Activation of transcription factors of nuclear factor kappa B, activator protein-1 and octamer factors in hyperalgesia

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Abstract

Involvement of c-fos and neuronal nitric oxide synthase (nNOS) in the hyperalgesia induced by complete Freund adjuvant (CFA) has been reported. In this paper, we attempted to investigate whether the transcription factors regulating the gene expression of c-fos and nNOS, including activator protein-1 (AP-1), nuclear factor kappa B (NF-κB), and octamer factors (Oct), are activated by CFA during the development of hyperalgesia. The electrophoretic mobility shift assay (EMSA) was used to determine whether there were changes in the transcription factors in the lumbar spinal cord of adult rats following subcutaneous injection of CFA in one hindpaw of the rats. Maximum binding of AP-1, NF-κB and Oct was found at 0.5, 1 and 2 h after CFA injection, respectively. These findings suggest that the activation of these transcription factors is pivotal for the expression of c-Fos and nNOS proteins, which reached a peak at 3 and 48 h after CFA injection, respectively. The behavioral testing of hyperalgesia demonstrated that CFA reduced the thresholds for mechanical and thermal algesia, reaching a minimum at 6 h. The thresholds had only partially recovered after 96 h. Based on these findings, we conclude that AP-1, NF-κB and Oct are crucial for the expression of c-Fos proteins at an early stage (at 3 h) and for the expression of nNOS at a late stage of hyperalgesia (48 h post-injection) induced by CFA.

Introduction

An experimental model to study “chronic” pain is adjuvant-induced arthritis in the rat, which resembles human rheumatoid poly-arthritis. A parallel clinical and behavioral study of adjuvant-induced arthritis in the rat showed four stages in the time course of the disease: pre-clinical (first week), acute (weeks 2–4), post-acute (weeks 5–8) and recovery (weeks 9–11) (Lantéri-Minet et al., 1993). Arthritis was induced by subcutaneous injection of Freund's adjuvant (killed Mycobacterium butyricum suspended in mineral oil) into one hindpaw. In the model of adjuvant-induced arthritis, an initial inflammatory response develops within hours. There is a dramatic modification in the activity of both superficial (I and II) and deeper (V and VI) laminae of the dorsal horn neurons receiving noxious inputs.

There is evidence that various nociceptive peripheral stimuli result in increased c-fos expression in the spinal cord Hunt et al., 1987, Menétrey et al., 1989, Herdegen et al., 1991, Abbadie and Besson, 1992, Abbadie et al., 1994, Yashpal et al., 1998. C-Fos expression in spinal cord neurons occur not only after acute stimulation and inflammation, but also in chronic pain diseases such as poly- or mono-arthritis (Abbadie and Besson, 1992). Nitric oxide (NO) mediates the actions of glutamate and is involved in sustained changes in neuronal activity such as long-term suppression and long-term potentiation Bredt and Snyder, 1992, Shibuki, 1993. Evidence suggests that nitric oxide synthase (NOS) activity in the spinal cord may participate in nociception and in the responses of spinal neurons to electrically evoke C-fiber input (Meller and Gebhart, 1993). These events may also lead to the establishment of persistent pain and hyperalgesia in a concerted manner. It is well known that NOS gene expression is promoted by activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB)Kleinert et al., 1998, Todoroki et al., 1998, Marks-Konczalik et al., 1998. Recently, the octamer factor-2 (Oct-2) transcription factor has been identified which can also activate neuronal nitric oxide synthase (nNOS) gene expression in neuronal cells (Deans et al., 1996). Moreover, both aspirin and sodium salicylate inhibit the effects of glutamate through blockade of NF-κB activation (Grilli et al., 1996).

We attempted to investigate whether complete Freund adjuvant (CFA) could activate the transcription factors AP-1, NF-κB and octamer factors (Oct) and what their relation is to the induction of c-Fos, nNOS and hyperalgesia. Using electrophoretic DNA mobility shift assays (EMSA), we examined the activity of nuclear protein binding to the AP-1 site or the promoter regions of nNOS after CFA injection. Our results demonstrated that the transcription factors AP-1, NF-κB and Oct were immediately activated by CFA prior to the expression of c-fos and nNOS. This may contribute to persistent hyperalgesia following CFA injection.

Section snippets

Injection of CFA

CFA (0.1%, 50 μl, Sigma, St. Louis, MO, USA) was injected subcutaneously into the plantar surface of the proximal portion of the foot in awake male Wistar rats (250–350 g) with a 29-gauge needle (n=45). A vehicle containing saline was injected into the proximal portion of the plantar surface of the foot of three additional control animals. All experiments were approved by the Institutional Animal Care Subcommittee of National Taiwan University Hospital and are in accordance with guidelines for

The behavioral profiles of hyperalgesia

The time course of two behavioral correlates of CFA-induced hyperalgesia was determined with Von Frey filaments and radiant heat. As seen in Fig. 1, the mechanical threshold of CFA-injected paws had decreased significantly (P<0.01) 3 h after CFA injection, to reach its minimal value at 6 h (12.7±5.6% of control). At 24 h, the mechanical threshold of the paw started increasing, and hyperalgesia was still detected even at 96 h (40.4±16.6% of control, P<0.01). The thermal threshold of CFA-treated

Intracellular signaling induced by CFA

It may be the best strategy to diminish pain by blocking the expression of target genes in hyperalgesia. In a previous study, formalin-induced nociceptive behavior was decreased when we administered c-fosantisense oligodeoxynucleotide and cycloheximide to rats (Hou et al., 1997a). We further investigated the regulation of the transcription of target genes (c-fos and nNOS) associated with hyperalgesia. The recently found transcription factor Oct-2, a member of the POU (a 60-amino acid

References (31)

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