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In addition to many important roles for Cdk5 in brain development and synaptic function, we reported previously that Cdk5 regulates inflammatory pain signaling, partly through phosphorylation of transient receptor potential vanilloid 1 (TRPV1), an important Na+/Ca2+ channel expressed in primary nociceptive afferent nerves. Because TGF-β regulates inflammatory processes and its receptor is expressed in TRPV1-positive afferents, we studied the cross-talk between these two pathways in sensory neurons during experimental peripheral inflammation. We demonstrate that TGF-β1 increases transcription and protein levels of the Cdk5 co-activator p35 through ERK1/2, resulting in an increase in Cdk5 activity in rat B104 neuroblastoma cells. Additionally, TGF-β1 enhances the capsaicin-induced Ca2+ influx in cultured primary neurons from dorsal root ganglia (DRG). Importantly, Cdk5 activity was reduced in the trigeminal ganglia and DRG of 14-day-old TGF-β1 knock-out mice, resulting in reduced Cdk5-dependent phosphorylation of TRPV1. The decreased Cdk5 activity is associated with attenuated thermal hyperalgesia in TGF-β1 receptor conditional knock-out mice, where TGF-β signaling is significantly reduced in trigeminal ganglia and DRG. Collectively, our results indicate that active cross-talk between the TGF-β and Cdk5 pathways contributes to inflammatory pain signaling.
is a proline-directed serine/threonine kinase that belongs to the family of cyclin-dependent protein kinases. Cdk5 kinase activity is mainly present in postmitotic neurons where its activators, p35 and p39, are predominantly expressed (for review see Refs.
). Although earlier studies focused on delineating the molecular roles of Cdk5 in brain development, more recent work has implicated Cdk5 in many other functions in the mature brain such as memory, learning, and cellular processes leading to neurodegeneration (
). Furthermore, we observed that peripheral inflammation increased Cdk5-mediated phosphorylation of transient receptor potential vanilloid 1 (TRPV1), a ligand-gated ion channel critically involved in thermal and inflammatory pain (
). Pharmacological modulation of Cdk5 can produce analgesia or anti-hyperalgesia. We observed that resveratrol, a polyphenolic compound with known analgesic activity, can inhibit Cdk5 activity through decreased expression of p35 (
). These findings suggest that Cdk5 plays an important role in multiple molecular mechanisms involved in pain signaling and modulation.
Transforming growth factor-β1 (TGF-β1) is an important member of a superfamily of multifunctional growth factors involved in many cellular processes including cell proliferation, differentiation, migration, and apoptosis (
). TGF-β1−/− mice developed a rapid wasting syndrome and died by 3–4 weeks of age. As early as 2 weeks, these mice displayed multifocal inflammation with massive infiltration of lymphocytes and macrophages into several organs, but principally into the heart, lungs, and salivary glands (
). Multiple actions of TGF-β1 have been reported in the CNS. TGF-β1 is normally present at low levels in healthy adult CNS cells, but it is rapidly up-regulated following injury and directly induces expression of several injury-related genes (
). However, the molecular mechanisms underlying its involvement in nociceptive signaling are still far from clear. Therefore, the main purpose of this study was to explore a possible cross-talk between Cdk5 and TGF-β signaling pathways, and the influence of the cross-talk on inflammation-induced nociceptive responses.
Here we used in vitro and in vivo approaches to study the role of TGF-β1 in the regulation of Cdk5 activity and its involvement in inflammatory pain signaling. We found that TGF-β1 increases Cdk5 activity in B104 neuroblastoma cells and causes increased Cdk5-dependent TRPV1 phosphorylation and capsaicin-induced Ca2+ influx in dorsal root ganglia (DRG) primary cultures. Likewise, a deficiency of TGF-β signaling in TGF-β1−/− mice, or where transforming growth factor-β receptor 1 (Tgfbr1) is conditionally knocked out in TG and DRG, resulted in reduced Cdk5 activity and attenuated thermal hyperalgesia, suggesting an active cross-talk between TGF-β and Cdk5 pathways in sensory afferents during peripheral inflammatory states.
In this study, we investigated cross-talk between two important signaling pathways involved in inflammation and how this cross-talk could affect nociceptive signaling. Our investigations demonstrate that in rat neuroblastoma B104 cells, TGF-β1 treatment increased Cdk5 and p35 protein levels with a concomitant increase in Cdk5 activity. Notably, we found TGF-β1 treatment increased Cdk5-mediated phosphorylation of TRPV1 and increased capsaicin-induced calcium influx in primary DRG neuronal cultures. We also report here that Cdk5 activity was decreased in TG and DRG of Tgf-β1−/− mice, resulting in decreased Cdk5-mediated phosphorylation of TRPV1 in DRG. We confirmed these findings in Tgfbr1 cKO mice (SNS-Cre; Tgfbr1f/f) and again observed decreased Cdk5 activity in TG and DRG with attenuated thermal hyperalgesia after peripheral inflammation. Thus, our results indicate that an active cross-talk between the TGF-β and Cdk5 pathways directly influences pain signaling.
We and others have demonstrated that Cdk5 plays an important role in pain signaling (
). Previously, we discovered that p35 knock-out mice exhibit significantly decreased Cdk5 activity and show delayed responses to acute noxious thermal stimulation as compared with control mice. In contrast, mice overexpressing p35 exhibit elevated levels of Cdk5 activity and are more sensitive to noxious thermal stimuli than controls. Furthermore, carrageenan-induced (
). In addition to TGF-β1 activation of canonical Smad pathways, recent reports have shown that TGF-β1 also induces other (no canonical) pathways, including the RhoA and mitogen-activated protein kinase (MAPK) cascades, the latter include extracellular signal-regulated kinases, ERK1/2 (
). Our results show that B104 cells express Cdk5 and p35 protein, and also display Cdk5 kinase activity. Most importantly, we report here that TGF-β1 treatment significantly increases p35 promoter activity. In addition, we found that the TGF-β1 induced an increase of phospho-ERK1/2, and increased Egr-1 and p35 mRNA expression levels, with a subsequent increase of p35 protein levels resulting in an increase of Cdk5 kinase activity. We also noticed that Egr-1 mRNA peaks at about 1 h and returned to basal level at 6 h after TGF-β treatment, whereas the p35 protein level remained higher at 24 h following the treatment. This lag could be attributed to complex transcriptional regulation of Egr-1 and its involvement in multiple transduction cascades (
). However, the functional significance of TGF-β action was reinforced by our observation that the TGF-β1 inhibitor (SB431542) significantly blocked the TGF-β1-mediated increase of Cdk5 kinase activity. Likewise, MEK1/2 inhibitor U0126 alone, or in combination with TGF-β1, significantly decreased Cdk5 kinase activity, suggesting that the ERK1/2 signaling pathway is a key pathway mediating the cross-talk between TGF-β1 and Cdk5 kinase pathways. Together these results show that TGF-β1 directly activates ERK1/2 leading to an increase in Egr-1 expression and subsequent elevation of p35 expression. This results in an increase in Cdk5 kinase activity.
We also found that Cdk5-mediated phosphorylation of TRPV1 was regulated by TGF-β1 in DRG primary cultures. Treatment with TGF-β1 increased, whereas treatment with SB431542 decreased the phosphorylation of TRPV1 in DRG cultures. This phosphorylation of TRPV1 has a functional consequence, because treatment with TGF-β1 increased the number of DRG neurons responding to capsaicin. In contrast, treatment with SB431542 blocked the TGF-β1 effect, suggesting that the TGF-β1 signaling pathway needs to be active to sensitize DRG neurons. It is likely, however, that TGF-β1 directly regulates TRPV1 through post-translational mechanisms in these cells, possibly via regulation of Cdk5 activity because TRPV1 mRNA levels remain unchanged in DRG cultures treated with TGF-β1. Although some cells appear hyper-responsive after TGF-β1 treatment in our experiment, the largest observed affect was an increase in the overall number of cells responding to capsaicin. This suggests that Cdk5-mediated phosphorylation of TRPV1 could regulate trafficking of TRPV1 to the membrane. But location of the Cdk5 phosphorylation site on the N-terminal region of TRPV1 also suggests it may regulate protein-protein interactions that influence TRPV1 activity. Collectively, these observations point toward a role for TGF-β1 in the development of inflammation-induced hyperalgesia through direct regulation of Cdk5 activity and Cdk5-mediated phosphorylation of TRPV1.
TGF-β has been implicated in pain signaling but its precise role is not completely clear. For instance, TGF-β family members negatively modulate pain perception, specifically in mouse models for neuropathic pain (
) of patients with migraines. To evaluate in vivo the role of TGF-β1 in nociceptive processes and its involvement in the regulation of Cdk5 activity, we removed TG and DRG from the Tgf-β1−/− mice and assessed the p35 expression levels and Cdk5 kinase activity in those tissues. A lack of TGF-β1 resulted in decreased p35 protein levels in TG and DRG, along with a decrease in Cdk5 activity. Most importantly, we found a parallel decrease in Cdk5 activity and Cdk5-mediated phosphorylation of TRPV1 in TG and DRG from Tgf-β1−/− mice. To circumvent systemic inflammation associated with TGF-β1−/− mice, we generated cKO mice that specifically lack Tgfbr1 in a subpopulation of nociceptive primary afferents of the TG and DRG and evaluated whether a blunted TGF-β signaling pathway could affect Cdk5 activity and pain sensation. Interestingly, we found a decrease in Cdk5 kinase activity and an associated attenuation of behavioral response to thermal stimulation following inflammation in Tgfbr1 cKO mice, corroborating our observations in Tgf-β1−/− mice. Taken together, these results identify a novel molecular mechanism based on cross-talk between TGF-β1 and Cdk5 that appears to contribute to the sensitization of nociceptive signaling.
It was reported earlier that TGF-β1, among other members of this family, could be negatively modulating pain perception (
). In contrast, our results on Tgf-β1−/− mice are consistent with a positive modulation of pain signaling by TGF-β1. It should be noted that the Cdk5 kinase activity was unchanged in the spinal cords of Tgf-β1−/− mice (data not shown), suggesting that TGF-β1 may play dual roles in different types of persistent pain conditions. At the level of the nociceptive primary afferent neuron, TGF-β may negatively modulate sensitivity by a Cdk5-independent mechanism in neuropathic nerve injury models, and modulate it positively by a Cdk5-dependent mechanism in peripheral inflammation (as examined in this report). In support of our proposal that TGF-β1 plays a positive role in inflammation-induced hyperalgesia, activin A, another member of the TGF-β family, is released during peripheral inflammation. This increases the expression of the calcitonin gene-related peptide and pain sensation (
). Basal responses to Aδ-fiber and C-fiber stimulation were not changed in Tgfbr1 cKO mice, but after the induction of inflammation with carrageenan we found decreased hyperalgesia at 5 h but not at 24 h after injection, suggesting that deletion of Tgfbr1 in DRG produced a temporally discrete phenotype characterized by reduced nociceptive transmission following the onset of inflammation. As the inflammation progresses, multiple parallel processes are engaged that lead to pain sensitization but are independent of TGF-β1-mediated Cdk5 activity in peripheral afferents. In conclusion, we have shown that an active cross-talk exists between TGF-β and Cdk5 in primary sensory neurons and affects nociceptive signaling during inflammation (Fig. 6). Thus, TGF-β activates Smad-dependent and Smad-independent pathways, which could regulate ERK1/2 activity and in turn induce Egr-1 expression. This results in elevated levels of Cdk5 and p35 mRNA and proteins, as well as a concomitant increase in Cdk5 activity leading to an increase in Cdk5-mediated phosphorylation of TRPV1. When DRG neurons are sensitized or activated, the increased intracellular Ca2+ could also regulate calpain activity, which could generate more p25, resulting in a stronger induction of Cdk5 activity. Similarly, a lack of TGF-β1 or Tgfbr1 decreased p35 protein expression, which in turn decreases Cdk5 activity. Subsequently, we found an associated decrease in Cdk5-mediated phosphorylation of TRPV1. Our in vitro finding suggests that Cdk5 phosphorylation increases the amount of active TRPV1 in the membrane of DRG neurons. We believe our findings on the active cross-talk between Cdk5 and TGF-β pathways in primary afferents and the influence of the cross-talk on nociceptive processing will enhance our understanding of the complex nature of inflammatory pain transduction and primary afferent sensitization.
We thank Bradford Hall and Zhijun Sun for critical reading of the manuscript, Alfredo Molinolo for analysis of stained section of Tgfr1 cKO mice, and Shelagh Johnson for expert editorial assistance.