The major findings in the current study demonstrated that intradermal capsaicin injection causes an increase in Ser-133 CREB phosphorylation in the dorsal horn and that the enhancement of CREB phosphorylation is regulated by the activity of MAP kinase, PKA and PKC.
A number of studies have shown that enhanced phosphorylation of CREB transcription factor could account for the potential contribution to long-term changes in the spinal processing of nociceptive information [6, 13]. As one of important transcription factors, CREB is phosphorylated in response to stimuli-triggered calcium influx into post-synaptic neurons [14, 21, 22]. Our previous findings reported the role of nitric oxide system and CaM kinase II in the CREB regulation [13, 25]. In this investigation, we report that noxious stimulation with capsaicin injection induces phosphorylation of CREB protein (Ser-133) in the spinal cord. This finding is similar to what we reported previously  and studies of other investigators using different stimulation regimens. In addition, we assessed the regulating mechanism by which intracellular protein kinase mediates CREB phosphorylation. The data demonstrated that the intrathecal treatment with inhibitors of MAP kinase, PKA or PKC blocked capsaicin-triggered CREB phosphorylation (at the Serine 133 site), confirming that not only c-AMP-dependent protein kinase A (PKA), but also ERK/MAP kinase and PKC, play roles in the mediation of CREB phosphorylation during the central processing of nociception.
Peripheral noxious stimulation, such as capsaicin injection, causes an increased responsiveness in spinal nociceptive neurons that involve the activation of glutamate receptors. This event produces a large influx of calcium into the nociceptive neurons, activating multiple intracellular protein kinase cascades, such as CaM kinase II, PKC, as well as MAP kinase [2, 4, 6, 18, 30–34]. The PKA, PKG and nitric oxide synthase systems were also found to be activated following increased cAMP and cGMP in nociceptive neurons during painful stimulation [2, 22, 35]. Enhanced phosphorylation of CREB through the activation of glutamate receptors and the above kinase cascades during central sensitization suggest a connection between CREB phosphorylation and the molecular mechanisms governing stimuli-induced CREB activation by protein kinase pathways. Investigators from other laboratories also noted that increased CREB phosphorylation was found in animals following different kinds of noxious stimulation [7, 8, 11, 17].
Like other transcription factors, such as AP-1 proteins, c-fos, and c-Jun, phosphorylated CREB may participate in the downstream signal transduction cascade in the longer lasting changes in synaptic plasticity. CREB phosphorylation was reported in the transcription regulation of nociception-related genes, such as dynorphin, enkephalin and opioid receptors, during the activation of nociceptive neurons. A CREB binding site is found in the promoter regions of the dynorphin, enkephalin and opioid receptor genes following painful stimulation [14, 16, 20, 28, 35]. CREB phosphorylation is required for prolonged synaptic plasticity strengthening during central sensitization [2, 21, 24].
Recently, mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK 1/2) was reported to play a critical role in spinal dorsal horn neurons in response to various painful stimuli [5, 18, 30–34]. Following noxious peripheral stimuli or c-Fiber volleys, an increased phosphorylation of ERK was observed in nociceptive neurons in rats and an inhibitor of ERK reduced the formalin-injection-induced nociceptive behavior [5, 36]. Peripheral nerve injuries or noxious stimulation with various inflammatory agents, such as carrageenan, capsaicin, Complete Freund's Adjuvent (CFA), mustard oil and melittin (a major toxic peptide of whole bee venom) were reported to active the MAP/MEK kinase pathway [5, 9, 18, 20, 31, 33, 34]. Furthermore, the activation of MAP kinase pathways was related to the activation of several types of glutamate receptors (NMDA, AMPA and mGlu-R) in dorsal horn neurons, as well as other intracellular protein kinase cascades in the pathogenesis of nociceptive sensitization [9, 18]. The activation of MAP kinase signaling was also reported to be involved in the transcriptional regulation of prodynorphin and neurokinin-1 (NK-1) gene products [5, 14, 16, 35]. In our present study, we found that U0126, a MAP kinase inhibitor, significantly blocked CREB phosphorylation at Ser-133 in response to capsaicin injection. Another investigator reported a similar effect of U0126 in a different spinal cord slice preparation following C-fiber stimulation . In an investigation involving in vitro preparation of cultured striatal neurons, the NMDA- and AMPA/kainate-induced CREB phosphorylation was blocked by MAP kinase inhibitor, U0126 . These findings provide supportive evidence that the MAP kinase pathway is one of the important intracellular elements and may contribute to the nociceptive plasticity in central sensitization.
It is well recognized that both the PKA and the PKC pathways are involved in the central neurotransmission of nociception. In experimental animals with capsaicin injection induced-central sensitization, activation of the PKA or the PKC pathways by their activators enhanced the response of spinothalamic (STT) cells to mechanical stimuli [15, 19] and inhibitors of PKA or PKC blocked the hypersensitivity of STT neurons. Experiments from behavioral studies also support the roles of PKA and PKC in the generation of spinal cord central sensitization [2, 15, 19]. Our study further demonstrates the roles of PKA and PKC in molecular mechanism of regulation of gene transcription in response to capsaicin injection, in which they mediate the phosphorylation of the transcription factor, CREB. However, as important regulators, both PKA and PKC actively contribute to the activity-dependent hippocampal synaptic plasticity by regulating CREB-dependent target genes. The present study supports the results from our earlier work on central sensitization, which is believed to be a spinal cord form of LTP .
In summary, the findings from our investigation provide strong evidence that MAP kinase, PKA and PKC participate in the regulation of CREB phosphorylation status in the spinal cord following peripheral noxious stimulation. These results also suggest a possible explanation for how the activated intracellular kinase cascades convey extracellular signals into the nucleus for subsequent transcription of plasticity-associated genes in the spinal cord.