www.nature.com/scientificreports
OPEN
received: 02 November 2015 accepted: 04 May 2016 Published: 24 May 2016
GCN5 modulates osteogenic differentiation of periodontal ligament stem cells through DKK1 acetylation in inflammatory microenvironment Bei Li1,2,*, Jin Sun1,3,*, Zhiwei Dong4,*, Peng Xue1,2, Xiaoning He1,2, Li Liao1,2, Lin Yuan3 & Yan Jin1,2 Periodontal ligament stem cells (PDLSCs) from periodontitis patients showed defective osteogenic differentiation. However, the mechanism of impaired osteogenic differentiation of PDLSCs in inflammatory microenvironments is still unclear. In this study, we found that inflammation in the microenvironment resulted in downregulation of histone acetyltransferase GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Previous study showed activated Wnt/βcateinin pathway of PDLSCs resulted in defective osteogenic differentiation. Here we found knockdown of GCN5 decreased the expression of DKK1, an inhibitor of Wnt/β-cateinin pathway, thus activated Wnt/β-catenin pathway of PDLSCs. Mechanistically, GCN5 regulated DKK1 expression by acetylation of Histone H3 lysine 9 (H3K9) and Histone H3 lysine 14 (H3K14) at its promoter region. Interestingly, we found that in vivo injection of aspirin rescued the periodontitis of rats through inhibiting inflammation and upregulating GCN5 expression. Furthermore, aspirin treatment of PDLSCs upregulated GCN5 expression and increased osteogenic differentiation of PDLSCs. In conclusion, GCN5 plays a protective role in periodontitis through acetylation of DKK1 and applying drugs targeting GCN5, such as aspirin, could be a new approach for periodontitis treatment. Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of self-renew, multilineage differentiation, and immunomodulation1,2. MSCs maintain the bone homeostasis due to their multilineage differentiation potentials including osteogenesis, adipogenesis and chondrogenesis3,4. Microenvironment or the niche often governs the lineage commitment and cell differentiation of stem cells5. MSCs exhibited defective osteogenic differentiation in inflammatory microenvironments6–8. Periodontal ligament stem cells (PDLSCs) are a group of MSCs derived from periodontal ligament and defective osteogenic differentiation of PDLSCs were documented being closely related to periodontitis9,10. Besides, osteogenic deficit could not be recovered from ex vivo culture and expansion. Such deficiency of stem cells seem to retain a “memory” of abnormal microenvironment, which suggests the epigenetic modification may be involved. However, it remains unknown if epigenetic modification regulated the osteogenic differentiation of PDLSCs in inflammatory microenvironments. The differentiation potential in MSCs often relies on orchestrated activation or repression of lineage specific genes. Previous studies have been focused on identifying numerous extrinsic regulators and their downstream transcription factors that control cell differentiation11–13. However, cell differentiation can be regulated more specifically at the epigenetic level14. Upon differentiation of MSCs, the epigenetic marks change to reflect the 1 State Key Laboratory of Military Stomatology, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032, China. 2Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi 710032, China. 3Department of Stomatology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510140, China. 4Department of Oral and Maxillofacial surgery, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110840, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.Y. (email: [email protected]) or Y.J. (email: [email protected])
Scientific Reports | 6:26542 | DOI: 10.1038/srep26542
1
www.nature.com/scientificreports/
Figure 1. Down-regulated expression of GCN5 in PDLSCs in inflammatory microenvironments. (a) Western blot analysis showed the protein expression of GCN5 of freshly isolated H-PDLSCs and P-PDLSCs, and GAPDH was used as the internal control. (b) Relative gene expression of GCN5 of 2, 4, 6 passage H-PDLSCs and P-PDLSCs was analyzed by qRT-PCR. Relative gene expression of GCN5 was determined based on the threshold cycle (CT) values. The expression levels of the target genes were normalized to that of the housekeeping gene GAPDH. (c) Western blot analysis showed the protein expression of GCN5 of 2, 4, 6 passage H-PDLSCs and P-PDLSCs, and GAPDH was used as the internal control. (d) Western blot analysis showed the protein expression of GCN5 of 2 passage H-PDLSCs after either alone or in combination of LPS, TNF-αand IL-1β treatment. (e,f) Western blot analysis showed the protein expression of GCN5 of 4 and 6 passage H-PDLSCs, which were passed by 2 passage H-PDLSCs treated with either alone or in combination of LPS, TNF-αand IL-1β. GAPDH was used as the internal control. Data represent the means ± SD. *p
OPEN
received: 02 November 2015 accepted: 04 May 2016 Published: 24 May 2016
GCN5 modulates osteogenic differentiation of periodontal ligament stem cells through DKK1 acetylation in inflammatory microenvironment Bei Li1,2,*, Jin Sun1,3,*, Zhiwei Dong4,*, Peng Xue1,2, Xiaoning He1,2, Li Liao1,2, Lin Yuan3 & Yan Jin1,2 Periodontal ligament stem cells (PDLSCs) from periodontitis patients showed defective osteogenic differentiation. However, the mechanism of impaired osteogenic differentiation of PDLSCs in inflammatory microenvironments is still unclear. In this study, we found that inflammation in the microenvironment resulted in downregulation of histone acetyltransferase GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Previous study showed activated Wnt/βcateinin pathway of PDLSCs resulted in defective osteogenic differentiation. Here we found knockdown of GCN5 decreased the expression of DKK1, an inhibitor of Wnt/β-cateinin pathway, thus activated Wnt/β-catenin pathway of PDLSCs. Mechanistically, GCN5 regulated DKK1 expression by acetylation of Histone H3 lysine 9 (H3K9) and Histone H3 lysine 14 (H3K14) at its promoter region. Interestingly, we found that in vivo injection of aspirin rescued the periodontitis of rats through inhibiting inflammation and upregulating GCN5 expression. Furthermore, aspirin treatment of PDLSCs upregulated GCN5 expression and increased osteogenic differentiation of PDLSCs. In conclusion, GCN5 plays a protective role in periodontitis through acetylation of DKK1 and applying drugs targeting GCN5, such as aspirin, could be a new approach for periodontitis treatment. Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of self-renew, multilineage differentiation, and immunomodulation1,2. MSCs maintain the bone homeostasis due to their multilineage differentiation potentials including osteogenesis, adipogenesis and chondrogenesis3,4. Microenvironment or the niche often governs the lineage commitment and cell differentiation of stem cells5. MSCs exhibited defective osteogenic differentiation in inflammatory microenvironments6–8. Periodontal ligament stem cells (PDLSCs) are a group of MSCs derived from periodontal ligament and defective osteogenic differentiation of PDLSCs were documented being closely related to periodontitis9,10. Besides, osteogenic deficit could not be recovered from ex vivo culture and expansion. Such deficiency of stem cells seem to retain a “memory” of abnormal microenvironment, which suggests the epigenetic modification may be involved. However, it remains unknown if epigenetic modification regulated the osteogenic differentiation of PDLSCs in inflammatory microenvironments. The differentiation potential in MSCs often relies on orchestrated activation or repression of lineage specific genes. Previous studies have been focused on identifying numerous extrinsic regulators and their downstream transcription factors that control cell differentiation11–13. However, cell differentiation can be regulated more specifically at the epigenetic level14. Upon differentiation of MSCs, the epigenetic marks change to reflect the 1 State Key Laboratory of Military Stomatology, Center for Tissue Engineering, School of Stomatology, Fourth Military Medical University, Xi’an, Shaanxi 710032, China. 2Research and Development Center for Tissue Engineering, Fourth Military Medical University, Xi’an, Shaanxi 710032, China. 3Department of Stomatology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510140, China. 4Department of Oral and Maxillofacial surgery, General Hospital of Shenyang Military Area Command, Shenyang, Liaoning 110840, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to L.Y. (email: [email protected]) or Y.J. (email: [email protected])
Scientific Reports | 6:26542 | DOI: 10.1038/srep26542
1
www.nature.com/scientificreports/
Figure 1. Down-regulated expression of GCN5 in PDLSCs in inflammatory microenvironments. (a) Western blot analysis showed the protein expression of GCN5 of freshly isolated H-PDLSCs and P-PDLSCs, and GAPDH was used as the internal control. (b) Relative gene expression of GCN5 of 2, 4, 6 passage H-PDLSCs and P-PDLSCs was analyzed by qRT-PCR. Relative gene expression of GCN5 was determined based on the threshold cycle (CT) values. The expression levels of the target genes were normalized to that of the housekeeping gene GAPDH. (c) Western blot analysis showed the protein expression of GCN5 of 2, 4, 6 passage H-PDLSCs and P-PDLSCs, and GAPDH was used as the internal control. (d) Western blot analysis showed the protein expression of GCN5 of 2 passage H-PDLSCs after either alone or in combination of LPS, TNF-αand IL-1β treatment. (e,f) Western blot analysis showed the protein expression of GCN5 of 4 and 6 passage H-PDLSCs, which were passed by 2 passage H-PDLSCs treated with either alone or in combination of LPS, TNF-αand IL-1β. GAPDH was used as the internal control. Data represent the means ± SD. *p
