptide, CTX) and formation (procollagen type I N-propeptide, PINP; and osteocalcin, OCN) were measured before each ICI application (PD1 inhibitors, PD1i or PD-L1 inhibitors, PD1i) for 6 months or until disease progression in patients with advanced cancer and no evidence of bone metastases. To validate the in vivo results, we evaluated osteoclast (OC) and osteoblast (OB) differentiation upon treatment with ICI. In addition, their effect on bone remodeling was assessed by immunohistochemistry, immunofluorescence and proteomics analysis in a dynamic 3D model of the bone multicellular unit (BMU). Results The longitudinal assessment of BTM revealed a significant decrease in CTX levels after 3 weeks, followed by a return to baseline levels within 3 months. In contrast, serum levels of PINP and OCN gradually increased from baseline to the last postbaseline assessment. In vitro, ICI impaired the maturation of preosteoclasts by inhibiting STAT3/NFATc1 signaling, but not JNK, ERK and AKT; while lacking any direct effect on osteogenesis. However, using our novel bioengineered 3D bone model, which enables the simultaneous differentiation of OB and OC precursor cells, we confirmed the uncoupling of the OC/OB activity upon ICI treatment by demonstrating impaired OC maturation along with increased OB differentiation. Conclusion Our study indicates that the inhibition of the PD1/PD-L1 signaling axis interferes with the OB/OC coupling of bone turnover and may potentially exert a protective effect on bone by impairing the differentiation OCs and indirectly promoting osteogenesis.

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Gene expression analysis in the zebrafish fractured scale

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