Table 1.
Primer sequences used for RT-PCR.
Figure 1.
TGF-β induces vascular smooth muscle cells differentiation of mesenchymal stem cells through nuclear translocation of Smad3.
A) Rat mesenchymal cells treated with TGF-β for 14 days were stained for phospho-Smad3 immunofluorescence (red) and counterstained with DAPI (blue) to determine phospho-Smad3 subcellular localization. In TGF-β treated cells, positive phospho-Smad3 immunofluorescence was localized into the nucleus. Original magnification: 40x. B) Vascular smooth muscle actin (VSM-actin, green) was stained and the nuclei were counter-stained with DAPI showing cytoskeleton organization in Control cells and TGF-β treated cells. Original magnification: 20x.C) After 7 and 14 days, TGF-β induced the expression of vascular smooth muscle cells markers such as VSM-actin, SM22α, Myocardin and Myosin heavy chain with respect to control cells (a p<0.001 vs. control cells). Images are representative of three experiments.
Figure 2.
TGF-β administration prevents osteogenic effects induced by high phosphate.
A) High phosphate (P) increased the expression of BMP-2 while TGF-β or the combination of TGF-β plus high phosphate decreased significantly the expression of this osteogenic marker (a p<0.001 vs. all groups). Results are expressed as fold change vs. Control cells. B) High phosphate (P) decreased significantly SM22α and myocardin expression with respect to Control cells (b p<0.01 for SM22α and a p<0.001 for myocardin) and TGF-β group (c p<0.001). The combination of TGF- β and high phosphate (TGF-β + P) decreased the expression of SM22α and Myocardin although less than high phosphate alone (c p <0.001vs. TGF-β group). C) TGF-β alone did not change significantly the alkaline phosphatase activity. This activity increased after high phosphate treatment (a p<0.001 vs. all others groups). The combination of TGF-β and high phosphate for 14 days significantly decreased this activity when compared with high phosphate group. D) Calcium content was significantly increased after high phosphate treatment (a p<0.001 vs other groups). The combination of TGF-β and high phosphate prevented this increase of calcium induced by high phosphate alone.
Figure 3.
TGF-β addition inhibits nuclear translocation of Smad 1/5/8 induced by high Phosphate.
A) Rat mesenchymal stem cells treated with high phosphate showed nuclear localization of phospho-Smad1/5/8 (Red) (a p<0.001 vs. all groups). Cells treated with TGF-β (alone or plus high phosphate) were negative for phospho-Smad1/5/8. Merged images of phospho-Smad1/5/8 immunofluorescence and DAPI staining are shown. Original magnification: 40x. Image is representative of three experiments. Colocalization Finder plugging from Image J software was carried out to analyse nuclear localization of Smad 1/5/8 showing a submask with white areas specific to nuclear colocalization with DAPI. Original magnification: 40x. B) Quantification of confocal immunofluorescence was performed with Image J software.
Figure 4.
BMP2 inhibition prevents the osteogenic effects of high phosphate.
A and B) Noggin administration (200 ng/ml) prevented the expression of osteogenic markers such as Osterix and Runx2 (a p<0.001 vs. high phosphate treated cells) and reduced calcium deposition. C) Alkaline phosphatase activity was not modified after Noggin administration. The figures are representative of at least three experiments.
Figure 5.
High phosphate activates Wnt/β-catenin pathway.
A) Rat mesenchymal cells treated with TGF-β and/or high phosphate were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. High phosphate induced nuclear translocation of β-catenin while the addition of TGF-β inhibited this translocation. Original magnification: 40x. Image is representative of three experiments. B) Quantification of β-catenin confocal immunofluorescence was performed with Image J software (a p<0.001 vs. all groups). C) With respect to control cells high phosphate decreased the expression of Dkk1 (b p<0.001) and Gsk3β (b p<0.001) while increased the expression of Lrp5 with respect to other groups (d p<0.001). These differences were also significant respect to TGF-β treated groups (c p<0.001 vs. TGF groups). TGF-β alone increased the expression of Dkk1 and Gsk3β (a p<0.001).
Figure 6.
Dkk-1 inhibits the high phosphate-induced osteogenic-like characteristics in rat mesenchymal stem cells.
A) Rat mesenchymal cells treated with high phosphate and Dkk-1 were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Dkk-1 administration reduced nuclear translocation of β-catenin. Original magnification: 40x. B) BMP-2 mRNA expression in rat mesenchymal stem cells treated with high phosphate and Dkk-1 was determined by RT-PCR (a p<0.001 vs high phosphate treated cells). C) Calcium content and alkaline phosphatase activity (Units/mg protein) in rat mesenchymal stem cells treated with high phosphate and Dkk-1 (a p<0.001 vs high phosphate alone). Image is representative of three experiments.
Figure 7.
Wnt/β-catenin pathway activation enhances the high phosphate-induced osteogenic-like characteristics in rat mesenchymal stem cells.
A) Rat mesenchymal cells treated with high phosphate and CHIR98014 (0.4 µM) or lithium chloride (5 mM) were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Both Wnt activators (CHIR98014 and lithium chloride) increased nuclear translocation of β-catenin. Original magnification: 40x. B) BMP-2 protein and C) mRNA expression in rat mesenchymal stem cells treated with high phosphate and CHIR98014 or lithium chloride was determined by western blot and RT-PCR respectively (a p < 0.001 vs high phosphate alone). D) Calcium content and E) alkaline phosphatase activity in rat mesenchymal stem cells treated with high phosphate and CHIR98014or lithium chloride (a p<0.001 vs. high phosphate alone). Image is representative of three experiments.