A critique on nuclear factor-kappa B and signal transducer and activator of transcription 3: The key transcription factors in periodontal pathogenesis.

Periodontal disease is initiated by microorganisms in dental plaque, and host immunoinflammatory response to the microbial challenge helps in disease progression. Conventional periodontal therapy was mainly targeted on the elimination of microbial component. However, a better understanding of molecular aspects in host response will enable the clinicians to formulate effective host modulation therapy (HMT) for the periodontal management. Inflammatory mediators were the main targets for HMT in the past. Transcription factors can regulate the production of multiple mediators simultaneously, and inhibition of these factors will be more beneficial than blocking individual molecule. Two important transcription factors implicated in chronic inflammatory diseases are nuclear factor kappa B (NF-κB) and signal transducers and activators of transcription 3. The role of these factors in periodontal disease is a less explored area. This comprehensive review is aimed at unveiling the critical role of NF-κB and signal transducers and activators of transcription 3 in periodontal pathogenesis. An online search was performed using MEDLINE/PubMed database. All publications till 2016 related to NF-κB, signal transducer and activator of transcription 3 (STAT3), and inflammation were included in writing this review. A total of 27,390 references were published based on the search terms used. Out of these, 507 were related to the periodontal research published in English till 2016. Relevant papers were chosen after carefully reading the abstract. This review has attempted to comprehend the existing knowledge regarding the role of transcription factors NF-κB and STAT3 in periodontal disease. Moreover, it also provides a connecting molecular link for the periodontal medicine concept.

INTRODUCTION

Periodontal disease is a common, chronic inflammatory disease of multifactorial etiology. Microbes in the dental plaque initiate periodontal inflammation, but host response to these microbial insults is mainly responsible for the progression of the disease.[1] Periodontal inflammation is a defense mechanism of the host against infection. Inflammatory mediators are produced from the host tissues to limit the activity of periodontopathogens, but paradoxically, it will create destruction of the periodontium. Genetic and environmental risk factors deregulate this host response further aggravating the disease process.

Microbial factors such as microbe-associated molecular patterns (MAMP) and host proteins like cell damage-associated molecular patterns (DAMP) communicate with periodontal cell surface receptors such as toll-like receptors (TLR), nod-like receptors, retinoic acid-inducible gene I-like receptors, and C-type lectin receptors.[2] When MAMPs and DAMPs are recognized by these receptors, a series of cellular signaling events take place which ultimately results in the production of various inflammatory mediators. The most important inflammatory mediators in periodontal disease are cytokines, prostaglandins, and matrix metalloproteinases.[1] Receptor activator nuclear factor kappa ligand (RANKL) is a critical molecular mediator of bone resorption. Along with these factors, oxidative stress also participates in both hard- and soft-tissue destruction of periodontium. During the cellular signaling event, transcription factors act as intermediate players to bring about activation of target genes. Epigenetic alterations in the host produced by polymicrobial synergy and dysbiosis also lead to activation of transcription factors as described in a recent study.[2]

Transcription factors are the factors present in cytoplasm of many cells and on activation, they are transported to nucleus to regulate the production of multiple inflammatory mediators. Even though lot of information is available on the inflammatory mediators of periodontal tissue destruction, little is known about the role of transcription factors in periodontal pathogenesis. This comprehensive review is aimed at unveiling the importance of two pivotal transcription factors, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3) in periodontal pathogenesis.

NUCLEAR FACTOR KAPPA B

NF-κB is a family of ubiquitous transcription factors first described by Sen and Baltimore in 1986 as the regulator of kappa light chain gene in murine B lymphocytes.[3] The family members are NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and cRel.[4] Any homo or heterodimer combination of these members is considered as NF-κB, but the classic form of NF-κB is the combination of p50 and p65.[4] Inside the cytoplasm, NF-κB exists as a complex with a protein known as inhibitor κB (IκB) and different types of IκB are IκBa, IκBβ, IκBέ, IκB-r, p100, p102, and BCL3.[4] When cells are stimulated with various activators such as tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), lipopolysaccharide (LPS), oxidants and viruses phosphorylation, ubiquitination, and subsequent degradation of IκB occurs. Thus, NF-κB is made free and is transported to the nucleus where it activates target genes [Figure 1].[4]

Figure 1

Activation of nuclear factor kappa B and signal transducer and activator of transcription 3 and possible cross talk (indicated by dotted red arrow) in their signaling in periodontal disease. TLR – Toll like receptors, LPS – Lipopolysaccharide, JAK – Janus activated kinase, STAT3 – Signal signal transducer and activator of transcription 3, MYD88 – Myeloid differentiation primary response 88, IXB – its not X but kappa (κ)- the expansion is inhibitor kappa B, P50 – PROTEIN 50, P65 – PROTEIN 65, NFB – nuclear factor-kappa B

SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling is mainly activated by cytokines and control expression of genes related to inflammation. STATs are cytoplasmic proteins which are characterized by SH2 (Src Homology-2) domain. When the receptors associated with STAT signaling are stimulated by appropriate cytokines, initially dimerization of the receptors occur. Then, tyrosine kinases JAK1, JAK2, JAK3, and Tyk2 which are associated with these receptors are activated.[5] This receptor-kinase complex interacts with and activates members of the STAT family through tyrosine phosphorylation resulting in the formation of homo or heterodimers of STAT. These dimeric molecules are transported to the nucleus by exposure of nuclear localization signal to stimulate transcription of the responsive genes [Figure 1].[5] At present, seven STAT family members have been identified, that is, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. They have similarity in their molecular structure and function but play diverse physiological roles in a wide variety of biological processes.[5]

STAT3 was known earlier as acute-phase response factor (APRF), and Zhongin (1994) included it as a member of STAT family.[6] Gp130 cytokines such as IL-6, oncostatin M (OSM), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor mediates STAT3 signaling.[6] STAT3 phosphorylation takes place at tyrosine (Tyr)-705, but maximal transcriptional activation requires additional phosphorylation at serine (Ser)-727.[7] SH2-containing phosphatases (SHPs), protein inhibitors of activated STAT3 (PIAS3), and suppressors of cytokine signaling (SOCS) are the endogenous regulators of STAT3 which prevent its activation in normal cells.[8]

CROSS TALK BETWEEN NUCLEAR FACTOR KAPPA B AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3

NF-κB and STAT3 synergistically control a common set of genes encoding for cytokines and chemokines.[9] NF-κB family members physically interact with STAT3 resulting in either transcriptional synergy or repression of NF-κB/STAT3 regulated genes, and many mechanisms for this have been suggested.[9] Unphosphorylated STAT3 bind to NF-κB/IκB complex and facilitate NF-κB activation. STAT3 may interact with p65 in the nucleus and increase its nuclear retention through acetylation and ensure constitutive NF-κB activation. NF-κB/STAT3 complex can activate target genes which cannot be induced by either factor alone.[9] STAT3 is an upstream signaling factor for NF-κB activation.[10] NF-κB activation will result in the production of IL-6 which in turn will activate STAT3.[11] Simultaneous activation may be beneficial to mediate effective inflammatory response in many diseases. NF-κB and STAT3 signaling cross talk was observed in many inflammatory conditions and cancers.[910] Even though functional studies have not yet identified such a cross talk in periodontal disease, there are every possibility for the same [Figure 1].

THE IMPORTANCE OF NUCLEAR FACTOR KAPPA B AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3 IN PERIODONTAL PATHOGENESIS

Among the 150 activators of NF-κB listed, majority are seen in abundance in periodontal disease, for example, bacterial LPS, prostaglandin E2, IL-1β, TNF alpha, stress, viruses, etc.[12] The keystone pathogen Porphyromonas gingivalis is used as an activator of NF-κB in many in vitro experiments.[1314] NF-κB activation by P. gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Fusobacterium nucleatum, and P. intermedia induced apoptosis of monocytes and neutrophils.[15] Target genes of NF-κB including cytokines, matrix metalloproteinases (MMP), cyclooxygenase 2 (COX 2), inducible nitric oxide synthase (iNOS), RANKL, etc., are beneficial to the progression of periodontal disease.[12] NF-κB activation by various activators such as IL-1β, TNF α has been observed in cultured periodontal cells which lead to production of inflammatory mediators such as prostaglandins and MMP.[1617] Ninety-one genes which represented biological processes associated with periodontitis were upregulated by NF-κB activation induced by periodontopathogens P. gingivalis and F. nucleatum in H400 oral epithelial cell lines.[15]

The hallmarks of periodontal destruction are inflammation of the soft tissues, collagen degradation, and bone resorption. The major tissue destructive enzyme in periodontal disease is MMP and its production is controlled by NF-κB activation as evidenced by in vitro experiments.[17] Periodontal tissue remodeling is also mediated by the activation of NF-κB-dependent genes encoding inducible forms of COX-2 and iNOS enzymes participating in production of prostaglandins, nitric oxide, and nitric oxide metabolites.[12] Vascular endothelial growth factor activated by NF-κB in fibroblasts promotes formation of new blood vessels in inflamed periodontal tissues.[18]

NF-κB plays an important role in bone destruction as it can activate osteoclasts according to scientific evidence.[1920] NF-κB-dependent cytokines such as IL-1α and IL-1 β, TNF-α, IL-6, and IL-17 induce osteoclast differentiation and activation mediated by RANK.[19] Osteoclastogenesis is controlled by NF-κB activation followed by c-Fos and NFATc1 (nuclear factor for activated T-cells) activation.[20] NF-κB activation impair Fos-related antigen-1 which is a transcription factor involved in bone matrix formation, thereby impairing bone formation.[21] Impairment of osteogenic differentiation of inflamed periodontal ligament stem cells is mediated through NF-κB signaling and inhibition of NF-κB activation can reverse this phenomenon.[22]

Activation of NF-κB in diseased human periodontal tissues has been documented in a few studies.[2324] Increased activation of p50 and p65 subunits of NF-κB and decreased expression of IκB were expressed in diseased periodontal tissues compared to healthy ones. Increased formation of nuclear p50 homodimers were observed in the human gingiva from chronic periodontitis patients and activation of dendritic cells by P. gingivalis LPS resulted in increase in p50/p65 ratios.[25] Cytoplasmic expression of NF-κB was strong in all cell types in inflamed periodontal ligament, and intense nuclear expression was noted in immune and endothelial cells.[14]

STAT3 pathway is activated mainly by IL-6 and IL-6 family of cytokines which signal through gp130 receptors.[6] The importance of IL-6 in periodontal disease is well documented.[1] Other activators of STAT3 such as IL-22, INF-γ, TNF-α, IL-1, IL-4, IL-10, IL-17/23, and LPS, etc., are also seen associated with periodontal disease.[1] Signaling target of STAT3 include INF-γ, TNF-α, IL-1, IL-4, IL-6, and IL-10, MMP-2 and MMP-9, iNOS, VEGF, COX-2, etc., and many of these molecules have biologically significant role in the periodontal pathogenesis.[1]

Even though STAT3 activation has not been evaluated in human periodontal tissues; activation of STAT3 in human periodontal ligament cells was observed when they were stimulated with IL-1 β and IL-6.[26] The activated STAT3 lead to the expression of CC chemokine ligand 20 which recruit Th17 cells that play a central role in periodontal bone destruction.[26]

A few experimental studies in periodontal disease models also demonstrated STAT3 activation. Activation of STAT3 was observed in a ligature-induced periodontitis model in winstar rats.[27] Rapid and transient activation of extracellular-regulated kinases and p38 mitogen-activated protein kinase as well as NF-κB was also noted in the same specimens. This experiment indicates a cross talk and involvement of many signaling pathways in cytokine-mediated periodontal destruction. Upregulated expression of activated STAT3 was also induced by LPS in experimental animals.[28]

Risk factors of periodontal disease such as diabetes, smoking, and obesity activate STAT3 and NF-κB leading to persistent inflammation.[29303132] Nicotine and LPS-treated periodontal ligament cells increased the number of osteoclasts and inhibition of hypoxia inducible factor-2α reversed the effects through multiple signaling pathways including STAT3 and NF-κB.[33] STAT3 and NF-κB activation has also been reported in many chronic inflammatory diseases such as rheumatoid arthritis which share similarities with periodontal disease in pathogenesis.[34]

Microbial etiology is insufficient to explain the periodontal disease progression in a subgroup of patients where molecular mechanisms such as STAT3 and NF-κB activation can be considered as logical explanations for the exaggerated host response. It is evident that activation of these transcription factors is possible in periodontal disease and their cross talk and target gene upregulation can favor the rapid progression of the disease.

NUCLEAR FACTOR KAPPA B AND SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION 3: A MOLECULAR LINK IN PERIODONTAL MEDICINE CONCEPT

Recent studies have identified association between periodontal disease and cardiovascular disease, diabetes mellitus, preterm low birth weight, and many other systemic diseases.[35] Many mechanisms to explain this association has been proposed, including systemic dissemination of pathogens and stimulation of host response. However, none of them were able to give satisfactory explanation for this interrelation. NF-κB and STAT3 activation can be considered as an important molecular mechanism to explain the link between periodontal disease and systemic diseases. NF-κB as a connecting link between atherogenesis and periodontitis has been already proposed.[36] The role of activated STAT3 in atheroma formation also has been clearly understood from in vivo as well as in vitro studies.[37] STAT3 mediates inhibition of Pseudomonas aeruginosa and P. gingivalis-induced apoptosis in respiratory epithelial cells, thus contributing to the pathogenesis of respiratory disease.[38] In a recent study, placental tissues of preeclampsic women exhibited NF-κB upregulation giving supportive evidence for the role of NF-κB in the association between preeclampsia and periodontal disease.[39] NF-κB activation produce IL-6 which is an important activator of STAT3, and this molecular mechanism operates in association between obesity, aging, chronic inflammation, and cancer.[40]

Chronic infections and inflammation are found to be associated with about 20% of human cancers.[41] Chronic periodontal disease is the most abundant source of low-grade inflammation in the body. Studies have found out an association between chronic periodontal inflammation and cancer of oral cavity, pancreas, lungs, kidney, blood, and gastric cancers.[42] However, molecular mechanism pertaining to this association is still under investigation. NF-κB and STAT3 pathways may be the pivotal signaling mediators in inflammation associated tumor development.[43] NF-κB and STAT3 together activate FAT10 which inhibit tumor suppressor p53 facilitating tumor progression, and it can also upregulate oncogenes such as cyclin D1.[4445] A recent study has demonstrated that P. gingivalis and F. nucleatum interact with oral epithelial cells through toll-like receptors and signal along IL-6/STAT3 resulting in oral squamous cell carcinoma in murine periodontitis model.[46]

CLINICAL IMPLICATIONS

Traditional periodontal therapy was focused on eliminating or reducing microorganisms in plaque biofilm using mechanical and chemical methods. Extended knowledge of the role of host response in periodontal pathogenesis has opened up a new treatment approach called host modulation therapy (HMT). HMT has proved as a beneficial adjunct to conventional periodontal therapy individuals who are susceptible to periodontal disease.[1] Drugs mainly utilized for host modulation were subantimicrobial dose doxycycline, NSAIDs, and bone-sparing drugs like bisphosphonates. Anticytokine therapy targeting IL-1, IL-6, and RANKL when administered either systemically or locally was also found to be beneficial for periodontal therapy. However, most of these drugs had unwanted side effects and inhibition of cytokines can affect its protective functions in the immune system.

Chronic diseases are connected with approximately 500 gene products. Thus, prevention and treatment of such diseases require inhibition of multiple molecular mediators. A more recent approach for HMT is by inhibiting major signaling pathways of inflammation.[47] Here, the advantage is that many molecules can be simultaneously targeted. Signaling blockade is more beneficial because when individual molecule is inhibited there can be compensating mechanism with another molecule. Rapid and transient activation of signaling can result in long-lasting modulation of cytokine gene expression. Hence, a short-term blockade without affecting normal physiological processes may be beneficial for periodontal disease.

Signaling blockade has been attempted and found to be useful in experimental periodontitis and also in other similar chronic inflammatory diseases.[4849] NF-κB inhibitors include a wide variety of molecules such as antioxidants, proteasome inhibitors, IκB phosphorylation/degradation blockers, upregulators of IκB, inhibitors of Rel/NF-κB nuclear transport, inhibitors of Rel/NF-κB DNA binding, and transactivation. Genetically engineered proteins that block specific steps in NF-κB activation were also developed including I-TRAF (TNF receptor-associated factor interacting protein), IKK complex mutants, and IκBα superrepressor.[50] NF-κB is inhibited by drugs such as aspirin, NSAID's, glucocorticoids, cyclosporine, and tacrolimus different mechanisms. Recent research has shown promising results in preventing the progression of bone loss with topical application of NF-κB decoy oligodeoxynucleotides in experimental periodontitis in beagle dogs.[51] Clinical efficacy of these agents for periodontal therapy can be further established with human clinical trials.

STAT3 inhibition was attempted by targeting tyrosine kinases, and several inhibitors were developed. JAK2 inhibitors are already in the market.[52] JAK inhibitors such as tofacitinib and ruxolitinib are found to be useful in patients with rheumatoid arthritis.[53] Inhibitors of JAK3 and Tyk2 were mainly tried for the drug development because deficiency of JAK1 or JAK2 can be lethal. No Tyk2 inhibitor is developed so far, but JAK3 antagonist CP-690550 (Pfizer) showed potential benefits in the treatment of rheumatoid arthritis.[54]

STAT3 inhibition can also be achieved using peptidomimetics, STAT3 dimerization inhibiting molecules, competitive binding molecules for STAT3, antisense oligonucleotides, and small inhibitory RNA.[55] Cell-specific modulation of STAT3/SOCS3 decreases pro-inflammatory response in many inflammatory conditions like arthritis.[56] Photodynamic therapy which is an emerging treatment concept in periodontal disease produce cross-linking of STAT3, and cross-linked STAT3 is unable to enter the nucleus. The cells will be unresponsive to cytokines such as IL-6, oncostatin M at least for 24 h.[57]

Inhibition of one signaling pathway allows cells to survive using some other mechanism. Dual inhibition of STAT3–NF-κB may overcome problems associated with inhibition of either pathway. Dual inhibition has been reported to be beneficial in many studies. Blocking STAT3 activity preferentially inhibits LPS-mediated IL-1 β and IL-6 production, but not TNF-α, in RAW264.7 cells which require inhibition of NF-κB pathway.[58] Hence, agents that simultaneously block NF-κB and STAT3 may be more beneficial for inflammatory bone diseases like periodontal disease. Inhibitors of STAT3, NF-κB, or both were successfully used in treating many immune inflammatory conditions like arthritis.[59]

Inhibition of toll-like receptor 4 (TLR4) and STAT3 by 25-hydroxyvitamin D3 was utilized in experimental periodontitis.[60] Fasting blood glucose, glycosylated hemoglobin, and TNF-α levels were reduced in diabetic rats by 25-OHD3 intraperitoneal injection and correspondingly reduction in alveolar bone loss was also noted. In the same animals, increased expression of vitamin D receptor and reduction in TLR4, JAK1, STAT3, and their phosphorylation were also noted in gingival epithelia.

Basal activity of both NF-κB and STAT3 are required for many normal physiological functions. Moreover, drugs with NF-κB and STAT3 inhibiting property possess many side effects. Specific and potent inhibitors that provide an effective treatment while sparing the host from the side effects are actually required. The route of drug delivery might also affect the outcome of therapy. Local delivery of the agents in periodontal therapy reduces the side effects associated with systemic exposure and minimize the risk of general immunosuppression. Gene delivery also may appear promising for periodontal therapy.

Extracts of many medicinal plants can inhibit NF-κB and STAT3 signaling.[5961] Curcumin which has proven to be effective in managing periodontal disease interfere with NF-κB STAT3 cross talk.[16] Nutritional modulation is a significant and emerging concept in periodontal management. Omega 3 fatty acid, one of the important nutritional supplements recommended for periodontal disease also reduce the activation of inflammatory transcription factors NF-κB and STAT3.[62] Specific inhibitors of these transcription factors for systemic administration, local drug delivery as well as HMT have to be identified for periodontal disease. Future controlled clinical trials are required to identify safe and effective NF-κB and STAT3 inhibitors that can revolutionize the existing periodontal treatment strategies and thereby help to improve patient's oral as well as systemic health.

CONCLUSION

Periodontal disease is a multifactorial disease, and the major role in its pathogenesis is played by immune inflammatory response of the host. So along with controlling microbial factors, host modulation is an integral part in periodontal therapy. NF-κB and STAT3 are two crucial transcription factors in chronic inflammatory diseases. The role of NF-κB and STAT3 in periodontal disease is clearly evident from the available literature. Activation and cross talk between STAT3 and NF-κB is possible in periodontal disease environment, and it can favor the disease progression if allowed to continue. Inhibition of these factors has been utilized successfully in the treatment of similar chronic inflammatory diseases. Simultaneous inhibition of NF-κB and STAT3 may constitute the next generation therapeutic modality for periodontal disease.

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Conflicts of interest

There are no conflicts of interest.