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RESEARCH REPORT |
1 Department of Operative and Preventive Dentistry and Endodontics, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany;
2 Department of Molecular and Cellular Sport Medicine, German Sport University, Cologne, Germany;
3 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany;
4 Department of Operative Dentistry and Periodontology, University of Cologne, Germany; and
5 Department I of Anatomy, University of Cologne, Germany
* corresponding author, yueksel.korkmaz{at}uni-duesseldorf.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: eNOS • phosphorylation of eNOS • Akt/PKB • ERK1/2 • odontoblasts
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In endothelial cells, the phosphorylation of human eNOS at Ser1177 (equivalent to Ser1179 bovine eNOS amino acid sequence) results in the increase of eNOS activity, whereas the phosphorylation of human eNOS at Ser114 (Ser116 bovine eNOS) and Thr495 (Thr497 bovine eNOS) decreases activity of eNOS (Fleming et al., 2001; Harris et al., 2001; Michell et al., 2001; Kou et al., 2002; Fleming and Busse, 2003).
In the growth of the ameloblastoma cell line, an involvement of ERK1/2 and Akt/PKB has been described (Sandra et al., 2004). It has been reported that eNOS activated at Ser1177 by Akt/PKB-dependent phosphorylation leads to the release of NO in endothelial cells (Dimmeler et al., 1999; Fulton et al., 1999). The role of MAP kinase was shown in the regulation of biomineralization in odontoblast-like cells (Narayanan et al., 2001). The bradykinin (BK)-induced activation of the mitogen-activated protein (MAP) kinases and extracellular signal-regulated kinases 1/2 (ERK1/2) leads to eNOS phosphorylation and enzyme inhibition (Bernier et al., 2000). The regulation of eNOS in endothelial cells has been well-characterized, whereas there are only a few reports about the regulation of eNOS in different types of cells.
The localization of eNOS in odontoblasts indicates that eNOS may be critical for odontoblast homeostasis (Felaco et al., 2000; Korkmaz et al., 2005); however, the regulation of eNOS in odontoblasts is not clear. BK receptor 2 has been detected in inflammation of the dental pulp (Goodis et al., 2000). In the present study, we examined the phosphorylation sites of eNOS in odontoblasts, and the effects of BK and the BK receptor 2 antagonist HOE 140 on the phosphorylation of eNOS, as well as the phosphorylation of Akt/PKB and ERK1/2 in odontoblasts.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, anti-phospho-Akt1/PKB (Thr308), and anti-ERK1/2 were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Bradykinin (BK), BK receptor 2 (B2) antagonists D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin (HOE 140), mouse monoclonal anti-diphospho-ERK1/2, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). Biotinylated goat anti-rabbit IgG, biotinylated anti-mouse IgG, normal goat serum (NGS), and Vectastain-ABC Kit were purchased from Vector Laboratories (Burlingame, CA, USA). Alexa Fluor 546-conjugated goat anti-rabbit IgG was from Molecular Probes Inc. (Eugene, OR, USA). The specificity of each primary antibody was confirmed by immunoblot analysis (Sommer et al., 2002; Fleming et al., 2005; Li et al., 2005).
Tissue Preparation
Male Wistar rats (n = 12; 3 mos old, each weighing from 250 to 280 g) were deeply anesthetized with a mixture of Ketamine (100 mg/kg) and Xylazine (5 mg/kg) and transcardially perfused with a fixative containing 4% paraformaldehyde. The molars were demineralized in 4 N formic acid and frozen-sectioned at 40 µm. The animal procedures were carried out in compliance with guidelines of the University of Cologne.
Bradykinin and HOE 140 Treatment
To test the stimulation of BK on phosphorylation sites of eNOS, Akt/PKB, ERK1/2 and the effects of HOE 140 on eNOS, p-eNOS at Ser1177 and at Thr495, we performed organ bath experiments. All 3 molars from one half of a rat jaw (n = 11) were dissected free from the roots, by means of forceps, and placed in Tyrode solution (pH 7.4) of the following composition (in mM): CaCl2 x 2H2O 1.8; MgCl2 x 6H2O 1.1; KCl 5.4; NaCl 136.9; NaH2PO4 0.4; Glucose 10.1; and NaHCO3 23.8 (all reagents were purchased from Merck, Darmstadt, Germany). In separate experiments, the molars were treated with a 10 mL Tyrode solution containing 10–7 M BK for 1 (n = 3), 3 (n = 3), 5 (n = 4), and 10 (n = 4) min and with 10–6 M HOE 140 for 5 min (n = 3) at 37°C. The control molars were treated only with Tyrode solution without BK or HOE 140. The molars were immersion-fixed in 4% paraformaldehyde, decalcified, and frozen-sectioned at 20 µm.
Immunohistochemistry
The free-floating sections were incubated for 48 hrs at 4°C with the following antibodies: anti-eNOS (1:800), anti-p-eNOS at Ser1177 (1:800) (human sequence), anti-p-eNOS at Thr495 (1:800) (human sequence), anti-p-eNOS at Ser116 (1:800) (bovine sequence), anti-Akt1/PKB (1:1000), anti-p-Akt1/PKB (Thr308) (1:1000), anti-ERK1/2 (1:1000), and anti-diphospho-ERK1/2 (1:1000). Then, sections were incubated with biotin-conjugated goat anti-rabbit IgG (1:500) and biotinylated anti-mouse IgG (1:500), followed by avidin-biotin-peroxidase complex (1:100). The signal was visualized with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma). Incubations without the primary or secondary antisera and pre-absorption were carried out as negative controls (Appendix 1).
Immunofluorescence and Confocal Scanning Laser Microscopy
The free-floating sections (20 µm) of rat (n = 4) molars (n = 12) were incubated with eNOS (1:400), p-eNOS at Ser1177 (1:500), Ser116 (1:400), and at Thr495 (1:400). Immunofluorescence signal was visualized with Alexa Fluor 546-conjugated goat anti-rabbit IgG. Secondary antibody alone was used as a negative control. The confocal images of odontoblasts within dentin tubules were visualized under a Zeiss LSM 510 Meta confocal, laser-scanning, inverted microscope (Carl Zeiss Inc., Jena, Germany) (Appendix 2).
Densitometric Analysis
The densitometric staining intensities of the eNOS, p-eNOS at Ser1177, Ser116, Thr495, Akt1/PKB, p-Akt1/PKB (Thr308), ERK1/2, and p-ERK1/2 in treated and in control sections were measured by image analysis of grey values of immunostaining (Appendix 3).
Statistical Analysis
Statistical differences in staining intensities between control-BK treatment and control-HOE 140 treatment were analyzed by the two-tailed Student’s t test for paired samples, as implemented in the software package SPSS for Windows, Version 11.0. All data presented are mean ± standard deviation (SD). Significance was considered at a P value < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The phosphorylation sites of eNOS at Ser1177 (Fig. 1B) and at Ser116 (Fig. 1C) were identified in odontoblasts, while phosphorylated (p)-eNOS at Thr495 was not detectable in odontoblasts (Fig. 1D). Localization of t-Akt/PKB (Fig. 1E) and p-Akt/PKB (Fig. 1F) was detected in odontoblasts. In odontoblasts, t-ERK1/2 was identified (Fig. 1G), while a subpopulation of odontoblasts revealed a signal for p-ERK1/2 (Fig. 1H). Controls resulted in the absence of the signal product (Appendix 4).
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), p-eNOS at Ser1177 (Fig. 1J), and at Ser116 (Fig. 1K) in odontoblasts and in their processes in predentin and dentin. Phosphorylation of eNOS at Thr495 was not detectable in odontoblasts (Fig. 1L). In the control sections, Alexa Fluor 546 alone resulted in the absence of the signal (data not shown).
Effects of BK on the Phosphorylation of eNOS in Odontoblasts
In odontoblasts, there was no significant difference in signal intensities of t-eNOS in control and after 1 (Fig. 2M), 3 (Fig. 2N), 5 (Fig. 2O), and 10 min (Fig. 2P) of BK treatment. The signal intensities for p-eNOS at Ser1177 in control odontoblasts (Fig. 2A) increased after 1 (Figs. 2B, 2M), 3 (Fig. 2N), and 5 min (Fig. 2O) treatment with BK. There was no significant difference for 10 min in signal intensities of Ser1177 between control (Fig. 2G) and BK-treated odontoblasts (Figs. 2H, 2P). Immunoreactivity of p-eNOS at Thr495 was absent or present at low levels in control odontoblasts for 1 (Figs. 2C, 2M), 3 (Fig. 2N), 5 (Fig. 2O), and 10 min (Figs. 2I, 2P), while the signal intensities of p-eNOS at Thr495 in odontoblasts were significantly increased after 1 (Figs. 2D, 2M), 3 (Fig. 2N), 5 (Fig. 2O), and 10 (Figs. 2J, 2P) min of treatment with BK. The absence of primary antibodies in incubations for 1 (control, Fig. 2E; BK, Fig. 2F), 3, 5, and 10 min (data not shown) resulted in a lack of specific signal in control and BK-treated odontoblasts. In all instances, odontoblast cell nuclei were positive for p-eNOS at Ser116 (Fig. 2K, 10 min). The signal intensities of p-eNOS at Ser116 in untreated odontoblasts (Fig. 2K) were not changed in sections after 10 min of treatment with BK (Fig. 2L).
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) and 10 min (Figs. 3A, 3J) and BK-treated molars for 5 (Fig. 3I) and 10 min (Figs. 3B, 3J). In comparison with the control for 5 (Fig. 3I) and 10 (Figs. 3C, 3J) min, BK increased acute staining intensities of p-Akt/PKB in odontoblasts after 5 (Fig. 3I) and 10 min (Figs. 3D, 3J). In odontoblasts, there were no differences in staining intensities of t-ERK1/2 for 5 (Fig. 3I), 10 (Figs. 3E, 3J) min in the controls, and after 5 (Fig. 3I) and 10 (Figs. 3F, 3J) min of treatment by BK. In all instances, odontoblasts were negative for p-ERK (Figs. 3G, 3H, 3I, 3J).
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), treatment with HOE 140 attenuated faint signal intensities for t-eNOS in odontoblasts after 5 min (Fig. 4B). Treatment of odontoblasts with HOE 140 after 5 min resulted in a decrease of p-eNOS at Ser1177 (Figs. 4C, 4D) and at Thr495 (Figs. 4E, 4F). Incubations without primary antibodies for 5 min of HOE 140 treatment resulted in an absence of signal in control (Fig. 4G) and HOE-140-treated (Fig. 4H) odontoblasts. HOE-140-treated odontoblasts revealed no significant signal intensities for t-Akt/PKB in odontoblasts (Figs. 4I, 4J). The signal intensities of p-Akt/PKB in odontoblasts (Fig. 4K) were decreased after 5 min of treatment with HOE 140 (Fig. 4L). HOE 140 did not affect signal intensities of t-ERK1/2 (Figs. 4M, 4N) and p-ERK1/2 (Figs. 4O, 4P) in odontoblasts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The regulatory roles of the Akt/PKB (Borgatti et al., 2000) and ERK1/2 (Jiang et al., 2002) in osteoblast cell proliferation and the existence of Akt/PKB and ERK1/2 in odontoblasts suggest an involvement of Akt/PKB and ERK1/2 in the regulation of odontoblasts. Involvement of MAP kinase in the biomineralization of odontoblast-like cells (Narayanan et al., 2001) supports a regulatory role for ERK1/2 in odontoblastic differentiation and dentin biomineralization. Localization of p-ERK1/2 in a subpopulation of odontoblasts is compatible with a physiological activity-dependent regulation of odontoblasts. The p-eNOS at Ser1177 and p-Akt/PKB in non-stimulated odontoblasts suggest that p-Akt/PKB influences the regulation of p-eNOS at Ser1177 in odontoblasts. The lack of p-eNOS at Thr495 and distinct p-ERK1/2 localization in a subpopulation of non-stimulated odontoblasts make the regulation of p-eNOS at Thr495 by ERK1/2 unlikely.
In pulpitis, the action of BK is linked to BK B2 receptors (Goodis et al., 2000). The signal intensities for p-eNOS at Ser1177 in odontoblasts were increased after 1, 3, and 5 min of treatment with BK, but after 10 min of treatment with BK, there was no significant difference in signal intensities of Ser1177 between control and by BK-treated odontoblasts. BK increased signal intensities of p-Akt/PKB in odontoblasts, while treatment of odontoblasts by the BK B2 receptor antagonist HOE 140 resulted in a decrease of signal intensities for p-eNOS at Ser1177 as well as for p-Akt/PKB. Analysis of these data indicates that BK treatment may induce the transient phosphorylation of eNOS at Ser1177 through the phosphorylation of Akt/PKB. It has been reported that treatment of endothelial cells with BK results in Akt/PKB-mediated phosphorylation of eNOS at Ser1177 (Venema, 2002). The signal intensities of p-eNOS at Thr495 in odontoblasts were increased after 1, 3, 5, and 10 min of BK treatment. HOE 140 reduced signal intensities of p-eNOS at Thr495 in odontoblasts. In endothelial cells, BK phosphorylated eNOS through the activation of ERK, and decreased the activity of eNOS (Bernier et al., 2000). In our results, however, there was no effect of BK treatment on the phosphorylation of ERK1/2 in odontoblasts. Analysis of these data suggests that BK induced the phosphorylation of eNOS at Thr495 in ERK1/2 independent mechanisms throughout the entire period of BK treatment in odontoblasts.
The phosphorylation of eNOS at Ser116 decreases eNOS activity, while dephosphorylation of eNOS at Ser116 results in an increase of eNOS activity of stimulated endothelial cells (Kou et al., 2002). The signal intensities of p-eNOS at Ser116 in non-treated odontoblasts were not significantly changed after 5 and 10 min of BK treatment in treated odontoblasts. BK treatment of odontoblasts appears to have no effect on the phosphorylation of eNOS at Ser116 residue in odontoblasts. In contrast, BK has been reported to stimulate the enhanced phosphorylation of Ser116 residue in endothelial cells (Fleming and Busse, 2003). This gives evidence for cell-type-specific regulation of eNOS phosphorylation at Ser116.
It has been reported that dephosphorylation of eNOS at Thr495 results in increased enzyme activity with increased production of NO and/or a switch of eNOS catalytic activity from NO to theproduction of superoxide anions (O2– ) (Lin et al., 2003). BK induces phosphorylation of eNOS at Thr495 in odontoblasts and may reduce eNOS catalytic activity and production of NO and/or O2– (Fleming and Busse, 2003; Lin et al., 2003). The localization of p-eNOS at Ser116 within the oxygenase domain and the proximity of Ser116 to the H4B binding sites support the notion that p-eNOS at Ser116 is involved in a switch of catalytic activity of eNOS from NO to O2– production (Fleming and Busse, 2003). Localization of the Ser116 residue in odontoblasts indicates that another signaling pathway may be a key point in the control of O2– production by p-eNOS at Ser116 in odontoblasts. However, the role for an alteration of catalytic activity of eNOS from NO to O2– production by phosphorylation of eNOS at Ser116 and Thr495 in odontoblasts remains to be elucidated.
In conclusion, analysis of our data indicates that BK has dual effects on the activation of eNOS in odontoblasts, the Akt/PKB-dependent up-regulation of eNOS by the transient phosphorylation of eNOS at Ser1177, and the ERK1/2-independent down-regulation of eNOS by the phosphorylation of eNOS at Thr495. The cell-specific regulation of p-eNOS at Thr495 and Ser116 may be a key point for the control of NO and/or O2– production in odontoblasts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received August 26, 2004; Last revision January 21, 2006; Accepted February 21, 2006
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Borgatti P, Martelli AM, Bellacosa A, Casto R, Massari L, Capitani S, et al. (2000). Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors. FEBS Lett 477:27–32.[ISI][Medline]
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM (1999). Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601–605.[Medline]
Felaco M, Di Maio FD, De Fazio P, D’Arcangelo C, De Lutiis MA, Varvara G, et al. (2000). Localization of the e-NOS enzyme in endothelial cells and odontoblasts of healthy human dental pulp. Life Sci 68:297–306.[ISI][Medline]
Fleming I, Busse R (2003). Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 284:R1–R12.
Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R (2001). Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88:E68–E75.
Fleming I, Mohamed A, Galle J, Turchanowa L, Brandes RP, Fisslthaler B, et al. (2005). Oxidized low-density lipoprotein increases superoxide production by endothelial nitric oxide synthase by inhibiting PKCalpha. Cardiovasc Res 65:897–906.
Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, et al. (1999). Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399:597–601.[Medline]
Goodis HE, Bowles WR, Hargreaves KM (2000). Prostaglandin E2 enhances bradykinin-evoked iCGRP release in bovine dental pulp. J Dent Res 79:1604–1607.
Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, et al. (2001). Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 276:16587–16591.
Jiang GL, White CR, Stevens HY, Frangos JA (2002). Temporal gradients in shear stimulate osteoblastic proliferation via ERK1/2 and retinoblastoma protein. Am J Physiol Endocrinol Metab 283:E383–E389.
Korkmaz Y, Baumann MA, Steinritz D, Schröder H, Behrends S, Addicks K, et al. (2005). NO-cGMP signaling molecules in cells of the rat molar dentin-pulp complex. J Dent Res 84:618–623.
Kou R, Greif D, Michel T (2002). Dephosphorylation of endothelial nitric-oxide synthase by vascular endothelial growth factor. Implications for the vascular responses to cyclosporin A. J Biol Chem 277:29669–29673.
Li C, Harris MB, Venema VJ, Venema RC (2005). Endostatin induces acute endothelial nitric oxide and prostacyclin release. Biochem Biophys Res Commun 329:873–878.[ISI][Medline]
Lin MI, Fulton D, Babbitt R, Fleming I, Busse R, Pritchard KA Jr, et al. (2003). Phosphorylation of threonine 497 in endothelial nitric-oxide synthase coordinates the coupling of l-arginine metabolism to efficient nitric oxide production. J Biol Chem 278:44719–44726.
Michell BJ, Chen ZP, Tiganis T, Stapleton D, Katsis F, Power DA, et al. (2001). Coordinated control of endothelial nitric-oxide synthase phosphorylation by protein kinase C and the cAMP-dependent protein kinase. J Biol Chem 276:17625–17628.
Narayanan K, Srinivas R, Ramachandran A, Hao J, Quinn B, George A (2001). Differentation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1. Proc Natl Acad Sci USA 98:4516–4521.
Sandra F, Harada H, Nakamura N, Ohishi M (2004). Midkine induced growth of ameloblastoma through MAPK and Akt pathways. Oral Oncol 40:274–280.[ISI][Medline]
Sommer F, Klotz T, Steinritz D, Schmidt A, Addicks K, Engelmann U, et al. (2002). MAP kinase 1/2 (Erk 1/2) and serine/threonine specific protein kinase Akt/PKB expression and activity in the human corpus cavernosum. Int J Impot Res 14:217–225.[ISI][Medline]
Venema RC (2002). Post-translational mechanisms of endothelial nitric oxide synthase regulation by bradykinin. Int Immunopharmacol 2:1755–1762.[ISI][Medline]
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