Protein kinase D isoforms are expressed in rat and mouse primary sensory neurons and are activated by agonists of protease‐activated receptor 2

Abstract

Serine proteases generated during injury and inflammation cleave protease-activated receptor 2 (PAR 2 ) on primary sensory neurons to induce neurogenic inflammation and hyperalgesia. Hyperalgesia requires sensitization of transient receptor potential vanilloid (TRPV) ion channels by mechanisms involving phospholipase C and protein kinase C (PKC). The protein kinase D (PKD) serine/threonine kinases are activated by diacylglycerol and PKCs, and can phosphorylate TRPV1. Thus, PKDs may participate in novel signal transduction pathways triggered by serine proteases during inflammation and pain. However, it is not known whether PAR 2 activates PKD, and the expression of PKD isoforms by nociceptive neurons is poorly characterized. Using HEK293 cells transfected with PKDs, we found that PAR 2 stimulation promoted plasma membrane translocation and phosphorylation of PKD1, PKD2 and PKD3, indicating activation. This effect was partially dependent on PKCε. Using immunofluorescence and confocal microscopy, with antibodies against PKD1/PKD2, PKD3 and neuronal markers, we found that PKDs were expressed in rat and mouse dorsal root ganglia (DRG) neurons, including nociceptive neurons that expressed TRPV1, PAR 2 and neuropeptides. PAR 2 agonist induced phosphorylation of PKD in cultured DRG neurons, indicating PKD activation. Intraplantar injection of PAR 2 agonist also caused phosphorylation of PKD in neurons of lumbar DRG, confirming activation in vivo. Thus, PKD1, PKD2 and PKD3 are expressed in primary sensory neurons that mediate neurogenic inflammation and pain transmission, and PAR 2 agonists activate PKDs in HEK293 cells and DRG neurons in culture and in intact animals. PKD may be a novel component of a signal transduction pathway for protease-induced activation of nociceptive neurons and an important new target for anti-inflammatory and analgesic therapies.

Feeding-dependent activation of enteric cells and sensory neurons by lymphatic fluid: evidence for a neurolymphocrine system

“…Soybean trypsin inhibitor (Sigma) was added to neutralize trypsin. Neurons were pelleted, suspended in DMEM containing 10% FBS, 10% horse serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine, plated on glass coverslips coated with poly-L-lysine and laminin, and cultured for 2-3 days before use in the studies as previously described (1).…”

The G Protein–Coupled Receptor–Transient Receptor Potential Channel Axis: Molecular Insights for Targeting Disorders of Sensation and Inflammation

“…Similarly, small PKC sequence peptides fused to membrane-permeating TAT sequences can selectively inhibit specific PKC isoforms and have been shown to reduce ischemic heart damage (Chen et al, 2001). Although these kinase inhibitors may selectively prevent inflammatory pathways (e.g., PKC« is critical for PAR 2 sensitization of TRPV1), penetrating peptides directed toward the b-strands, linker region, or ankyrins may similarly affect interaction sites in TRP channels (Amadesi et al, 2009).…”

Neutrophil Elastase Activates Protease-activated Receptor-2 (PAR2) and Transient Receptor Potential Vanilloid 4 (TRPV4) to Cause Inflammation and Pain

“…Trypsin-activated PAR 2 couples to G␣ q , leading to mobilization of intracellular Ca 2ϩ and activation of second messenger kinases such as protein kinase C (PKC) and D (PKD) (8,13,14). Trypsin-activated PAR 2 also recruits G protein receptor kinase-2 (GRK2) and ␤-arrestins, which uncouple PAR 2 from G proteins and mediate clathrin-and dynamin-dependent receptor endocytosis (15,16).…”