Role Of Apoptotic Marker Caspase 3 In Periodontal Disease

Keywords

Apoptosis; Caspase-3; Chronicperiodontitis; Gene Expression; CASP 3 Gene.

Introduction

Caspases are cysteine protease enzymes they play a vital role in many processes like inflammation, programmed cell death (apoptosis), necroptosis, pyroptosis [1]. Depending on function caspases are subcategorized into initiator caspases and executioner caspases and inflammatory caspases [2, 3]. Initiator caspases, (caspase- 2,8 &-9) initiates the apoptosis signal while the executioner caspases (caspases 3,6&7) degrade into cellular components in order to induce the morphological changes [4] and inflammatory caspases (caspases 1, 4, 5, 11, 12, 13, and 14) are involved in inflammatory cytokine signalling [5]. In inflammatory conditions like gingivitis and periodontitis, oral micro-organisms induce apoptosis via activation of caspases. Evidence suggests that toxins released by keystone pathogens as well as other bacterial factors directly activates caspases leading to apoptosis [6-8]. Galluzi L et al concluded that till date 14 caspases were discovered, out of which 12 caspases were found in humans [9].

However, caspase 12 is found only in few humans. Caspases are activated either by homoactivation (requires recruitment of the zymogens to the adapter proteins) or by heteroactivation (requires action of another protease upon the caspase zymogen) [10]. These activated caspases are involved in apoptosis in two pathways commonly known as intrinsic and extrinsic pathways.

Caspase 3 is activated by both intrinsic and extrinsic pathway. In periodontitis, binding of Fas with FasL allows recruitment of Fas-associated death domain (FADD) and it leads to activation of the extrinsic pathway. Since caspase 3 is activated by both intrinsic and extrinsic pathway, caspase 3 is most commonly involved in periodontal disease progression than other caspases. Apart from inflammatory conditions like periodontitis and gingivitis, literature evidence also reveals that caspase 3 plays a role in orthodontic movements [11, 12] and as well as in premalignant conditions like oral submucous fibrosis and squamous cell carcinoma [13].

The levels of caspases have been studied in GCF and tissue samples and observed that caspases levels are increased in periodontitis subjects compared to healthy subjects. Numerous studies have also concluded that hyperglycaemic status elevates caspases activity, levels and as well as caspases gene levels in periodontitis [14, 15]. Thus, the role of Caspase 3 as a biomarker in periodontal diseases is established.

This literature review explains about caspase 3 and its role in periodontal diseases like gingivitis, periodontitis, apical periodontitis etc, as well as the influence of oral microbial organisms on caspase 3 and gene expression in periodontal disease conditions.

Caspase 3

Galluzi L et al concluded that till date 14 caspases were discovered, out of which 12 caspases were found in humans 41 (as shown in Fig1).

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Figure 1:

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Figure 1. Shows different types of caspases.

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Caspase 3 protein is encoded by the CASP3 gene. Caspase3 is active even under normal cellular conditions due to its basic nature. Since, caspase 3 is activated by both intrinsic and extrinsic pathway of apoptosis it is considered an executor enzyme (Fig 2). Caspase 3 is inhibited through the IAP (inhibitor of apoptosis) with protiens like c-IAP1, c-IAP2, XIAP, and ML-IAP [16].

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Figure 2:

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Figure 2. Shows caspase 3 is activated by both intrinsic and extrinsic pathways.

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Caspase 3 in periodontitis

Extrinsic pathway is most relevant in periodontitis.Fas receptor and Fas ligand (FasL) play an important role in activation of caspases in periodontitis. Fas is a protein encoded by Fas gene and it is also called as CD95. Fas receptors are present of gingival fibroblasts [17, 18]. Binding of Fas with Fas ligand (FasL) allows the recruitment of Fas-associated death domain (FADD) and leads to activation of death-inducing signalling complex (DISC) and this DISC activates caspases like 3, 6 and 7 leading to apoptosis of gingival cells.

Several oral microorganisms activate caspases either directly or indirectly resulting in periodontitis. For example: - A.actinomytemcomitans induces apoptosis in epithelial cells through leukotoxin [19, 20], P.gingivalis induces apoptosis in T-cells and fibroblasts through gingipains [21, 23]. Messina et al [24] observed that upregulated Fas and FasL levels in A.actinomycetemcomitans induced periodontitis rats.

Based on above literature evidence it can be concluded that oral keystone pathogens alter immune response and enhances caspase activity in periodontitis.

Caspase 3 protein in GCF, saliva and serum

Caspase 3 expression increases with progression of gingival inflammation, probing depth and clinical attachment level in chronic periodontitis. Pradeep A R et al 2016 observed serum and GCF levels of caspase 3 in chronic periodontitis subjects and healthy subjects and observed that caspase 3 levels were higher in patients with periodontitis and correlated with clinical periodontal parameters compared to healthy individuals [25]. Aral K et al in 2018 studied GCF levels of p53, caspase3, TNFa, TRAIL, IL1ß, and IL10 in subjects with GAgP before and after non-surgical periodontal treatment, ELISA analysis results showed that TRAIL, TNFa, and IL10 were similar at baseline and 3 and 6 months but caspase3 and IL1ß levels were decreased at 3 months [26].

Caspase 3 in human gingival tissue

Immunohistochemistry studies have revealed that increased expression of caspase 3 could be involved in the inflammatory process associated with gingival tissue destruction in patients with periodontitis.

In an immunohistochemistry analysis, Das et al 2009 [27] observed intrinsic and extrinsic pro-apoptotic markers in inflamed human gingival samples and also observed that, increased cytochrome c and caspase-3 levels were detected in periodontal disease groups when compared to healthy group.

In a human gingival tissue immunohistochemistry analysis done by Bantel H et al., 2005 [28] observed that expression of apoptosis associated genes increased when exposed to pathogenic periodontal bacteria and expression of caspase 3 is increased in subjects with periodontitis than healthy subjects. In an immunohistochemistry study published by Lucas et al, 2010 [29] concludedthatapoptosis in periodontitis may be inhibited by elevated expression of TRAIL decoy receptors and cleaved caspase-3 inhibitors. Gomonal et al 2001 [30] studied apoptotic events in the gingival tissue of chronic adult periodontitis patients. After electron microscopic and immunohistochemistry analysis they observed positive staining for active caspase-3, Fas, Fas L, BCL2 and p53 is more in chronic periodontitis group when compared to control group.

Based on the above studies it can be concluded that caspase 3 is involved in periodontitis.

Influence of periodontitis on CASP3 gene

Caspase 3 gene is significant proapoptotic gene which has been observed consistently upregulated in gingival tissue sites.

Gonzalez et al 2011 studied 88 genes related to apoptotic pathway in gingival biopsies of healthy and periodontitis sites from non-human primates and comparison between healthy and periodontitis gingival tissues showed that the up or down regulated apoptotic genes in diseased gingival tissue are different in adults compared with aged animals. Furthermore, they observed that caspase 3 gene is 1.5 fold highly expressed in periodontitis compared to healthy, suggesting a potential role of caspase 3 in pathogenesis of periodontal disease [31].

Gene polymorphism i.e. variation at a single nitrogen base pair in gene, also plays a role in periodontitis disease progression by upregulatingcaspase levels even in systematically healthy subjects. Kang et al 2015 [32] recently, in a Korean population observed polymorphism in caspase 3 gene by assessing 201 SNP’s of CASP3, out of which only 3 genotypes that is rs12108497, rs4647602 and rs113420705 were significantly associated with chronic periodontitis.

Considering the above data, it has been established that association of caspase 3 in chronic periodontitis however the link between periodontitis and CASP 3 gene polymorphism needs further research in larger samples and different populations. Several periodontopathogenic microorganisms and their products act on the cellular constituents of the gingival tissues and activates cellular processes that induce the destruction of the connective tissue as well as bone, for example A. actinomycetemcomitans expresses virulent factors which helps in formation of inflammasomes and these inflammasomes activates caspases like caspase 1 which leads to formation of inflammatory cytokines mainly interleukins like IL-1 and IL-18 [33].

Influence of Diabetes mellitus on caspases and its influence on periodontitis

In systemic diseases like diabetes mellitus, caspases are activated in other pathways as well like TNF pathway, polyol pathway, reactive oxygen species (ROS) etc. Graves et al 2006evaluated periodontal disease pathogenesis in diabetic patients and concluded that diabetes activates several pathways and activates caspases leading to apoptosis stimulation [34, 35]. Alikhani et al concluded that, diabetes mellitus activates polyol pathway and leads to elevation of Advanced Glycation End Products (AGEs). In periodontitis, AGEs induce fibroblast apoptosis by activating caspase 8,9 as well as caspase 3 [36].Joussen et al concluded that diabetes mellitus enhances TNF and Fas/FasL as well as caspase levels and activates extrinsic pathway of apoptosis [37] and similarly. Hesham et al in 2006 observed expression of Fas mRNA levels in diabetic periodontitis mice and also observed that inhibiting apoptosis improved healing and increased mRNA levels of collagen I and III in diabetic mice [38]. Yousef M et al 2016 evaluated caspase 3 activity in type II DM controls with and without periodontitis, after ELISA analysis concluded that caspase-3 play a role as a biomarker in periodontitis [39]. Manosudprasit et al 2017evaluated caspase-3, -8, and -9 levels of PMN in patients with chronic periodontitis and type 2 Diabetes mellitus (T2DM). After TUNEL, Annexin V assay and colorimetric assays they observed caspase-3 was significantly higher in T2DM patients with and without CP compared to healthy controls [40].

Discussion

Caspase-3 is activated by several other caspases so it is considered as executioner enzyme.

Above literature studies concluded that caspase3 levels were increased in GCF, saliva, serum and tissue samples are positively correlated with clinical parameters like probing depth, clinical attachment loss and caspases concentrations were increased proportionally with the severity of periodontal disease. CASP genes levels were upregulated in periodontal diseases. Caspases such as caspase 3 can be activated in several aspects, in addition to inflammatory conditions such as keystone pathogens, premalignant conditions, systemic diseases and age. Systemic diseases like diabetes mellitus also influences caspase3 levels, studies published by Graves DT et al [34, 35] concluded that diabetes mellitus influences caspases like caspase 3 and involves in periodontal disease progression. Yousef MK et al [39] concluded that caspase 3 can play as role of biomarker in chronic periodontitis. Apart from gingivitis and periodontitis, caspase 3 also plays a role in premalignant conditions.

However further studies in relation to caspase3 with other factors such as smoking, stress etc in periodontal diseases are less. Further, longitudinal studies involving larger population, meta-analysis are needed and also genetic studies like gene polymorphisms of CASP3 gene are much required for better understanding confirming its function and role in pathogenesis of periodontal diseases.

Acknowledgements

We sincerely thank SRM Dental College, Ramapuram, Chennai for providing the study participants.

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