Figure 1.
Characterization of canine ASCs.
Representative images show positive immunofluorescent staining of stromal markers CD29, CD44, and CD 90 on almost all passage 2 ASCs (A). Representative histograms from flow cytometry analysis reveal dominant expression of CD 44 and CD90 on passage 6 ASCs (B). The mRNA expression of hematopoietic markers CD14 and CD34 in ASCs was dramatically reduced with progressive passages (C). The population doubling time (PDT) of canine ASCs was is shown through passage 6 (D).
Figure 2.
BMP12 and BMP14 induce tenogenic gene expression in canine ASCs.
The mRNA expression of the tendon markers SCX and TNMD was significantly increased in canine ASCs treated with BMP12. In contrast, the expression of the bone marker BGLAP was dose- and time-dependently reduced by BMP12. Similar changes in gene expression were detected in BMP14-treated ASCs. The results were determined by quantitative real-time RT-PCR and are shown as fold change related to the expression level of control ASCs (dashed lines). Log scales are used in (E) and (F). The legend in (A) applies to all of the panels.
Figure 3.
BMP12 increases SCX and TNMD protein expression in ASCs.
Representative Western blots (A and C) and semi-quantifications (B and D) of band volumes of the corresponding bands are shown.
Figure 4.
BMP12 induces scleraxis promoter-driven GFP expression in mouse ASCs.
GFP was expressed by all the TFs from the ScxGFP transgenic mice in the cell nuclei (A). The number of GFP-positive cells was increased in BMP12-treated ASCs in a dose- (B) and time- (C) dependent manner. Representative fluorescent and phase contrast images are shown. ASCs in (B) and (C) were treated with BMP12 for 14 days and at a concentration of 1000 ng/ml, respectively. The scale bar = 50 µm and applies to all of the panels.
Figure 5.
BMP12 increases TNMD protein expression in mouse ASCs.
The expression of TNMD protein (in red) in ASCs from ScxGFP transgenic mice was dose-dependently increased after a 14-day treatment with BMP12. Representative fluorescent images are shown. Cell nuclei were counterstained with H33258 (in blue). The scale bar = 50 µm and applies to all panels.
Figure 6.
BMP12 activates Smad1/5/8 signaling pathway in ASCs.
The interaction of BMPs with type I and type II receptors leads to the transphosphorylation of type I receptors by type II receptors (A). The type I receptors are the signaling receptors that activate Smad signaling pathways. There are seven known type I receptors, namely ALK1 through ALK7. They are typically separated into two groups: ALK1, 2, 3, and 6 phosphorylate Smad1/5/8; ALK4, 5, and 7 phosphorylate Smad2/3. LDN-193189 and SD-505124 are selective inhibitors for ALK2, 3, 6 and ALK4, 5, 7, respectively (A). For simplicity, ALK1 is not shown in the figure. Smad1/5/8 (B) but not Smad2 (C) and p38 (D) in ASCs was phosphorylated by BMP12 (1000 ng/ml) as detected by Western blot. BMP2 (200 ng/ml) and TGFbeta3 (10 ng/ml) were used as positive controls for the induction of phosphorylation of Smad1/5/8 and Smad2, respectively.
Figure 7.
ALK2, 3, and 6 receptors are involved in BMP12 signaling.
The BMP12-induced phosphorylation of Smad1/5/8 was dose-dependently blocked by the selective ALK2, 3, and 6 inhibitor LDN-193189 (A) but not by the ALK4, 5, and 7 inhibitor SB-505124 (B). The latter blocked the phosphorylation of Smad2 induced by TGFbeta3. Representative Western blots are shown. The Smad1 blots were used as sample loading controls.