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.

Evidence for an association with type 2 diabetes mellitus at the PPARG locus in a South Indian population

Peroxisome proliferator-activated receptor-gamma2 (PPARG2) is a nuclear hormone receptor of ligand-dependent transcription factor involved in adipogenesis and a molecular target of the insulin sensitizers thiazolidinediones. We addressed the question of whether the 3 variants (-1279G/A, Pro12Ala, and His478His) in the PPARG2 gene are associated with type 2 diabetes mellitus and its related traits in a South Indian population. The study subjects (1000 type 2 diabetes mellitus and 1000 normal-glucose-tolerant subjects) were chosen randomly from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The variants were screened by single-stranded conformational variant, direct sequencing, and restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. The -1279G/A, Pro12Ala, and His478His variants of the PPARG2 gene were not associated with type 2 diabetes mellitus. However, the 2-loci analyses showed that, in the presence of Pro/Pro genotype of the Pro12Ala variant, the -1279G/A promoter variant showed increased susceptibility to type 2 diabetes mellitus (odds ratio, 2.092; 95% confidence interval, 1.22-3.59; P = .008), whereas in the presence of 12Ala allele, the -1279G/A showed a protective effect against type 2 diabetes mellitus (odds ratio, 0.270; 95% confidence interval, 0.15-0.49; P < .0001). The 3-loci haplotype analysis showed that the A-Ala-T (-1279G/A-Pro12Ala-His478His) haplotype was associated with a reduced risk of type 2 diabetes mellitus (P < .0001). Although our data indicate that the PPARG2 gene variants, independently, have no association with type 2 diabetes mellitus, the 2-loci genotype analysis involving -1279G/A and Pro12Ala variants and the 3-loci haplotype analysis have shown a significant association with type 2 diabetes mellitus in this South Indian population.

CLEC-2 activates Syk through dimerization

The C-type lectin receptor CLEC-2 activates platelets through Src and Syk tyrosine kinases, leading to tyrosine phosphorylation of downstream adapter proteins and effector enzymes, including phospholipase-C gamma2. Signaling is initiated through phosphorylation of a single conserved tyrosine located in a YxxL sequence in the CLEC-2 cytosolic tail. The signaling pathway used by CLEC-2 shares many similarities with that used by receptors that have 1 or more copies of an immunoreceptor tyrosine-based activation motif, defined by the sequence Yxx(L/I)x(6-12)Yxx(L/I), in their cytosolic tails or associated receptor chains. Phosphorylation of the conserved immunoreceptor tyrosine-based activation motif tyrosines promotes Syk binding and activation through binding of the Syk tandem SH2 domains. In this report, we present evidence using peptide pull-down studies, surface plasmon resonance, quantitative Western blotting, tryptophan fluorescence measurements, and competition experiments that Syk activation by CLEC-2 is mediated by the cross-linking through the tandem SH2 domains with a stoichiometry of 2:1. In support of this model, cross-linking and electron microscopy demonstrate that CLEC-2 is present as a dimer in resting platelets and converted to larger complexes on activation. This is a unique mode of activation of Syk by a single YxxL-containing receptor.

GPVI and CLEC-2 in hemostasis and vascular integrity

The glycoprotein VI (GPVI)-FcR gamma-chain complex initiates powerful activation of platelets by the subendothelial matrix proteins collagen and laminin through an immunoreceptor tyrosine-based activation motif (ITAM)-regulated signaling pathway. ITAMs are characterized by two YxxL sequences separated by 6-12 amino acids and are found associated with several classes of immunoglobulin (Ig) and C-type lectin receptors in hematopoietic cells, including Fc receptors. Cross-linking of the Ig GPVI leads to phosphorylation of two conserved tyrosines in the FcR gamma-chain ITAM by Src family tyrosine kinases, followed by binding and activation of the tandem SH2 domain-containing Syk tyrosine kinase and stimulation of a downstream signaling cascade that culminates in activation of phospholipase Cgamma2 (PLCgamma2). In contrast, the C-type lectin receptor CLEC-2 mediates powerful platelet activation through Src and Syk kinases, but regulates Syk through a novel dimerization mechanism via a single YxxL motif known as a hemITAM. CLEC-2 is a receptor for podoplanin, which is expressed at high levels in several tissues, including type 1 lung alveolar cells, lymphatic endothelial cells, kidney podocytes and some tumors, but is absent from vascular endothelial cells and platelets. In this article, we compare the mechanism of platelet activation by GPVI and CLEC-2 and consider their functional roles in hemostasis and other vascular processes, including maintenance of vascular integrity, angiogenesis and lymphogenesis.