TRPV4-mediated calcium influx into human bronchial epithelia upon exposure to diesel exhaust particles.
OBJECTIVE: We investigated a link between chronic obstructive pulmonary disease (COPD) pathogenesis and molecular mechanisms underlying [Ca.sup.2+] influx into human airway epithelia elicited by diesel exhaust particles (DEP).
METHODS AND RESULTS: Using primary cultures of human respiratory epithelial (HRE) cells, we determined that these cells possess proteolytic signaling machinery, whereby proteinase-activated receptor-2 (PAR-2) activates [Ca.sup.2+]-permeable TRPV4, which leads to activation of human respiratory disease--enhancing matrix metalloproteinase-1 (MMP-1), a signaling cascade initiated by diesel exhaust particles (DEP), a globally relevant air pollutant. Moreover, we observed ciliary expression of PAR-2, TRPV4, and phospholipase-C[beta]3 in human airway epithelia and their DEP-enhanced protein-protein complex formation. We also found that the chronic obstructive pulmonary disease (COPD)--predisposing [TRPV4.sub.P19S] variant enhances [Ca.sup.2+] influx and MMP 1 activation, providing mechanistic linkage between man-made air pollution and human airway disease.
CONCLUSION: DEP evoked protracted [Ca.sup.2+] influx via TRPV4, enhanced by the COPD-predisposing human genetic polymorphism [TRPV4.sub.P19S]. This mechanism reprograms maladaptive inflammatory and extracellular-matrix-remodeling responses in human airways. The novel concept of air pollution--responsive ciliary signal transduction from PAR-2 to TRPV4 in human respiratory epithelia will accelerate rationally targeted therapies, possibly via the inhalatory route.
KEY WORDS: air pollution, COPD, DEP, human bronchial epithelia, MMP-1, PAR-2, PI3-kinase, PLCp3, TRPV4, [TRPV4.sub.P19S]. Environ Health Perspect 119:784-793 (2011). doi:10.1.289/ehp.1002807 [Online 18 January 2011]
Human airway epithelia function in barrier formation, defense against pathogens, and mucociliary clearance (Hogg 2004). They represent the first barrier against airborne environmental pollutants, and they coordinate recruitment of pivotal inflammatory cells in several pathologies, including chronic obstructive pulmonary disease (COPD) (Li et al. 2009; Mercer et al. 2006; Yang et al. 2005). The inhalation of diesel exhaust particles (DEP), produced by vehicular traffic contributing to urban smog, leads to serious respiratory diseases (e.g., COPD, emphysema, bronchial cancer, chronic asthma) (Tortes-Duque et al. 2008). The particles' carbonaceous cores are coated with thousands of organics and heavy metals. Because large numbers of hazardous chemicals are present on DEP, its pathological effects on human airways are pleiotropic. We and others have found that DEP evokes the secretion of matrix metalloproteinase-1 (MMP-1) from human bronchial epithelia (Amara et al. 2007; Li et al. 2009). Matrix metalloptoteinase-1 (MMP-1) plays a role in tissue remodeling during development, inflammation, migration of inflammatory and malignant cells, and COPD and emphysema pathogenesis (Segura-Valdez et al. 2000). It also has neurotropic effects, possibly enhancing sensitization of airway-innervating sensory neurons, contributing to airway hvpersensitization and chronic cough (Conant et al. 2004). We recently identified a novel pathway that results in DEP-induced MMP-1 activation and entails activation of RAS-RAF-MEK-exttacellular signal-regulated kinase (ERK) signaling, dependent on [beta]-arrestins (Li et al. 2009). From a global health perspective, one important finding was that the human MMP-1 polymorphism at position -1607(lG/2G) of the MMP-1 promoter yielded, after DEP exposure, either a diminutive (1G) or large (2G) response. The 2G polymorphism is found in 75% of humans.
Against this background, we sought to identify critical elements upstream of RAS in human airways in response to DEP. The pathogenic component of DEP that activates MMP-1 is primarily retained in its organic extract (OE), such that DEP carbonaceous core particles shuttle water-insoluble OE to the ciliary plasma membrane. The DEP/OE initially activates proteinase-activated receptor 2 (PAR-2), which, via [G.sub.[i/o]] G-protein, phospholipase-C[beta]3 (PLC[beta]3), and phosphatidyl inositol 3 kinase (PI3-K), activates [Ca.sup.2+]-permeable TRPV4 (transient receptor potential vanilloid, family member 4) ion channels (Liedtke et al. 2000; Lorenzo et al. 2008; Sidhaye et al. 2008; Strotmann et al. 2000). A uniquely protracted [Ca.sup.2+] influx through TRPV4 follows, which is critical for mitogen-activated protein kinase (MAPK)--mediated MMP-1 activation. Localization studies show that PAR-2, PLCp3, and TRPV4 colocalize to cilia of human differentiated airway epithelia. DEP exposure greatly enhances protein-protein complex formation between these signaling molecules and calmodulin. Importantly, we observed that [TRPV4.sub.P19S], a human genetic polymorphism previously identified as a COPD susceptibility locus (Zhu et al. 2009), increases MMP-1 activation via increased [Ca.sup.2+] influx, providing a mechanistic link between human airway epithelia signaling, airway disease, and air pollution.
Materials and Methods
DEP. Particles were generated at the U.S. Environmental Protection Agency (EPA; Research Triangle Park, NC) from a Deutz four-cylinder diesel engine, running at three defined engine loads before collection, as described previously (Li et al. 2009). For experiments, we used DEP at 100 [micro]g/ mL. DEP organic extract (OE) was prepared by washing organic chemicals off of DEP using methylene chloride, followed by solvent exchange with dimethyl sulfoxide (DMSO). In experiments, we used 20 [micro]g/mL OE, which is equivalent to the organic compounds contained in 100 [micro]g DEP. We used Degussa Printex 90 carbon nanospheres (P90; provided by W. Moeller, GSF, Munich, Germany) as controls.
Chemicals. We used the following compounds: pertussis toxin ([G.sub.[i/o]] inhibitor; Sigma Chemical Co., St. Louis, MO), U73122 (PLC inhibitor; Tocris, Ellisville, MO), LY294002 and PI828 (PI3-K inhibitors; Tocris), 4[alpha]-phorbol 12,13 didecanoate (4[alpha]-PDD; TRPV4 activator; Tocris), GSK205 [TRPV4 blocker (Phan et al. 2009)], ruthenium red [TRP(V) blocker; Tocris], gadolinium (III) chloride [[GdCl.sub.3] (Sigma); inhibitor of store-operated calcium entry (SOCE) at 5 [micro]M (Bird et al. 2008)], thapsigargin ([Ca.sup.2+]-store depletion; Tocris), GM1489 and Z-PDLDA-NHOH (pan-MMP inhibitors; Endogen, Rockford, IL), and W-7 (calmodulin blocker at 10 [micro]M; Sigma).
Cell culture. BEAS-2B cells were obtained from ATCC (Rockville, MD), maintained as previously described (Li et al. 2009), and used for stimulation with DEP or OE and for all DNA and small interfering RNA (siRNA) transfection experiments. Primary human bronchial epithelial (HBE) cells were tracheobronchial cells derived from healthy, nonsmoking adult volunteers. We obtained institutional review board approval for this study from the participating institutions, and volunteer donors provided informed consent for use of the cells in research. Additional details on cell culture are available in Supplemental Material (doi:10.1289/ehp.1002807).
MMP-1 reporter gene assays were conducted as described previously (Li et al. 2009). A set of MMP-1 promoters of different lengths was available for both human polymorphisms, -l607G and-1607GG.
siRNA was transfected into BEAS-2B cells following previously published methods (Li et al. 2009). siRNA was directed against PAR-2, PAR-1, [beta]-arrestins 1 and 2, and TRPV4. Scrambled controls were used as provided by the manufacturer (Dharmacon, Lafayette, CO). siRNA efficiency was confirmed by quantitative reverse-transcriptase polymerase chain reaction (PCR) and Western blotting.
TRPV3 and TRPV4 dominant-negative (DN) isoforms were generated by isolating a truncated form of each channel, from 10 amino-acids N-terminal to the fifth transmembrane domain to 10 amino-acids C-terminal to the sixth ttansmembrane domain. In addition, two point mutations were generated as M680K and D682K for TRPV4 and as L619K and D621K for TRPV3 in order to render the channel fragments [Ca.sup.2+] impermeable. Both of these constructs were C-terminally fused to monomeric red fluorescent protein (RFP).
Dominant-negative isoforms of STIM1 and ORAI1, -2 and -3 were provided by L. Birnbaumer and S. Muallem. These cDNAs were driven by CMV promoters in eukaryotic expression plasmids, the coding region fused to eGFP. DN-STIM1 and DN-ORAI1-3 have been shown to specifically interfere with function of their cognate wildtype isoforms and inhibit them. Enzyme-linked immunosorbent assays (ELISAs) for MMP-1, RANTES (regulated on activation, normal T-expressed and secreted), and IP-10 (interferon-[gamma]-induced protein 10 kDa, CXCL10) were conducted using commercially available kits. For MMP-1 secretion, we previously demonstrated its correlation to the specific proteolytic activity of MMP-1 (Li et al. 2009).
[Ca.sup.2+] imaging of BEAS-2B cells was conducted using 2 [micro]M fura-2 acetoxymethyl ester for loading and following a protocol for ratio-metric [Ca.sup.2+] imaging using 340/380 nm blue light for dual excitation, recording emissions with specific filter sets. Ratios of the emissions were acquired every 5 sec. [DELTA]R/[R.sub.0] is the fraction of the increase of a given ratio over the baseline ratio, divided by baseline ratio. For stimulation of cells with DEP, we used particles at 100 [micro]g/mL and analyzed only cells with microscopically verified contact with particles. For stimulation with OE, all cells were analyzed. To stimulate TRPV4, hvpotonicity was used at 260 mOsmol/L, and 4[alpha]-PDD at 10 [micro]M; [Ca.sup.2+] stimulation was accomplished by switching from 0 to 2 mM. [Ca.sup.2+] imaging of primary HBE cells was conducted by excising the air-liquid interface matrix with a scalpel and affixing it to the opening of a glass-bottom dish, with other procedures as for BEAS-2B cells.
Electrophysiological recordings. Extracellular [Ca.sup.2+] was precipitated by addition of EGTA. We conducted PI3-K Forster resonance energy transfer (FRET) imaging based on FRET of membrane-targeted enhanced green fluorescent protein (eGFP; donor) and the pleckstrin homology domain of Bruton's tyrosine kinase, fused to mCherry fluorescent protein (acceptor). With low phosphatidylinositol (3,4,5)-trisphos-phate (PIP3) levels, the mCherry is cytoplasmic; with increased PIP3 levels, it translocates to the membrane leading to FRET, which we quantified using two-photon fluorescence lifetime imaging. Additional details for electrophysiological recordings are given in Supplemental Material (doi: 10.1289/ehp.1002807).
The phosphorylated ERK (phospho-ERK) trafficking assay was performed as described previously (Li et al. 2009). Briefly, BEAS-2B cells were stimulated either with DEP or with OE and fixed (4% paraformaldehyde) at 5-, 10-, 20-, and 30-min time points. Phospho-[ERK.sub.1/2] was verified by fluorescent immunodetection and quantified densitometrically ([greater than or equal to] 75 cells/time point), corrected for background, in the nuclear area using Image] software (version 1.42q; Rasband 2009).
Confocal imaging was conducted after immunocytochemical staining for acetylated [alpha]-tubulin, PLC[beta]3, TRPV4, and PAR-2. Fluorescently labeled sections were visualized using a Zeiss LSM710 confocal imaging suite with lasers tuned to the emission spectra of the secondary fluorescent antibodies.
Coimmunoprecipitation studies were conducted using [10.sup.6] BEAS-2B cells per experiment; cells were harvested in lysis buffer (1% NP40 detergent). Exactly 100 [micro]g protein was incubated with rabbit anti-PLC-[beta]3, rabbit anti-TRPV4, or mouse monoclonal anti-PAR-2 overnight at 4[degrees]C, and then solutions were exposed to 15 [micro]L protein-A/G-Sepharose for 4 hr (4[degrees]C). After stringency washing, complexes were investigated by Western blotting using antibodies specific for TRPV4, PAR-2, or calmodulin. Normal rabbit or mouse isotype antibodies were used as controls. Western blotting was performed following standard methodology with chemoluminescence detection.
Statistical analysis. We compared mean and SE of quantified outcome parameters after stimulation with their respective controls. Group comparisons were performed using Student's t-test or analysis of variance with post hoc Scheffe test for multigroup comparison, applying the statistics program StatPlus:mac (AnalystSoft, Vancouver, British Columbia, Canada). Minimum significance was set at p < 0.05.
The OE of DEP contains the active component to activate MMP-1. To understand which component(s) of DEP activate MMP-1, we investigated effects of DEP and its OE in human BEAS-2B and primary HBE cells, the latter exposed at air-liquid interface [see Supplemental Material, Figure 1 (doi:10.1289/ehp.1002807)]- Our findings suggest that the carbonaceous core of DEP, by size a carbon nanoparticle, acts as a vehicle carrier for delivery of the highly active, water-insoluble organic fraction to the plasma membrane of human airway epithelia to elicit MMP-1 activation.
Extracellular [Ca.sup.2+] influx is necessary for activation of MMP-1. Previous studies in lung cells and neurons have shown that particulate matter evokes [Ca.sup.2+] transients (Agopyan et al. 2004); other studies have shown that [Ca.sup.2+] increases activated RAS (Lee and Yasuda 2009) and that DEP activates RAS (Li et al. 2009). Therefore, we examined whether DEP and/or OE causes [Ca.sup.2+] influx and whether this can activate MMP-1.
We found that DEP and OE evoke extracellular [Ca.sup.2+] influx (Figure 1A-D), as indicated by curtailing of the response by addition of EGTA (Figure 1C,D). P90 control carbon nanoparticles had no effect on [Ca.sup.2+] (Figure 1B), whereas DEP activated a uniquely protracted and monotonically increasing response with a peak at approximately 60 min that gradually declined (data not shown). In comparison, the response to OE increased more rapidly, reaching a maximum at approximately 20 min and decreasing to baseline in the next 10 min (Figure 1B), indicating that the particle core retarded [Ca.sup.2+] influx by slowing delivery of the organic fraction to the plasma membrane.
To determine whether DEP-induced [Ca.sup.2+] influx was necessary for transcriptional activation of MMP-1, we exposed cells to DEP in the presence and absence of extracellular [Ca.sup.2+] and measured MMP-1 transcriptional activation at 2 and 24 hr and the appearance of nuclear phospho-ERK at 30 min (Figure 1E--I). These experiments indicated that extracellular [Ca.sup.2+] was necessary for both nuclear translocation of phospho-ERK and MMP-1 activation in response to DEP or OE. In primary HBE cells, EGTA eliminated MMP-1 secretion in response to DEP or OE (Figure 1H), thus confirming the validity of this mechanism.
We were unable to demonstrate functionality of SOCE in DEP/OE--evoked [Ca.sup.2+] influx [see Supplemental Material, Figure 2A-C (doi: 10.1289/ehp.l002807)]. Preincubation with 5 [micro]M thapsigargin or 5 [micro]M [GdCl.sub.3] (Bird et al. 2008) did not markedly change DEP-evoked [Ca.sup.2+] increase. Furthermore, cotransfecrion of STIM1-DN and ORAI1-3-DN, both known to function in SOCE, did not significantly alter [Ca.sup.2+] responses evoked by DEP or OE.
Finally, we found that DEP and OE also caused the [Ca.sup.2+]-dependent secretion of proinflammatory mediators RANTES (Jeffery 2004) and IP-10 (Torvinen et al. 2007) [see Supplemental Material, Figure 2D,E (doi:10.1289/ehp.1002807)]. Thus, in human lung cells, [Ca.sup.2+] influx is necessary for DEP/OE-evoked activation of MMP-1, IP-10, and RANTES.
PAR-2 is a DEP-sensitive G-protein-coupled receptor (GPCR) that activates [G.sub.[i/o]], PLC[beta]3, and PI3-K. We focused on PAR-2 because an earlier low-throughput proteomics screen revealed that, compared with DEP alone, DEP plus PAR-2-acrivating peptide (PAR-2-AP) increased MMP-1 secretion (data not shown). We first conducted experiments in BEAS-2B cells by siRNA-mediated knockdown of PAR-2. PAR-2 mRNA was significantly reduced by PAR-2 siRNA bur not by the scrambled control [see Supplemental Material, Figure 3A (doi:10.1289/ehp.1002807)]. Cells treated with PAR-2 siRNA exhibited significantly reduced [Ca.sup.2+] influx, MMP-1 reporter gene activation, and MMP-1 secretion (Figure 2A; see also Supplemental Material, Figure 3B,C). This demonstrates that PAR-2 functions upstream of [Ca.sup.2+]-mediated MMP-1 activation. In addition to scrambled siRNA controls, PAR-1--specific siRNA had no effect on MMP-1 activation (data not shown).
Costimulation of BEAS-2B and primary HBE cells with DEP or OE and PAR-2-AP potentiated MMP-1 activation [Figure 2B; see also Supplemental Material, Figure 3D,E (doi:10.1289/ehp.1002807)]. To boost its moderate expression level in BEAS-2B cells, we overexpressed PAR-2. This led to increased baseline and DEP-evoked MMP-1 activation, indicating that PAR-2 overexpression is sufficient to increase MMP-1 expression and to render the cell more responsive to DEP (see Supplemental Material, Figure 3F). Thus, specific activation and inhibition of PAR-2 imply that this receptor is critical in DEP-mediated [Ca.sup.2+] influx that leads to MMP-1 activation.
We next investigated whether secreted MMP-1 activates PAR-2 proteolytically, as
it docs for PAR-1 (Boire et al. 2005), which might explain the protracted [Ca.sup.2+] influx in response to DEP. This was not the case, because MMP inhibitors accelerated MMP-1 reporter gene activity in response to DEP [see Supplemental Material, Figure 3G (doi:10.1289/ehp.l002807)].
We addressed whether [beta]-arrestins are necessary for PAR-2-mediated [Ca.sup.2+] influx in response to DEP or OE (Cottrell et al. 2003). This was not the case in view of [Ca.sup.2+] increase in the absence of [beta]-arrestins 1 and 2 [siRNA-mediated knockdown; sec Supplemental Material, Figure 3H (doi: 10.1289/ehp.1002807)]. We previously verified elimination of MMP-1 activation by siRNA-mediated [beta]-arrestin knockdown (Li et al. 2009). Together, these results implicate [beta]-arrestins as MAPK scaffolds necessary for the DEP-MMP-1 response yet dispensable for PAR-2-mediated [Ca.sup.2+] influx in response to DEP or OE.
We examined [G.sub.[i/o]] signaling because of PAR-2's known signal transduction mechanisms via this G-protein (Olianas et al. 2007). We found that the DEP-MMP-1 response, namely, [Ca.sup.2+] influx, MMP-1 transcription, and MMP-1 secretion, depends on [G.sub.[i/o]], which we targeted specifically with pertussis toxin in both BEAS-2B and primary HBE cells [Figure 2A,B; see also Supplemental Material, Figure 4A (doi: 10.1289/ehp.1002807)]. Because [G.sub.[i/o]] is known to activate PLC (Exton 1996), we next treated cells, with PLC-selective inhibitor, U73122, which led to a marked DEP-MMP-1 response (Figure 2A,B; see also Supplemental Material, Figure 4B). PLC has several isoforms; we Investigated the [beta]-isoforms because of PLC[beta]'s link to GPCRs, specifically PLC[beta]3, in view of its previously established link to [G.sub.[i/o]] (Senyshyn et al. 1998). When we immuno-labeled for PLC[beta]1-4, we found the most robust expression for PLCp3 in primary HBE cells (data not shown). Interestingly, using a phospho-specific antibody against PLC[beta]3, we documented phospho-PLC[beta]3 up-regulation within 30 min after DEP application (Figure 2C). This finding can help explain the protracted [Ca.sup.2+] influx because PLC[beta]3, being upstream of extracellular [Ca.sup.2+] influx, was previously demonstrated to be attenuated by phosphorylation (Yue et al. 2000).
Another phospholipid-metabolizing enzyme that signals downstream of [G.sub.[i/o]] and upstream of TRP channel [Ca.sup.2+] conductances is PI3-K (Zhuang et al. 2004). We identified its critical role in response to DEP or OE using the PI3-K inhibitor LY294002 by documenting significant reduction of [Ca.sup.2+] influx and subsequent MMP-1 activation in both BEAS-2B and primary HBE cells [Figure 2D; see also Supplemental Material, Figure 4C (doi:10.1289/ehp.l002807)], suggesting the signaling position of PI3-K upstream of [Ca.sup.2+] influx. Moreover, using a novel FRET-based assay, we could visualize the enzymatic activity of PI3-K (change in PIP3) in BEAS-2B cells in response to DEP or OE, which indicated PI3-K activity as an early signaling event (Figure 2D). Furthermore, in addition to time-scale resolution after DEP or OE exposure, this method illustrates the confinement of PI3-K signaling to the plasma membrane.
TRPV4 forms a DEP-sensitive [Ca.sup.2+] pathway downstream of PI3-K/PLC-[beta]3. PAR-2 has been shown to sensitize TRP channels, including TRPV1, TRPV4, and TRPA1 (Amadesi et al. 2006; Gatti et al. 2006; Grant et al. 2007). Because TRPV4 is expressed in tracheobronchial epithelia (Lorenzo et al. 2008), we addressed whether it functions downstream of the above signaling cascade, initially by inhibiting its function in BEAS-2B cells expressing TRPV4-DN [see Supplemental Material, Figure 5A-C (doi:10.1289/ehp.1002807)], which produced strong reduction of [Ca.sup.2+] influx in response to DEP yet no reduction for TRPV3-DN (Figure 3A). We also knocked down TRPV4 using specific siRNA, which effectively down-regulated TRPV4 mRNA and protein (see Supplemental Material, Figure 5B,C). Compared with the scrambled control, the siRNA-TRPV4 knockdown reduced [Ca.sup.2+] influx in response to DEP or OE (Figure 3B). Thus, TRPV4 is necessary for DEP-evoked [Ca.sup.2+] influx.
Next we addressed whether DEP-evoked, TRPV4-mediated [Ca.sup.2+] influx activates MMP-1. Using MMP-1 reporter assays, we found that TRPV4-specific siRNA decreased MMP-1 transcriptional activation, thus implying that TRPV4 is critical for DEP/ OE-evoked [Ca.sup.2+] influx, which then activates MMP-1 [see Supplemental Material, Figure 5D (doi:10.1289/ehp.1002807)1. Based on these results, we used the MMP-1 reporter platform to determine that TRPV4 functions downstream of PAR-2 because siRNA-mediated TRPV4 knockdown virtually eliminated potentiated MMP-1 activation by DEP or OE plus PAR-2-AP (see Supplemental Material, Figure 5E). This effect of TRPV4-specific siRNA was recapitulated for DEP-evoked MMP-1 secretion in BEAS-2B cells (Figure 3C). Finally, we found that ruthenium red, an unspecific TRP(V) blocker, decreased MMP-1 activation (see Supplemental Material, Figure 5F).
TRPV4 activation by 4[alpha]-PDD or hypotonicity strongly increased MMP-1 secretion, indicating that in airway epithelia, TRPV4 activation is sufficient to up-regulate MMP-1 (Figure 3D). Furthermore, TRPV4 transfection in BEAS-2B cells increased MMP-1 reporter activation. Because these findings were obtained in the BEAS-2B cell line, we also tested TRPV4 function in primary HBE cells. First, we were able to significantly attenuate the DEP-evoked [Ca.sup.2+] response by GSK205, a specific small-molecule TRPV4 inhibitor (Phan et al. 2009), in a dose-dependent manner (Figure 3E). In addition, secreted MMP-1 in response to DEP was significantly reduced by two concentrations of GSK205 (Figure 3F). Thus, the cornerstones of TRPV4's involvement in the DEP-MMP-1 response, namely, dependence of the [Ca.sup.2+] response and MMP-1 secretion on TRPV4, were recapitulated in primary HBE cells. Whenever possible, we performed the same experiment in human primary HBE cells as in permanent human BEAS-2B cells.
Taken together, these findings point toward critical functioning of TRPV4 in [Ca.sup.2+] influx into human airway epithelia evoked by DEP, a globally relevant air pollutant.
TRPV4 signaling complex is located on motile cilia of primary human airway epithelia.
Because TRPV4 channels have been found in primary motile cilia of mouse tracheal epithelia (Lorenzo et al. 2008), we determined TRPV4's subcellular location in human ciliated airway epithelia. Primary HBE cells were differentiated in culture until they became ciliated. They showed ciliary location of TRPV4, PAR-2, and PLC[beta]3 (Figure 4). Thus, critical DEP-responsive membrane-bound components all localize to motile cilia of primary human HBE cells.
DEP-facilitated recruitment to a membrane-associated receptor-signa . multicomplex. Given the ciliary localization of the DEP-evoked transduction cascade, we asked whether these membrane-associated signaling molecules coaggregate in response to DEP or OE (Figure 5). In aggregate, DEP-responsive membrane-bound signaling is characterized by a nonincremental interaction between PLC[beta]3 and TRPV4, with subsequent recruitment of PAR-2 and calmodulin caused by DEP exposure. Calmodulin's response to DEP is inhibitory, because specific inhibition of calmodulin with W-7 increased [Ca.sup.2+] signaling and MMP-1 activation (Figure 5B,C). This suggests as explanatory mechanism(s) an increase in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) via disinhibited TRPV4 and/or activation of PLC[beta]3, both of which have previously been shown to bind calmodulin.
COPD-associated [TRPV4.sub.P19S] increases MMP-1 activation in response to DEP or OE. For human TRPV4, a number of genetic polymorphisms enhance susceptibility for COPD; one of them, P19S, is located in the coding region (Zhu et al. 2009). In another study, [TRPV4.sub.P19S] was reported as a DN channel in transfected HEK cells in response to weak but not strong hypotonicity (Tian et al. 2009). Because our finding that DEP-evoked [Ca.sup.2+] influx via TRPV4 causes MMP-1 activation rationalizes airway injury by MMP-1 as caused by TRPV4 channel activity, not by DN channels, we attempted to resolve these seemingly contradictory concepts.
Compared with wild-type TRPV4 ([TRPV4.sub.wt]), [TRPV4.sub.P19S] exhibited gain-of-function effects in [Ca.sup.2+] influx, patch clamp, MMP-1 reporter gene activation, and MMP-1 secretion [Figure 6; see also Supplemental Material, Figure 6 (doi:10.1289/ehp.1002807)]. For MMP-1 transcriptional activation, [TRPV4.sub.P19S] gain-of-function effects were strictly dependent on [Ca.sup.2+] influx. This was evidenced by inhibitory effects of [TRPV4.sub.[P19S/M680K]], where the selectivity-filter--blocking M680K mutation causes [Ca.sup.2+] impermeability, leading to elimination of gain of function (Figure 6D). Furthermore, [Ca.sup.2+] influx in response to changes in Ca concentration and to DEP or OE were significantly increased in [TRPV4.sub.P19S] versus [TRPV4.sub.wt], as was nonstimulated [[[Ca.sup.2+]].sub.i]- [extracellular [Ca.sup.2+] concentration, 2 mM) (Figure 6A-C; see also Supplemental Material, Figure 6). Thus, in a human airway epithelium-derived cell line with robust similarity to primary HBE cells, [TRPV4.sub.P19S] functions as [Ca.sup.2+]-permeable gain-of-function channel to hyperactivate the pathogenic mediator gene MMP-1 in response to the common air pollutant DEP.
We identified a novel DEP-activated signaling pathway in human airway epithelia that consists of a GPCR (PAR-2) signaling to a TRP channel (TRPV4). Specific activation of PAR-2 by OE leads to [Ca.sup.2+] influx mediated by TRPV4 channels, via membrane phospholipid signaling involving PLC[beta]3 and PI3-K. This pathway can be used to develop effective medical therapy for human airway injury caused by airborne particulate pollution, a well-recognized global health problem chat contributes to development of COPD and other respiratory illnesses. Regarding COPD, we identified a molecular, cellular, and subcellular mechanism for how the COPD-predisposing nonsynonymous genetic polymorphism [TRPV4.P19S] may potentiate DEP-evoked [Ca.sup.2+] influx and activation of airway-pathogenic MMP-1. A schematic of our findings is presented Figure 7.
In the context of our results, three qualifiers should be mentioned. First, TRPV4 may not be the only critical [Ca.sup.2+] conductance that is activated. Second, regarding generation of phospholipid molecules with modulatory activity on TRPV4 (e.g., by PI3-K and PLC[beta]3), we speculate that ratios of active small molecules in the immediate vicinity of the channel are critical for channel function. These two aspects deserve further study. Third, mice do not have an MMP- 1--orthologous gene, thus restricting the direct translatability of our findings into a rodent model that is amenable to genetic engineering.
The critical participants of the DEP response, PAR-2, PLC[beta]3, and TRPV4, were localized to motile cilia of differentiated primary human respiratory epithelia. Motile cilia represent a cellular extension that enhances transfer of DEP organics to ciliary membranes via autogenic exposure, given the enormous increase in cellular surface (Figure 7A). This delivery mechanism can be viewed as "slow-release" delivery of organic chemicals from coated particles to lipid membranes.
We also intend to address the question of how this novel signaling pathway has been shaped by evolution. PAR-2 signaling, as yet another variant of sensory signaling (Schmidlin and Bunnett 2001), has evolved in respiratory cilia likely as a sentinel for activation by microbial proteases, such as granzyme and chitinase. PAR-2 signaling thus would have conferred survival benefits in host defenses of airway integrity yet has subsequently been hijacked by man-made air pollution, a detrimental turn that occurred very recently, during the last milliseconds of the evolutionary clock.
A recently published landmark report showed that bitter taste receptors and PLC[beta]2 localized to motile cilia of human primary airway epithelia (Shah et al. 2009); however, we found a different isoform, PLC[beta]3, that localized to respiratory cilia. Whether bitter taste receptor-mediated [Ca.sup.2+] increase will trigger a different set of responses compared with PAR-2/TRPV4-mediated [Ca.sup.2+] influx remains to be established. Our results in the present study suggest that TRPV4-mediated [Ca.sup.2+] influx in response to DEP or OE leads to maladaptive, propathogenic reprogramming of gene-regulatory mechanisms in human airway epithelia.
In the novel signaling mechanism in human airway epithelia described here, [Ca.sup.2+] influx is characterized by uniquely slow kinetics. Two possible causes for the slow kinetics are a) DEP-dependent PLC[beta]3 phosphorylation, and b) DEP-enhanced calmodulin binding to a receptor-signaling multiplex containing TRPV4 and PLC[beta]3 (Figures 5, 7), both of which attenuate signaling via known properties of the modified phospholipase or channel. The attractive hypothesis of proteolytic activation of PAR-2 by MMP-1 has not been not corroborated.
This study is relevant for global human health because of the global presence of DEP. However, we also discovered a possibly novel mechanism of airway injury that is caused by DEP yet enhanced by the human COPD-suscepcibility polymorphism [TRPV4.sub.P19S]. Our identification of [TRPV4.sub.P19S] as a gain-of-function [Ca.sup.2+]-permeable channel in a human respiratory epithelial cell line, in response to DEP, links COPD pathogenesis to pathologically increased [Ca.sup.2+] influx into human airway epithelia elicited by a globally relevant air pollutant. Furthermore, out results imply that two human genetic polymorphisms are linked to respiratory health, [TRPV4.P19S] and MMP-1 (-1607G/GG), thus highlighting the concept of disease susceptibility as a function of genetic "makeup" combined with environmental insults. Finally, we note yet another translational medical implication: The novel pathway described here can be targeted by inhalation of compounds that can specifically inhibit critical signaling molecules. In other words, although DEP injures respiratory epithelia via a luminal--apical unloading mechanism of DEP organics delivered by carbonaceous nanoparticles, this very same route could become the avenue for safe and effective therapy now that key participants are known.
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Address correspondence to W. Liedtke, Duke University Center for Translational Neuroscience, Box 2900, Durham, NC 27710 USA. Telephone: (919) 684-0058. Fax: (919) 684-6514. E-mail: firstname.lastname@example.org
Supplemental Material is available online (doi:10. 1289/ehp. 1002807 via http://dx.doi.org/).
(*) These authors contributed equally to this work.
R. Lefkowitz, (Duke University) provided invaluable discussions and suggestions regarding signaling and [beta]-arrestin-related reagents, and J. Putney [National Institute of Environmental Health Sciences (NIEHS)] provided enlightened discourse on [Ca.sup.2+] entry mechanisms and their pharmacology. The following reagents were kindly supplied by the following colleagues: P90 carbon particles, W. Moeller (GSF, Munich. Germany); MMP-1 reporter gene constructs, C.E. Brinckerhoff (Dartmouth University); dominant-negative ORAI constructs, L. Birnbaumer (NIEHS); and dominant-negative STIM1 construct, S. Muallem (University of Texas Southwestern Medical Center.
This research was supported by startup funds from Duke University and financial support from R.J. Reynolds, Inc., to W.L.; by a grant from Philip Morris USA Inc. and Philip Morris International to S.A.S.; and by U.S. Environmental Protection Agency (EPA) internal funds to A.G. Fellowship support for J.L. (Duke University Integrated Toxicology and Environmental Health Program, Leon-Goldberg Fellowship) and S.H.C. (Research Participation Program in U.S. EPA administered by the Oak Ridge Institute for Science and Education) is acknowledged.
S.A.S. and W.L. received funding from Philip Morris USA and Philip Morris International, and W.L. received funding from R.J. Reynolds. The other authors declare they have no actual or potential competing financial interests.
Received 30 July 2010; accepted 20 December 2010.
Jinju Li, (1) Patrick Kanju, (1), (2) * Michael Patterson, (2) * Wei-Leong Chew, (2) Seung-Hyun Cho, (3) Ian Gilmour, (3) Tim Oliver, (4) # Ryohei Yasuda, (2), (5) Andrew Ghio, (6) Sidney A. Simon, (2) and Wolfgang Liedtke (1), (2)
(1) Department of Medicine, and (2) Department of Neurobiology, Duke University, Durham, North Carolina, USA; (3) U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; (4) Department of Cell Biology, Duke University, Durham, North Carolina, USA; (5) Howard Hughes Medical Institute, Chevy Chase, Maryland, USA; (6) U.S. Environmental Protection Agency, Chapel Hill, North Carolina, USA
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|Author:||Li, Jinju; Kanju, Patrick; Patterson, Michael; Chew, Wei-Leong; Cho, Seung-Hyun; Gilmour, Ian; Olive|
|Publication:||Environmental Health Perspectives|
|Date:||Jun 1, 2011|
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