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RGS5 pericytes contribute to bone formation under homeostasis and fracture repair

RGS5 (Regulator of G Protein Signaling 5) is a GPTase activator for heterotrimeric G-protein α-subunits and shown to be a marker of pericytes. Existing studies point to a potential role of RGS5 in bone and bone remodeling. Goal of our study is to generate a mouse model using RGS5 suitable for lineage tracing of osteoprogenitors. Mice were generated using Bacterial Artificial Chromosome (BAC) containing full-length murine RGS5 BAC vector and CreERT2sequence. Germ line founders were confirmed for Cre and F1 progeny crossed with Ai9 reporter mice where upon tamoxifen (Tx) treatment, RGS5 cells and their progeny will express tdTomato (Tom). Tom+ expression was confirmed in blood vessels of heart and kidney and absent in lung. Histological analysis of femurs under homeostatic conditions 2 days post Tx treatment showed RGS5/Tom+ cells are perivascular within the bone marrow (BM), periosteum, endosteal, trabecular, and cortical channel niches surrounding CD31 vasculature. Expansion of RGS5/Tom+ cells was most evident within the trabecular area at 20 days post Tx where they are positioned between mineralized matrix and CD31 vasculature, some lining the bone surface co-expressing osterix and embedded in bone. Few RGS5/Tom+ chondrocytes were seen in the growth plate. Using stabilized femoral fractures to study bone regeneration, histological sections of lineage tracing at 7 days post-fracture (dpf) demonstrated a heterogenous population of Tom+ cells including pericyte, fibroblastic cells and chondrocytes within the fracture callus expanded mostly in the BM. FACS analysis of callus tissue and intact periosteum was done to phenotype RGS5/Tom+ cells using bone progenitor markers. 9.3% and 6.1% RGS5/Tom+ cells are present in callus and periosteal tissue and do not express endothelial CD31 or hematopoietic CD45 markers. Within the Tom+ gates, both populations were 99% CD51+ with equivalent levels of PDGFRß expression (callus 77% and periosteum 80%). However, CD51+Sca1+ cells decreased from 72.4% in periosteum to 36.9% in callus digests indicating these Tom+ progenitors have become more committed cell type during fracture repair at 7 dpf. RGS5/Tom+Osterix+ cells 10 dpf, are most notably seen around newly formed bone within the BM cavity. Under homeostatic conditions, lineage traced RGS5 pericytes within the trabecular area are demonstrating osteoprogenitor capacity. In an injury model, this osteoprogenitor potential is more pronounced and mostly evident within the BM niche.

osteoprogenitors, periosteum, perivascular, Regulator of G Protein Signaling 5

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