Lentiviral delivery of PPARγ shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.
BACKGROUND
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 (PPARγ) 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 PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model.


METHODS
PPARγ 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 PPARγ 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 (PPARγ, fatty acid binding protein 4 [FABP4]), osteoblasts (alkaline phosphatase [ALP], osteocalcin [OCN]), and osteoclasts (tartrate-resistant acid phosphatase [TRAP], Cathepsin K).


RESULTS
In vitro, PPARγ 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 PPARγ shRNA treatment.


CONCLUSIONS
These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and it may help determine whether PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.
1 Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California , Los Angeles, Los Angeles, California.
Aaron W
AW
James
Jia
J
Shen
Kevork
K
Khadarian
Shen
S
Pang
Greg
G
Chung
Raghav
R
Goyal
Greg
G
Asatrian
Omar
O
Velasco
Jung
J
Kim
Xinli
X
Zhang
Kang
K
Ting
Chia
C
Soo
D050156:Adipogenesis; D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis / Q000235:genetics; D005269:Femur / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016086:Lentivirus; D008297:Male; D051379:Mice; D016513:Mice, SCID; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis; D047495:PPAR gamma / Q000096:biosynthesis* / Q000235:genetics; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000235:genetics
D000943:Antigens, Differentiation; D047495:PPAR gamma; D034741:RNA, Small Interfering