- Research Article
- Open Access
G Protein-Coupled Receptor Kinase 6 Acts as a Critical Regulator of Cytokine-Induced Hyperalgesia by Promoting Phosphatidylinositol 3-Kinase and Inhibiting p38 Signaling
© The Author(s) 2012
- Received: 18 October 2011
- Accepted: 3 February 2012
- Published: 6 February 2012
The molecular mechanisms determining magnitude and duration of inflammatory pain are still unclear. We assessed the contribution of G protein-coupled receptor kinase (GRK)-6 to inflammatory hyperalgesia in mice. We showed that GRK6 is a critical regulator of severity and duration of cytokine-induced hyperalgesia. In GRK6−/− mice, a significantly lower dose (100 times lower) of intraplantar interleukin (IL)-1β was sufficient to induce hyperalgesia compared with wild-type (WT) mice. In addition, IL-1β hyperalgesia lasted much longer in GRK6−/− mice than in WT mice (8 d in GRK6−/− versus 6 h in WT mice). Tumor necrosis factor (TNF)-α-induced hyperalgesia was also enhanced and prolonged in GRK6−/− mice. In vitro, IL-1β-induced p38 phosphorylation in GRK6−/− dorsal root ganglion (DRG) neurons was increased compared with WT neurons. In contrast, IL-1β only induced activation of the phosphatidylinositol (PI) 3-kinase/Akt pathway in WT neurons, but not in GRK6−/− neurons. In vivo, p38 inhibition attenuated IL-1β- and TNF-α-induced hyperalgesia in both genotypes. Notably, however, whereas PI 3-kinase inhibition enhanced and prolonged hyperalgesia in WT mice, it did not have any effect in GRK6-deficient mice. The capacity of GRK6 to regulate pain responses was also apparent in carrageenan-induced hyperalgesia, since thermal and mechanical hypersensitivity was significantly prolonged in GRK6−/− mice. Finally, GRK6 expression was reduced in DRGs of mice with chronic neuropathic or inflammatory pain. Collectively, these findings underline the potential role of GRK6 in pathological pain. We propose the novel concept that GRK6 acts as a kinase that constrains neuronal responsiveness to IL-1β and TNF-α and cytokine-induced hyperalgesia via biased cytokine-induced p38 and PI 3-kinase/Akt activation.
Proper functioning of the nociceptive system is essential to protect the body from tissue damage. Inflammation sensitizes the nociceptive system, leading to a lower threshold to painful stimuli (hyperalgesia) (1). This process is thought to serve adaptive purposes, but becomes maladaptive when hyperalgesia persists after resolution of inflammation. The proinflammatory cytokine interleukin (IL)-1β directly sensitizes nociceptors, leading to transient hyperalgesia (2,3). Peripheral injection of other inflammatory mediators such as prostaglandin E2 (PGE2) also increases the sensitivity of nociceptors and the response to painful stimuli (4). Moreover, there is evidence that proinflammatory cytokines such as IL-1β and tumor necrosis factor (TNF)-α contribute to the genesis of neuropathic pain (5,6).
Many of the signals involved in inflammatory hyperalgesia are generated via activation of G protein-coupled receptors (GPCRs) expressed in sensory neurons. The activity of GPCRs is regulated by the family of GPCR kinases (G protein-coupled receptor kinase [GRK] 1–7). Agonist-activated GPCRs are phosphorylated by GRKs, inducing rapid uncoupling from the G protein, a process called homologous receptor desensitization. GRK-mediated GPCR phosphorylation facilitates binding of arrestin proteins, promoting GPCR internalization (7,8). GRKs are also capable of interacting with a variety of downstream signaling molecules, thereby regulating cellular signaling independently of GPCRs (8,9).
GRK6 plays a crucial role in inflammatory pathologies. For example, GRK6 deficiency increases acute inflammatory arthritis as well as colitis in male mice (10,11). Recently, we showed that post-inflammatory visceral hyperalgesia is enhanced in female GRK6−/− mice without affecting inflammation (12). This previous study indicated that GRK6 plays a role in regulating visceral post-inflammatory pain, but did not give insight into the mechanisms involved.
Although most of the substrates of GRK6 are probably still unknown, it has been shown that GRK6 regulates desensitization of the chemokine receptor CXCR4, the BLT1 receptor for the leukotriene B4 (LTB4) and the calcitonin gene-related peptide (CGRP) receptor (13–16). Furthermore, GRK6 binds and phosphorylates PDZ domains in Na+/H+ exchanger regulatory factor (NHERF) and binds to downstream regulatory element antagonistic modulator (DREAM), both regulators of ion channels, indicating that GRK6 can also regulate cellular signaling via mechanisms independent of GPCR desensitization (17,18).
We aimed to determine the contribution of GRK6 to somatic inflammatory hyperalgesia. As a model, we induced local hyperalgesia by a single injection of carrageenan or the proinflammatory cytokines IL-1β or TNF-α into the paw. In search of the mechanism via which GRK6 regulates hyperalgesia, we analyzed the consequences of GRK6 deficiency for cytokine signaling to p38 and PI 3-kinase.
Female GRK6-deficient C57BL/6 and wild-type (WT) control littermates were bred in the Utrecht University Central Animal Facility (12) and genotyped by polymerase chain reaction (PCR) analysis on genomic DNA. All experiments were performed in accordance with international guidelines and approved by the University Medical Center Utrecht experimental animal committee or were approved under the United Kingdom Home Office Animals (Scientific Procedures) Act 1986.
Induction of Cytokine-Induced Thermal Hyperalgesia
Mice received an intraplantar injection of 5 µL 1% λ-carrageenan (Sigma-Aldrich, St. Louis, MO, USA), 5 µL recombinant mouse IL-1β (0.2–200 ng/mL; Preprotech, Rocky Hill, NC, USA) or TNF-α (20 ng/mL; Preprotech) in saline or 5 µL saline as a control (19).
Heat withdrawal latency times were determined using the Hargreaves test (IITC Life Science, Woodland Hills, CA, USA) as described (20). Mechanical allodynia was measured using von Frey hairs (Stoelting, Wood Dale, IL, USA), and the 50% paw withdrawal threshold was calculated using the up-and-down method (21). The observers were blinded to genotype.
The p38 inhibitor SB239063 (5 µg/paw; Sigma-Aldrich) or the PI 3-kinase inhibitor LY249002 (10 µg/paw; Sigma-Aldrich) was injected intraplantarly, 20 min before cytokine injection (22).
Chronic Pain Models
L5 transection (neuropathic). In isoflurane anesthetized mice, the transverse processes at the L4-S1 levels was accessed, the L5 transverse process was removed and the left L5 spinal nerve was cut. Dorsal root ganglions (DRGs) were isolated 4 wks later.
Carrageenan (inflammatory). Carrageenan (2%, 20 µL; Sigma-Aldrich) was injected in both hindpaws. DRGs were isolated 6 d later.
Dorsal Root Ganglia Cell Culture
DRGs were digested in collagenase (Type XI, 0.6 mg/mL; Sigma-Aldrich), protease (Streptomycis Griseus, 0.4 mg/mL; Sigma-Aldrich) and glucose (1.8 mg/mL; Sigma-Aldrich) in Ca2+ and Mg2+-free phosphate-buffered saline. Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Gibco), 2 mmol/L glutamine (Gibco), 10,000 IU/mL penicillin-streptomycin (Gibco) and 100 ng/mL nerve growth factor (NGF) (Sigma-Aldrich) and poly-L-lysine- and laminin-coated wells. Cells were stimulated with IL-1β for 5 min 15–25 h after plating.
Western Blot Analysis
Cells were homogenized in lysis buffer (200 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 10% glycerol, 1% NP-40, 2mmol/L sodium orthovanadate, 2mmol/L phenylmethylsulfonyl fluoride [PMSF], 2 µmol/L leupeptin, protease inhibitor mix [p3840, 1:200; Sigma-Aldrich]). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Blots were stained with rabbit-anti-p-p38, rabbit-anti-p38, rabbit-anti-p-Akt and rabbit-anti-Akt (Cell Signaling Technology Inc., Danvers, MA, USA) followed by goat anti-mouse-peroxidase (Jackson Laboratories) or donkey anti-rabbit-peroxidase (Amersham International) and developed by enhanced chemiluminescence plus (Amersham International). Band density was quantified using a GS-700 Imaging Densitometer (Bio-Rad, Hercules, CA, USA).
mRNA Isolation and Real-Time PCR
Lumbar DRGs were homogenized in Trizol (Invitrogen, Paisley, UK). Total RNA was isolated with RNeasy Mini Kit (Qiagen) and reverse-transcribed using an iScript™ Select cDNA Synthesis Kit (Invitrogen). Real-time quantitative PCR was performed with an iQ™ SYBR® Green Supermix (Invitrogen). Primer pairs used were as follows:
GRK6: CTTGG TCTCA TAGGC GTAGG (forward), GCGGA TAAAG AAGCG AAAGG (reverse); GRK2: CGGGACTTC TGCCT GAACC ATCTG (forward), CTCGG CTGCG GACCA CACG (reverse); β-arrestin1: AAGGG ACACG AGTGT TCAAG A (forward), CCCGC TTTCC CAGGT AGAC (reverse); β-arrestin2: GGCAA GCGCG ACTTT GTAG (forward), GTGAG GGTCA CGAAC ACTTT C (reverse); β-actin: TTCTT TGCAG CTCCT TCGTT (forward), ATGGA GGGGA ATACA GCCC-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): TGCGA CTTCA ACAGC AACTC (forward), CTTGC TCAGT GTCCT TGCTG (reverse).
Data are expressed as mean ± standard error of the mean (SEM) and analyzed using the Student t test, one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni analysis. A P value <0.05 was considered statistically significant.
All supplementary materials are available online at https://doi.org/www.molmed.org.
Increased IL-1β-Induced Thermal and Mechanical Hyperalgesia in GRK6−/− Mice
Intraplantar injection of IL-1β dose-dependently increased thermal hyperalgesia in WT mice, as determined at 2 h after injection (Figure 1C). In GRK6−/− mice, the dose-response curve was sharply shifted to the left (see Figure 1C). Notably, the lowest dose of IL-1β (100 pg) that induced detectable thermal hyperalgesia in WT mice was 100-fold higher than the lowest dose that induced thermal hyperalgesia in GRK6−/− mice (1 pg).
To determine whether GRK6 also regulates duration of IL-1β-induced thermal hyperalgesia, we followed intraplantar IL-1β-induced thermal hyperalgesia over time. Hyperalgesia induced by intraplantar injection of either a low (10 pg/paw) or high (1,000 pg/paw) dose of IL-1β was markedly prolonged in GRK6-deficient mice (3 or 8 d after injection of a low or high dose of IL-1β in GRK6−/− mice versus 6 h or 1 d in WT mice; Figures 1D, E). Mechanical allodynia induced by IL-1β (1,000 pg/paw) was also markedly prolonged in GRK6−/− mice compared with WT mice (4–6 d after injection in GRK6−/− mice versus <1 d in WT mice; Figure 1F). At 24 h after intraplantar IL-1β (1,000 pg/paw), there were no differences in expression of COX2, IL-6 and TNF-α mRNA, indicating that the prolongation of hyperalgesia was independent of inflammatory activity (Supplementary Figure 1).
In Vitro Response of GRK6−/− DRG Neurons to IL-1β
These in vitro data indicate that GRK6 deficiency prevents activation of PI 3-kinase/Akt while facilitating p38 activation, thereby inducing a switch in IL-1β signaling from activation of both the p38 and PI 3-kinase/Akt pathways toward activation of p38 only.
In Vivo Role of p38 and PI 3-Kinase in the IL-1β-Induced Hyperalgesia
To determine the in vivo relevance of the shift in IL-1β-induced activation of the p38 and PI 3-kinase/Akt pathway in DRG neurons of GRK6−/− mice, we compared the effect of intraplantar administration of the p38 inhibitor SB239063 and the PI 3-kinase inhibitor LY249002 on IL-1β-induced hyperalgesia in WT and GRK6−/− mice.
In contrast, intraplantar administration of the PI 3-kinase inhibitor LY249002 (10 µg/paw) significantly enhanced the magnitude of acute (0.5–6 h) IL-1β-induced hyperalgesia in WT mice but did not have any effect on acute IL-1β hyperalgesia in GRK6−/− mice (Figure 4B). Additionally, IL-1β-induced hyperalgesia in WT mice was significantly prolonged after inhibition of PI 3-kinase. LY249002 did not have any effect on the duration of IL-1β-induced hyperalgesia in GRK6−/− mice. Injection of SB239063 or LY249002 alone did not have any effect on heat withdrawal latencies in WT or GRK6−/− mice (Figure 4C). These findings indicate that GRK6−/− mice lack an inhibiting signal that is provided in WT mice by activation of the PI 3-kinase pathway.
PGE2- and TNF-α-Induced Hyperalgesia in WT and GRK6−/− Mice
GRK6 mRNA Expression Levels in DRG of Mice with Neuropathic or Inflammatory Pain
To investigate whether changes in GRK6 do occur in conditions of chronic pain, we investigated GRK6 mRNA expression levels in DRGs of mice with chronic neuropathic or inflammatory pain. Four weeks after unilateral L5 nerve transection (L5 SNT), mice were more sensitive to mechanical stimulation of the ipsilateral paw (Figure 6A). Importantly, at this same time point, GRK6 mRNA levels were significantly reduced in ipsilateral DRGs compared with contralateral DRGs from sham-operated mice (Figure 6B). L5 SNT did not induce changes in mRNA levels for GRK2, β-arrestinl or β-arrestin2 (Supplementary Figures 2A–C). Chronic inflammatory pain was induced by intraplantar injection of carrageenan. Six days after carrageenan injection, heat-withdrawal latencies were reduced (Figure 6C). At this time, GRK6 mRNA levels were significantly decreased in the DRGs of carrageenan-treated mice compared with vehicle-treated mice (Figure 6D). mRNA levels for GRK2 and β-arrestin2 did not differ between carrageenan- and vehicle-treated mice, whereas α-arrestin1 mRNA levels were slightly reduced (Supplementary Figures 2D–F).
Unfortunately, we were unable to test whether the decrease in GRK6 mRNA was associated with a reduction in GRK6 protein, since no reliable GRK6 antibodies were available.
In this study, we present the novel concept that the kinase GRK6 plays a pivotal role in regulating the duration and intensity of inflammatory hyperalgesia. GRK6 deficiency strongly enhanced and prolonged thermal hyperalgesia and mechanical allodynia induced by intraplantar injection of either IL-1β or TNF-α. Similarly, hyperalgesia induced by intraplantar injection of carrageenan was markedly prolonged in GRK6−/− mice. We also show that GRK6 deficiency promotes activation of p38 while the activation of PI 3-kinase/Akt is dampened. Thus, the novelty and significance of these results is that GRK6 emerges here as a kinase that constrains neuronal responsiveness to IL-1β and TNF-α and ultimately cytokine-induced hyperalgesia via biased cytokine-induced p38 and PI 3-kinase/Akt activation (Figure 6E). The potential pathophysiological significance of these findings is substantiated by our finding that DRG GRK6 expression levels are reduced in a model of chronic neuropathic pain and inflammatory pain.
Recent evidence indicates that hyperalgesia induced by intraplantar administration of IL-1β is mediated via activation of p38 and subsequent modulation of the activity of tetrodoxin-resistant sodium channels (2). TNF-α is also known to signal to p38 and is capable of inducing hyperalgesia via mechanisms that involve increased transient receptor potential cation channel subfamily V member 1 (TRPV1) expression and modulation of tetrodoxin-resistant sodium channels (26,27). We show here for the first time that GRK6 constrains IL-1α-induced p38 activation in sensory neurons. This conclusion is on the basis of our in vitro finding that IL-1β-induced p38 phosphorylation was enhanced in GRK6−/− DRG cultures. Additionally, our in vivo studies showed that p38 activation is required for IL-1β and TNF-α hyperalgesia both in WT and GRK6−/− mice. Finally, we show that in vivo IL-1β- and TNF-α-induced hyperalgesia is increased in GRK6-deficient mice. Collectively, these findings support a central role of p38 in cytokine-induced hyperalgesia and position GRK6 as a pivotal regulator of cytokine-induced hyperalgesia.
Our findings indicate that GRK6 deficiency enhances IL-1α signaling to p38 in nociceptors. The IL-1 receptor is expressed in virtually all sensory neurons (28). Moreover, IL-1β injection into the rat paw enhances p-p38 in peripherin-positive sensory nerves (2). Finally, in mice in which Nav1.8-positive nociceptors are deleted, peripherin-positive neurons are reduced >85% and inflammatory pain does not develop (29). Therefore, we propose that GRK6 deficiency enhances IL-1β-induced hyperalgesia via promoting p38 signaling in peripherin-positive nociceptors.
Our experiments identified GRK6 as a prerequisite for IL-1β-induced activation of the PI 3-kinase/Akt pathway in primary sensory neurons. This conclusion is on the basis of our finding that IL-1β induced a significant increase in Akt phosphorylation in WT mice, but was unable to do so in GRK6−/− mice. Second, we show here that IL-1β- and TNF-α-induced PI 3-kinase activity is required to constrain cytokine-induced hyperalgesia in magnitude as well as duration. Inhibition of PI 3-kinase enhanced and prolonged IL-1β and TNF-α hyperalgesia in WT mice, whereas inhibition of PI 3-kinase did not affect hyperalgesia in GRK6−/− mice. Thus, our studies in GRK6-deficient mice reveal a completely novel role of the PI 3-kinase/Akt signaling cascade in attenuating the severity and duration of cytokine-induced hyperalgesia. Interestingly, recent evidence shows that PI 3-kinase also mediates a negative feedback loop in preventing neuronal hyperexcitability in the Drosophila neuromuscular junction (30). In mice, it was shown that PI 3-kinase inhibition blocks capsaicin- and NGF-induced increases in pain sensitivity (hyperalgesia) (31), whereas the morphine-induced reduction in pain sensitivity (analgesia) is mediated via PI 3-kinase (32). Our present data demonstrate that PI 3-kinase inhibition increases and prolongs cytokine-induced hyperalgesia in WT mice. The differential effects of PI 3-kinase activity in determining pain sensitivity may depend on the fact that the isoform of PI 3-kinase that is activated in response to NGF is of a different subtype than the PI 3-kinase activated by cytokines or morphine, PI3Kγ (PI 3-kinase γ) (32). Thus, if GRK6 is only required for activation of the PI3Kγ isoform of PI 3-kinase activated by TNF-α and IL-1β, one would expect that NGF-induced hyperalgesia would not be affected by GRK6. Indeed, we show that NGF-induced hyperalgesia was similar in GRK6−/− and WT mice (Supplementary Figures 3A, B).
Opposing effects of the mitogen-activated protein kinase p38 and PI 3-kinases on cellular functioning have been shown previously. Lipopolysaccharide (LPS)-induced IL-6 release is inhibited when p38 is blocked, whereas inhibition of the PI 3-kinase/Akt pathway enhances IL-6 production (33). Additionally, TNF-α and IL-1β can both induce a PI 3-kinase/Akt-dependent decrease in potassium currents (34), whereas p38 activation enhances potassium currents (35). The exact molecular mechanisms that explain how p38 and PI 3-kinase define the balance in the effects on cytokine-induced hyperalgesia remain to be elucidated (Figure 6E).
The question arises through which mechanisms GRK6 is required for the activation of PI 3-kinase and how GRK6 inhibits p38 activation. IL-1β-induced activation of PI 3-kinase requires recruitment of PI 3-kinase to the interleukin-1 receptor (IL-1R) (36). Another member of the GRK family, GRK2, was implicated in facilitating agonist-induced PI3Kγ recruitment to the β2-adrenergic receptor (37). Similarly, it is possible that GRK6 regulates recruitment of PI3Kγ to the IL-1β receptor, enabling IL-1β-induced activation of PI 3-kinase. In addition, GRK2 inhibits p38 activation by phosphorylation of p38 at Thr-123, a residue located at its docking groove (38). Importantly, the central serine/threonine kinase catalytic domain as well as the N-terminal regulator of G protein signaling (RGS)-like domain are highly conserved in all GRKs (39). Thus, it is possible that GRK6 interferes with p38 activation via binding and phosphorylation of p38 similar to what has been described for GRK2. Future studies will have to unravel the precise mechanisms.
We recently described that a partial reduction in GRK2 increases hyperalgesia induced by carrageenan and the GPCR ligands CCL3 and PGE2 (24,40). However, reduced GRK2 did not affect the magnitude of IL-1β-induced acute hyperalgesia (40,41). Vice versa, we show here that GRK6 deficiency does not affect PGE2-induced hyperalgesia. These findings indicate that GRK6 and GRK2 regulate hyperalgesia induced by inflammatory mediators via separate mechanisms. These distinct effects of GRK6 and GRK2 on inflammatory hyperalgesia conform to the notion that the different GRKs have specific and often contrasting effects on signaling pathways (42).
GRK6 and other members of the GRK family have been originally identified because of their capacity to phosphorylate agonist-occupied GPCRs (43). However, to the best of our knowledge, there is no evidence that IL-1β and TNF-α signal directly via a GPCR. One possible explanation for our findings could be that IL-1β induces the local production of GPCR ligands (for example, PGE2) and that GRK6 regulates signaling via this receptor with consequences for hyperalgesia. However, inhibition of COX2 did not affect IL-1β-induced hyperalgesia in WT or GRK6−/− mice (Supplementary Figure 4). Additionally, we showed that the magnitude and duration of PGE2 hyperalgesia was similar in WT and GRK6−/− mice. We cannot exclude that other GPCR ligands are produced at the site of injection of the cytokines. However, this explanation seems unlikely in view of our finding that the change in phosphorylation of p38 and Akt in sensory neurons was already observed 5 min after stimulation with IL-1β in vitro. Overall, these data point to a role of GRK6 in regulating cytokine-induced events that are independent of GPCR signaling.
The data presented herein indicate that GRK6 deficiency enhances the sensitivity to IL-1β-induced hyperalgesia. It may therefore be that GRK6 deficiency also facilitates other inflammatory processes involving IL-1β. Indeed, this hypothesis is supported by our earlier studies. For example, we have shown that increased inflammatory visceral hyperalgesia in GRK6−/− mice is characterized by increased neuronal and behavioral responses to noxious colonic stimulation with capsaicin. We also showed that overexpression of GRK6 attenuated IL-1β-induced TRPV1 sensitization (12). Interestingly, blocking TRPV1 with capsazepine partially inhibited the ongoing IL-1β hyperalgesia 24 h after IL-1β administration (Supplementary Figure 5), suggesting enhanced TRPV1 sensitization may underlie the enhanced intraplantar IL-1β-induced heat hyperalgesia as well. Finally, we demonstrated that in vivo, GRK6 deficiency leads to increased severity as well as duration of colitis (11). In these models of visceral hyperalgesia and colon inflammation, IL-1β is produced in the colon, and thus increased sensitivity to IL-1β-induced events may contribute to the increased and prolonged pain and colitis. In addition, Tarrant et al. (10) showed that GRK6−/− mice develop more severe joint inflammation in the K/BxN model of acute inflammatory arthritis (10). These authors showed that serum IL-1 levels are normal, but IL-6 levels are elevated in GRK6−/− mice in this model of inflammatory arthritis. Because IL-1 is a potent IL-6 inducer, it may be that increased IL-1 signaling also underlies the higher levels of IL-6 and increased joint inflammation in this model.
The pathophysiological relevance of our finding that GRK6 deficiency increasesseverity and duration of cytokine-induced hyperalgesia is underlined by our findings on GRK6 expression in DRGs in two different chronic pain models. We show that sensory neuron GRK6 expression is reduced in DRGs innervating the sensitized paw of mice with chronic neuropathic or inflammatory pain. Moreover, chronic inflammatory diseases that are accompanied by pain, such as rheumatoid arthritis, are associated with a decrease in intracellular GRK6 levels (44).
In summary, here we have identified for the first time GRK6 as a crucial kinase that is required to constrain cytokine signaling and cytokine-induced hyperalgesia. This contribution of GRK6 to hyperalgesia is likely to be mediated by regulating the balance of cytokine-induced p38 and PI 3-kinase/Akt activation in normal mice. This important relevant function of GRK6 in modulating cytokine-induced signaling events may also play a role in chronic inflammatory conditions.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank Richard T Premont and Robert J Lefkowitz, Duke University, Durham, North Carolina, for providing the GRK6−/− mice. This study was supported in part by National Institutes of Health grants RO1-NS073939 to R Dantzer, KW Kelley and A Kavelaars and R01-NS074999 to A Kavelaars. Part of the work of N Eijkelkamp was supported by a Rubicon fellowship of the Netherlands Organization for Scientific Research.
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.
The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this license, visit (https://doi.org/creativecommons.org/licenses/by-nc-nd/4.0/)
- Marchand F, Perretti M, McMahon SB. (2005) Role of the immune system in chronic pain. Nat. Rev. Neurosci. 6:521–32.View ArticleGoogle Scholar
- Binshtok AM, et al. (2008) Nociceptors are interleukin-1beta sensors. J. Neurosci. 28:14062–73.View ArticleGoogle Scholar
- Schafers M, Sorkin L. (2008) Effect of cytokines on neuronal excitability. Neurosci. Lett. 437:188–93.View ArticleGoogle Scholar
- Hucho T, Levine JD. (2007) Signaling pathways in sensitization: toward a nociceptor cell biology. Neuron. 55:365–76.View ArticleGoogle Scholar
- Myers RR, Campana WM, Shubayev VI. (2006) The role of neuroinflammation in neuropathic pain: mechanisms and therapeutic targets. Drug Discov. Today 11:8–20.View ArticleGoogle Scholar
- Wolf G, Gabay E, Tal M, Yirmiya R, Shavit Y. (2006) Genetic impairment of interleukin-1 signaling attenuates neuropathic pain, autotomy, and spontaneous ectopic neuronal activity, following nerve injury in mice. Pain. 120:315–24.View ArticleGoogle Scholar
- Zhang J, et al. (1997) Molecular mechanisms of G protein-coupled receptor signaling: role of G protein-coupled receptor kinases and arrestins in receptor desensitization and resensitization. Receptors Channels. 5:193–9.PubMedGoogle Scholar
- Ribas C, et al. (2007) The G protein-coupled receptor kinase (GRK) interactome: role of GRKs in GPCR regulation and signaling. Biochim. Biophys. Acta. 1768:913–22.View ArticleGoogle Scholar
- Reiter E, Lefkowitz RJ. (2006) GRKs and beta-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol. Metab. 17:159–65.View ArticleGoogle Scholar
- Tarrant TK, et al. (2008) Granulocyte chemotaxis and disease expression are differentially regulated by GRK subtype in an acute inflammatory arthritis model (K/BxN). Clin. Immunol. 129:115–22.View ArticleGoogle Scholar
- Eijkelkamp N, et al. (2007) G protein-coupled receptor kinase 6 controls chronicity and severity of dextran sodium sulphate-induced colitis in mice. Gut. 56:847–54.View ArticleGoogle Scholar
- Eijkelkamp N, et al. (2009) G protein-coupled receptor kinase 6 controls post-inflammatory visceral hyperalgesia. Brain Behav. Immun. 23:18–26.View ArticleGoogle Scholar
- Vroon A, et al. (2004) GRK6 deficiency is associated with enhanced CXCR4-mediated neutrophil chemotaxis in vitro and impaired responsiveness to G-CSF in vivo. J. Leukoc. Biol. 75:698–704.View ArticleGoogle Scholar
- Kavelaars A, et al. (2003) Increased acute inflammation, leukotriene B4-induced chemotaxis, and signaling in mice deficient for G protein-coupled receptor kinase 6. J. Immunol. 171:6128–34.View ArticleGoogle Scholar
- Fong AM, et al. (2002) Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 99:7478–83.View ArticleGoogle Scholar
- Aiyar N, et al. (2000) Involvement of G protein-coupled receptor kinase-6 in desensitization of CGRP receptors. Eur. J. Pharmacol. 403:1–7.View ArticleGoogle Scholar
- Ruiz-Gomez A, et al. (2007) G protein-coupled receptor kinase 2-mediated phosphorylation of downstream regulatory element antagonist modulator regulates membrane trafficking of Kv4.2 potassium channel. J. Biol. Chem. 282:1205–15.View ArticleGoogle Scholar
- Hall RA, et al. (1999) G protein-coupled receptor kinase 6A phosphorylates the Na(+)/H(+) exchanger regulatory factor via a PDZ domainmediated interaction. J. Biol. Chem. 274:24328–34.View ArticleGoogle Scholar
- Fukuoka H, Kawatani M, Hisamitsu T, Takeshige C. (1994) Cutaneous hyperalgesia induced by peripheral injection of interleukin-1 beta in the rat. Brain Res. 657:133–40.View ArticleGoogle Scholar
- Hargreaves K, Dubner R, Brown F, Flores C, Joris J. (1988) A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain. 32:77–88.View ArticleGoogle Scholar
- Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL. (1994) Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods. 53:55–63.View ArticleGoogle Scholar
- Woolf CJ, Allchorne A, Safieh-Garabedian B, Poole S. (1997) Cytokines, nerve growth factor and inflammatory hyperalgesia: the contribution of tumour necrosis factor alpha. Br. J. Pharmacol. 121:417–24.View ArticleGoogle Scholar
- Wajant H, Pfizenmaier K, Scheurich P. (2003) Tumor necrosis factor signaling. Cell Death Differ. 10:45–65.View ArticleGoogle Scholar
- Eijkelkamp N, et al. (2010) Low nociceptor GRK2 prolongs prostaglandin E2 hyperalgesia via biased cAMP signaling to Epac/Rap1, protein kinase Cepsilon, and MEK/ERK. J. Neurosci. 30:12806–15.View ArticleGoogle Scholar
- Aley KO, Levine JD. (1999) Role of protein kinase A in the maintenance of inflammatory pain. J. Neurosci. 19:2181–6.View ArticleGoogle Scholar
- Constantin CE, et al. (2008) Endogenous tumor necrosis factor alpha (TNFalpha) requires TNF receptor type 2 to generate heat hyperalgesia in a mouse cancer model. J. Neurosci. 28:5072–81.View ArticleGoogle Scholar
- Jin X, Gereau RW. (2006) Acute p38-mediated modulation of tetrodotoxin-resistant sodium channels in mouse sensory neurons by tumor necrosis factor-alpha. J. Neurosci. 26:246–55.View ArticleGoogle Scholar
- Obreja O, Rathee PK, Lips KS, Distler C, Kress M. (2002) IL-1 beta potentiates heat-activated currents in rat sensory neurons: involvement of IL-1RI, tyrosine kinase, and protein kinase C. FASEB J. 16:1497–503.View ArticleGoogle Scholar
- Abrahamsen B, et al. (2008) The cell and molecular basis of mechanical, cold, and inflammatory pain. Science. 321:702–5.View ArticleGoogle Scholar
- Howlett E, Lin CC, Lavery W, Stern M. (2008) A PI3-kinase-mediated negative feedback regulates neuronal excitability. PLoS. Genet. 4:e1000277.View ArticleGoogle Scholar
- Zhuang ZY, Xu H, Clapham DE, Ji RR. (2004) Phosphatidylinositol 3-kinase activates ERK in primary sensory neurons and mediates inflammatory heat hyperalgesia through TRPV1 sensitization. J. Neurosci. 24:8300–9.View ArticleGoogle Scholar
- Cunha TM, et al. (2010) Morphine peripheral analgesia depends on activation of the PI3Kgamma/AKT/nNOS/NO/KATP signaling pathway. Proc. Natl. Acad. Sci. U. S. A. 107:4442–7.View ArticleGoogle Scholar
- Qian Y, et al. (2009) Regulation of TLR4-induced IL-6 response in bladder cancer cells by opposing actions of MAPK and PI3K signaling. J. Cancer Res. Clin. Oncol. 135:379–86.View ArticleGoogle Scholar
- Diem R, Meyer R, Weishaupt JH, Bahr M. (2001) Reduction of potassium currents and phosphatidylinositol 3-kinase-dependent AKT phosphorylation by tumor necrosis factor-(alpha) rescues axotomized retinal ganglion cells from retrograde cell death in vivo. J. Neurosci. 21:2058–66.View ArticleGoogle Scholar
- Redman PT, et al. (2007) Apoptotic surge of potassium currents is mediated by p38 phosphorylation of Kv2.1. Proc. Natl. Acad. Sci. U. S. A. 104:3568–73.View ArticleGoogle Scholar
- Marmiroli S, et al. (1998) Phosphatidylinositol 3-kinase is recruited to a specific site in the activated IL-1 receptor I. FEBS Lett. 438:49–54.View ArticleGoogle Scholar
- Naga Prasad SV, et al. (2002) Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the receptor/beta-arrestin complex. J. Cell Biol. 158:563–75.View ArticleGoogle Scholar
- Peregrin S, et al. (2006) Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK. Curr. Biol. 16:2042–7.View ArticleGoogle Scholar
- Willets JM, Challiss RA, Nahorski SR. (2003) Non-visual GRKs: are we seeing the whole picture? Trends Pharmacol. Sci. 24:626–33.View ArticleGoogle Scholar
- Eijkelkamp N, et al. (2010) GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain. J. Neurosci. 30:2138–49.View ArticleGoogle Scholar
- Willemen HL, et al. (2010) Microglial/macrophage GRK2 determines duration of peripheral IL-1beta-induced hyperalgesia: contribution of spinal cord CX3CR1, p38 and IL-1 signaling. Pain. 150:550–60.View ArticleGoogle Scholar
- Gurevich EV, Tesmer JJ, Mushegian A, Gurevich VV. (2012) G protein-coupled receptor kinases: more than just kinases and not only for GPCRs. Pharmacol. Ther. 133:40–69.View ArticleGoogle Scholar
- Lefkowitz RJ. (1998) G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization. J. Biol. Chem. 273:18677–80.View ArticleGoogle Scholar
- Lombardi MS, et al. (1999) Decreased expression and activity of G-protein-coupled receptor kinases in peripheral blood mononuclear cells of patients with rheumatoid arthritis. FASEB J. 13:715–25.View ArticleGoogle Scholar