Cyclin-dependent kinase 5 (Cdk5), a proline-directed serine/threonine kinase, plays a pivotal role in inflammatory pain. Cdk5 kinase activity is predominant in post-mitotic neurons where its activators, p35 and p39, are expressed, although recently Cdk5 activity has also been detected in non-neuronal tissues. The increased expression of p35, which occurred after experimentally-induced inflammation, was associated with elevated Cdk5 activity in the murine nociceptive primary afferent neurons. We also discovered that the Cdk5-mediated phosphorylation of TRPV1 at Thr407 is involved in thermal nociception and inflammatory pain. We have further demonstrated that tumor necrosis factor-alpha;(TNF-alpha) increases Cdk5 activity, while resveratrol, a polyphenolic compound with known analgesic activity, inhibits Cdk5 activity. Most importantly, we recently discovered that TGF-beta 1 is a key regulator of Cdk5 activity in nociceptive neurons, indicating that the active crosstalk between the TGF-beta 1 and Cdk5 pathways plays an important role in inflammation-induced pain signaling. The precise role of similar crosstalk between the TGF-beta 1 and Cdk5 has not been studied in relation to tooth pain, which is frequently induced by inflammation associated with an infection that affects the odontoblast cells. Therefore, we have evaluated the expression of Cdk5 and p35 in both an odontoblast-enriched preparation from murine teeth and in the odontoblast-like MDPC-23 cell line. To evaluate its possible involvement in tooth pain, we then characterized the regulation of Cdk5 activity by TGF-beta 1, detected the Cdk5-mediated phosphorylation of TRPV1, and finally measured its effect on proton- and capsaicin-induced TRPV1 activation. To determine whether or not mouse odontoblasts express Cdk5 and p35, we extracted total RNA from an odontoblast-enriched primary preparation from mouse incisors and performed RT-PCR analysis. As a positive control for Cdk5 and p35 expression, we used mouse brain and trigeminal ganglia (TG). We found that Cdk5 and p35 mRNAs are expressed in the odontoblast-enriched preparation at levels similar to TG, but less compared to the brain. To evaluate the purity of our odontoblast-enriched preparation, we analyzed the expression of dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1), which are two well-characterized odontoblast markers. Interestingly, we found DSPP expression only in the odontoblast-enriched preparation, whereas DMP1 was found not only in our odontoblast-enriched preparation but also in the brain, thus confirming an earlier report. Moreover, we detected the expression of Tau, a neuronal marker, in our odontoblast-enriched preparation. Interestingly, we found detectable expression of TRPV1 in our odontoblast-enriched preparation, but it was less robust compared to TG. Taken together, these results confirm the expression of Cdk5 and p35, as well as TRPV1, in our odontoblast-enriched preparation from mouse incisors, thus implicating a potential role for Cdk5 in tooth pain. We previously reported that Cdk5 is a key player in pain signaling, and that both TNF-alpha and TGF-beta 1 regulate Cdk5 activity in sensory neurons by modulating the expression of p35, a co-activator of Cdk5. To investigate whether or not Cdk5 plays a similar role in tooth pain signaling, we examined its expression and kinase activity in MDPC-23 cells, which is an odontoblast-like cell line derived from rodent dental papilla cells. We observed that differentiation of MDPC-23 cells induces the expression of Cdk5 and p35, with a subsequent increase in Cdk5 kinase activity. Interestingly, we also found that TGF-beta 1 signaling is activated during the differentiation of MDPC-23 cells, and that TGF-beta 1 treatment increases p35 protein levels and Cdk5 kinase activity in these cells. Increased levels of p35 and Cdk5 activity during differentiation are associated with activated ERK1/2 signaling, which is known to regulate p35 expression. Odontoblasts have been linked to dental nociception due to the expression of functional TRPV1 channels that are found in cells from humans and mice. We previously reported that Cdk5 can phosphorylate TRPV1, specifically at Thr407. Therefore, we evaluated whether a TGF-beta 1-mediated increase in Cdk5 activity could regulate phospho-Thr407-TRPV1 in MDPC-23 cells. The expression of TRPV1 has not been previously reported in MDPC-23 cells, and we measured the expression of TRPV1 but we could not find detectable TRPV1 protein. Therefore, we transiently transfected the MDPC-23 cells with a CMV promoter-driven TRPV1-GFP vector, and found that TGF-beta 1 treatment significantly increased phospho-Thr407-TRPV1 levels. These results suggest that in the TRPV1-expressing MDPC-23 cells, p35 protein levels increase in response to TGF-beta 1, thus resulting in elevated Cdk5 activity and TRPV1 phosphorylation. To evaluate whether the increased TRPV1 phosphorylation in the MDPC-23 cells treated with TGF-beta 1 has a physiological effect, we examined proton- and capsaicin-induced calcium influx in these cells. Ca2+ influx was measured in the MDPC-23 cells stably transfected with rat TRPV1, and then these cells were activated either with low pH buffer or with 100 nM capsaicin in the presence of calcium-45. The confluent cells were pre-treated with TGF-beta 1 (0.1 to 3.0 ng/ml) alone, TGF-beta 1 in the presence of SB431542 (10 M), or TGF-beta 1 in the presence of roscovitine (25 M), and then the cells were assayed for calcium uptake at 24 hours. We found enhanced calcium uptake by the cells treated with TGF-beta1, compared to the untreated control cells, and this effect was blocked when the cells were co-treated with either roscovitine or SB431542. Therefore, these results suggest that the TGF-beta 1-mediated phosphorylation of TRPV1 potentiates the proton- and capsaicin-induced Ca2+ influx in the TRPV1-expressing MDPC-23 cells. There is accumulating evidence that supports our findings. From one study, the number of TGF-beta 1 positive cells increased significantly during pulpitis in the human odontoblast layer (ODL). Another report showed that several cytokines, chemokines, and their receptors, were upregulated in the human ODL during tooth caries caused by bacteria and yeast that colonize dentin and root cementum. Additionally, it was shown that immunoreactivity for TGF-beta 1 was significantly increased in the odontoblast and pulpal cells of carious teeth. These findings indicate that TGF-beta 1 is upregulated in normal pathological conditions, such as carious inflammation, further suggesting that TGF-beta 1 is vital not only in resolving inflammation and promoting wound healing, but also likely involved in pain signaling. Based on our studies, we propose a model where TGF-beta 1 is secreted during bacterial inflammation and promotes Cdk5 kinase activity in the odontoblasts. This in turn leads to the phosphorylation of TRPV1, other TRP ion channels, or TREK-1 channels, which then potentiates the calcium influx during stimulation. The subsequent depolarization caused by the calcium influx activates downstream effectors, which possibly leads to the transmitter release from the odontoblasts and pain signal transmission. In summary, primary odontoblasts and the odontoblast-like MDPC-23 cells express functional Cdk5/p35. TGF-beta 1 treatment increases Cdk5 activity in the TRPV1-expressing MDPC-23 cells. This subsequently leads to increased TRPV1 phosphorylation, thereby potentiating proton- and capsaicin-induced calcium influx in these cells. These findings suggest that odontoblasts are directly involved in dental nociception.

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Rozas, Pablo; Lazcano, Pablo; Piña, Ricardo et al. (2016) Targeted overexpression of tumor necrosis factor-α increases cyclin-dependent kinase 5 activity and TRPV1-dependent Ca2+ influx in trigeminal neurons. Pain 157:1346-62
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