Abstract
Determining how extracellular matrix (ECM) components influence cytokine-induced growth and differentiation of cells in malignancies and other diseases is critical for understanding disease pathophysiology and for developing novel treatment strategies. Bone morphogenetic proteins (BMPs) regulate the growth, differentiation and apoptosis of cells in the brain, bone, bone marrow and diverse tissues. ECM glycosaminoglycans (GAGs) such as heparan sulfate (HS) interact with and influence the biological activity of a number of proteins including BMPs. We examined if heparin, endogenous HS in malignant cells and the structurally abnormal HS accumulated in Hurler cells influence BMP signaling and activity. First we showed using real-time quantitative RT-PCR (qRT-PCR) that the BMP signaling pathway including BMPs 2–7, BMP and activin receptors and Smad-1 and -5 are expressed by SaOS-2 human osteosarcoma cells. Western immunoblotting showed that BMP-4 induced Smad-1 phosphorylation, activation and nuclear translocation. Optimal Smad-1 activation was achieved by 25 ng/ml BMP-4 at 30–60 min, and blocked by the extracellular BMP antagonist chordin. BMP-4 also induced a concentration-dependent increase in alkaline phosphatase activity, indicative of induction of osteogenic differentiation in these malignant cells. Soluble heparin directly inhibited BMP-4 induced Smad-1 phosphorylation, and also markedly augmented the inhibitory effect of chordin. Similar effects were seen with N-desulfated, N-re-acetylated heparin but to a lesser degree than with heparin, indicating that N-sulfation of glucosamine residues in heparin/HS contributes to the effect of GAGs on BMP signaling. Inhibition of sulfation of endogenous GAGs by sodium chlorate augmented BMP-4 mediated increase in alkaline phosphatase, suggesting that endogenous sulfated GAGs themselves block BMP-4 mediated malignant cell differentiation. Because BMPs play a critical role in neurogenesis and osteogenesis, we also examined if GAGs that accumulate in Hurler syndrome impair BMP-4 signaling. Neurological dysfunction and skeletal abnormalities are among the most devastating manifestations of Hurler syndrome, an inborn metabolic disorder due to lack of lysosomal GAG-degrading α-L-iduronidase (IDUA) enzyme that leads to HS and dermatan sulfate GAG accumulation. We recently showed that HS in Hurler syndrome cells are structurally and functionally abnormal, and have impaired capability to bind and mediate FGF-2 signaling (Pan C et al. Blood2005;106:1956–64). In the present study, using Affymetrix microarrays we found that expression of the BMP signaling cascade including BMPs 1–8, -10 and -15, BMP and activin receptors, Smads 1–8, chordin and inhibitors of DNA binding (IDs) 1–4 is equivalent in normal and Hurler bone marrow derived multipotent progenitor cells. In Hurler cells, BMP-4 did induce a concentration- and time-dependent activation and nuclear translocation of Smad-1 (confocal immunofluorescent microscopy). However, BMP-4 activity was significantly enhanced following clearance of the abnormally accumulated GAGs in Hurler cells by recombinant IDUA enzyme, indicating that GAGs in Hurler cells impair BMP-4 activity. Thus, both endogenous GAGs and exogenous (soluble) heparin, via N- and O-sulfated disaccharide residues, inhibit BMP-4 activity. These findings have implications for understanding the pathobiology of diverse diseases, and for developing novel therapeutic agents that may restore BMP signaling and activity.
A Survey of Strategies to Modulate the Bone Morphogenetic Protein Signaling Pathway: Current and Future Perspectives