The transmission of signals from cell membrane into the nucleus requires coordinated action of diverse signaling proteins. In this study we identified the key signaling molecules involved in the induction of innate immunity in human oral keratinocytes in response to PAR1 and PAR2 activation. PAR1 and PAR2 have been demonstrated to activate members of the MAPK signaling cascade in the induction of IL-8 and IL-1b in epithelial cells from different tissue origin [21
]. In agreement with these reports, our findings indicated both p38 and ERK1/2 were phosphorylated by PAR1 and PAR2 activation. Our findings further reveal that the induction of additional innate immune markers, CXCL3, CXCL5 and CCL20, upon activation of PAR1 and PAR2 signals via p38 and ERK1/2. However, we observed divergent role for ERK1/2 and p38 MAPK in transducing signals for innate immunity by PAR1 and PAR2. PAR1 signals via both p38 and ERK1/2, whereas the induction of similar chemokines by PAR2 is primarily via p38. We also showed that PI3K activation had a negative regulatory role for both PAR1 and PAR2 signaling and thus may limit proinflammatory responses induced by proteases in the environment.
Our kinetics studies demonstrated transient ERK1/2 phosphorylation by PAR1 and PAR2 activation, which was followed by a distinct pattern of ERK1/2 dephosphorylation. This was more prominent with PAR2 activation compared to PAR1 activation. This may explain the minimum effect of inhibition of ERK1/2 for PAR2-mediated innate immune responses. On the other hand, PAR2 activation, compared to PAR1, resulted in more effective phosphorylation of p38. These data suggest that dephosphorylation of ERK1/2 following PAR2 activation may be a protective mechanism against excess innate immune responses via p38 and ERK1/2. A similar protective effect by down-regulation of MAPK signaling downstream of PAR2 activation is reported in acute pancreatitis induced by an intraperitoneal injection of caerulein in rats [25
]. However, the mechanism of ERK dephosphorylation by PAR2 activation is still unclear, and we are investigating whether PAR2 signaling mediates activation of phosphatases or if other mechanisms are involved.
Inhibition of p38 also differentially affected the expression of selected markers induced by PAR1 and PAR2 activation with different sensitivity to the presence of inhibitor for each marker. This may be related to the involvement of different p38 subunits (α,β,γ,δ) with differential downstream signaling and also to the lack of the equipotency of the current inhibitor against all subunits [26
Our studies suggest PI3K has an inhibitory effect on PAR signaling in HOKs. This effect was shown most clearly at the mRNA level and also for CXCL5 at protein level. We did not observe this effect in the secretion of CCL20, which may be related either to the peptide structure of CCL20 which is vulnerable to proteolytic activity of enzymes, or to involvement of other mechanisms that affect CCL20 expression at the post-transcriptional level. Little information is available about PAR-mediated PI3K signaling in normal human keratinocytes with comparable cellular function, but our results indicate HOKs have a unique signaling system. It has been shown that thrombin signals via PI3K to induce osteoprotegerin in human periodontal ligament and VEGF in human pigment retinal epithelial cells [23
]. In a recent study Minhajuddin et al
. showed that PI3K/Akt is involved in modulation of NF-κB and expression of ICAM-1 induced by thrombin in endothelial cells. Their study suggested that activation of PI3K/Akt leads to activation of mTOR. While the over-expression of the catalytic domain of Akt increases activation of NF-κB in the absence of mTOR activity, restoring mTOR signaling dampens activation of NF-κB and induction of ICAM-1 [28
]. In an earlier study by this group it was reported that thrombin-mediated ICAM-1 induction relies on parallel activation of PI3K and PKC that converges at Akt and induces activation of NF-κB [29
]. In contrast to their findings, our results suggest a direct inhibitory role for PI3K/Akt. These discrepancies may be explained by cell type-specific signaling mechanisms or different markers that have been investigated. Our finding in HOKs suggests PI3K acts as a compensatory mechanism which suppresses inflammatory responses. A similar inhibitory role for PI3K signaling in response to TLR2 and TLR5 activation has been reported in monocytes, dendritic cells and epithelial cells [30
], suggesting that PI3K may act as a balancing point to prevent excessive innate immune responses. It has been reported that PI3K knockout mice compared to their heterozygous littermates displayed increased levels of IL-6, IL-8 and nitrite in response to TLR5 activation [32
]. Results from our study suggest that inhibition of PI3K/Akt resulted in the up-regulation of innate immune markers CXCL3, CXCL5 and CCL20 via PAR activation in HOKs.
Our results suggest that the mechanism of crosstalk between PI3K and PAR signaling is via effect on phosphorylation of p38 and ERK1/2. We observed inhibition of PI3K resulted in increased p38 phosphorylation even in the absence of external stimulants (thrombin and tryspin), and this effect was significantly greater when cells were stimulated with active enzyme versus inactive form of thrombin and trypsin. This finding suggested a specific role of PAR activation in the induction of a crosstalk between PI3K and p38. This interaction between p38 and PI3K signaling pathways downstream of PARs activation may serve as a protective strategy HOKs utilize to keep innate immune responses in balance. Activation of PI3K inhibits the induction of proinflammatory chemokines possibly by suppression of p38 MAPK activation. When TLR5 is activated by flagellin in intestinal cells [32
] and in VEGF-induced tissue factor in endothelial cells [33
], suppressive effect of PI3K has been observed. Although we expected to see a similar relationship between ERK1/2 and PI3K activation, our studies showed blocking PI3K limited ERK1/2 activity and suggest that PI3K and ERK signaling pathways are acting in series. Other studies showed that inhibition of PI3K induced phosphorylation of ERK1/2 in intestinal epithelial cells stimulated with flagellin [32
] and in hepatic stellate cells activated with platelet-derived growth factor [34
]. These studies may reflect that the interaction between PI3K and ERK signaling varies depending on the stimulus and cell type.
Our studies suggested that PAR1 signals via both ERK1/2 and p38, but that ERK1/2 has a more prominent role. However, there was no role for either p38 or ERK1/2 in the induction of CCL20 by PAR1 activation, although its expression was increased when PI3K and Akt were inhibited. It is likely that an alternative mechanism, which is independent of the ERK1/2 and p38 pathways, but still blocked by PI3K, is involved in the induction of CCL20 by PAR1 activation. This is consistent with our previous study showing that CCL20 induction by thrombin may occur via a mechanism other than PAR1 [12
Induction of cytokines and chemokines by PAR activation leads to infiltration of mononuclear cells in the microenvironment of periodontal tissue [5
]. This process is part of the initial recognition of danger in the environment and serves as an important protective function. While this primary immune response can protect the body against pathogenic factors, over-activity of these responses can become destructive and lead to progressive diseases. In periodontal diseases, exaggerated immune responses lead to excess inflammation, thus it is potentially important that oral keratinocytes keep immune responses in balance by shutting down the expression of proinflammatory genes. It is likely that crosstalk between p38-MAPK and PI3K/Akt signaling pathways plays a role in this process. Downstream of PAR activation, PI3K has a suppressive effect on the regulation of chemokines, thus may act to minimize the potential negative consequences of over-activity of inflammatory responses. However, bacterial pathogens with ability to activate PAR could take advantage of this mechanism in gingival epithelium and dampen innate immune responses to increase the survival of pathogens, which will result in sustained infection. Thus, it is necessary to consider both sides of the role of PI3K/Akt in evaluating possible therapeutic targets. Furthermore, understanding the molecular events associated with PAR signaling in keratinocytes may open new possibilities of intervention for mucosal inflammation such as periodontal diseases.