Protease-Activated Receptor 2 (PAR2) Expressed in Sensory Neurons Contributes to Signs of Pain and Neuropathy in Paclitaxel Treated Mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common, dose-limiting side effect of cancer therapy. Protease-activated receptor 2 (PAR2) is implicated in a variety of pathologies, including CIPN. In this study, we demonstrate the role of PAR2 expressed in sensory neurons in a paclitaxel (PTX)-induced model of CIPN in mice. PAR2 knockout/wildtype (WT) mice and mice with PAR2 ablated in sensory neurons were treated with PTX administered via intraperitoneal injection. In vivo behavioral studies were done in mice using von Frey filaments and the Mouse Grimace Scale. We then examined immunohistochemical staining of dorsal root ganglion (DRG) and hind paw skin samples from CIPN mice to measure satellite cell gliosis and intra-epidermal nerve fiber (IENF) density. The pharmacological reversal of CIPN pain was tested with the PAR2 antagonist C781. Mechanical allodynia caused by PTX treatment was alleviated in PAR2 knockout mice of both sexes. In the PAR2 sensory neuronal conditional knockout (cKO) mice, both mechanical allodynia and facial grimacing were attenuated in mice of both sexes. In the DRG of the PTX-treated PAR2 cKO mice, satellite glial cell activation was reduced compared to control mice. IENF density analysis of the skin showed that the PTX-treated control mice had a reduction in nerve fiber density while the PAR2 cKO mice had a comparable skin innervation as the vehicle-treated animals. Similar results were seen with satellite cell gliosis in the DRG, where gliosis induced by PTX was absent in PAR cKO mice. Finally, C781 was able to transiently reverse established PTX-evoked mechanical allodynia. PERSPECTIVE: Our work demonstrates that PAR2 expressed in sensory neurons plays a key role in PTX-induced mechanical allodynia, spontaneous pain, and signs of neuropathy, suggesting PAR2 as a possible therapeutic target in multiple aspects of PTX CIPN.

Alternaria alternata-induced airway epithelial signaling and inflammatory responses via protease-activated receptor-2 expression.

Inhalation of the fungus Alternaria alternata is associated with an increased risk of allergic asthma development and exacerbations. Recent work in acute exposure animal models suggests that A. alternata-induced asthma symptoms, which include inflammation, mucus overproduction and airway hyperresponsiveness, are due to A. alternata proteases that act via protease-activated receptor-2 (PAR2). However, because other active components present in A. alternata may be contributing to asthma pathophysiology through alternative signaling, the specific role PAR2 plays in asthma initiation and maintenance remains undefined. Airway epithelial cells provide the first encounter with A. alternata and are thought to play an important role in initiating the physiologic response. To better understand the role for PAR2 airway epithelial signaling we created a PAR2-deficient human bronchial epithelial cell line (16HBEPAR-/-) from a model bronchial parental line (16HBE14o-). Comparison of in vitro physiologic responses in these cell lines demonstrated a complete loss of PAR2 agonist (2at-LIGRL-NH2) response and significantly attenuated protease (trypsin and elastase) and A. alternata responses in the 16HBEPAR-/- line. Apical application of A. alternata to 16HBE14o- and 16HBEPAR2-/- grown at air-liquid interface demonstrated rapid, PAR2-dependent and independent, inflammatory cytokine, chemokine and growth factor basolateral release. In conclusion, the novel human PAR2-deficient cell line allows for direct in vitro examination of the role(s) for PAR2 in allergen challenge with polarized human airway epithelial cells.

Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2).

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of ß-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

Beta-arrestins as regulators of signal termination and transduction: how do they determine what to scaffold?

Over the last decade ß-arrestins have emerged as pleiotropic scaffold proteins, capable of mediating numerous diverse responses to multiple agonists. Most well characterized are the G-protein-coupled receptor (GPCR) stimulated ß-arrestin signals, which are sometimes synergistic with, and sometimes independent of, heterotrimeric G-protein signals. ß-arrestin signaling involves the recruitment of downstream signaling moieties to ß-arrestins; in many cases specific sites of interaction between ß-arrestins and the downstream target have been identified. As more information unfolds about the nature of ß-arrestin scaffolding interactions, it is evident that these proteins are capable of adopting multiple conformations which in turn reveal a specific set of interacting domains. Recruitment of ß-arrestin to a specific GPCR can promote formation of a specific subset of available ß-arrestin scaffolds, allowing for a higher level of specificity to given agonists. This review discusses recent advances in ß-arrestin signaling, discussing the molecular details of a subset of known ß-arrestin scaffolds and the significance of specific binding interactions on the ultimate cellular response.

Beta-arrestin inhibits CAMKKbeta-dependent AMPK activation downstream of protease-activated-receptor-2.

BACKGROUND: Proteinase-activated-receptor-2 (PAR2) is a seven transmembrane receptor that can activate two separate signaling arms: one through Gαq and Ca2+ mobilization, and a second through recruitment of ß-arrestin scaffolds. In some cases downstream targets of the Gαq/Ca2+ signaling arm are directly inhibited by ß-arrestins, while in other cases the two pathways are synergistic; thus ß-arrestins act as molecular switches capable of modifying the signal generated by the receptor. RESULTS: Here we demonstrate that PAR2 can activate adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent Kinase Kinase ß (CAMKKß), while inhibiting AMPK through interaction with ß-arrestins. The ultimate outcome of PAR2 activation depended on the cell type studied; in cultured fibroblasts with low endogenous ß-arrestins, PAR2 activated AMPK; however, in primary fat and liver, PAR2 only activated AMPK in ß-arrestin-2-/- mice. ß-arrestin-2 could be co-immunoprecipitated with AMPK and CAMKKß under baseline conditions from both cultured fibroblasts and primary fat, and its association with both proteins was increased by PAR2 activation. Addition of recombinant ß-arrestin-2 to in vitro kinase assays directly inhibited phosphorylation of AMPK by CAMKKß on Thr172. CONCLUSIONS: Studies have shown that decreased AMPK activity is associated with obesity and Type II Diabetes, while AMPK activity is increased with metabolically favorable conditions and cholesterol lowering drugs. These results suggest a role for ß-arrestin in the inhibition of AMPK signaling, raising the possibility that ß-arrestin-dependent PAR2 signaling may act as a molecular switch turning a positive signal to AMPK into an inhibitory one.

beta-Arrestins scaffold cofilin with chronophin to direct localized actin filament severing and membrane protrusions downstream of protease-activated receptor-2.

Protease-activated receptor-2 (PAR-2) mediates pro-inflammatory signals in a number of organs, including enhancing leukocyte recruitment to sites of injury and infection. At the cellular level, PAR-2 promotes activation of the actin filament-severing protein cofilin, which is crucial for the reorganization of the actin cytoskeleton and chemotaxis. These responses require the scaffolding functions of beta-arrestins; however, the mechanism by which beta-arrestins spatially regulate cofilin activity and the role of this pathway in primary cells has not been investigated. Here, using size-exclusion chromatography and co-immunoprecipitation, we demonstrate that PAR-2 promotes the formation of a complex containing beta-arrestins, cofilin, and chronophin (CIN) in primary leukocytes and cultured cells. Both association of cofilin with CIN and cell migration are inhibited in leukocytes from beta-arrestin-2(-/-) mice. We show that, in response to PAR-2 activation, beta-arrestins scaffold cofilin with its upstream activator CIN, to facilitate the localized generation of free actin barbed ends, leading to membrane protrusion. These studies suggest that a major role of beta-arrestins in chemotaxis is to spatially regulate cofilin activity to facilitate the formation of a leading edge, and that this pathway may be important for PAR-2-stimulated immune cell migration.

Beta-arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2.

Beta-arrestins are pleiotropic molecules that mediate signal desensitization, G-protein-independent signaling, scaffolding of signaling molecules, and chemotaxis. Protease-activated receptor-2 (PAR-2), a Galpha(q/11)-coupled receptor, which has been proposed as a therapeutic target for inflammation and cancer, requires the scaffolding function of beta-arrestins for chemotaxis. We hypothesized that PAR-2 can trigger specific responses by differential activation of two pathways, one through classic Galpha(q)/Ca(2+) signaling and one through beta-arrestins, and we proposed that the latter involves scaffolding of proteins involved in cell migration and actin assembly. Here we demonstrate the following. (a) PAR-2 promotes beta-arrestin-dependent dephosphorylation and activation of the actin filament-severing protein (cofilin) independently of Galpha(q)/Ca(2+) signaling. (b) PAR-2-evoked cofilin dephosphorylation requires both the activity of a recently identified cofilin-specific phosphatase (chronophin) and inhibition of LIM kinase (LIMK) activity. (c) Beta-arrestins can interact with cofilin, LIMK, and chronophin and colocalize with them in membrane protrusions, suggesting that beta-arrestins may spatially regulate their activities. These findings identify cofilin as a novel target of beta-arrestin-dependent scaffolding and suggest that many PAR-2-induced processes may be independent of Galpha(q/11) protein coupling.