Divergent activities of osteogenic BMP2, and tenogenic BMP12 and BMP13 independent of receptor binding affinities

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.

Figure 1

Characterization of purified rhBMP2, rhBMP12, and rhBMP13. (a) The purified proteins were collected and analyzed under reducing (RED) and nonreducing (NR) conditions using 12% polyacrylamide slab gels. (b) SDS-PAGE analysis of rhBMP12 purified from E. coli or CHO cells reduced with b-mercaptoethanol. (c) CD spectroscopy of BMP12 purified from E. coli or CHO cells.

Figure 2

Histology and X-ray analysis of BMP-treated implants after 14 days. Saffranin O stain of representative sections of implants containing (a) 25 µg of rhBMP2, (b) 25 µg of rhBMP12, or (c) 25 µg of rhBMP13. Faxitron X-ray images of representative implants containing (d) 25 µg of rhBMP2, (e) 25 µg of rhBMP12, or (f) 25 µg of rhBMP13. The implants containing rhBMP2 consists of mostly bone, whereas the implants containing rhBMP12 or rhBMP13 consists of only small little bone spicules (arrows).

Figure 3

Expression of bone, tendon, and cartilage markers by BMPs in C3H10T1/2 cells. mRNA levels were determined by Taqman for the bone markers (a) Ocn (osteocalcin), (b) Runx2 (runt-related transcription factor 2, (c) Alpl (ALP), and (d) Sp7 (osterix), the tendon markers (e) Thbs4 (thrombospondin 4) and (f) Tnmd, (tenomodulin) and the cartilage markers (g) Sox9 (SRY-box containing gene 9), (h) Col2a1 (collagen, type II, alpha 1), (i) Acan (Aggrecan 1) and (j) Col11a1 (collagen, type 11, alpha 1).

Figure 4

BMP activity in C3H10T1/2 cells. (a) C3H10T1/2 cells infected with BRE-Luc construct were treated with increasing concentration of rhBMP2, rhBMP12, or rhBMP13. (b) ALP activity in C3H10T1/2 cells following 2-day incubation with 0, 1, 10, or 100nM of rhBMP2, rhBMP12, or rhBMP13.

Figure 5

Induction of SMAD signaling by BMPs in C3H10T1/2 cells. Cell lysates from rhBMP2, rhBMP12, or rhBMP13 treated C3H10T1/2 cells were analyzed by western blot with antibodies to phosphorylated SMAD 1/5/8 and to β-actin or tubulin. Cells were treated with (a)10 nM BMPs for 0 to 30 min, (b) 10 nM BMPs for 0 to 120 min or (c) treated with 100 nM BMPs for 0 to 3 h.

Figure 6

Kinase dead ALK3 blocks Thbs4 induction. (a) mRNA levels for Thbs4 were determined by Taqman after treatment with kinase dead ALK3 and rhBMP1 2. Error bars represent SD (N = 3). (b) anti-HA western of protein extract from the infected cells to detect expression of the kinase dead ALK3.