Exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity.

PURPOSE: The purpose of this study was to explore the effect of heparin on bone morphogenetic protein 6 (BMP6) osteogenic activity. METHODS: Western blot analysis was used to confirm the binding of BMP6 to heparin and to observe its effect on BMP6 signaling in C2C12-BRE-Luc myoblasts. Real-time RT-PCR was performed for the expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) in C2C12 myoblasts treated with BMP6 and heparin for 72 hours. Rat ectopic bone formation assay was performed to explore the effect of heparin on BMP6 osteogenic activity. Two weeks following implantation the implants were analysed morphologically and histologically. A mouse osteoporotic model was used to test the ability of BMP6 to improve the bone quality in vivo in the presence of heparin, followed by DEXA and µCT analyses. Blood coagulation was tested in rats previously treated with BMP6. RESULTS: BMP6 specifically bound to heparin and induced Smad1/5/8 phosphorylation which was inhibited by heparin. After 48 and 72 hours of treatment, heparin inhibited BMP6-induced ALP and OC expression in C2C12 cells. Heparin dose dependently inhibited BMP6-induced new bone and cartilage formation in the rat ectopic bone formation assay, while in osteoporotic mice heparin inhibited the BMP6 potential to improve the bone quality as evidenced by decreased bone mineral density and trabecular bone parameters. Interestingly, BMP6 prevented the effect of heparin on the blood coagulation parameters. CONCLUSION: The interaction of BMP6 with heparin might contribute to the heparin-induced osteoporosis and blood coagulation.

Circulating bone morphogenetic protein 1-3 isoform increases renal fibrosis.

Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography-mass spectrometry, we identified BMP6, GDF15, and the BMP1-3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1-3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1-3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1-3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFß1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1-3 increased the transcription of collagen type I, TGFß1, ß-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin ß1 subunit. The profibrotic effect of BMP1-3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1-3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD.