Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1–modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth

• Transforming growth factor beta 1 (TGF-β1) affects growth, collagen turnover, and differentiation of apical papilla cells.
• TGF-β1 stimulates Smad2 and ERK phosphorylation.
• The effects of TGF-β1 on apical papilla cells are related to Smad2 and ERK signaling.
• TGF-β1 and stem cells from apical papilla are crucial for engineering of pulpal regeneration and apexogenesis.

IntroductionTransforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-β1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2.MethodsSCAPs were exposed to TGF-β1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho–enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA).ResultsTGF-β1 stimulated the growth and collagen content of cultured SCAPs. TGF-β1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-β1–induced cell growth and collagen content in SCAPs. TGF-β1 stimulated ALP activity at lower concentrations (0.1–1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-β1–induced ALP activity but showed little effect on 10 ng/mL TGF-β1–induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-β1.ConclusionsTGF-β1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-β1 and SCAP for engineering of pulpal regeneration and apexogenesis.