Protease-activated receptor 2 sensitizes TRPV1 by protein kinase Cepsilon- and A-dependent mechanisms in rats and mice

Abstract

Proteases that are released during inflammation and injury cleave protease-activated receptor 2 (PAR2) on primary afferent neurons to cause neurogenic inflammation and hyperalgesia. PAR2-induced thermal hyperalgesia depends on sensitization of transient receptor potential vanilloid receptor 1 (TRPV1), which is gated by capsaicin, protons and noxious heat. However, the signalling mechanisms by which PAR2 sensitizes TRPV1 are not fully characterized. Using immunofluorescence and confocal microscopy, we observed that PAR2 was colocalized with protein kinase (PK) Cepsilon and PKA in a subset of dorsal root ganglia neurons in rats, and that PAR2 agonists promoted translocation of PKCepsilon and PKA catalytic subunits from the cytosol to the plasma membrane of cultured neurons and HEK 293 cells. Subcellular fractionation and Western blotting confirmed this redistribution of kinases, which is indicative of activation. Although PAR2 couples to phospholipase Cbeta, leading to stimulation of PKC, we also observed that PAR2 agonists increased cAMP generation in neurons and HEK 293 cells, which would activate PKA. PAR2 agonists enhanced capsaicin-stimulated increases in [Ca2+]i and whole-cell currents in HEK 293 cells, indicating TRPV1 sensitization. The combined intraplantar injection of non-algesic doses of PAR2 agonist and capsaicin decreased the latency of paw withdrawal to radiant heat in mice, indicative of thermal hyperalgesia. Antagonists of PKCepsilon and PKA prevented sensitization of TRPV1 Ca2+ signals and currents in HEK 293 cells, and suppressed thermal hyperalgesia in mice. Thus, PAR2 activates PKCepsilon and PKA in sensory neurons, and thereby sensitizes TRPV1 to cause thermal hyperalgesia. These mechanisms may underlie inflammatory pain, where multiple proteases are generated and released.

Figure 1. Localization of immunoreactive PAR₂, PKCɛ and PKA_C in DRG neurons

A and D, PAR₂; B, PKCɛ; E, PKA_C; C and F, merge. G, PKCɛ preabsorption control; H, PKA_C preabsorption control. Neurons expressing PAR₂ also expressed PKCɛ and PKA_C (arrows). Scale bar = 20 μm.

Figure 2. Effects of PAR₂ agonist on the subcellular distribution of PKCɛ determined by confocal microscopy

A, HEK 293 cells expressing PKCɛ-EGFP (enhanced green fluorescent protein). In unstimulated cells (non-treated, NT), PKCɛ-EGFP was cytosolic and in vesicles (arrows). PMA (phorbol 12-myristate 13-acetate) induced translocation to the plasma membrane at 5 min (arrowheads). PAR₂-AP induced translocation to the plasma membrane at 1 min (arrowheads). PAR₂-RP did not affect the subcellular location of PKCɛ-EGFP, which remained in the cytosol and vesicles (arrows). Scale bar = 10 μm. B, rat DRG neurons in culture. In unstimulated neurons (NT, non-treated), PKCɛ was cytosolic and in vesicles (arrows). PMA induced translocation of PKCɛ to the plasma membrane at 5 min (arrowheads). PAR₂-AP also induced translocation of PKCɛ to the plasma membrane at 0.5 and 1 min (arrowheads). PKCɛ was in the cytosol and vesicles of cells treated with PAR₂-RP (arrows). Control shows staining with preabsorbed PKCɛ antibody. Scale bar = 10 μm. C, fluorescence intensity (in arbitrary units) measured in a line bisecting the neuronal soma (e.g. see dashed line in B). In untreated cells (NT) or cells incubated with PAR₂-RP (1 min), the signal was mostly cytosolic. In cells treated with PMA (5 min) or PAR₂-AP (1 min), the signal in the cytosol diminished and was more prominent at the plasma membrane. Each trace is an average of 4–6 cells. D, the effect of PMA, PAR₂-AP and PAR₂-RP on the percentage of the total number of observed neurons expressing PKCɛ with PKCɛ either in the cytosol or at the plasma membrane of the soma. The proportion of cells with PKCɛ at the plasma membrane was increased after treatment with PMA and PAR₂-AP. Observations from n > 100 cells.

Figure 3. Effects of PAR₂ agonist on the subcellular distribution of PKCɛ in HEK 293 cells determined by subcellular fractionation and Western blotting

A, Western blot, and B, densitometric analysis of PKCɛ-EGFP (enhanced green fluorescent protein) in cytosolic (c) and membrane (m) fractions of HEK-PKCɛ-EGFP cells. PAR₂-AP increased PKCɛ-EGFP in membrane fractions and decreased PKCɛ-EGFP in cytosolic fractions. C, Western blot, and D, densitometric analysis of immunoreactive PKCɛ in cytosolic and membrane fractions of HEK-TRPV1 cells. PAR₂-AP increased PKCɛ in membrane fractions and decreased PKCɛ in cytosolic fractions. *P < 0.05 compared to 0 min, n = 4 experiments.

Figure 4. Effects of PAR₂ agonist on the subcellular distribution of PKA_C determined by confocal microscopy in rat DRG neurons in culture

A, unstimulated neuron (non-treated, NT), PKA_C (PKA catalytic subunit) was in vesicles and uniformly distributed throughout the cytosol (arrows). Forskolin (FSK) induced redistribution of PKA_C from the central to the peripheral region of the soma at 0.5 min (arrows). PAR₂-AP also induced redistribution of PKA_C to the peripheral region of the cell at 1 min and 5 min (arrows). PKA_C was uniformly distributed in the cytosol and in vesicles of cells treated with PAR₂-RP (arrows). Control shows staining with preabsorbed PKA_C antibody. Scale bar = 10 μm. B, fluorescence intensity (in arbitrary units) measured in a line bisecting the neuronal soma (e.g. see dashed line in A). In untreated cells (NT) or cells incubated with PAR₂-RP (5 min), the signal was mostly cytosolic. In cells treated with FSK (0.5 min) or PAR₂-AP (5 min), the signal in the cytosol diminished and was more prominent in superficial regions of the soma. Each trace is an average of 4–6 cells. C, the effects of FSK, PAR₂-AP and PAR₂-RP on the percentage of the total number of observed neurons expressing PKA_C with PKA_C either in the cytosol or near the plasma membrane of the soma. The proportion of cells with PKA in a superficial region of the cytosol was increased after treatment with FSK and PAR₂-AP. Observations from n > 100 cells.

Figure 5. Effects of PAR₂ agonists on the subcellular distribution of PKA_C (catalytic subunit) in HEK 293 cells and on cAMP levels

A, Western blot, and B, densitometric analysis of PKA_C in cytosolic (c) and membrane (m) fractions of HEK-TRPV1 cells. PAR₂-AP caused an increase in PKA_C in membrane fractions and a decrease in cytosolic fractions. *P < 0.05 compared to 0 min, n = 4 experiments. C, effects of PAR₂ agonists on cAMP level in HEK-PAR₂ cells (left) and DRG neurons (right). Try. = trypsin. PAR₂-AP and trypsin caused an increase in cAMP levels after 1 and 5 min. n = 3 experiments. *P < 0.05 compared to non-treated cells (NT), ***P < 0.05 compared to PAR₂-RP.

Figure 6. Effects of PAR₂ agonists on capsaicin-induced Ca²⁺ signalling in HEK-TRPV1 cells

Cells were exposed to PAR₂-AP or PAR₂-RP for 5 min, and then challenged with capsaicin. A, changes in [Ca²⁺]_i, with each line the average trace from n = 31–35 cells. B, the effects of antagonists on PAR₂-induced sensitization of capsaicin responses (100%). Veh = vehicle control. *P < 0.05 compared to PAR₂-AP/vehicle cells. n = 70–80 cells from 3 experiments.

Figure 7. PKCɛ and PKA antagonists inhibit the PAR₂-mediated sensitization of TRPV1 currents in HEK-TRPV1 cells

A, whole-cell inward currents induced by capsaicin (300 nm) before (left trace) and 3 min after (right trace) application of PAR₂-AP for 2 min. B, summary of mean TRPV1 currents over time following activation of PAR₂ with PAR₂-AP (100 μm; n≥ 5 for each time point). PAR₂-AP sensitized the capsaicin-induced current in a time-dependent manner; reaching its maximum value 3 min after PAR₂-AP treatment (P < 0.01; n = 10). Membrane current values are expressed as percentage of the control current recorded prior to PAR₂-AP application (second control bar). In five control cells (first control bar), capsaicin was applied for ∼20 s and re-applied 3 min later (second control bar), demonstrating the consistency of the capsaicin response. In another five control cells, capsaicin was only applied once before application of PAR₂-AP. C, TRPV1 sensitization by PAR₂-AP (Veh = vehicle control n = 10, at 3 min) was prevented by PKCɛ (PKCɛI, 200 μm, n = 5) and PKA (H89, 3 μm, n = 5) inhibitors. *P < 0.05.

Figure 8. Mechanisms of PAR₂-induced potentiation of TRPV1-mediated thermal hyperalgesia

Compounds were injected into the paws of mice, and hyperalgesia was measured as a significant decrease in withdrawal latency in response to a thermal stimulus, compared to basal (time 0). A, effects of the PKCɛ inhibitor (PKCɛl) alone or on PAR₂-AP and capsaicin-induced hyperalgesia. B, effects of the PKA inhibitor (WIPTIDE) alone or on PAR₂-AP and capsaicin-induced hyperalgesia. Controls include use of inactive PKCɛl-sc or the WIPTIDE vehicle. n = 8 mice per group, *P < 0.05 compared to basal values.