Salivary histatin 3 inhibits heat shock cognate protein 70-mediated inflammatory cytokine production through toll-like receptors in human gingival fibroblasts

Background Salivary histatins are bioactive peptides related to the innate immune system associated with antimicrobial activities. However, very little is known about the physiological and biological functions of histatins against host cells or their role in oral cell inflammation. Histatin 3 binds to heat shock cognate protein 70 (HSC70, a constitutively expressed heat shock protein (HSP)). It is unclear whether HSC70 is involved in the inflammatory response in oral cells. Injured oral cells release some intracellular proteins including HSC70. It is possible that released HSC70 induces toll-like receptor (TLR) activation, just as extracellular HSP70 (a stress inducible HSP) does, and that histatin 3 affects this process. Therefore, we tested the hypothesis that HSC70 activates TLR signaling and histatin 3 inhibits this activation and inflammatory cytokine production. Methods A nuclear factor (NF)-κB-dependent luciferase reporter plasmid was transfected into HEK293 cells stably expressing TLR2 with coreceptor CD14 (293-TLR2/CD14 cells) or stably expressing TLR4 with CD14 and the accessory molecule MD2 (293-TLR4/MD2-CD14 cells). The cells were stimulated with HSC70 in the presence or absence of histatin 3, and examined using luciferase assays. We also stimulated human gingival fibroblasts (HGFs) with HSC70 with or without histatin 3. Then, we analyzed the levels of inflammatory cytokines (interleukin (IL)-6 and IL-8) in the culture media. Cell proteins were analyzed using enzyme-linked immunosorbent assay and Western blotting with antibodies of mitogen-activated protein kinases and NF-κB inhibitor IκB-α, respectively. Histatin 3-bound form of HSC70 was analyzed using limited V8 protease proteolysis. Results HSC70 induced NF-κB activation in a dose-dependent manner in 293-TLR2/CD14 and 293-TLR4/MD2-CD14 cells, and histatin 3 inhibited this process and when histatin 3 binding to HSC70 was precluded by 15-deoxyspergualin, which augmented NF-κB-triggered activation. In HGFs, histatin 3 also inhibited HSC70-induced inflammatory cytokine production, extracellular signal-regulated protein kinase phosphorylation, and degradation of IκB-α. Moreover, HSC70 in the presence of histatin 3 was relatively resistant to digestion by V8 protease compared with HSC70 in the presence of control peptide. Conclusions Histatin 3 may be an inhibitor of HSC70-triggered activation of TLR signaling and inflammatory cytokine production and may be involved in inflammation processes noted in oral cells.


Background
Histatins constitutively secreted by the salivary glands are associated with innate immunity processes in the oral cavity. These peptides have antimicrobial properties and protect oral tissues from pathogens [1]. The histatin family comprises 12 histidine-rich cationic peptides found in healthy adults at concentrations of 50-425 μg/ml, corresponding to approximately 10% of total protein in saliva [2][3][4]. Histatins 1 and 3 are full-length peptides of 38 and 32 amino acid residues, respectively; other characterized members of the histatin family are proteolytic products formed during secretion [2,5]. Histatins 3 and 5 are the heat shock protein (HSP)-binding proteins that are most abundant in saliva and are active against Candida albicans and Porphyromonas gingivalis (the pathogen of periodontitis) [6][7][8][9][10]. In addition, histatins 3 and 5 are also involved in proliferation of human gingival fibroblasts (HGFs) and rabbit costal chondrocytes, respectively [7,11].
HSPs are induced by a wide variety of stresses in prokaryotic and eukaryotic cells, including environmental, pathological, and physiological stimuli [12]. HSPs function as ATPase activity-dependent molecular chaperones that assist in the correct folding of proteins, the assembly of various protein complexes, transport of proteins across membranes into organelles, and the degradation of proteins by the lysosome [13][14][15]. Heat shock cognate protein 70 (HSC70), an HSP family member, is a cytosolic protein that is abundantly, constitutively, and ubiquitously expressed in most cells [16]. HSC70 consists of an ATPase domain (amino acid residues 1-384), a substrate (peptides, including histatin 3)-binding domain (amino acid residues 385-543), and a lid domain (amino acid residues 544-646) [7,17]. Three-dimensional structure of the ATPase domain has been determined by X-ray crystallography; the structure of this domain is similar to that of hexokinase and actin [18,19].
Toll-like receptors (TLRs) have been identified as human homologs of Drosophila Toll receptors, which are involved in innate immunity [20,21]. TLRs recognize their respective pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs, such as intracellular proteins released from damaged and necrotic cells) [22]. TLR2, a TLR family member, recognizes peptidoglycan (PGN, a major cell-wall component of gram-positive bacteria), lipopolysaccharide (LPS) from P. gingivalis, and HSP70 (a stress-induced HSP) [23][24][25]. TLR4 is another TLR family member, which recognizes LPS from the outer membranes of gramnegative bacteria and HSPs such as HSP70, HSP60, and Gp96 [25][26][27][28]. TLR2 and TLR4 require their interacting proteins for the recognition of some PAMPs and DAMPs. TLR2 can induce nuclear factor (NF)-κB activation in response to HSP70 stimulation, but only in the presence of CD14, a glycosylphosphatidylinositol-anchored protein [25]. In order for TLR4 to function satisfactorily as a receptor for LPS, both MD2 and CD14 must be coexpressed [29,30]. Once TLR2 and TLR4 recognize their respective ligands, the specific response initiated by these TLRs depends on the recruitment of adaptor proteins (e.g., myeloid differentiation primary response protein 88 or Tollinterleukin (IL)-1 receptor domain-containing adaptor protein). These adaptor proteins transmit signals that result in the activation of mitogen-activated protein kinases (MAPKs) and NF-κB and the induction of inflammatory cytokines [31].
It is not known whether HSC70 is involved in the inflammatory responses in oral cells, such as the production of inflammatory cytokines. It has been proposed that oral diseases accompanying damage or oral injuries cause the release of some intracellular proteins, including HSC70. If HSC70, like HSP70, functions as DAMPs, HSC70 could be a putative inducible factor in the inflammatory response in oral cells through TLRs. HGFs, which constitute the major cellular population of gingival tissue, express TLR2, TLR4, MD2, and CD14 [32][33][34]. In addition, histatin 3 in saliva may associate with the released HSC70. Therefore, we can infer that histatin 3 inhibits TLR-mediated HSC70 function, reducing the production of inflammatory cytokine in oral cells.
In this study, we identified HSC70 as a putative ligand of TLR2 and TLR4. We observed the inhibitory effect of histatin 3 on inflammatory cytokine production in HGFs and on NF-κB activation in HEK293 cells expressing TLR2 or TLR4 as a result of HSC70 stimulation. These findings represent our current knowledge of physiological functions of salivary proteins in oral cavity.

Statistical analysis
All quantitative data were statistically analyzed using either one-way analysis of variance (ANOVA) or two-way ANOVA using the StatMate software (ATMS). Differences were considered statistically significant at P < 0.05.

NF-κB activation by stimulation with exogenous HSC70 through TLR2 and TLR4
HSP70 stimulates inflammatory cytokine production (e.g., IL-6 and tumor necrosis factor-α) in monocytes and dendritic cells through TLR2 and TLR4 [25,37]. However, the activation of inflammatory cytokine production by HSC70, even though it belongs to the same HSP family as HSP70, is poorly understood. Therefore, we investigated whether cytokine production by HSC70 is mediated by TLRs and their interacting proteins such as HGFs, particularly in oral cells. Because HGFs express TLR2 and TLR4, it is necessary to perform systematic experiments investigating the dependence of HSC70 stimulation on TLR2 or TLR4. It has been reported that HSP70 stimulation induces NF-κB activation in 293T cells transiently expressing TLR4 and MD2, but not in the cells expressing TLR4 alone [38]. In contrast, till now there has been no evidence that HSC70 activates NF-κB in HEK293 cells expressing TLRs. Therefore, we examined NF-κB activation in the stable cell line 293-TLR4 (HEK293 cells constitutively expressing TLR4) transiently transfected with an MD2 expression vector and an NF-κB-dependent luciferase reporter plasmid. The results indicated that HSC70 stimulated NF-κBdependent promoter activity through TLR4/MD2 signaling pathway. Moreover, when CD14 and MD2 were both expressed in 293-TLR4 cells, the promoter activation by HSC70 was more effective than that in 293-TLR4 cells transiently expressing MD2 alone (data not shown). We then examined whether this activation was dosedependent. The stable cell line 293-TLR4/MD2-CD14 (HEK293 cells constitutively expressing TLR4, MD2, and CD14) transfected with the NF-κB-dependent reporter plasmid (293-TLR4/MD2-CD14/NF-κB) was stimulated with HSC70, and luciferase assays were performed. As shown in Figure 1A, both HSC70 and HSP70 (control) induced promoter activation in a dose-dependent manner, whereas BSA did not. Promoter activity levels with heated HSC70 and HSP70 were similar to those in unstimulated cells. When 293-TLR4/MD2-CD14/NF-κB cells were stimulated with the heated or unheated HSC70 ATPase fragments (HSC70 with deletions of the substrate-binding and lid domains), the promoter activation was at a lower level ( Figure 1B). These results suggest that HSC70 as well as E. coli LPS induces NF-κB activation through TLR4/MD2/CD14 system. HSP70 also induces NF-κB activation in stable HEK293-TLR2 cells transiently expressing CD14, but not in HEK293-TLR2 without CD14 [25]. Therefore, we examined NF-κB activation by HSC70 in the stable cell line 293-TLR2/CD14 (HEK293 cells constitutively expressing TLR2 and CD14) transfected with the NF-κB-dependent reporter plasmid (293-TLR2/CD14/NF-κB). As shown in Figure 1C, HSC70 as well as HSP70 (control), but not BSA, induced the promoter activation through TLR2/ CD14 in a dose-dependent manner. Heated and unheated P. gingivalis LPSs induced promoter activation, whereas heated HSC70 and heated HSP70 reduced activation compared with the respective unheated HSPs. When the cells were stimulated with heated and unheated HSC70 ATPase fragments, only low levels of the promoter activation were observed ( Figure 1D). These results suggest that both HSC70 and P. gingivalis LPS induce NF-κB activation through TLR2/CD14.
Effect of DSG on HSC70-induced NF-κB activation through TLR2 and TLR4 in the presence of histatin 3 We investigated whether the specific interaction of histatin 3 and HSC70 was necessary for inhibition of HSC70-induced NF-κB activation. The NF-κB-driven luciferase assays were performed on 293-TLR4/MD2-CD14/NF-κB and 293-TLR2/CD14/NF-κB cells treated with HSC70 along with or without histatin 3 in the presence or absence of DSG. DSG is a synthetic analog of spergualin, a natural product from Bacillus laterosporus, and has a peptidomimeric structure. It binds particularly well to HSC70 and is considered to preclude peptide binding to HSC70 [40,41]. The results demonstrated that in the presence of histatin 3 and DSG HSC70-stimulated NF-κB-dependent promoter activity was significantly higher than with histatin 3 alone in both 293-TLR4/ MD2-CD14 ( Figure 3A, P < 0.001) and 293-TLR2/CD14 ( Figure 3B, P < 0.001) cells. These results suggest that the specific binding of histatin 3 to HSC70 is important for inhibition of HSC70-mediated activation of TLR2 and TLR4 signaling activation.

Production of inflammatory cytokines by HSC70 stimulation in HGFs
It is unknown whether HSC70 is involved in inflammatory response in HGFs, especially in the production of inflammatory cytokines. To examine this possibility, HGFs were stimulated with HSC70 for 24 h, and the amounts of IL-6 and IL-8 in the culture media were measured by ELISAs. As shown in Figures 4A and 4B, unheated HSC70 significantly induced cytokine production in HGFs in a dose-dependent manner, whereas heated HSC70 did not induce cytokine production (HSC70, 7 nM (P < 0.01) and 70 nM (P < 0.001) in Figure 4A; 7 nM (P < 0.05) and 70 nM (P < 0.01) in Figure 4B). When HGFs were stimulated with the unheated and heated HSC70 ATPase fragments, the levels of IL-6 and IL-8 were similar to those in unstimulated cells. These results suggest that HSC70 induces inflammatory cytokine production in HGFs.

Inhibitory effect of anti-TLR antibodies on inflammatory cytokine production stimulated by HSC70 in HGFs
To examine whether inflammatory cytokine production stimulated by HSC70 was dependent on TLR2 or TLR4 in HGFs, we performed experiments using anti-TLR2 and anti-TLR4 antibodies. As shown in Figure 5A, levels of HSC70-stimulated IL-6 production in HGFs with anti-TLR4, anti-TLR2, and both antibodies significantly decreased (P < 0.001). LPS-stimulated IL-6 production also significantly decreased in the presence of anti-TLR4 and both anti-TLR2 and anti-TLR4 antibodies (P < 0.001).
The patterns of HSC70-and LPS-stimulated IL-8 production in the presence of the antibodies were similar to those of IL-6 production under the same condition ( Figure 5B). Next, we examined the effect of anti-CD14 antibody on HSC70-and LPS-stimulated inflammatory cytokine production in HGFs. HSC70-stimulated IL-6 and IL-8 production significantly decreased in the presence of this antibody (P < 0.001) ( Figures 5C and 5D). The antibody also caused a decrease in cytokine production in LPSstimulated HGFs. The levels of IL-6 and IL-8 production under HSC70 stimulation were approximately1.6-fold higher than those by LPS stimulation. This behavior is similar to the previously reported findings for HSP70 stimulation of cytokine production through a CD14dependent pathway [37]. These results suggest that HSC70-stimulated inflammatory cytokine production in HGFs is dependent on TLR2, TLR4, and CD14.

Inhibitory effect of histatin 3 on HSC70-stimulated inflammatory cytokine production in HGFs
Histatin 3 was found to inhibit NF-κB activation through TLR2 and TLR4 (Figure 2). We then examined the effect of histatin 3 on HSC70-stimulated inflammatory cytokine production in HGFs. HSC70 and histatin 3 were added to HGFs, and the amounts of IL-6 and IL-8 released into the culture media were measured by ELISAs. As shown in Figures 6A  and 6B, HSC70-stimulated cytokine production significantly decreased in the presence of histatin 3, in a dose-dependent manner (histatin 3, 0.15 μM (P < 0.001) and 1.5 μM (P < 0.001)). The control peptide and P3a did not appreciably affect the cytokine production. We then investigated whether other members of the histatin family were capable of inhibiting HSC70-stimulated cytokine production in HGFs. Histatins 3, 4, and 5 were mixed with HSC70 and used to stimulate HGFs, and the amounts of IL-6 and IL-8 in the culture media were measured. As shown in Figures 6C and 6D, the inhibitory effect of histatin 3 on the cytokine production was significantly higher than that of histatin 5 (IL-6, P < 0.01; IL-8, P < 0.001). These results suggest that histatin 3 is an inhibitory factor of HSC70-stimulated inflammatory cytokine production in HGFs.
To examine whether HSC70-induced ERK phosphorylation and IκB-α degradation were inhibited by histatin 3, HGFs were stimulated with HSC70 in the presence of histatin 3, and Western blotting was performed. As shown in Figures 7C and 7D, p42/44 phosphorylation induced by HSC70 in the presence of histatin 3 was lower than that in the absence of histatin 3 or in the presence of the control peptide. Stimulation of either the control peptide or histatin 3 alone decreased levels of p42/44 phosphorylation. NF-κB activation by stimulation with the control peptide or histatin 3 alone was also diminished in 293-TLR4/MD2-CD14 and 293-TLR2/CD14 cells (Figures 2A and 2B). HSC70 stimulated IκB-α degradation in the presence of the control peptide, but not in the presence of histatin 3. These results suggest that histatin 3 inhibits HSC70-mediated MAPK phosphorylation and IκB-α degradation in HGFs.

Histatin 3 binding-induced protease-resistant conformation of HSC70
Histatin 3 prevented HSC70-stimulated NF-κB activation through TLR2 and TLR4 ( Figure 2) and inflammatory cytokine production in HGFs ( Figure 6). Therefore, we assumed that histatin 3 binding to HSC70 had some effect on the conformation of HSC70. To examine this assumption, HSC70 was mixed with histatin 3, and the mixture was analyzed by limited V8 protease proteolysis. As shown in Figure 8, HSC70 with histatin 3 was relatively more resistant to digestion by V8 protease with HSC70 with the control peptide or P3a. Histatin 3 did not inhibit the activity of V8 protease (data not shown). These results suggest that histatin 3 may affect the conformation of HSC70 upon binding.

Discussion
It has been reported that extracellular HSP70 induces inflammatory cytokine production through TLR2 and TLR4 pathway in human monocytes [25]. However, it is not known whether HSC70 activates TLR2 and TLR4 signaling in oral cells, and if so, whether intravital (bioactive) factors that inhibit HSC70 function exist in saliva. In this study, we found that HSC70, along with MD2/CD14, stimulated TLR4 and that HSC70, along with CD14, stimulated TLR2, resulting in the induction of NF-κB-dependent activation. HSC70 also induced inflammatory cytokine production, MAPK phosphorylation, and IκB-α degradation in HGFs. Histatin 3 inhibited those effects of HSC70. Moreover, we believe that histatin 3 may affect conformation of HSC70 upon binding, presumably inhibiting HSC70 function.
In experiments related to TLR stimulation, stimulating reagents might be contaminated with endotoxin. Our studies showed that boiling (95°C, 20 min) abrogated the effects of HSC70 induction, but not of LPS induction ( Figure 1A). Moreover, polymyxin B, an LPS antagonist, abrogated the effects of LPS induction, but not of HSC70 induction (data not shown). In addition, endotoxin activity in the HSC70 reagents was analyzed by the limulus amebocyte lysate (LAL) assay and was found to be low. A recent study has revealed that TLR4 activation is induced by the HSP70 reagents which may include a small amount of endotoxin. The results of the study suggest that the stimulatory effect depends on HSP70, even in the presence of a small amount of endotoxin, and the structural integrity of HSP70 is essential [42]. Our results showed that histatin 3 significantly inhibits HSC70stimulated NF-κB activation and inflammatory cytokine production, despite a slight contamination of HSC70 reagents with endotoxins (Figures 2A and 6). Consequently, we can conclude that HSC70 provably affects NF-κB activation and inflammatory cytokine production and histatin 3 may inhibit those effects upon its binding to HSC70. It is also possible that reagents used in stimulating experiments were contaminated with lipoproteins. We observed a decrease in the levels of NF-κB activation after stimulation with heated HSC70 ( Figure 1C). Furthermore, the levels of inflammatory cytokine production after treatment with heated HSC70 were very low ( Figure 4). In addition, histatin 3 significantly inhibited NF-κB activation and inflammatory cytokine production caused by HSC70 stimulation (Figures 2B and 6), although HSPs possess an affinity for lipids [43,44]. It is also difficult to precisely quantify the small amount of HSC70 associated with lipids in the HSC70 reagents. Consequently, if the HSC70 reagents contain HSC70 associated with lipids (even to a very small extent), our results might reflect the function of HSC70 in various physiological forms (for example, HSC70 that exists under the various physiological conditions of the oral cavity), because HSC70 derived from the cells might form lipoproteins [42,43]. We can conclude that histatin 3 binding to HSC70 may inhibit HSC70 activity.
A previous study has reported that HSC70 is released from injured cells [45]. The release of HSC70 from glial and K562 erythroleukemic cells has been also observed [46,47]. Our findings show that extracellular HSC70 stimulates TLR2 and TLR4 and increases the production of inflammatory cytokines in HGFs (Figure 4). Therefore, we suggest that HSC70 as well as other HSPs (e.g., HSP70 and HSP60) may function as a DAMP for TLRs and elicit inflammatory responses. The release of HSC70 has also been observed in the heart, contributing to the postischemic myocardial inflammatory response and to cardiac dysfunction [48]. Inflammatory response in the oral cavity is also observed in oral diseases and injuries. It is tempting to speculate that HSC70 released from the damaged cells may stimulate oral cells such as HGFs.
Our present findings suggest that inflammatory cytokine production stimulated by the released HSC70 might be inhibited by histatin 3 in saliva in HGFs, histatin 3 may be involved in inflammatory processes in the oral cavity. Our previous study demonstrated the HSC70-binding ability of histatins [7]. The study showed that histatin 3 bound to the substrate-binding domain of HSC70 more strongly than histatin 5 and that histatin 4 did not bind to HSC70. Our present findings indicate that the inhibitory effects of histatin 5 on HSC70-stimulated NF-κB-dependent activation and inflammatory cytokine production significantly reduced compared with those of histatin 3 ( Figures 2C, 2D, 6C, and 6D). Consequently, the strength of the association between various histatins and HSC70 may be related to the function of the complex.
In addition, it seems very likely that the primary structure of HSC70 is necessary for the function of HSC70 in TLR-mediated processes. Our findings showed that fulllength HSC70 and not the HSC70 ATPase fragment, can stimulate TLRs (Figures 1 and 4). In fact, a previous study reported that the substrate-binding domain of HSC70 is required to induce the myocardial inflammatory response [48]. In addition, conformation of HSC70 is also important for its correct functioning. Our findings show that a relatively protease-resistant conformation is formed upon histatin 3 binding to HSC70, but not in the presence of the control peptide, P3a (Figure 8), or DSG (data not shown). These results imply the possibility that there are some effects on conformation of HSC70. In fact, previous studies have reported that the peptide-binding domain of HSC70, as well as the ATPase domain of DnaK (the E. coli homolog of HSC70) is capable of undergoing conformational changes [49][50][51]. Thus, both the primary structure and other conformations of HSC70 may contribute to the activation of TLR signaling.
The innate host defense system recognizes foreign substances and tries to decrease their effects. One of the various host defense factors, pulmonary surfactant protein A downregulates the activation of TLR2 signaling by PGN [52]. An inhibitory peptide of TLR signaling, P13, inhibits both in vitro and in vivo LPS-induced inflammatory responses [53]. However, the precise mechanisms of this action have not been clarified. Histatin 3 is a peptide that binds directly to HSC70 and inhibits HSC70-induced TLR2 and TLR4 cell signaling (Figures 2, 6, and 7). Therefore, the results presented here provided the first evidence that histatin 3 is a salivary bioactive molecule. This molecule may prevent early-stage TLR signaling activation by interacting with TLR stimulators (ligands), such as HSC70, a putative ligand found in the oral cells.

Conclusion
In this study, we demonstrated the production of inflammatory cytokines in oral cells constitutively expressing HSP and the inhibition of this production by a salivary protein. Thus, this salivary protein has anti-inflammatory activity as well as already reported antimicrobial activity. It is possible that this salivary protein may resolve the HSPmediated inflammatory response in the oral cavity. The present findings further our understanding of the functions and mechanisms of actions of salivary proteins.