PMC

PEDF directs MSC fate toward osteoblasts and away from adipocytes. This occurs through its action as a Wnt-β-catenin agonist that suppresses PPARγ.

PEDF expression is markedly suppressed during adipogenesis. A) PEDF expression from FACS-sorted adipocyte precursors and mature adipocytes derived from subcutaneous fat. B) Analysis of unbiased microarray analyses of SVCs undergoing adipogenesis was interrogated for PEDF expression. PEDF expression was reduced by 90% after SVC conversion to mature adipocytes.

A) PEDF activates Wnt signaling in hMSCs. hMSCs were treated with Wnt3a (50 ng/ml) and PEDF (500 ng/ml) and immunoblotted for phosphorylated LRP6. B) hMSCs were treated with IWP-2 (2 μM) for 24 and 48 h and then challenged with PEDF (500 ng/ml in basal medium). Blots are representative of n = 4 experiments/condition. Vehicle was PBS for Wnt3a and PEDF experiments; DMSO for IWP-2 experiments. C) Committed preadipocytes, 3T3-L1 cells, transfected with vector and shRNA targeting LRP6 were assessed for knockdown of LRP6. D) Adding PEDF significantly suppressed PPARγ in vector-transfected cells, while LRP6 knockdown resulted in increased PPARγ expression.

PEDF enhances osteoblast mineralization and differentiation. A) PEDF KO SVCs were placed in osteoblast differentiation medium for 21 d and stained with Alizarin Red (top panels). KO SVCs were treated with vehicle or PEDF (500 ng/ml/d) starting on d 2 of osteoblast differentiation, and gene expression evaluated on d 21. Differences in Runx2 or collagen 1a1 expression were not seen with PEDF, while PEDF significantly suppressed TGF-β and PPARγ expression. B) hMSCs demonstrate increased alkaline phosphatase staining with PEDF. C) Osteoblast progenitors from WT and PEDF KO mice display increased alkaline phosphatase staining in response to PEDF. Os, osteoblast differentiation medium.

PEDF suppresses adipogenesis. A) MSCs derived from SVCs from the subcutaneous fat pads of WT and PEDF KO mice undergo adipogenesis by d 8, as demonstrated by Oil Red O staining. B) MSCs from WT mice were treated with vehicle or PEDF (500 ng/ml/d) and assessed for adipocyte differentiation, represented by Oil Red O staining, on d 8. C) Treatment with PEDF starting on d 1 of differentiation significantly suppressed multiple proadipogenic transcription factors and adipocyte-specific markers. D) Proadipogenic transcription factor PPARg and its cofactor, PGC1a, were significantly higher in PEDF KO compared with WT MSCs at baseline. PEDF inhibited PPARg and PGC1a expression in WT and KO SVCs when PEDF was initiated on d 3 of differentiation. E) PEDF inhibits adipogenesis of hMSCs. PEDF was started on d 0 of differentiation, and cells were differentiated in adipogenic medium for 10 d. Ad, adipocyte differentiation medium; vehicle, PBS.

PEDF deletion is associated with increased total body adiposity and reduced bone mineral content in mice. A) Body weight and percentage of total body fat by MR spectroscopy of 12-wk-old WT and PEDF KO mice. B) Representative images of WT and PEDF KO subcutaneous (top left panel) and epididymal (bottom left panel) fat pads, and corresponding quantification of subcutaneous white adipose tissue (SWAT), epididymal white adipose tissue (EWAT), and retroperitoneal white adipose tissue (RWAT) under normal feeding and 1 wk of a high-fat diet (right panels). C) MicroCT-obtained images of trabecular, dorsal, and lateral surface bone morphology of distal femurs: left panels, cross-section; left center panels, dorsal frontal view; right center panels, left lateral surface; right panels, cut left lateral view. D) Quantification of trabecular bone volume (BV), total volume (TV), and BV/TV, demonstrating diminished trabecular volumes in PEDF KO bones. E) Low-power (4×) and high-power (10×) images of Goldner's stained tibiae and femurs from WT and PEDF KO mice. Decreased epiphyseal and chondro-osseous mineral content in 14-d-old KO compared with WT mice (arrows). Older (26-d-old) mice demonstrate hypomineralization in the epiphysis (short arrows) and chondro-osseous junction (longer arrows) with a diminished proliferative (P) zone; n = 6–9 mice for adipose tissue determination and n = 3–4 mice for bone imaging.