TGF-β1 sensitizes TRPV1 through Cdk5 signaling in odontoblast-like cells

Background: Odontoblasts are specialized cells that form dentin and they are believed to be sensors for tooth pain. Transforming growth factor-β1 (TGF-β1), a pro-inflammatory cytokine expressed early in odontoblasts, plays an important role in the immune response during tooth inflammation and infection. TGF-β1 is also known to participate in pain signaling by regulating cyclin-dependent kinase 5 (Cdk5) in nociceptive neurons of the trigeminal and dorsal root ganglia. However, the precise role of TGF-β1 in tooth pain signaling is not well characterized. The aim of our present study was to determine whether or not in odontoblasts Cdk5 is functionally active, if it is regulated by TGF-β1, and if it affects the downstream pain receptor, transient receptor potential vanilloid-1 (TRPV1).Results: We first determined that Cdk5 and p35 are indeed expressed in an odontoblast-enriched primary preparation from murine teeth. For the subsequent analysis, we used an odontoblast-like cell line (MDPC-

Differential steady state levels of Sox6 in WT and Cdk5^−/− mice.

<p>The mRNA expression of Sox6 in Cdk5 WT and Cdk5^−/− mouse brain. Total RNA was extracted from WT and Cdk5^−/− mice brain on embryonic day 17 (E17) and cDNA was prepared using reverse transcription. A) Quantitative PCR of Cdk5 expression in WT and Cdk5^−/− mice brain. The Cdk5^−/− brain shows complete abrogation of Cdk5 message. B, Sox6 expression in WT and Cdk5^−/− mice brain. There is no change in the Sox6 expression in WT and Cdk5^−/− mice n = 3 and p<0.001 (C) Lysates from embryonic day 17 (E17) age-matched wild-type (WT) and Cdk5^−/− mouse (KO) brain were separated by SDS-PAGE and transferred onto nitrocellulose. Cdk5 was detected with polyclonal C-8 antibody. Sox6 was immunodetected using Sox6 specific antibody. Equal loading was confirmed by immunodetection of β-actin. Cdk5^−/− mouse brain lysates showed elevated steady state levels of Sox6 protein. D) Densitometry analysis of Sox6 expression obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089310#pone-0089310-g005" target="_blank">Figure 5C</a>. n = 3 and <i>*p<0.001</i> of Sox6 expression in Cdk5^−/− brain compared to WT.</p

Sox6 associates with Cdk5/p35.

<p>(A) Sox6 was immunoprecipitated (IP) from postnatal day 1 (PN1) rat brain lysate and subjected to Western blotting using Sox6, Cdk5 and p35 antibodies. (B) Co-IP of Cdk5 and Sox6. The Cdk5 was immunoprecipitated from PN1 rat brain lysate and immunodetected with Sox6. (C) Colocalization of Sox6 with Cdk5. The cortical neurons were dissociated from embryonic stage 17 (E17) of rat and were cultured <i>in vitro</i> for 5 days and subjected to immunofluorescence. The Cdk5 was visualized using the monoclonal antibody (red) and Sox6 was visualized using polyclonal antibody (green). (D) Co-localization of Sox6 and p35 in nucleus. E17 dissociated rat cortical neurons were cultured <i>in situ</i> for 7 days and subjected to immunofluorescence. The Sox6 was immunostained with polyclonal antibody (green) and p35 was stained with monoclonal antibody (red). Sox6 co localizes with p35 in the nucleus.</p

Sox6 is a phosphoprotein and a substrate of Cdk5.

<p>(A) A schematic representation of domains of Sox6 protein. Domain structure of Sox6 protein reveals that Sox6 is a phospho protein with potential Cdk5 phosphorylation sites (Ser 98 and Thr119). (B) Alignment of human, mouse, and rat Sox6 amino acid sequences. The conserved Cdk5 phosphorylation sites [(S/T)PX(K/H/R)], Ser 98 and Thr119 are boxed. The Cdk5 consensus phosphorylation site is conserved among all three species. (C) Rat brain lysate from postnatal day 1 (PN1) was subjected to calf intestinal alkaline phosphatase (CIAP) treatment followed by Western blot analysis with Sox6 antibodies. CIAP treatment resulted in faster migration of Sox6, suggesting that Sox6 is a phosphoprotein. <i>Lane 1</i>, untreated, <i>Lane 2</i>, CIAP treated lysate (D) Sox6 was immunoprecipitated from postnatal day 1 and subjected to Western blot analysis with phospho-Ser and MPM2 (phospho-Ser/ThrPro) antibodies. Lysates with no antibody immunoprecipitation was used as control. (E) Sox6 protein immunoprecipitated from postnatal day 1 (PN1) and subjected to phosphorylation assay with recombinant Cdk5/p35 (<i>Lanes 1, 2 and 3</i>). Immune complex Cdk5 assay with no antibody does not show Sox6 phosphorylation (<i>Lane 1</i>). The Cdk5 inhibitor roscovitine was added in the reaction mixture inhibited the Sox6 phosphorylation by Cdk5 (<i>Lane 3</i>). (F) The GST-Sox6 was phosphorylated with Cdk5/p35 (<i>lane 2</i>). <i>Lane 1</i> shows the negative control (without Cdk5/p35). (G) Cdk5 phosphorylates Sox6 at Thr¹¹⁹. The Sox6 protein was phosphorylated <i>in vitro</i>, digested with trypsin, and phosphopeptides were isolated using an IMAC column before separation by HPLC. Elution of the peptide T*PER (MS/MS) is shown, suggesting that Thr119 is one of the site phosphorylated by Cdk5/p35.</p

Sox6 is developmentally regulated in brain.

<p>A) Developmental expression of Sox6 (upper panel) and Cdk5 (middle panel) in embryonic brain (E18), postnatal days 0, 7, 14, 21, 60 and 365 days. The lower panel shows the β-actin staining as loading control. B) Densitometry analysis of Sox6 obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089310#pone-0089310-g002" target="_blank">Figure 2A</a>. C) Sections of mouse brain during embryonic development were fixed and immunostained with Sox6-specific antibodies from embryonic day 12, E12 (b) and E16 (c). C<i>a</i>, Negative control shows the section of rat brain (E12) that were fixed and immunostained with Sox6 antibodies preabsorbed with Sox6 protein. Immunostaining with pre-immune serum as well as with no primary antibody showed no signal. <i>Cb</i>, The E12 rat brain section was fixed and immunostained with Sox6-specific antibodies. Sox6 staining is observed in the mitotic nuclei of E12 rat brain. C<i>c</i>, Sox6 staining in the E16 brain. The neocortex-cortical plate and the sub ventricular zone-neuroepithelium (svz) are shown in E16.</p

Inhibition of Cdk5 activity by DN Cdk5 increases Sox6 expression.

<p>A) We transfected the 5 DIC primary cortical neurons either with WT Cdk5 (lane 2), DN Cdk5 (lane 3) and p35 (lane 4) and then Western blot analysis was performed with Sox6 antibodies to analyze the endogenous Sox6 levels, 2-days after transfection (7DIC). Transfection of WT Cdk5 (lane 2) reduced the Sox6 levels, while transfection of DN Cdk5 (lane 3) increased Sox6 expression. The middle panel shows the transfected Cdk5 and endogenous Cdk5 levels. The lower panel corresponds to the β-actin levels. B) Densitometry analysis of Sox6 expression shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089310#pone-0089310-g006" target="_blank">Figure 6A</a>. Sox6 expression compared to non-tranfected neurons, n = 3 and <i>*p<0.01</i>.</p