FIG. 6.

Small molecule inhibition of TGFβ signaling using SB431542 on primary human POb cells enhances osteogenic capacity and leads to increased BMP signaling through cross-talk. (A) Mineralization of the extracellular matrix detected by alizarin red staining demonstrates a significant increase in osteogenesis in primary hPOb cells treated with SB431542 as compared with controls. (B) Histogram represents alizarin red staining quantification. (C) PCR analysis indicates that treatment with SB431542 upregulates RUNX2 and BGLAP expression. The relative mRNA level in each sample is normalized to GAPDH and values are given relative to GAPDH expression. *p ≤ 0.05. (D) and (E) Immunoblotting analysis of pSMAD2/3 and pSMAD1/5 performed on cells during osteogenic differentiation demonstrates that SB431542 treatment effectively inhibits TGFβ signaling while increasing BMP signaling in hPOb cells at the 24, 48, and 72 h time points. Histograms represent quantification of pSMAD2/3 and pSMAD1/5 electrophoretic bands using densitometry analysis by ImageJ software. Membranes were stripped and reprobed with α−tubulin antibody to control for equal loading. Total SMAD2 and SMAD5 proteins were detected by specific pan SMAD2/3 and SMAD1/5 antibodies. (F) PCR analysis reveals that SB431542-treated cells express significantly more BMP2. (G) PCR showing that increased expression of BMP2 is paralleled by upregulation of SMAD6 in SB431542-treated cells with noggin treatment abrogating this upregulation. *p ≤ 0.05. hPOb, human parietal osteoblasts. Color images available online at www.liebertpub.com/tea