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. 2010 Sep 22;30(38):12806-15.
doi: 10.1523/JNEUROSCI.3142-10.2010.

Low nociceptor GRK2 prolongs prostaglandin E2 hyperalgesia via biased cAMP signaling to Epac/Rap1, protein kinase Cepsilon, and MEK/ERK

Niels Eijkelkamp  1 , Huijing Wang, Anibal Garza-Carbajal, Hanneke L D M Willemen, Fried J Zwartkruis, John N Wood, Robert Dantzer, Keith W Kelley, Cobi J Heijnen, Annemieke Kavelaars
Affiliations

Low nociceptor GRK2 prolongs prostaglandin E2 hyperalgesia via biased cAMP signaling to Epac/Rap1, protein kinase Cepsilon, and MEK/ERK

Niels Eijkelkamp et al. J Neurosci. .

Abstract

Hyperexcitability of peripheral nociceptive pathways is often associated with inflammation and is an important mechanism underlying inflammatory pain. Here we describe a completely novel mechanism via which nociceptor G-protein-coupled receptor kinase 2 (GRK2) contributes to regulation of inflammatory hyperalgesia. We show that nociceptor GRK2 is downregulated during inflammation. In addition, we show for the first time that prostaglandin E2 (PGE2)-induced hyperalgesia is prolonged from <6 h in wild-type (WT) mice to 3 d in mice with low GRK2 in Nav1.8+ nociceptors (SNS-GRK2+/- mice). This prolongation of PGE2 hyperalgesia in SNS-GRK2+/- mice does not depend on changes in the sensitivity of the prostaglandin receptors because prolonged hyperalgesia also developed in response to 8-Br-cAMP. PGE2 or cAMP-induced hyperalgesia in WT mice is PKA dependent. However, PKA activity is not required for hyperalgesia in SNS-GRK2+/- mice. SNS-GRK2+/- mice developed prolonged hyperalgesia in response to the Exchange proteins directly activated by cAMP (Epac) activator 8-pCPT-2'-O-Me-cAMP (8-pCPT). Coimmunoprecipitation experiments showed that GRK2 binds to Epac1. In vitro, GRK2 deficiency increased 8-pCPT-induced activation of the downstream effector of Epac, Rap1, and extracellular signal-regulated kinase (ERK). In vivo, inhibition of MEK1 or PKCε prevented prolonged PGE2, 8-Br-cAMP, and 8-pCPT hyperalgesia in SNS-GRK2+/- mice. In conclusion, we discovered GRK2 as a novel Epac1-interacting protein. A reduction in the cellular level of GRK2 enhances activation of the Epac-Rap1 pathway. In vivo, low nociceptor GRK2 leads to prolonged inflammatory hyperalgesia via biased cAMP signaling from PKA to Epac-Rap1, ERK/PKCε pathways.

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Figures

Figure 1.
Figure 1.
Chronic carrageenan hyperalgesia is associated with reduced GRK2 in DRG neurons. A, Percentage decrease in heat-withdrawal latency time after intraplantar carrageenan injection (20 μl, 2%) in WT mice (n = 8–12 per genotype). B, GRK2 expression in dorsal root ganglia isolated 6 d after intraplantar carrageenan or saline administration was compared by immunofluorescence analysis. Representative pictures of GRK2 staining of dorsal root ganglia. C, Bar graph represents average GRK2 immunofluorescence intensity of ∼40 neurons on two to three different slides per animal (n = 4 animals per group). Data are expressed as mean ± SEM fluorescence intensity. *p < 0.05.
Figure 2.
Figure 2.
Reduced nociceptor GRK2 prolongs PGE2 hyperalgesia. A, Percentage decrease in heat-withdrawal latency time after intraplantar PGE2 injection (100 ng/paw) in SNS–GRK2 +/+ control animals and in SNS–GRK2 −/− mice (n = 8 per genotype). B, Percentage decrease in heat-withdrawal latency times after intraplantar PGE2 injection in SNS–GRK2 +/+ control animals and SNS–GRK2 +/− mice (n = 8 per genotype). C, Percentage decrease in heat-withdrawal latency time after intraplantar PGE2 injection (100 ng/paw) in WT and GRK2 +/− mice (n = 8 per genotype). D, GRK2 levels in dorsal root ganglia of inducible GRK2 +/− and WT mice after tamoxifen treatment (n = 3). E, Percentage decrease in heat-withdrawal latency time after intraplantar PGE2 injection in WT mice and inducible GRK2 +/− (n = 7–8 per genotype). F, Percentage decrease in heat-withdrawal latency time after intraplantar PGE2 injection in WT and GRK6 −/− mice (n = 7–8 per genotype). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3.
Figure 3.
PGE2 hyperalgesia is PKA independent in SNS–GRK2 +/− and GRK2 +/− mice. Percentage change in heat-withdrawal latencies after intraplantar injection of the PKA inhibitor H89 (27 μg/paw) before PGE2 injection in SNS–GRK2 +/+ (WT) animals and SNS–GRK2 +/− mice (n = 4 per group) (A) or WT and GRK2 +/− mice (n = 4 per group) (B). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 SNS–GRK2 +/+ (WT) versus SNS–GRK2 +/+ (WT) treated with inhibitor.
Figure 4.
Figure 4.
Reduced nociceptor GRK2 only affects hyperalgesia induced by an EP4 agonist. A, Percentage decrease in heat-withdrawal latency times after intraplantar sulprostrone (EP1/3 agonist) injection in SNS–GRK2 +/+ (WT) and SNS–GRK2 +/− mice (n = 8 per genotype). B, Percentage decrease in heat-withdrawal latency time after intraplantar L-902668 (EP4 agonist) injection in SNS–GRK2 +/+ control animals and in SNS–GRK2 +/− (n = 8 per genotype). Data are expressed as mean ± SEM. **p < 0.01, ***p < 0.001.
Figure 5.
Figure 5.
Reduced nociceptor GRK2 specifically prolongs cAMP- and Epac-mediated hyperalgesia but not PKA-mediated hyperalgesia. A, Percentage change in heat-withdrawal latencies after intraplantar injection of 8-Br-cAMP (10 ng/paw) in SNS–GRK2 +/+ (WT) animals and SNS–GRK2 +/− mice (n = 8 per genotype). B, Percentage change in heat-withdrawal latencies after intraplantar injection of the PKA inhibitor H89 (27 μg/paw) before 8-Br-cAMP injection in SNS–GRK2 +/+ (WT) animals and SNS–GRK2 +/− mice (n = 4 per group). C, Percentage decrease in heat-withdrawal latency time after intraplantar injection of 8-pCPT; 12.6 ng/paw) in SNS–GRK2 +/+ control animals and in SNS–GRK2 +/− (n = 12 per genotype). D, Percentage decrease in heat-withdrawal latency times after intraplantar injection of 6-Bnz-cAMP (cAMP analog that specifically activates PKA; 11.2 ng/paw) in SNS–GRK2 +/+ control mice and SNS–GRK2 +/− mice (n = 8 per genotype). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6.
Figure 6.
Low primary sensory neuron GRK2 enhances 8-pCPT-induced Rap1 and ERK1/2 activation in vitro. A, Epac1 and Epac2 expression levels in isolated primary sensory neurons of WT and GRK2 +/− mice (n = 5 per genotype). B, Rap1 expression levels in isolated primary sensory neurons of WT and GRK2 +/− mice (n = 4 per genotype). C, 8-pCPT-induced Rap1 activation in splenocytes from WT and GRK2 +/− mice. Bar graphs show quantification of Rap1 activation after 8-pCPT (100 μm) treatment (n = 4–6 per genotype). D, HA–Epac1, HA–TSC1, or GRK2 was immunoprecipitated (IP) from lysates of HEK293 cells that were transfected with HA–Epac or with HA–TSC1 as a control. 8-pCPT (100 μm) was added during the precipitation to keep Epac in an activated conformation. E, Immunoprecipitates of GRK2 from lysates of either spinal cord or DRG immunoblotted for Epac1. F, 8-pCPT-induced ERK1/2 phosphorylation in splenocytes from WT and GRK2 +/− mice. Bar graphs show quantification of activated p-ERK1/2 after 8-pCPT treatment (100 μm; n = 3–6 per genotype). G, 8-pCPT-induced (100 μm) ERK1/2 phosphorylation in primary sensory neurons of WT and GRK2 +/− mice. H, cAMP-induced (1 mm) ERK1/2 phosphorylation in primary sensory neurons of WT and GRK2 +/− mice. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01.
Figure 7.
Figure 7.
PGE2, 8-Br-cAMP, and 8-pCPT-induced hyperalgesia in SNS–GRK2 +/− mice is biased to MEK/ERK. Percentage change in heat-withdrawal latencies after intraplantar injection of the MEK inhibitor PD98059 (2.5 μg/paw) before PGE2 (A), 8-Br-cAMP (B), or 8-pCPT (C) injection in SNS–GRK2 +/+ (WT) animals and SNS–GRK2 +/− mice (n = 8 per group). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 SNS–GRK2 +/− versus SNS–GRK2 +/− treated with inhibitor. ## p < 0.01 SNS–GRK2 +/+ (WT) versus SNS–GRK2 +/+ (WT) treated with inhibitor.
Figure 8.
Figure 8.
PGE2, 8-pCPT-induced hyperalgesia in SNS–GRK2 +/− mice is biased to PKCε. Percentage change in heat-withdrawal latencies after intraplantar injection of the PKC inhibitor PKCεv1–2 (2.5 μg/paw) before PGE2 injection (A) or 8-pCPT (B) in SNS–GRK2 +/+ (WT) animals and SNS–GRK2 +/− mice (n = 4 per group). As a control, mice were treated with scrambled peptide before PGE2 injection (scr). Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 SNS–GRK2 +/− versus SNS–GRK2 +/− treated with inhibitor. # p < 0.05, ## p < 0.01 SNS–GRK2 +/+ (WT) versus SNS–GRK2 +/+ (WT) treated with inhibitor. C, Model of the role of reduced nociceptor GRK2 in prolonged PGE2 hyperalgesia. Low levels of nociceptor GRK2 promote cAMP-to-Epac signaling, whereas in nociceptor with “normal” GRK2 levels cAMP signals mainly to PKA. Additionally, low levels of GRK2 enhance ERK1/2 activation at the MEK/ERK1/2 interface. The effect of low nociceptor GRK2 levels ultimately lead to enhanced cAMP-to-Epac and ERK/PKCε signaling resulting in prolonged hyperalgesia.
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