Protease‐activated receptor 4 activation increases the expression of calcitonin gene‐related peptide mRNA and protein in dorsal root ganglion neurons

Abstract

Accumulating evidence demonstrates that nociceptor activation evokes a rapid change in mRNA and protein levels of calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons. Although the colocalization of CGRP and protease-activated receptor-4 (PAR4), a potent modulator of pain processing and inflammation, was detected in DRG neurons, the role of PAR4 activation in the expression of CGRP has not been investigated. In the present study, the expression of CGRP and activation (phosphorylation) of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in rat DRG neurons were measured by immunofluorescence, real-time PCR, and Western blotting after AYPGKF-NH2 (selective PAR4-activating peptide; PAR4-AP) intraplantar injection or treatment of cultured DRG neurons. The expression of CGRP in cultured DRG neurons was also assessed after treatment with AYPGKF-NH2 with preaddition of PD98059 (an inhibitor for ERK1/2 pathway). Results showed that PAR4-AP intraplantar injection or treatment of cultured DRG neurons evoked significant increases in DRG cells displaying CGRP immunoreactivity and cytoplasmic and nuclear staining for phospho-ERK1/2 (p-ERK1/2). Percentages of total DRG neurons expressing both CGRP and PAR4 or p-ERK1/2 also increased significantly at 2 hr after PAR4-AP treatment. Real-time PCR and Western blotting showed that PAR4-AP treatment significantly increased expression of CGRP mRNA and protein levels in DRG neurons. The PAR4 activation-evoked CGRP expression both at mRNA and at protein levels was significantly inhibited after p-ERK1/2 was inhibited by PD98059. These results provide evidence that activation of PAR4 upregulates the expression of CGRP mRNA and protein levels in DRG neurons via the p-ERK1/2 signal pathway.

New insights into protease-activated receptor 4 signaling pathways in the pathogenesis of inflammation and neuropathic pain: a literature review

“…Accumulating evidence demonstrates that nociceptor activation and inflammation evoke a rapid change in the levels of CGRP mRNA and protein in DRG neurons, [26][27][28] and CGRP expression in these neurons contributes to the regulation of neurogenic inflammation and pain. [29][30][31][32] In a previous study, the PAR4 agonist did not induce a calcium signal in DRG neurons; it reduced the calcium signal of DRG neurons in response to KCl, 16 suggesting that PAR4 activation could inhibit the nociceptive signal in DRG neurons. Furthermore, in an in vivo experiment, the PAR4 agonist was able to increase the nociceptive threshold to thermal and mechanical stimuli, and to reduce thermal and mechanical inflammatory hyperalgesia and allodynia.…”

“…The percentages of total DRG neurons expressing both CGRP and PAR4 or p-ERK1/2 also increased significantly at 2h after PAR4-AP treatment. 32,103,104 Moreover, previous studies have reported that noxious stimulation immediately increases p-ERK1/2 expression in the cytoplasm and nucleus of DRG neurons, all of which are features of small-and medium-sized nociceptive neuronal cells. 16,33,105 Furthermore, membrane depolarization and calcium influx evoked by noxious stimulation lead to ERK1/2 activation, 106 which triggers CREB-induced transcription and upregulation of CGRP mRNA levels in DRG neurons.…”

“…101,102 PAR4 activation induced by the PAR4-AP AYPGKF-NH 2 is associated with an increase in CGRP expression in DRG neurons and indicates that PAR4 activation evokes a remarkable increase in the number of CGRP-labeled neurons and CGRP mRNA and protein levels in DRG neurons. 31,32 There is a close correlation between CGRP mRNA and protein levels; therefore, it seems more likely that the effect of PAR4 on CGRP expression results from a change in transcription. 31 Several studies have shown that these rapid changes in CGRP mRNA and protein levels were evoked by nociceptor activation and inflammation.…”

“…26,28 PAR4-AP induces the upregulation of CGRP expression in primary sensory neurons via activation of the ERK1/2 signaling pathway. Notably, this upregulation was significantly inhibited after inhibition of ERK1/2 phosphorylation, 32 intraplantar injection of PAR4-AP, or treatment of cultured DRG neurons with PAR4-AP evoked a significant increase in the number of DRG cells expressing CGRP and cytoplasmic and nuclear staining for phospho-ERK1/2 (p-ERK1/2). The percentages of total DRG neurons expressing both CGRP and PAR4 or p-ERK1/2 also increased significantly at 2h after PAR4-AP treatment.…”

Protease-Activated Receptor 4: A Critical Participator in Inflammatory Response

“…PAR4-AP evokes the overexpression of CGRP via activation of the ERK1/2 signaling pathway, and PAR4 activation notably enhances the levels of CGRP messenger RNA (mRNA) and protein in DRG neurons in the presence of AYPGKF-NH2 both in vivo and in vitro [72]. Remarkably, this overexpression is significantly attenuated after inhibiting ERK1/2 phosphorylation.…”