Nigel W. Bunnett

Proteinase-activated receptors: novel mechanisms of signaling by serine proteases

Although serine proteases are usually considered to act principally as degradative enzymes, certain proteases are signaling molecules that specifically regulate cells by cleaving and triggering members of a new family of proteinase-activated receptors (PARs). There are three members of this family, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, a receptor for trypsin and mast cell tryptase. Proteases cleave within the extracellular NH2-terminus of their receptors to expose a new NH2-terminus. Specific residues within this tethered ligand domain interact with extracellular domains of the cleaved receptor, resulting in activation. In common with many G protein-coupled receptors, PARs couple to multiple G proteins and thereby activate many parallel mechanisms of signal transduction. PARs are expressed in multiple tissues by a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation. Because the cleaved receptor is physically coupled to its agonist, efficient mechanisms exist to terminate signaling and prevent uncontrolled stimulation. These include cleavage of the tethered ligand, receptor phosphorylation and uncoupling from G proteins, and endocytosis and lysosomal degradation of activated receptors.

4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1

TRPA1 is an excitatory ion channel expressed by a subpopulation of primary afferent somatosensory neurons that contain substance P and calcitonin gene-related peptide. Environmental irritants such as mustard oil, allicin, and acrolein activate TRPA1, causing acute pain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also activated downstream of one or more proalgesic agents that stimulate phospholipase C signaling pathways, thereby implicating this channel in peripheral mechanisms controlling pain hypersensitivity. However, it is not known whether tissue injury also produces endogenous proalgesic factors that activate TRPA1 directly to augment inflammatory pain. Here, we report that recombinant or native TRPA1 channels are activated by 4-hydroxy-2-nonenal (HNE), an endogenous α,β-unsaturated aldehyde that is produced when reactive oxygen species peroxidate membrane phospholipids in response to tissue injury, inflammation, and oxidative stress. HNE provokes release of substance P and calcitonin gene-related peptide from central (spinal cord) and peripheral (esophagus) nerve endings, resulting in neurogenic plasma protein extravasation in peripheral tissues. Moreover, injection of HNE into the rodent hind paw elicits pain-related behaviors that are inhibited by TRPA1 antagonists and absent in animals lacking functional TRPA1 channels. These findings demonstrate that HNE activates TRPA1 on nociceptive neurons to promote acute pain, neuropeptide release, and neurogenic inflammation. Our results also provide a mechanism-based rationale for developing novel analgesic or anti-inflammatory agents that target HNE production or TRPA1 activation.

Role for protease activity in visceral pain in irritable bowel syndrome

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

Protease-Activated Receptor 2 Sensitizes the Capsaicin Receptor Transient Receptor Potential Vanilloid Receptor 1 to Induce Hyperalgesia

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Protease-activated receptors in inflammation, neuronal signaling and pain.

The ability of proteases to regulate cell function via protease-activated receptors (PARs) has led to new insights about the potential physiological functions of these enzymes. Several studies suggest that PARs play roles in both inflammation and tissue repair, depending on the cellular environment in which they act. The recent detection of PARs on peripheral and central neurons suggests that neuronal PARs might be involved not only in neurogenic inflammation and neurodegenerative processes, but also in nociception. Thus, the list of potential roles for PARs has lengthened considerably and their physiological course of action might be much broader than initially anticipated.

Induction of Intestinal Inflammation in Mouse by Activation of Proteinase-Activated Receptor-2

Proteinase-activated receptor (PAR)-2, a G-protein-coupled receptor for trypsin and mast cell tryptase, is highly expressed in the intestine. Luminal trypsin and tryptase are elevated in the colon of inflammatory bowel disease patients. We hypothesized that luminal proteinases activate PAR-2 and induce colonic inflammation. Mice received intracolonically PAR-2 agonists (trypsin, tryptase, and a selective PAR-2-activating peptide) or control drugs (boiled enzymes, inactive peptide) and inflammatory parameters were followed at various times after this treatment. Colonic administration of PAR-2 agonists up-regulated PAR-2 expression and induced an inflammatory reaction characterized by granulocyte infiltration, increased wall thickness, tissue damage, and elevated T-helper cell type 1 cytokine. The inflammation was maximal between 4 and 6 hours and was resolved 48 hours after the intracolonic administration. PAR-2 activation also increased paracellular permeability of the colon and induced bacterial trans-location into peritoneal organs. These proinflammatory and pathophysiological changes observed in wild-type mice were not detected in PAR-2-deficient mice. Luminal proteinases activate PAR-2 in the mouse colon to induce inflammation and disrupt the integrity of the intestinal barrier. Because trypsin and tryptase are found at high levels in the colon lumen of patients with Crohns disease or ulcerative colitis, our data may bear directly on the pathophysiology of human inflammatory bowel diseases.

Neuropeptides in the skin: interactions between the neuroendocrine and the skin immune systems

Abstract: The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. It has been observed that certain skin diseases such as psoriasis and atopic dermatitis have a neurogenic component. Neuropeptides released by sensory nerves that innervate the skin and often contact epidermal and dermal cells can directly modulate functions of keratinocytes, Langerhans cells (LC), mast cells, dermal microvascular endothelial cells and infiltrating immune cells. Among these neuropeptides the tachykinins substance P (SP) and neurokinin A (NKA), calcitonin gene related peptide (CGRP), vasoactive intestinal peptide (VIP) and somato statin (SOM) have been reported to effectively modulate skin and immune cell functions such as cell proliferation, cytokine production or antigen presentation under physiological or pathophysiological conditions. Expression and regulation of their corresponding receptors that are expressed on a variety of skin cells as well as the presence of neuropeptidespecific peptidases such as neutral endopeptidase (NEP) or angiotensinconverting enzyme (ACE) determine the final biological response mediated by these peptides on the target cell or tissue. Likewise, skin cells like keratinocytes or fibroblasts are a source for neurotrophins such as nerve growth factor that are required not only for survival and regeneration of sensory neurons but also to control responsiveness of these neurons to external stimuli. Therefore, neuropeptides, neuropeptide receptors, neuropeptidedegrading enzymes and neurotrophins participate in a complex, interdependent network of mediators that modulate skin inflammation, wound healing and the skin immune system. This review will focus on recent studies demonstrating the role of tachykinins, CGRP, SOM and VIP and their receptors and neuropeptide‐degrading enzymes in mediating neurogenic inflammation in the skin.

Protease-Activated Receptor 2 Mediates Eosinophil Infiltration and Hyperreactivity in Allergic Inflammation of the Airway

Trypsin and mast cell tryptase can signal to epithelial cells, myocytes, and nerve fibers of the respiratory tract by cleaving proteinase-activated receptor 2 (PAR2). Since tryptase inhibitors are under development to treat asthma, a precise understanding of the contribution of PAR2 to airway inflammation is required. We examined the role of PAR2 in allergic inflammation of the airway by comparing OVA-sensitized and -challenged mice lacking or overexpressing PAR2. In wild-type mice, immunoreactive PAR2 was detected in airway epithelial cells and myocytes, and intranasal administration of a PAR2 agonist stimulated macrophage infiltration into bronchoalveolar lavage fluid. OVA challenge of immunized wild-type mice stimulated infiltration of leukocytes into bronchoalveolar lavage and induced airway hyperreactivity to inhaled methacholine. Compared with wild-type animals, eosinophil infiltration was inhibited by 73% in mice lacking PAR2 and increased by 88% in mice overexpressing PAR2. Similarly, compared with wild-type animals, airway hyperreactivity to inhaled methacholine (40 μg/ml) was diminished 38% in mice lacking PAR2 and increased by 52% in mice overexpressing PAR2. PAR2 deletion also reduced IgE levels to OVA sensitization by 4-fold compared with those of wild-type animals. Thus, PAR2 contributes to the development of immunity and to allergic inflammation of the airway. Our results support the proposal that tryptase inhibitors and PAR2 antagonists may be useful therapies for inflammatory airway disease.

Protease‐activated receptor 2 sensitizes the transient receptor potential vanilloid 4 ion channel to cause mechanical hyperalgesia in mice

Exacerbated sensitivity to mechanical stimuli that are normally innocuous or mildly painful (mechanical allodynia and hyperalgesia) occurs during inflammation and underlies painful diseases. Proteases that are generated during inflammation and disease cleave protease‐activated receptor 2 (PAR2) on afferent nerves to cause mechanical hyperalgesia in the skin and intestine by unknown mechanisms. We hypothesized that PAR2‐mediated mechanical hyperalgesia requires sensitization of the ion channel transient receptor potential vanilloid 4 (TRPV4). Immunoreactive TRPV4 was coexpressed by rat dorsal root ganglia (DRG) neurons with PAR2, substance P (SP) and calcitonin gene‐related peptide (CGRP), mediators of pain transmission. In PAR2‐expressing cell lines that either naturally expressed TRPV4 (bronchial epithelial cells) or that were transfected to express TRPV4 (HEK cells), pretreatment with a PAR2 agonist enhanced Ca2+ and current responses to the TRPV4 agonists phorbol ester 4α‐phorbol 12,13‐didecanoate (4αPDD) and hypotonic solutions. PAR2‐agonist similarly sensitized TRPV4 Ca2+ signals and currents in DRG neurons. Antagonists of phospholipase Cβ and protein kinases A, C and D inhibited PAR2‐induced sensitization of TRPV4 Ca2+ signals and currents. 4αPDD and hypotonic solutions stimulated SP and CGRP release from dorsal horn of rat spinal cord, and pretreatment with PAR2 agonist sensitized TRPV4‐dependent peptide release. Intraplantar injection of PAR2 agonist caused mechanical hyperalgesia in mice and sensitized pain responses to the TRPV4 agonists 4αPDD and hypotonic solutions. Deletion of TRPV4 prevented PAR2 agonist‐induced mechanical hyperalgesia and sensitization. This novel mechanism, by which PAR2 activates a second messenger to sensitize TRPV4‐dependent release of nociceptive peptides and induce mechanical hyperalgesia, may underlie inflammatory hyperalgesia in diseases where proteases are activated and released.

Cigarette smoke-induced neurogenic inflammation is mediated by α,β-unsaturated aldehydes and the TRPA1 receptor in rodents

Cigarette smoke (CS) inhalation causes an early inflammatory response in rodent airways by stimulating capsaicin-sensitive sensory neurons that express transient receptor potential cation channel, subfamily V, member 1 (TRPV1) through an unknown mechanism that does not involve TRPV1. We hypothesized that 2 alpha,beta-unsaturated aldehydes present in CS, crotonaldehyde and acrolein, induce neurogenic inflammation by stimulating TRPA1, an excitatory ion channel coexpressed with TRPV1 on capsaicin-sensitive nociceptors. We found that CS aqueous extract (CSE), crotonaldehyde, and acrolein mobilized Ca2+ in cultured guinea pig jugular ganglia neurons and promoted contraction of isolated guinea pig bronchi. These responses were abolished by a TRPA1-selective antagonist and by the aldehyde scavenger glutathione but not by the TRPV1 antagonist capsazepine or by ROS scavengers. Treatment with CSE or aldehydes increased Ca2+ influx in TRPA1-transfected cells, but not in control HEK293 cells, and promoted neuropeptide release from isolated guinea pig airway tissue. Furthermore, the effect of CSE and aldehydes on Ca2+ influx in dorsal root ganglion neurons was abolished in TRPA1-deficient mice. These data identify alpha,beta-unsaturated aldehydes as the main causative agents in CS that via TRPA1 stimulation mediate airway neurogenic inflammation and suggest a role for TRPA1 in the pathogenesis of CS-induced diseases.