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Naati Fakatava, 御手洗 裕美, 祐田 明香, 原口 晃、長谷川 大学、前田 英史、和田 尚久, ACTA2 regulates human PDL function via interaction with or without TGF-β1, 日本歯科保存学会2022年度春季学術大会（第156回）, 2022.06, [OBJECTIVE] ACTA2 (alpha-smooth muscle actin; α-SMA), one of the cytoskeleton protein, is known to be expressed in periodontal ligament (PDL) tissue. It might be involved in PDL function such as proliferation and migration. Also ACTA2 is upregulated with transforming growth factor-β1 (TGF-β1) which exists in PDL tissue predominantly (Fujii et al, Cell Tissue Res. 2010). So it is assumed that ACTA2 is involved in TGF-β1-dependent function in human PDL cells, but little is known about them. Therefore, we focused on ACTA2 and examined its role within the PDL function via interaction with or without TGF-β1 to find its role in the remodeling of the PDL.
[MATERIALS AND METHODS] Human PDL cell line 2-23, which was isolated from heterogeneous immortalized Human PDL cells (Hasegawa et al. J Cell Physiol. 2018) was used. (1) Western blotting analysis was performed to examine the protein expression of ACTA2. (2) ACTA2 or scramble siRNA were transfected to 2-23 for 48 h, and performed the WST-1 assay and scratch wound healing assay to analyze the proliferation and migration. (3) 2-23 cells were cultured with or without human recombinant TGF-β1 (rhTGF-β1: 10 ng/ml) for 24 h in the presence of ACTA2 or scramble siRNA. qRT-PCR was performed to examined the mRNA expression of PDL related genes; collagen1A1 (COL1A1), periostin (POSTN), and fibrillin1 (FBN-1). Picro-sirius red staining and sircol collagen assay were performed to analyze the collagen production. Western blotting analysis was performed to examine the phosphorylation of TGF-β1-related molecules; Smad2, Smad3, and YAP. All procedures were performed in compliance with requirements of the Institutional Review Board for Human Genome / Gene Research (approval number: 30-167) and Research Ethics Committee (approval number: 27-76) at Kyushu University.
[RESULTS] ACTA2 protein expression was observed through Western blotting analysis in 2-23. After transfection with ACTA2 siRNA, cell proliferation and migration levels were significantly downregulated compared with scramble siRNA. The mRNA expression of ACTA2, COL1A1, POSTN, and FBN-1 was upregulated in 2-23 stimulated with TGF-β1. Those mRNA expression was significantly downregulated in the presence of ACTA2 siRNA stimulated with rhTGF-β1 compared with scramble siRNA. The amounts of collagen production were upregulated in 2-23 stimulated with rhTGF-β1 which were analyzed by picro-sirius red staining and sircol collagen assay. But after ACTA2 knockdown, the collagen production stimulated with rhTGF-β1 were significantly downregulated compared with scramble siRNA. We revealed that phosphorylation of Smad2 and Smad3 in 2-23 were observed at 15-min time point with TGF-β1, and phosphorylation of YAP was observed at 30-min time point with TGF-β1 by Western blotting analysis. However, after ACTA2 knockdown, at each time point, the phosphorylation of Smad2, Smad3, and YAP was downregulated after TGF-β1 stimulation.
[DISCUSSION] In this research, ACTA2 was involved in proliferation and migration of human PDL cells. These results suggest that as cytoskeleton protein, ACAT2 itself is crucial for PDL function. In the presence of ACTA2 siRNA, upregulation of PDL related genes, collagen production, and phosphorylation of TGF-β1-related molecules were significantly downregulated, suggesting that ACTA2 might be a key factor for TGF-β1 function.
[CONCLUSION] ACTA2 regulates human PDL function via interaction with or without TGF-β1.
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Fakatava Naati、御手洗裕美、祐田明香、長谷川大学、前田英史、和田尚久, The Role of Acta2 in Periodontal Ligament cell stimulated with TGF-β1, 日本歯科保存学会, 2020.06. |
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