Fig. 6.
Hypersecretion of MMP-2/9 promotes excessive latent TGF-β1 activation. (A) Active TGF-β1, latency associated peptide (LAP)-bound TGF-β1, and β-actin were detected by Western blot in protein extracts from the fracture site of Nf1flox/−;Col2.3Cre mice with tibial fracture nonunion and WT controls. The bar graph represents the fold change in the active TGF-β1/LAP-TGF-β1 ratio. n = 5 to 6 mice per group. (B) Activity levels of MMP-2/9 were measured in WT and Nf1+/− myeloid cell conditioned medium by zymography. n = 3 biological replicates. (C) Osteoprogenitors transfected with a Smad luciferase reporter were stimulated with recombinant, active TGF-β1 (1 ng/mL) and latent, LAP-TGF-β1 (100 ng/mL) for 18 hours. n = 3 technical replicates. The experiment was repeated on two independent occasions with similar results. *p < 0.05, **< 0.01. (D) Working model of NF1 skeletal defects mediated by a pathological cycle of increased TGF-β1 synthesis, activation, and Smad signaling.