Rho plays a key role in TGF-β1-induced proliferation and cytoskeleton rearrangement of human periodontal ligament cells

Abstract Human periodontal ligament cells (hPDLCs) form specialised connective tissues that influence the lifespan of the tooth. Periodontal disease is a chronic infectious disease of the periodontal supporting tissues caused by a variety of factors, particularly the loss of hPDLCs. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine known to play an important role in periodontal disease, but little is known about the effects of TGF-β1 on human PDL cells. To determine how TGF-β1 mediates the changes in hPDLCs, we characterised the effects of TGF-β1 treatment on hPDLCs. We then elucidated the signalling pathway that mediates these effects. Serum-starved hPDLCs were incubated with 10 ng/mL TGF-β1, and their proliferation was examined using the Cell Counting Kit-8, while their morphological changes were examined by phase-contrast microscopy. F-actin reorganisation was visualised by phalloidin staining and confocal microscopy. Protein expression was analysed by western blotting. We found that TGF-β1 treatment induced proliferation and cytoskeletal reorganisation, decreased Rho-GDIa protein expression, activated ROCK protein expression, and increased the phosphorylation of LIM kinase and cofilin. Proliferation and cytoskeletal rearrangement were suppressed by pre-treatment with the ROCK inhibitor Y-27632; additionally, expression of ROCK protein and phosphorylation of LIM kinase and cofilin were decreased by Y-27632, while Rho-GDIa knockdown by targeted siRNA transfection causes opposite effects. Therefore, we propose that TGF-β1 induces proliferation and cytoskeletal rearrangement in hPDLCs via Rho GTPase-dependent pathways that modulate ROCK, LIM kinase, and cofilin activity.