Polymers of bacterial flagellin are organized into flagella, organelles whose whip-like action enhances bacterial motility. As part of their life cycle, bacteria degrade and renew flagellin; in doing so, they release flagellin near host cell membranes. In these studies, we have established a functional link between two previously described flagellin receptors, ASGM1 and TLR5. Having previously identified the distal effector mechanisms of ASGM1 (14
), we show here that they are Toll dependent. A surprising implication of this is that TLRs represent upstream effectors of a nucleotide receptor signaling network, specifically, one lying upstream of MAP kinase Erk1/2 activation.
Erk is a member of the mitogen-activated protein kinase (MAPK) family consisting of Erk, Jnk, and p38. Erk 1 and 2 are isoenzymes with molecular weights of 42 and 44 kD, respectively (25
). Upon phosphorylation, Erk translocates to the nucleus of the stimulated cell, where it can phosphorylate nuclear substrates, thereby activating them as transcription factors (e.g., c-fos
, Elk-1, Ets, RSK) (26
). Erk's ability to accumulate in the nucleus depends on its phosphorylation state (30
). The classical upstream effectors of Erk1/2 are Ras, Raf, and Mek. The mechanism by which Toll receptors communicate with Erk is unclear (7
). Tolls generally communicate with distal effector molecules through a pathway involving IRAK and TRAF6. Interestingly, TRAF6-induced activation of Erk (but not NF-κB) has been found to be Ras independent (31
), thereby showing that Toll receptors activate NF-κB and Erk via somewhat different mechanisms, consistent with our data in and . Moreover, recent work indicates that flagellin-induced IL-8 and MIP3α gene expression occurs via a TRAF6-mediated MEK-dependent (but NF-κB–independent) pathway (7
). Interestingly, this pathway appears to be independent of the evolutionarily conserved signaling intermediate in Toll pathways (ECSIT), which interacts with MEKK1 and thereby links TRAF6 to Erk1/2 signaling (7
). In sum, the available data are controversial and do not specify the mechanism by which Toll receptors activate Erk.
The Toll–Erk signaling pathway is of great significance to innate immune responses involving cytokine induction (7
). Cytokines are needed for the recruitment of inflammatory cells to infection sites and also to facilitate cell–cell communication at these sites, thereby optimizing pathogen clearance. It has been suggested that the induction of cytokines IL-8 and MIP3α by flagellin relies on an NF-κB–independent signaling pathway that requires Erk1/2 (7
). Our data indicate that whatever the precise mechanism mediating Erk activation by flagellin, it is dependent on both Toll receptors and extracellular ATP ().
ATP is released from cells as a result of multiple stressors, including: ischemia, injury, and inflammation. The mechanism of ATP release from intact cells has received considerable attention but remains poorly understood (reviewed in Refs. 33
). Upon release, extracellular ATP has been shown to act through specific cell surface receptors to regulate a variety of responses, including: platelet aggregation, smooth muscle contractility, neurotransmitter function, induction of cell death, mucociliary clearance, and mitogenic stimulation (reviewed in Refs. 35
). These effects are mediated via two different subfamilies of extracellular nucleotide-binding P2 receptors, P2X and P2Y. P2Y receptors are metabotropic receptors coupled to G proteins and P2X receptors are ligand-gated ion channels. Both our previous work and the current study suggest an important role for G protein–coupled P2Y receptor activation in flagellin-induced Erk1/2 phosphorylation (14
). It is important to consider, however, that P2X receptors may be equally involved along with P2Y receptors in this response. In both salivary acinar cells and astrocytes, Erk1/2 is activated via a PKC-dependent signaling pathway initiated by P2X7 (38
). Human T cells and mast cells also exhibit ATP-induced activation of Erk1/2 that is mediated through P2X7 (40
), whereas P2X4 invokes neuropathic pain via MAPK-induced production of IL-1β, IL-6, and TNF-α (42
). It is worth noting that ATP-initiated P2X/MAPK signaling in T cells leads to the activation of transcription factor AP-1, while NF-κB activation is reduced. This agrees with our current observation that flagellin-mediated Erk1/2 phosphorylation occurs via an ATP-dependent pathway that does not involve NF-κB. Moreover, because P2X/Erk activation of AP-1 is fully dependent on the influx of extracellular Ca2+
, it suggests that the release of calcium from both intracellular stores (mediated by P2Y receptors) and extracellular stores (mediated by P2X receptors) may be involved in the TLR signaling pathway activated by flagellin. Further studies will be necessary to define the ATP release mechanisms initiated by these receptors, as well as the specific molecular sequence of events that mediates MAPK activation in response to bacterial pathogens.
Earlier work clearly demonstrated that TLR5 recognizes a conserved site on flagellin required for protofilament formation and motility (18
). Interestingly, efforts to define the affinity constant for flagellin binding to TLR5 have been hampered by interactions of flagellin with lipid bilayers (19
). Precedent for a membrane lipid coreceptor can be found for LPS signaling via TLR4 and the GPI-linked membrane protein CD14, which functions to concentrate LPS at the host cell surface (43
). Gangliosides have also been shown to act as coreceptors with TLR5 for flagellin in the induction of hBD-2 (44
). This work suggests that the interaction of gangliosides such as GD1a, GD1b, and GT1b with flagellin and/or TLR5 promotes hBD-2 expression via an MAPK signaling pathway that involves AP-1. Moreover, both ganglioside and TLR5 expression are necessary for full hBD-2 upregulation in response to flagellin (44
). Thus, bacterial ligand may be captured by high-affinity interactions with lipid-anchored receptors, but signal transduction requires their interaction with Toll-like receptors. In our own studies, TLR5-deficient CHO cells overexpressing ASGM1 showed little if any NF-κB activation in response to flagellin (data not shown). This supports the notion that signaling downstream of ganglioside receptors is Toll dependent, and is consistent with a recent study showing that the inhibitory effect of gangliosides on flagellin signaling in macrophage-like cells occurs in the absence of an effect on flagellin binding to TLR5 (45
An important implication of Toll-dependent signaling downstream of ASGM1 is that TLRs represent upstream effectors of a nucleotide receptor signaling network. Direct examination of the relationship between TLRs and P2Y receptors has been complicated by difficulties in identifying the specific nucleotide receptor involved in flagellin signaling. Rank order efficiency testing in lung epithelial cells has not been consistent with the profile of any currently recognized nucleotide receptors, and a yet-unidentified P2Y receptor that mediates flagellin signaling cannot be ruled out. This possibility is the subject of further investigation as is the potential involvement of P2X receptors in TLR/ATP signaling.
In summary, our experiments show that host responses triggered at the flagellin receptor ASGM1 are dependent on the intact functioning of Toll-like receptors, specifically TLR5. The point of intersection of the two receptor pathways is at or above the level of ATP release (14
). This further reveals for the first time that certain aspects of TLR signaling are dependent on an autocrine ATP receptor loop in epithelial cells.