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The Protease-activated Receptor-2-specific Agonists 2-Aminothiazol-4-yl-LIGRL-NH2 and 6-Aminonicotinyl-LIGRL-NH2 Stimulate Multiple Signaling Pathways to Induce Physiological Responses in Vitro and in Vivo*
* This work was supported, in whole or in part, by National Institutes of Health Grant ES 04940 from the NIEHS Superfund (to S. B.), Grant R01NS065926 (to T. J. P.), and Training Grant T32-HL007249 (to A. N. F.). This work was also supported by Semiconductor Research Corp. Project 425.024 (to S. B.) and by the State of Arizona Technology and Research Initiative Fund awarded through Bio5 (to J. V.). This work is part of a multi-principal investigator collaboration between J. V., T. J. P., and S. B. The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and videos.
Protease-activated receptor-2 (PAR2) is one of four protease-activated G-protein-coupled receptors. PAR2 is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR2 reveals a tethered ligand that activates PAR2 and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca2+ signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR2 (e.g. SLIGRL-NH2 and SLIGKV-NH2) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH2 (2-f-LIGRLO-NH2) underscores the use of peptidomimetic PAR2 ligands as a mechanism to enhance pharmacological action at PAR2, 2-f-LIGRLO-NH2 has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH2 and two recently described pentapeptidomimetic PAR2-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at-LIGRL-NH2) and 6-aminonicotinyl-LIGRL-NH2 (6-an-LIGRL-NH2). All peptidomimetic agonists stimulated PAR2-dependent in vitro physiological responses, MAPK signaling, and Ca2+ signaling with an overall rank order of potency of 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ≫ SLIGRL-NH2. Because PAR2 plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca2+ signaling in nociceptors in vitro, and both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 stimulated PAR2-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR2 in a variety of systems and pathologies.
). Proteolytic cleavage of the N terminus results in exposure of a tethered ligand that quickly activates the receptor to induce cellular signaling. Cleaved PAR2 interacts with its tethered ligand to activate trimeric G-proteins (primarily Gq) and results in activation of phospholipase C, cleavage of phosphatidylinositol 4,5-bisphosphate, transient increases of intracellular calcium ion concentration ([Ca2+]i), and activation of several downstream signaling pathways. Following activation, PAR2 is quickly phosphorylated via a G-protein receptor kinase pathway that leads to β-arrestin binding and activation of MAPK signaling pathways (
). PAR2 has been implicated in several protease-associated conditions including allergic asthma, cancer, arthritis, and chronic pain. Stimulation of PAR2 in pain-sensing primary sensory neurons (or nociceptors) leads to the sensitization of the noxious heat and capsaicin receptor TRPV1 (
) and remains the most selective and potent activator of PAR2 described to date. However, 2-f-LIGRLO-NH2 has not been evaluated in vivo or with in vitro physiologic assays that allow for the direct comparison between peptides. Such peptides and peptidomimetics have specificity and affinity for PAR2 in the absence of proteolytic cleavage and have been used to both activate PAR2 and, in high concentrations, desensitize cells to subsequent PAR2 responses in model cells (
). Our goals in this report were to develop procedures to evaluate the specific, high affinity PAR2 hexapeptidomimetic 2-f-LIGRLO-NH2 and the newly developed pentapeptidomimetic agonists 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2 and their ability to act as full, partial, or signaling pathway-specific agonists in a high throughput fashion.
To better examine PAR2 physiology and downstream signaling, we describe in this study a three-tiered methodology to evaluate agonists to PAR2: a high throughput in vitro physiological assay, standard and high throughput MAPK evaluation, and single cell and population-based digital imaging microscopy of Ca2+ signaling. The most potent known PAR2 agonist, 2-f-LIGRLO-NH2, was evaluated for activity and selectivity along with two recently identified peptidomimetic agonists based on serine substitution of the SLIGRL-NH2 activating peptide, 2-aminothiazol-4-yl-LIGRL-NH2 (2-at-LIGRL-NH2) and 6-aminonicotinyl-LIGRL-NH2 (6-an-LIGRL-NH2) (see Fig. 1 and Ref.
). All of the tested agonists displayed full agonist responses in physiological assays including stimulating MAPK and Ca2+ signaling in model epithelial cells and Ca2+ signaling in primary trigeminal ganglion neuronal cultures. Furthermore, both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 were shown to induce PAR2-dependent thermal hyperalgesia in vivo. Hence, a selection of PAR2 peptidomimetic agonists can provide potent and efficacious tools to evaluate the role of PAR2 signaling and physiology in vitro and in vivo.
Minimum essential medium with Earle's salts, Dulbecco's modified Eagle's medium (DMEM), DMEM/Ham's nutrient mixture F-12, Lechner and LaVeck basal medium, Hanks' balanced saline solution, penicillin, hygromycin, and streptomycin were purchased from Invitrogen. Fibronectin, type I collagen, and cell strainers were purchased from BD Biosciences. GlutaMAX, 5-fluorodeoxyuridine (5-FdU), uridine, and thrombin were purchased from Sigma. E-plates, collagenase A, and collagenase D with papain were purchased from Roche Applied Science. 10% fetal bovine serum was purchased from Hyclone (Logan, UT) or Sigma. All other chemicals were from Fisher or Sigma and were molecular biology grade.
PAR2 agonists were prepared by semimanual solid-phase peptide synthesis (
) performed in fritted syringes using a Domino manual synthesizer obtained from Torviq (Niles, MI). Crude peptides were purified by HPLC and size exclusion chromatography. Purity of the peptides was ensured using analytical HPLC (Waters Alliance 2695 separation model with a dual wavelength detector, Waters 2487) with a reverse-phase column (Waters Symmetry, 4.6 × 75 mm, 3.5 μm; flow rate, 0.3 ml/min). Structures were characterized by electrospray ionization on a Thermoelectron (Finnigan) LCQ ion trap instrument (low resolution), a Bruker Ultraflex III MALDI-TOF/TOF (low resolution), or a Bruker 9.4 T Fourier transform ion-cyclotron resonance (high resolution accurate mass) mass spectrometer.
Epithelial Cell Culture
16HBE14o- cells are an SV40-transformed human bronchial epithelial cell line (
) and were obtained through the California Pacific Medical Center Research Institute (San Francisco, CA). PAR2-transfected (kNRK-PAR2) and vector control-transfected Kirsten virus-transformed kidney (kNRK) cells were kind gifts from Dr. Nigel Bunnett (University of California, San Francisco) and were grown in DMEM supplemented with 10% FBS, penicillin, streptomycin, and hygromycin (150 μg/ml) at 37 °C in a 5% CO2 atmosphere. All three cell lines were expanded in tissue culture flasks prior to transfer to cultureware for specific experiments. Flasks (16HBE14o-) and cultureware (16HBE14o-, PAR2-kNRK, and kNRK) were coated initially with matrix coating solution (88% Lechner and LaVeck basal medium, 10% bovine serum albumin (BSA; from 1 mg/ml stock), 1% bovine collagen type I (from 2.9 mg/ml stock), and 1% human fibronectin (from 1 mg/ml stock solution)) and incubated for 2 h at 37 °C after which the coating solution was removed and allowed to dry for at least 1 h. 16HBE14o- cells were plated onto the matrix-coated cultureware at a concentration of 1 × 105 cells/cm2. 16HBE14o- (minimum essential medium with Earle's salts), PAR2-kNRK, or kNRK (DMEM/Ham's nutrient mixture F-12) cells were cultured in appropriate growth medium supplemented with 10% FBS, 2 mm GlutaMAX, penicillin, and streptomycin at 37 °C in a 5% CO2 atmosphere. Medium was replaced every other day until the cells reached confluence (5–7 days).
Primary Neuronal Cultures
Male ICR mice (Harlan; 20–25 g) were used. All animal procedures were approved by the Institutional Animal Care and Use Committee of The University of Arizona and were in accordance with International Association for the Study of Pain guidelines. Trigeminal ganglia were excised aseptically and placed in Hank's balanced saline solution supplemented with 25 mm Hepes (HBSS) on ice. The ganglia were dissociated enzymatically with collagenase A (1 mg/ml; 25 min) and collagenase D (1 mg/ml) with papain (30 units/ml) for 20 min at 37 °C. To eliminate debris, 70-μm cell strainers were used. The dissociated cells were resuspended in DMEM/Ham's nutrient mixture F-12 containing 1× penicillin-streptomycin, 1× GlutaMAX, 3 μg/ml 5-FdU, 7 μg/ml uridine, and 10% fetal bovine serum. The cells were plated on glass coverslips and incubated at 37 °C in a humidified 5% CO2 incubator. 24 h later the cells were used for Ca2+ imaging.
Impedance Assay for Physiological Response in Vitro
16HBE14o- cells were grown on E-plates (Roche Applied Science) coated with matrix coating solution. Impedance from each well was measured overnight at 37 °C in a 5% CO2 atmosphere using the xCELLigence real time cell analyzer (RTCA; Roche Applied Science) system to assure proper growth to 95% confluence in each well. On the following day, growth medium was replaced with 100 μl of prewarmed HBSS (37 °C). Background readings were obtained for an additional 30 min while the machine and buffer were allowed to reach room temperature. Agonists, diluted in HBSS at 2× final concentration, were added to the E-plate in 100-μl aliquots for a final volume of 200 μl. Relative impedance, expressed as the cell index was measured every 15 s for 4 h. According to the manufacturer's definition,
where Zi is impedance at a given time point during the experiment (i.e. post-agonist addition) and Z0 is impedance before the addition of agonist. Ω represents ohms. Thus, a loss of adhesion would generate a lower cell index; an increase in cell adhesion, which is typically seen with G-protein-coupled receptor activation, would result in an increase in the cell index.
Individual traces of the cell index over time represent the average of four experiments from a single E-plate normalized at the point of agonist addition and against untreated cells to correct for cell proliferation according to the manufacturer's instruction. This method also allows for the direct comparison of the cell index across individual wells in the high throughput experiment. EC50 values were calculated using the peak responses (EC50Peak) or the area under the curve measurements (EC50AUC) by plotting cell indices derived from 4-h experiments (n = 12 wells except for 300 nm 2-at-LIGRL-NH2 where n = 8) from three different E-plates.
16HBE14o- cells were plated on matrix coating solution-coated 6-well plates. Once cells reached confluence (5–7 days), the culture medium was removed, and the cells were incubated with each agonist diluted in HBSS for 5 min. After the agonist was removed, cells were lysed in a modified radioimmune precipitation assay buffer (50 mm Tris (pH 7.4), 150 mm NaCl, 1 mm EDTA (pH 8.0), 1% Triton X-100). Lysates were then sonicated for 10 min at 4 °C, centrifuged for 15 min at 14,000 × g to pellet nuclei and debris, and stored at −80 °C. Extracellular signal-regulated kinase 1/2 (ERK 1/2), both activated (phosphorylated ERK (pERK); antibodies from Cell Signaling Technology) and non-activated (total ERK; antibodies from Cell Signaling Technology), were assayed by standard SDS-PAGE. Band densities were measured with ImageJ (National Institutes of Health), and pERK was standardized to total ERK to assess changes in ERK activity.
In-cell Western Studies
For in-cell Western (ICW) studies, 16HBE14o- cells were plated in fluorescence-adjusted, black-walled 96-well plates (catalog number 3603, Costar, Wilkes-Barre, PA) and grown to 95% confluence as described above. Cells were washed with HBSS and allowed to come to room temperature for 30 min. Cells were exposed to a dose of each agonist from 10 nm to 10 μm in ½ log steps for 10 min at room temperature. Cells were then fixed with 4% formaldehyde (Ted Pella Inc., Redding, CA) in PBS for 20 min. Cells were incubated in primary antibody (rabbit anti-pERK; Invitrogen), secondary antibodies (Cell Signaling Technology), and nuclear stain DRAQ5 (Cell Signaling Technology) and imaged on an Odyssey scanner (LI-COR Biosciences, Lincoln, NE) according to the manufacturers' instructions.
Coverslip cultures were washed with HBSS and loaded for 40–45 min in 5 μm fura 2-AM in HBSS. Cells were removed from fura 2-AM loading solution and placed back into HBSS for at least 20 min before Ca2+ imaging. Fura 2 fluorescence was observed on an Olympus IX70 microscope with a 40× oil immersion objective after alternating excitation between 340 and 380 nm by a 75-watt xenon lamp linked to a DeltaRAM V illuminator (Photon Technology International, Inc.) and a gel optic line. Images of emitted fluorescence above 505 nm were recorded by an intensified charge-coupled device camera (Photon Technology International, Inc.) and simultaneously displayed on a 21-inch Vivitron color monitor. The imaging system was under software control (ImageMaster, Photon Technology International, Inc.) and collected a ratio approximately every 0.6 s. [Ca2+]i for each individual cell in the field of view was calculated by ratiometric analysis of fura 2 fluorescence using equations originally published in Grynkiewicz et al. (
). A typical experiment consisted of a 20-s recording of cells in HBSS to determine resting [Ca2+]i (typically ≤75 nm for 16HBE14o-, PAR2-kNRK, and kNRK cells) followed by a 10-s wash to introduce agonist or thrombin. Cells were monitored for an additional 2 min 40 s for activation experiments or up to 10 min for desensitization experiments. Additional washes were added as required (e.g. see FIGURE 6, FIGURE 7). For evaluation of the percentage of cells activated by agonists, a change in [Ca2+]i was considered positive if the cell [Ca2+]i increased to 200 nm or more within the experimental time frame. For desensitization experiments that required population-based studies, the average [Ca2+]i for all cells in the field of view was plotted with S.E. over time.
Assessment of Thermal Hyperalgesia
Thermal hyperalgesia was measured using the method of Hargreaves et al. (
). ICR or PAR2−/− mice (The Jackson Laboratory Strain B6.Cg-F2rl1tm1Mslb/J; stock number 004993; a kind gift from Dr. Michael O. Daines (The University of Arizona)) were placed in plastic boxes and allowed to habituate for 45 min. A radiant heat source was placed under the paw of the animal, and when it was turned on, the latency to paw withdrawal was measured. The machine was set to 30% intensity giving a base-line thermal latency of about 10 s. A cutoff time of 15 s was set to avoid potential tissue injury. After obtaining a baseline measurement, PAR2 agonist or vehicle (physiological saline) was injected into the plantar left hind paw in a volume of 25 μl through a 30-gauge needle. Thermal latencies were measured over the ensuing 3 h.
All statistical analyses were evaluated with GraphPad software (San Diego, CA). Multivariate comparisons were done with a one-way or two-way analysis of variance with Tukey's or Bonferonni's multiple comparison post-test as appropriate for the individual experiment. Pairwise comparisons were done with a two-tailed Student's t test. A value of p < 0.05 was used to establish a significant difference between samples. Data in figures are graphed ±S.E. unless otherwise noted.
Peptidomimetic PAR2 Agonists Stimulate a Physiological Response in Model Epithelial Cells
Human airway epithelial cells (16HBE14o-) express PAR2 that can be activated by various proteases (
), using the RTCA. This system uses a high throughput format to detect changes in G-protein-coupled receptor activation through cell adhesion, morphology, and viability expressed as a change in the cell index (
). Baseline measurements were first obtained in growth medium overnight and then every 15 s for up to 4 h following application of agonists. We found each agonist stimulated an increase in the cell index in a dose-dependent manner with peak responses occurring within 30 min of application (Fig. 2, A, C, and E). The most robust responses were observed at 3 μm agonist concentration, ∼
the concentration needed for maximum response to SLIGRL-NH2 (supplemental data). At the higher concentrations tested, each agonist displayed a more rapid return to the resting cell index, which can be seen over a 4-h time course (Fig. 2, B, D, and F). We evaluated each agonist for efficacy and potency from two metrics readily available from the RTCA experiments: peak responses and area under the curve (AUC). Using the peak response from the RTCA, the calculated EC50 (EC50Peak) of 2-f-LIGRLO-NH2 was 235 nm (95% confidence interval (CI), 193–290 nm). 2-at-LIGRL-NH2 showed slightly less potency with this measure (EC50Peak = 314 nm) but with an overlapping 95% CI (244–403 nm). The EC50Peak of 6-an-LIGRL-NH2 was the highest of the three at 430 nm (95% CI, 350–529 nm), suggesting that this compound was the least potent of the three tested when using the peak response metric. All three PAR2 agonists showed equivalent peak responses indicating similar efficacy (Emax). These potency and efficacy measurements allow for insight into agonist ability to quickly activate the receptor and cause a physiological response. However, a better predictor of physiological capacity of these PAR2 agonists may be their continuous activity over the entire 4-h period of the experiment, estimated by the AUC. Using this measure as our experimental end point, 2-at-LIGRL-NH2 displayed an EC50AUC (106 nm; 95% CI, 82–137 nm) nearly identical to that observed for 2-f-LIGRLO-NH2 (EC50AUC = 114; 95% CI, 83–158 nm). 6-an-LIGRL-NH2 displayed a higher EC50AUC (254 nm; 95% CI, 197–328 nm) with a confidence interval fully outside the other agonists. Emax values were the same for all agonists. From these data, we conclude that newly described pentapeptidomimetic PAR2 agonists 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2 and the known hexapeptidomimetic agonist 2-f-LIGRLO-NH2 are full agonists at PAR2 with a rank order potency in this physiological response assay of 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ≫ SLIGRL-NH2. The RTCA analysis demonstrates potent physiological activation of model epithelial cells by several PAR2 agonists and demonstrates the utility of this assay for high throughput identification of PAR2 agonists.
Peptidomimetic PAR2 Agonists Activate MAPK Signaling in Epithelial Cells
Our physiological response data are consistent with full activation of PAR2 by all agonists but do not provide information on specific cell signaling pathways downstream of PAR2. To examine the ability of 2-f-LIGRLO-NH2 and the newly described pentapeptidomimetic agonists 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2 to stimulate MAPK signaling, 16HBE14o- cells were exposed to a dose (1–10 μm) of each agonist for 5 min (Fig. 3). We found that a 2.5 μm concentration of each agonist was sufficient to stimulate a significant increase in ERK 1/2 phosphorylation. Although both 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 induced full phosphorylation at a dose of 5 μm, 6-an-LIGRL-NH2 continued to increase phosphorylation up to the highest dose tested (10 μm).
To better quantify differences in MAPK activity for each agonist, the above experiments were repeated using a high throughput ICW assay (
). In these experiments, 16HBE14o- cells were exposed to a half-log stepped dose response (10 nm–10 μm) for 10 min, and EC50 values were calculated for each agonist. Consistent with the traditional immunoblots, 2-f-LIGRLO-NH2 displayed a lower EC50 (314 nm; 95% CI, 193–508 nm) when compared with 2-at-LIGRL-NH2 (458 nm; 95% CI, 204 nm–1.03 μm) with overlapping 95% CI (Fig. 3D and supplemental data). The EC50 for 6-an-LIGRL-NH2 (1.04 μm; 95% CI, 358 nm–3.03 μm) was higher but displayed overlapping 95% CI with the other agonists tested. These ICW findings support the immunoblot results and demonstrate a rank order potency similar to that observed with the RTCA assay: 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 ≥ 6-an-LIGRL-NH2.
Peptidomimetic PAR2 Agonists Stimulate Ca2+ Signaling in Model Epithelial Cells
In addition to initiating MAPK signaling, PAR2 activation also stimulates intracellular Ca2+ signaling. To investigate the effect of peptidomimetic agonists on Ca2+ signaling, agonists were applied to 16HBE14o- cells, and [Ca2+]i was followed over time in individual cells using digital imaging microscopy. Representative experiments of the addition of 5 μm concentrations of each agonist are shown with full dose responses in Fig. 4. There was an initial increase in [Ca2+]i within 30 s in response to a 5 μm concentration of each agonist. Furthermore, most cells in the field of view responded over the 3-min time course. Because our method of Ca2+ imaging allows for detection of [Ca2+]i changes of individual cells, we were able to uniquely evaluate individual agonist responses. We first evaluated the efficacy of each agonist by scoring the percentage of cells in the field of view undergoing [Ca2+]i transient increases of >200 nm. These results were then plotted in a dose-response curve to compare the effectiveness of all three peptidomimetic agonists. As observed with the RTCA and MAPK assays, all agonists achieved maximal activation of PAR2 in the Ca2+ activation assay, demonstrating that they are full agonists at the receptor. The calculated EC50 in this assay for 2-f-LIGRLO-NH2 (0.84 μm; 95% CI, 0.69–0.98 μm) was lower than that calculated for 2-at-LIGRL-NH2 (1.82 μm; 95% CI, 1.47–2.25 μm) (Fig. 4P). Furthermore, the calculated EC50 for 6-an-LIGRL-NH2 (2.60 μm; 95% CI, 2.04–3.32 μm) was outside of the 95% CI for both 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2. However, all agonists represent a 20–30-fold improvement over Ca2+ activation by SLIGRL-NH2 (supplemental videos).
Although there was a robust and similar increase in [Ca2+]i in response to 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 within 30 s, there was an obvious delay in response to 6-an-LIGRL-NH2. This lag in response to 6-an-LIGRL-NH2 continued throughout the experiment as nearly all the cells showed increases in [Ca2+]i within 60 s of 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 addition (Fig. 4, A–H), whereas a full response to 6-an-LIGRL-NH2 was not observed until 90 s following application of the agonist (Fig. 4, I–L). To further examine how each agonist may be activating PAR2, we calculated the mean time of agonist application to a threshold [Ca2+]i (200 nm) for each agonist across the dose response (Fig. 4Q). Concentrations of 2-f-LIGRLO-NH2 (800 nm; 95% CI, 620 nm–1.03 μm) and 2-at-LIGRL-NH2 (1.98 μm; 95% CI, 1.81–2.17 μm) required to achieve 50% time to threshold mirrored the observations from the analysis of the percentage of cells showing a Ca2+ response. The time to threshold activation curve for 6-an-LIGRL-NH2 displayed a reduced activity and could not be fit with non-linear regression. The combined results of these experiments demonstrate a rank order potency consistent with the RTCA and the MAPK data: 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ≫ SLIGRL-NH2. A summary of the rank order potency for each agonist is shown in Table 1. Across assays, we consistently found that 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2.
TABLE 1Summary of agonist EC50 values for each assay
Peptidomimetic PAR2 Agonists Are Specific for PAR2 Receptor
The findings described above are consistent with PAR2 activation by all three peptidomimetic agonists. However, because there are a variety of mechanisms that can lead to increases in MAPK or Ca2+ signaling in epithelial cells, a demonstration of specificity for PAR2 is required. Thus, we examined changes in [Ca2+]i in the kNRK cell system (
). In this system, we compared activation of kNRK cells transfected with human PAR2 (kNRK-PAR2) with responses observed in kNRK cells transfected with an empty vector plasmid control (kNRK). We found that 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 were able to stimulate an increase in [Ca2+]i in over 80% of the kNRK-PAR2 cells with minimal stimulation of kNRK cells at doses of 5 and 2.5 μm (Fig. 5, A and B). As observed with 16HBE14o- cells, 6-an-LIGRL-NH2 was less efficient and required a 5 μm concentration to fully induce an increase in [Ca2+]i in a significant number of transfected cells (Fig. 5C).
To further examine the specificity of the newly described agonists for PAR2, we used a series of desensitization assays (
). In these experiments, a population of 16HBE14o- cells was pretreated with a high concentration of 2-f-LIGRLO-NH2 to effectively eliminate PAR2-based signaling prior to application of the pentapeptidomimetic agonists. Thus, any increase in [Ca2+]i would indicate a response that was not specific to PAR2. Application of 10 μm 2-f-LIGRLO-NH2, 2-at-LIGRL-NH2, or 6-an-LIGRL-NH2 alone activated nearly 100% of the cells to a population-averaged peak [Ca2+]i between 400 and 600 nm within seconds (Fig. 6, A–C). Average [Ca2+]i returned toward base line within 3 min. These responses were then compared with stimulation of [Ca2+]i by the purinergic receptor type 2 agonist ATP (Ref.
and Fig. 6D). When 16HBE14o- cells were desensitized with 50 μm 2-f-LIGRLO-NH2, subsequent washes with 50 μm 2-f-LIGRLO-NH2 did not increase [Ca2+]i (Fig. 6, E and F). Furthermore, we found that even at doses up to 10 μm neither of the newly described pentapeptidomimetic agonists was able to stimulate a Ca2+ response following 2-f-LIGRLO-NH2 desensitization. This loss of response was not caused by loss of Ca2+ signaling itself as ATP remained an effective agonist following desensitization of PAR2. Finally, 50 μm 2-at-LIGRL-NH2 effectively desensitized PAR2 and prevented 10 μm 2-f-LIGRLO-NH2 from inducing an increase in [Ca2+]i (Fig. 6G). This demonstrates that both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 are sufficiently potent to desensitize PAR2.
The most likely targets for nonspecific responses to PAR2 agonists are the closely related PAR1, PAR3, and PAR4 family members (
). Similar to the experiments above, 16HBE14o- cells were pretreated with 100 nm thrombin to inactivate PAR1, PAR3, and PAR4 (Fig. 6, H–J). Although the initial application of thrombin stimulated a modest increase in [Ca2+]i, an additional application of 100 nm thrombin did not stimulate Ca2+ signaling, which is indicative of loss of PAR1, PAR3, and PAR4 responses. Thrombin inactivation treatments were followed by application of a 5 μm concentration of each of each agonist, which induced a robust change in [Ca2+]i consistent with PAR2-specific activation. To control for specificity, we performed the reciprocal experiment by first desensitizing PAR2 with a 50 μm concentration of each agonist and following with 100 nm thrombin to activate PAR1, PAR3, and PAR4 (Fig. 6, K–M). The thrombin response following PAR2 desensitization (12.4 ± 2.6%) was nearly identical to the thrombin response prior to PAR2 desensitization (13.6 ± 2.2%). Taken together, these data demonstrate that the newly described agonists 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2 are similar to 2-f-LIGRLO-NH2 with respect to activation of PAR2-specific cellular signaling.
Peptidomimetic PAR2 Agonists Induce Ca2+ Signaling in Trigeminal Neurons
PAR2-induced Ca2+ signaling is linked to nociceptor activation and sensitization. To address this signaling pathway downstream of PAR2 in a cell type relevant to nociception, we assessed the ability of each agonist to stimulate Ca2+ signaling in primary cultures of trigeminal ganglion neurons. Following the application of each agonist, cultures were washed with HBSS followed by a modified, high K+ (37 mm) HBSS solution to depolarize and identify neurons from the heterogenous population of cells (Fig. 7). We found that 25 μm concentrations of 2-f-LIGRLO-NH2, 2-at-LIGRL-NH2, and 6-an-LIGRL-NH2 all stimulated increases in [Ca2+]i in trigeminal neurons in culture. Although higher concentrations of the peptidomimetics were required to activate Ca2+ signaling, these data show that all three peptidomimetic agonists are efficacious compounds to be used as tools to explore the effect of PAR2 in primary cultured neurons.
Peptidomimetic PAR2 Agonists Cause Thermal Hyperalgesia
Previous studies using the peptide activator SLIGRL-NH2 and work in PAR2−/− mice suggest an important role for PAR2 in acute and chronic pain (
). We tested whether 2-f-LIGRLO-NH2 (which has not been evaluated previously in tests for thermal nociception) and 2-at-LIGRL-NH2 could be used as efficacious tools to examine thermal hyperalgesia in mice. Both 2-f-LIGRLO-NH2 (EC50 = 7.4 μg; 95% CI, 3.4–16.2 μg) and 2-at-LIGRL-NH2 (EC50 = 4.7 μg; 95% CI, 2.0–10.7 μg) caused thermal hyperalgesia with peak effects at 30-μg doses (Fig. 8), and significant thermal hyperalgesia was observed through 90 min postinjection. Hence, 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 displayed roughly equal potency in all assays with the exception of Ca2+ signaling where 2-f-LIGRLO-NH2 was more potent (Table 1).
We next utilized PAR2−/− mice to assess whether 2-f-LIGRLO and 2-at-LIGRL-NH2 display specificity for PAR2 in terms of thermal hyperalgesia. Maximal effective doses (30 μg) of either peptidomimetic PAR2 agonist failed to evoke thermal hyperalgesia at any time point in PAR2−/− mice. These data demonstrate the in vivo utility for the peptidomimetic compounds and suggest that both 2-at-LIGRL-NH2 and 2-f-LIGRLO-NH2 are potent, efficacious, and specific PAR2 agonists for evaluating PAR2-mediated physiological responses in vivo.
In this report, we systematically evaluate the known hexapeptidomimetic 2-f-LIGRLO-NH2 and two newly developed pentapeptidomimetics, 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2, as potent, specific PAR2 agonists. All three agonists were subjected to in vitro high throughput physiological assays (RTCA and ICW) and a sensitive individual cellular analysis of Ca2+ signaling. We demonstrate PAR2 specificity, potency, and efficacy by 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2 that eclipses known peptide agonists (SLIGRL-NH2 and SLIGKV-NH2) and by all measures is comparable with 2-f-LIGRLO-NH2. Thus, the newly described and smaller pentapeptidomimetic agonists can be used along with the previously known hexapeptidomimetic 2-f-LIGRLO-NH2 as important tools to study PAR2-dependent processes in vitro and in vivo. To demonstrate this point, we show, for the first time, that that both 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 can be utilized to probe physiological responses related to nociception, stimulating Ca2+ signaling in cultured trigeminal ganglion neurons and causing thermal hyperalgesia, in a PAR2-specific fashion, when administered locally to the hind paw.
Activation of PAR2, whether by traditional protease activation or by small molecule activation as demonstrated in this report, results in the activation of two major intracellular signaling responses, MAPK and Ca2+ signaling pathways. Both of these pathways contribute to cell type-specific physiological responses (for reviews, see Refs.
). In model epithelial cells, activation of PAR2 causes an interaction with G-proteins, which can then activate phospholipase C to stimulate transient increases in [Ca2+]i and activate protein kinase C (
). These major signaling events, along with cell-specific signaling targets, result in the cellular physiology that defines the PAR2 response. The in vitro model system of adherent 16HBE14o- cells expresses the PAR2 receptor but has little PAR1, PAR3, or PAR4 expression (
). Thus, these cells provide an ideal system to evaluate PAR2 agonists using in vitro physiological responses (e.g. RTCA) or signaling pathway-specific techniques (e.g. immunoblot/ICW analysis of MAPK or digital imaging microscopy for Ca2+ imaging).
We used both MAPK and Ca2+ signaling analyses to compare the known PAR2 hexapeptidomimetic agonist 2-f-LIGRLO-NH2 with the newly described pentapeptidomimetics 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2. Using a high throughput MAPK assay and a single cell Ca2+ imaging assay, we report a similar rank order potency among these assays: 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2. Our use of ICW for MAPK analysis allowed the calculation of EC50 values, which are difficult to obtain using a traditional immunoblot. Our use of digital imaging microscopy likewise allowed improved sensitivity to assess Ca2+ signaling pathways. Despite these noted improvements, drawbacks in individual cell signaling analysis are the limited ability to evaluate agonist-induced cross-talk in cellular signaling pathways that may mediate physiological responses and the limited ability to test agonist responses for extended time points (e.g. hours).
To overcome these limitations of single signaling pathway analyses and develop a method to screen PAR2 agonists in a high throughput fashion, we adapted the RTCA system to evaluate PAR2 physiological responses in vitro (
). Using this assay, we were able to continuously and non-invasively evaluate a physiologic cellular response to PAR2 agonists at both acute (e.g. 10–30 min) and more prolonged time points (e.g. 4 h). Through this evaluation, we determined that both 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 provided fast and complete physiological activation with the highest efficacy and potency, whereas 6-an-LIGRL-NH2 was slightly less potent. It is interesting to note that our acute RTCA measurements resulted in a rank order potency similar to that observed in the short term single pathway analyses: 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2. The RTCA assay provided additional information on cell activation. For example, we noted that at the doses of each agonist that displayed the highest activity (3 μm) the cell index displayed a faster return to base line than that observed at lower agonist doses. This faster return to base line was exacerbated at even higher doses (e.g. 10 μm; data not shown). A possible explanation for these results is a desensitization of PAR2 at higher agonist doses that prevents continued activation of cellular signaling over the extended periods examined in the RTCA experiments. Consistent with this finding is the extended response (i.e. higher area under the curve) observed with 6-an-LIGRL-NH2 compared with 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 where agonist application resulted in faster peak responses (Fig. 2). While we have compared specific small molecule activation of PAR2 in this report and have uncovered measurable changes in physiology, there is some discussion that untethered ligand activation may be different from protease activation of PAR2 (
). Although further work is required to uncover the mechanisms that underlies these observations, we can conclude that the information obtained from this system is an important first step in high throughput screening of PAR2 activation via novel agonists or native proteases. This system allows the PAR2 response to be continually evaluated and serves as a valuable screen to identify effective agonists prior to further defining their ability to activate individual signaling pathways and/or an in vivo response.
Previous work has shown that peptides designed to activate individual PARs can have nonspecific effects, and a primary nonspecific target for these agonists are other PARs (
). Although our model epithelial cells discouraged off-target PAR activation (i.e. non-PAR2) as a potential source of agonist-induced response, we used additional established techniques to confirm specificity of the pentapeptidomimetic ligands and 2-f-LIGRLO-NH2. Results with both the PAR2-expressing and non-expressing kNRK cells (
) using 2-f-LIGRLO-NH2 showed a specificity of action for the pentapeptidomimetic agonists. These data were complemented by the inability of thrombin to desensitize the Ca2+ signaling response of model epithelial cells to each agonist. We have further demonstrated specificity by showing a lack of PAR2 activation by serine substitution in SLIGRL-NH2 with aliphatic surrogates such as diglycolyl, 1-piperidinethyl, and 2-cyanomethyl (
). Finally, both 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 failed to evoke thermal hyperalgesia in PAR2−/− mice at maximally effective doses of each compound, suggesting a high degree of specificity in terms of nociceptive sensitization. These findings are all consistent with specific PAR2 activation by the known hexapeptidomimetic (2-f-LIGRLO-NH2) and newly described pentapeptidomimetic (2-at-LIGRL-NH2 and 6-an-LIGRL-NH2) agonists used in this report.
It is now increasingly evident that different G-protein-coupled receptor agonists may result in signaling pathway-specific activation that can lead to altered physiological responses (for a review, see Ref.
). In the same report, the peptide SLAAAA-NH2 was found to activate wild-type rat PAR2 MAPK signaling without an increase in [Ca2+]i. Consistent with a physiological role for signaling pathway-specific activation in PAR2, a specific role in migration pathways has been described for the β-arrestin-mediated-MAPK activation that is independent of Ca2+ signaling following PAR2 activation (
). Because of these findings, 2-f-LIGRLO-NH2, 2-at-LIGRL-NH2, and 6-an-LIGRL-NH2 were assayed for their ability to activate both Ca2+ signaling and MAPK signaling. Consistent with the findings from the in vitro physiological assay described above, all agonists were found to fully activate both pathways and thus act as full agonists to PAR2. Importantly, the activity of 2-f-LIGRLO-NH2 in all of these assays was consistent with previous reports using less sensitive end points (e.g. Ref.
Understanding the role of PAR2 in pathophysiology is currently limited by a lack of potent and efficacious pharmacologic tools that specifically target PAR2. Chronic pain is a major clinical problem with few viable treatment options (
). To evaluate the utility of 2-f-LIGRLO-NH2 and our newly described agonists as PAR2 probes for the pain pathway, we used them in in vitro and in vivo models of nociception. In an in vitro assay designed to test the ability of agonists to initiate PAR2 signaling in primary trigeminal neurons, both 2-at-LIGRL-NH2 and 6-an-LIGRL-NH2 initiated Ca2+ signaling similarly to the previously described 2-f-LIGRLO-NH2. Hence, all three agonists are capable of inducing PAR2-dependent signaling in trigeminal ganglion neurons. This stimulation presumably occurred in nociceptors because PAR2 expression in sensory ganglia overlaps with markers for these neurons (
). Because of the decreased efficacy of 6-an-LIGRL-NH2 over the full in vitro evaluation, we limited the in vivo evaluation to a comparison between 2-f-LIGRLO-NH2 (which has not been evaluated previously in tests of thermal hyperalgesia) and 2-at-LIGRL-NH2. Both 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 induced thermal hyperalgesia in vivo, in a PAR2-specific fashion, with 2-at-LIGRL-NH2 displaying equal potency to 2-f-LIGRLO-NH2. This overlapping potency is identical to that observed in the 4-h RTCA in vitro physiological assay. Hence, 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 represent potent and specific PAR2 ligands for mechanistic studies aimed at further understanding the role of PAR2 in acute and chronic pain.
In summary, we have described a unique, three-tiered in vitro paradigm to fully describe 2-f-LIGRLO-NH2, 2-at-LIGRL-NH2, and 6-an-LIGRL-NH2 as efficacious, potent, and specific agonists to PAR2. These agonists were 10–40-fold better than SLIGRL-NH2 (across assays), a peptide activator of PAR2. The pentapeptidomimetic agonists displayed either equal or slightly reduced potency when compared with 2-f-LIGRLO-NH2 in assays designed to evaluate relatively fast responses via intracellular signaling pathways or in vitro physiological responses and showed a rank order of 2-f-LIGRLO-NH2 ≈ 2-at-LIGRL-NH2 > 6-an-LIGRL-NH2 ≫ SLIGRL-NH2 (summarized in Table 1). However, for extended in vitro physiological responses (i.e. 4-h RTCA-AUC measurements) or under in vivo evaluation, 2-f-LIGRLO-NH2 and 2-at-LIGRL-NH2 were identical in evoking PAR2 responses. The methods we describe to evaluate PAR2 agonists can be utilized in high throughput screening efforts to identify novel agonists and antagonists to PAR2. Specifically, major advantages of our novel approach combining multiple high throughput assays are the ability to quickly identify signaling pathway-specific ligands that hold great promise and the ability to better predict physiological responses of novel agonists and antagonists. These will be essential to understanding and potentially treating PAR2-dependent pathophysiology.
We thank Renata Patek and Zhenyu Zhang for work in helping to prepare peptidomimetic compounds, Daniel X. Sherwood for the program that allowed for quick quantification of Ca2+ data, and Cara L. Sherwood for help in the laboratory in getting this work started.