Cdk5 regulates pain signaling We have recently reported a novel role of Cdk5 in pain signaling. We previously reported that expression of Cdk5 and p35, as well as Cdk5 kinase activity, was increased in the dorsal root ganglia (DRG) and the spinal cord (SC) after peripheral-inflammation. Inflammation induced by carrageenan injection in the hind paws of mice increased the mRNA and protein levels of Cdk5/p35 in nociceptive neurons, with a subsequent increase in Cdk5 kinase activity. Furthermore, we also identified that the elevated Cdk5 activity phosphorylates transient receptor potential vanilloid 1 (TRPV1), a key receptor that modulates agonist-induced calcium influx in neurons. Additionally, we found that inflammation triggers an increase in Cdk5 activity through activation of early growth response 1 (Egr-1) and p35 expression by TNF-alpha. These findings suggest that Cdk5 plays an important role in the molecular mechanisms involved in pain signaling. Validation of p35-Luc cell based assay for screening chemical libraries for Cdk5-targeted analgesics Resveratrol, a naturally occurring polyphenol found mainly in red wine, grapes, and berries, has been shown to have many therapeutic effects. It is known to protect against heart disease and cancers, promote anti-aging effects, suppress neuronal degeneration, and also act as an analgesic. Earlier studies indicated that intraperitoneal administration of resveratrol decreases hyperalgesia in the rat model of inflammatory pain induced by carrageenan injection in the hind paw. This was attributed to the previously reported inhibitory effects of resveratrol on COX2 expression. Intra-cerebral injections of resveratrol also suppressed hyperalgesia in a rat model of thermal pain with a concomitant inhibition of COX1 and COX2. Similar analgesic effects of resveratrol were also observed in the rat model of diabetic neuropathic pain. All of these studies indicate that resveratrol has analgesic properties against acute and chronic pain that is triggered either by inflammation, heat or a diabetic condition. Although some of these studies have linked this analgesic action of resveratrol to the altered expression of TNF-alpha and nitric oxide in a diabetic rat model as well as to the reduced expression of COX2 in the inflammatory pain model, it is still not clear as to how resveratrol brings about its analgesic action. To characterize a possible link between the analgesic effects of resveratrol and the role of Cdk5 in pain signaling, we set out to determine if resveratrol affects Cdk5 activity and, if it does, to characterize the mechanism by which it brings about this effect. For these studies, we used a cell-based assay in which a p35-luciferase construct was stably transfected in PC12 cells. Our studies demonstrate that resveratrol inhibits p35 promoter activity, and in combination with TNF-alpha significantly decreases p35 promoter activity, indicating that resveratrol can block the TNF-alpha mediated increase in Cdk5 activity. In the presence of resveratrol, the MEK inhibitor (U0126) decreased p35 promoter activity, whereas inhibitors of p38 MAPK (SB203580), JNK (SP600125), and NF-kappaB increased p35 promoter activity, suggesting that these pathways regulate p35 expression differently. The TNF-alpha-mediated increase in Egr-1 expression was decreased by resveratrol treatment with a concomitant reduction in p35 expression and protein levels, resulting in reduced Cdk5 kinase activity. This new molecular mechanism adds to the known analgesic effects of resveratrol, which are brought about mainly by resveratrol-mediated regulation of COX-1 and COX-2, and confirms the need for identification of new analgesics based on their ability to inhibit Cdk5 activity for effective treatment of pain. These findings validate our cell-based assay for its use in high-trhoughput screening of chemical libraries to identify potential analgesics for the treatment of pain. In collaboration with Dr. Toshio Ohshima of Waseda University in Tokyo, Japan, we continue to analyze the role of Cdk5 in myelination and neurodegeneration. In collaboration with Dr. Maria Valenzuela of the University of Chile, we have begun studying the involvement of Cdk5 in T-cell leukemia.
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