Regulatory mechanisms of GCN5 in osteogenic differentiation of MSCs in periodontitis

Abstract Objectives The regulatory mechanisms of GCN5 (General control non‐repressed protein5) in the osteogenic differentiation of mesenchymal stem cells (MSCs) in periodontitis are still unclear. The purpose of this review focuses on the regulating roles of GCN5 in bone metabolism and periodontitis, discusses the potential molecular mechanism and provides targets and new ideas for the treatment of periodontitis. Material and Methods The integrative review methodology was used. Data sources include PubMed, Cochrane Library, and additional sources. Results MSCs play an important role in the osteogenesis balance of periodontal tissue. Periodontal ligament stem cells (PDLSCs) from periodontitis patients exhibited defective osteogenic differentiation capacities. Histone acetylation is important in regulating the differentiation of different types of MSCs cells and is closely related to the reduced osteogenic differentiation of PDLSCs. GCN5, one of the first histone acetyltransferase linked to gene transcriptional activation, participates in many biological processes of mesenchymal stem cells. Downregulation of GCN5 expression and lack of GCN5 caused decreased osteogenic differentiation of PDLSCs. Intercellular information exchange may be an important way for MSCs to exert their regulatory and therapeutic functions. Conclusions GCN5 affects the function of cell metabolism‐related genes by regulating the acetylation status of histones or non‐histones, thereby regulating some important progress of MSCs such as PDLSCs' osteogenic differentiation and BMCS osteogenic differentiation.


| INTRODUCTION
Periodontitis is a kind of chronic inflammatory disease characterized by inflammation of the periodontal tissue and resorption of the alveolar bone. Periodontitis has been the primary cause of tooth loss because of its ability to cause continuous and irreversible destruction of periodontal tissue (Engebretson et al., 2013). Moreover, periodontitis can be associated with systemic diseases such as cardiovascular disease and diabetes mellitus thus affecting oral and general health (Katz et al., 2001). Conventional periodontal therapy is aimed at controlling inflammation and cannot regenerate or restore the function of periodontal tissues. Guided tissue regeneration and implantation of materials show limited ability in periodontal tissue repair (Chen et al., 2009;Zhang et al., 2009). Therefore, new therapeutic approaches for periodontal tissue regeneration are required. The development of stem-cell-based tissue engineering has facilitated the regeneration of functional periodontal tissues (Ding et al., 2010). However, the long-term inflammatory microenvironment can inhibit the proliferation, migration, and regenerative potential of stem cells (Pluchino et al., 2008). Previous studies suggest stem cell transplantation promotes periodontal regeneration, of which the mechanism other than direct differentiation remains unclear. Therefore, the activation of the regenerative potential of stem cells in tissues is crucial for tissue regeneration. Recent studies have shown that source cells can secrete exosomes to transport signaling molecules to regulate host cells.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells isolated from multiple tissues including the umbilical cord, bone marrow, and fat tissue using standardized criteria proposed by the International Society for Cellular Therapy (ISCT). MSCs maintain plastic adherence when kept under standard culture conditions and express certain markers (Dominici et al., 2006). MSCs have a high capacity for self-renew by dividing while exhibiting great potential to differentiate into a variety of cell types such as osteocytes, chondrocytes, adipocytes, myocytes, and cardiomyocytes, making them an appealing and promising candidate for cell-based therapeutic applications in regenerative medicine and tissue engineering, particularly in bone tissue regeneration (Bianco, 2014;Chamberlain et al., 2007). From self-renew to oriented differentiation, chromatin is remodeled into heritable states that allow activation or maintain the repression of regulatory genes, which means specific genes in selfrenew are switched off and lineage-specific genes in oriented differentiation are activated in response to environmental stimuli.
Progression from MSCs into different differentiated lineages requires long-lasting changes in gene expression. Though stem cell behavior is largely mediated by DNA sequence, a great number of studies have revealed that epigenetic mechanisms, namely epigenetics, would also be fundamentally important regulatory factors in stem cell fate determination, commitment, and differentiation (Ermolaeva et al., 2018;Mortada & Mortada, 2018;Teven et al., 2011). MSCs are able to secrete a greater amount of exosomes than many other cells, suggesting that exosome-mediated intercellular information exchange may be an important way for MSCs to exert their regulatory and therapeutic functions (Figure 1).
Epigenetics refers to changes in gene expression without the alteration of the underlying DNA nucleotide sequence, which could be inherited by offspring and play an important role in the promotion of appropriate transcriptional pathways during both embryonic development and adult tissue maintenance (Brack et al., 1978;Im & Shin, 2015;Monk, 1995). Gene expression would be regulated at the epigenetic level through modification of local chromatin configuration or nuclear architecture, thus altering the accessibility of genes to transcription factors and other modulators. Specifically, these modifications regulate gene expression would result in two different forms of chromatin: one is euchromatin which is the less condensed and more accessible form of chromatin facilitating the opening of DNA to permit gene transcription; the other one is heterochromatin which is the tightly packed form of chromatin restricting the access of transcription factors to their cognate NDA binding site to repress gene transcription (Frobel et al., 2014;Meyer et al., 2016). The dynamic balance between euchromatin and heterochromatin is regulated by several epigenetic mechanisms, including DNA methylation, histone modifications, microRNAs, and chromatin remodeling.
Reportedly, DNA methylation patterns are crucial for embryonic stem cell differentiation, while histone modifications and other chromatinbased mechanisms may serve a larger role in MSCs differentiation capacity (Boquest et al., 2006;Mortada & Mortada, 2018). Therefore, the role of epigenetics, particularly histone modifications, on MSCs F I G U R E 1 Regulatory mechanisms of Gcn5 in osteogenic differentiation of MSCs. MSCs, mesenchymal stem cells. biology and differentiation protocols will be described in detail in this review.
Histones, the main structural components of chromatin, are small proteins that contain numerous positively charged amino acids in their N-terminal domain, such as lysine and arginine (Bártová et al., 2008;Cheung et al., 2000). These positively charged amino acids enable histones to tightly wrap the negatively charged doublestranded DNA. On the other hand, they can be covalently modified to change the affinity between histones and DNA to induce gene transcription or silencing. Histone acetylation and deacetylation are the widespread and dynamic modification of chromatin structure associated with the regulation of gene expression. In histone acetylation, negatively charged acetyl groups are transferred to NH 2 -terminal lysine residues on histone proteins (Clayton et al., 2006;Huang et al., 2015). Conversely, histone deacetylation refers to the removal of the acetyl functional group from the lysine residues. In most cases, histone acetylation enhances gene transcription while histone deacetylation represses transcription (Wang et al., 2013).
Histone acetylation and deacetylation are regulated by the opposing action of histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively.
HATs can be categorized into three major families based on primary-structure homology: the Gcn5-related N-acetyltransferase (GNAT) family, the MYST family (containing MOZ, Ybf2/Sas3, Sas2, and Tip60), and the p300/CBP family (Dekker & Haisma, 2009;Wapenaar & Dekker, 2016) (Table 1). Several other HAT families have been identified, but they haven't been studied extensively. It should be noted that HATs are often part of large coactivator complexes that determine their binding preferences and histone acetyltransferase activity since they don't possess DNA binding sites.
Gcn5 or KAT2A, a member of the GNAT family of HATs, is the first nuclear HAT protein identified from yeast, which is a transcriptional coactivator with histone acetyltransferase activity and is conserved with regard to structure as well as its histone substrates throughout the eukaryotes (Dyda et al., 2000;Salah ud-Din et al., 2016). Gcn5 is mainly found in two functionally distinct coactivator complexes SAGA (Spt-Ada-Gcn5-acetyltransferase) and ATAC (Ada-two-Acontaining), which is important in transcription activation mediated by interactions with transcription activators and general transcription factors (Krebs et al., 2011;Nagy et al., 2010). Gcn5 is structurally conserved throughout evolution and typically functions in a conserved fashion through the acetylation of a conserved set of lysine residues in target proteins. Thus, Gcn5 is expected to play a distinct role in the differential expression of regulatory genes during the differentiation of MSCs. Therefore, in the present review, we'll provide a comprehensive overview of recent studies on the impact of Gcn5 on MSCs biology and differentiation protocols with a focus on periodontitis and osteocytic differentiation, offering possible directions for future research in this area and further development towards therapeutic applications.

| GCN5 AND PERIODONTITIS
Periodontitis is a type of chronic inflammatory disorder characterized by gingival inflammation and alveolar bone resorption, which can cause progressive destruction of periodontal supporting tissues and irreversible resorption of alveolar bone, thus leading to the loss of a tooth (Kinane et al., 2017). Bacterial stimuli are responsible for the main pathogenic factors of periodontitis. Under the stimulation of lipopolysaccharide (LPS), immune cells within the periodontal tissues could produce inflammatory factors, such as tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and IL-6, which then cause a series of inflammatory responses (Lindemann et al., 1988). Periodontitis is a complex immune-inflammatory response.
Compared with normal PDLSCs, PDLSCs from chronic periodontitis showed a low level of osteogenic differentiation in vitro, which even maintained during cell passage and seemed to have a "memory" of the previous inflammatory environment. Therefore, some researchers thought that this phenomenon may be contributed to the changes in epigenetic modification . In recent years, the function of Gcn5 involved in the relationship between the osteogenic differentiation capacities of PDLSCs and periodontitis has attracted more and more researchers' attention.
HAT Gcn5 can specifically acetylate histone H3K9 and H3K14 in the nucleosome of Dick-kopf-related protein 1 (DKK1) gene. Li et al. showed that chronic periodontitis could inhibit the expression of Gcn5 and active Wnt/β-catenin signaling pathway, resulting in reduced osteogenic differentiation capacities of PDLSCs . Compared with normal PDLSCs, it's found that the expressions of Gcn5 and MORF in periodontitis-derived PDLSCs were significantly decreased (Sun et al., 2017). When small interfering RNA was used to down-regulate the expression of these two genes, the osteogenic differentiation capacities of PDLSCs were inhibited.
Clinically, molecular methods or drugs can be used to specifically Further analysis showed that Gcn5 promoted the osteogenic differentiation of BMSC by increasing the acetylation of histone 3 lysine 9 on the Wnt gene promoter. Reduced Gcn5 expression inhibits Wnt signaling, leading to osteogenic defects in OVX mouse BMSCs. In addition, restoring Gcn5 levels can restore BMSC osteogenic differentiation and reduce bone loss in OVX mice.
In another study by Zhang et al., by experiments of OVXed and aged mouse models, they also found that the metabolic bone disease osteoporosis was associated with abnormal expression of Gcn5 . In addition, the study revealed that regulation of osteogenic differentiation by Gcn5 is NF-kB-dependent. NF-kB plays an important role in bone remodeling and bone homeostasis by controlling the differentiation of bone progenitor cells. Selective inhibition of NF-kB has been shown to block RANKL-induced osteoclastogenesis in vivo and in vitro and prevent inflammatory bone destruction in vivo. Therefore, targeting NF-kB may promote bone formation and inhibit bone resorption, and factors that affect its expression or transcriptional activity may be potential targets for regulating osteogenic differentiation. In the study, Gcn5 was found to inhibit NF-kB signaling in MSCs, and most importantly, the HAT activity of Gcn5 was determined to be not required for this process.