Abstract
Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.
Highlights
Bone morphogenic proteins (BMPs) are members of the transforming growth factor beta superfamily, which is a group of growth factors known to induce growth and differentiation of various cell types (Reddi 1998)
Purification of recombinant human bone morphogenetic protein 2 (rhBMP2), rhBMP12, and rhBMP13 The mature domain of rhBMP2 was purified from CHO cells overexpressing rhBMP2.The mature domains of rhBMP12 and rhBMP13 were purified from E. coli inclusion bodies and refolded
Distinct intracellular pathway activation with rhBMP2, rhBMP12, and rhBMP13 As C3H10T1/2 cells were responsive to all three BMPs and replicate many of the molecular changes observed in vivo after ectopic expression, we explored differences in intracellular signaling mechanisms in this cell line
Summary
Bone morphogenic proteins (BMPs) are members of the transforming growth factor beta superfamily, which is a group of growth factors known to induce growth and differentiation of various cell types (Reddi 1998). RNA expression analysis identified many tendon marker genes including Col1a1, Col1a2, Eya1, Eya2, Scx, Epha4, Tnmd, and Thbs4 that exhibited changes in relative level in rhBMP12- and rhBMP13-induced ectopic tissues (Table II).
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