It has been reported that extracellular HSP70 induces inflammatory cytokine production through TLR2 and TLR4 pathway in human monocytes . 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 . Our results showed that histatin 3 significantly inhibits HSC70-stimulated 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 . 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 . 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 . 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 full-length 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 . 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 [49–51]. 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 . An inhibitory peptide of TLR signaling, P13, inhibits both in vitro and in vivo LPS-induced inflammatory responses . 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.