Lentiviral Delivery of PPAR gamma shRNA Alters the Balance of Osteogenesis and Adipogenesis, Improving Bone Microarchitecture

Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is commonly considered the master transcriptional regulator of adipogenesis, however, it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPAR gamma shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model. Methods: PPAR gamma shRNA was delivered by a replication-deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control green fluorescent protein lentivirus or PPAR gamma shRNA expressing lentivirus were delivered by intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and postmortem analysis at 4 weeks. Postmortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, quantitative real time polymerase chain reaction analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPAR gamma, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K). Results: In vitro, PPAR gamma shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at 4 weeks postinjection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+osteoblasts and decrease in TRAP+multinucleated osteoclasts was observed with PPAR gamma shRNA treatment. Discussion: These findings suggest that acute loss of PPAR gamma in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPAR gamma shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPAR gamma shRNA on OB and OC cell types, and it may help determine whether PPAR gamma antagonists are potential therapeutic agents for osteoporotic bone loss.