Regulation of Cdk5 activity inflammation-induced pain: We have previously reported that Cdk5 participates in the regulation of nociceptive signaling, and the expression of Cdk5 and its activator, p35, are upregulated in nociceptive neurons during peripheral inflammation.
The aim of our current FY09 study was to identify the pro-inflammatory molecules that regulate Cdk5/p35 activity in response to inflammation. We constructed a vector that contains the mouse p35 promoter driving luciferase expression. We transiently transfected this vector in PC12 cells to test the effect of several cytokines on p35 transcriptional activity and Cdk5 activity. Our results indicate that Tumor Necrosis Factor-alpha (TNF-alpha) activates p35 promoter activity in a dose-and time-dependent manner, and concomitantly upregulates Cdk5 activity. Because TNF-alpha is known to activate ERK1/2, p38 MAPK, JNK and NF-alpha signaling pathways, we examined its involvement in the activation of p35 promoter activity. MEK inhibitor, which inhibits ERK activation, decreased p35 promoter activity, while inhibitors of p38 MAPK, JNK, and NF-alpha increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The mRNA and protein levels of early gene response 1 (Egr-1), a transcription factor, were increased by TNF-alpha treatment, and this increase was dependent on ERK signaling. In a mouse model of inflammation-induced pain in which a carrageenan injection into the hind paw caused hypersensitivity to heat stimuli, TNF-alpha mRNA was increased at the site of injection. These findings suggest that TNF-alpha-mediated regulation of Cdk5 activity plays an important role in inflammation-induced pain signaling. Phosphoproteomic analysis of Cdk5 targets: The human genome encodes over 500 different protein kinases, which are the key regulatory enzymes that catalyze the phosphorylation of proteins at about 100,000 different sites to reversibly control their functional activities. Defects in specific protein kinases have been linked to over 400 diseases, and about 25% of all pharmaceutical industry research and development is now focused on the discovery and evaluation of protein kinase inhibitors for therapeutic applications. Cdk5 has become a target of high interest to the drug industry because of its key role in neuronal homeostasis. So far more than 40 different Cdk5 substrates have been identified, and abnormal Cdk5 activity has been implicated in several disease processes, including neurodegenerative disorders, cancer, and diabetes. However, a global profiling of protein phosphorylation mediated by Cdk5 is still not available. Our current knowledge about such profiling comes from experiments that are performed in different laboratories and mainly based on 2-dimensional gel electrophoresis or yeast 2-hybrid screening. Because of the limitations of these techniques at the point of validation of targeted proteins, we took a different approach to resolve this issue. We compared the phosphorylation status and the total protein levels of 258 different proteins by simple Western blotting analysis of Cdk5-/- and WT wild-type brains. The antibodies used in this analysis were already proven to be highly specific for their targeted sites in different biochemical pathways. We based our selection of these proteins on some known and predicted functions of Cdk5, and further categorized them into 6 different groups. The first group contained 25 different proteins involved in apoptosis, the second consisted of 77 other different kinases, the third consisted of 39 different substrates for these kinases, the fourth consisted of 43 different proteins involved in the cell cycle, the fifth contained 37 different proteins involved in numerous neurobiological functions, and the sixth consisted of 37 different proteins controlling different kinase pathways. This phosphoproteomic screening gave us a broad overview of Cdk5 targets involved in these biochemical pathways and cellular processes. Cdk5 is an attractive drug target for the development of novel therapies for a number of diseases and disorders. Several classes of chemical inhibitors for Cdk5 have been identified. Most of these inhibitors target the ATP-binding site, resulting in a lack of specificity for cyclin-dependent kinases, as well as for other ATP-dependent kinases. It is therefore critical to develop more specific inhibitors directed specifically to Cdk5/p35. A promising approach is the use of a 125 amino acid peptide of p35, called Cdk5 inhibitor peptide (CIP), which has a higher affinity for Cdk5/p25. CIP was found to inhibit aberrant tau phosphorylation in cortical neurons, and to protect against amyloid beta peptide toxicity, suggesting that CIP, and smaller molecules derived from this endogenous inhibitor, will allow more selective targeting of Cdk5 hyperactivation. Conversely, regulation of the Cdk5 activators, p35 and p39, could be an alternative approach for inhibiting Cdk5 activity. Several inflammatory mediators have been reported to regulate p35 expression and subsequently regulate Cdk5 activity. Therefore, if it is possible to regulate the release, activity, receptor-binding, or signaling pathways of these modulators, we will be able to selectively regulate Cdk5 activity to treat chronic pain. It is clear now that Cdk5 plays an important role in pain signaling. Our findings provide a key molecular mechanism for functional regulation of TRPV1 by Cdk5, and suggest a new paradigm for developing analgesics that target Cdk5-mediated phosphorylation. In addition to the current uses for opioids and anti-inflammatory drugs, Cdk5-targeted analgesics will prove to be novel therapeutic options to effectively treat many painful conditions and disorders. Our overall strategy for future work is focused on delineating the molecular roles of Cdk5 in pain signaling, with an emphasis on the facial and oral region. We will use 3 major strategies to pursue these studies. First, we will identify and characterize the upstream regulators of Cdk5, particularly those which are principal players in inflammation-mediated pain signaling. We will develop an efficient cell-based assay for high throughput screening of such regulators, and extend this assay to evaluate the efficacy of Cdk5 inhibitors as analgesics. We will develop strategies to help us identify the role of Cdk5 in tooth pain and develop potential analgesics. Work on other potentially important questions involving Cdk5 has been shifted to ongoing major collaborations with a number of leading laboratories in neurobiology. This shift will benefit us in our increasingly predominant program on Cdk5 and pain signaling.
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