Wang Li, Wang Tingle, Song Meng, Pan Jinsong
Department of Stomatology, Shanghai First People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Stomatology, Central Hospital of Minhang District, Shanghai, China.
Arch Oral Biol. 2014 Feb;59(2):149-57. doi: 10.1016/j.archoralbio.2013.11.004. Epub 2013 Nov 16.
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 10ng/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.
人牙周膜细胞（hPDLCs）形成影响牙齿寿命的特殊结缔组织。牙周病是由多种因素引起的牙周支持组织的慢性感染性疾病，尤其是hPDLCs的丧失。转化生长因子-β1（TGF-β1）是一种多功能细胞因子，已知在牙周病中起重要作用，但关于TGF-β1对人牙周膜细胞的影响知之甚少。为了确定TGF-β1如何介导hPDLCs的变化，我们对TGF-β1处理对hPDLCs的影响进行了表征。然后我们阐明了介导这些影响的信号通路。将血清饥饿的hPDLCs与10ng/mL TGF-β1孵育，使用细胞计数试剂盒-8检测其增殖，同时通过相差显微镜检查其形态变化。通过鬼笔环肽染色和共聚焦显微镜观察F-肌动蛋白的重组。通过蛋白质印迹分析蛋白质表达。我们发现TGF-β1处理诱导增殖和细胞骨架重组，降低Rho-GDIa蛋白表达，激活ROCK蛋白表达，并增加LIM激酶和丝切蛋白的磷酸化。用ROCK抑制剂Y-27632预处理可抑制增殖和细胞骨架重排；此外，Y-27632降低了ROCK蛋白的表达以及LIM激酶和丝切蛋白的磷酸化，而通过靶向siRNA转染敲低Rho-GDIa则产生相反的效果。因此，我们提出TGF-β1通过调节ROCK、LIM激酶和丝切蛋白活性的Rho GTPase依赖性途径诱导hPDLCs的增殖和细胞骨架重排。
