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J Dent Res 84(3):234-239, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORTS
Biological

Expression of Protein Kinases C ßI, ßII, and VEGF during the Differentiation of Enamel Epithelium in Tooth Development

M. Aida*, T. Irié, T. Aida, and T. Tachikawa

Department of Oral Pathology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan;

* corresponding author, aida{at}dent.showa-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein kinase C (PKC) is an important molecule involved in various cell function, and mediates induced secretion of vascular endothelial growth factor (VEGF). It is hypothesized that PKC and VEGF may be associated with tooth development. Using the laser microdissection method and real-time reverse-transcription-polymerase chain-reaction (RT-PCR), we investigated the expression of PKC ßI and ßII, VEGF, and amelogenin (used as a marker of differentiation to ameloblasts) in the inner and outer enamel epithelia, stellate reticulum, and dental papilla in each stage of the dental germ. We found that the expression levels of PKC ßI and ßII were increased in the inner enamel epithelium during the early bell stage. In addition, the increased expression levels of PKC ßI and ßII were accompanied by increased VEGF expression. These results indicate that PKC ßI, ßII, and VEGF are closely associated with the differentiation of the inner enamel epithelium to ameloblasts.

KEY WORDS: laser microdissection • protein kinase C • vascular endothelial growth factor • odontogenesis • real-time reverse-transcription • polymerase chain-reaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tooth development occurs by the close interaction of the oral epithelial tissue and mesenchyme (Lumsden, 1988). Fibroblast growth factor (FGF), transforming growth factor (TGF), and hepatocyte growth factor (HGF) play important roles as signaling factors between cells in this epithelial-mesenchymal interaction (Wilkinson et al., 1989; Lyons et al., 1990; Niswander and Martin, 1993; Vainio et al., 1993; Vaahtokari et al., 1996). The importance of the signals between such cells in tooth development is well-recognized. However, the role of an intracellular signal transduction molecules remains to be clarified. Protein kinase C (PKC) is a molecule involved in cell differentiation, cell growth (Clemens et al., 1992), interactions between cells (Freed et al., 1989), secretions (Lord and Ashcroft, 1984), cytoskeleton control (Owen et al., 1996), transcriptional control (Berry and Nisizuka, 1990), apoptosis (Pongracz et al., 1994), etc. This molecule is a serine/threonine kinase. PKC isoforms have been divided into 3 groups: cPKC ({alpha}, ß, ßI, ßII, {gamma}), aPKC ({lambda}, {zeta}), and nPKC ({delta}, {varepsilon}, {eta}, {theta}).

The role of PKC in tooth development is not clear. However, PKC{alpha}, which is an isoenzyme of PKC, is involved in early dentin formation and amelogenesis (Bawden et al., 1994). PKC regulates expression of tumor necrosis factor (TNF)-{alpha} in the rat dental follicle (Yao and Wise, 2003) and is associated with tooth eruption through TNF-{alpha} by promoting the recruitment of mononuclear cells to dental follicle to form osteoclasts (Wise and Yao, 2003a). Association between PKC and vascular endothelial growth factor (VEGF), which is a factor that induces angiogenesis, has been shown in several reports. It has been demonstrated that PKC ßI and ßII increase VEGF-induced endothelial cell proliferation (Suzuma et al., 2002), and that PKC ß inhibitor has an anti-angiogenic effect (Teicher et al., 2001). PKC mediates induced secretion of VEGF (Tsai et al., 2003). Moreover, it has been shown that VEGF is expressed in dental follicle and participates in tooth eruption by promoting osteoclastogenesis, and that activation of PKC may up-regulate VEGF expression (Wise and Yao, 2003b). However, the role of VEGF, as well as PKC, in tooth development is not clear. We hypothesize that PKC and VEGF may be associated with tooth development. To clarify this hypothesis, we studied the localization and temporal transition of quantitative amounts of gene expression of PKC ßI, ßII, and VEGF in the dental germ in each stage of tooth development, using immunohistochemistry, laser microdissection, and real-time reverse-transcription (RT)-polymerase chain-reaction (PCR).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Sprague-Dawley (SD) rats were purchased from Japan SLC, Inc. (Tokyo, Japan). The jaws of SD rats ranging in age from embryonic day (E) 16 to post-natal day (PN) 2 were used for analysis of the dental germ. The jaws of SD rats of E16 were used for analysis of the cap stage, those of E19 were used for analysis of the early bell stage, and those of PN2 were used for analysis of the late bell stage. This study was approved by the Animal Research Committee of Showa University.

Immunohistochemical Staining and Antibodies
Jaws were dissected and fixed overnight in 4% paraformaldehyde in phosphate-buffered saline, pH 7.3 (PBS). Immunohistochemical analysis with avidin-biotin complex (the ABC method) was performed. The tissues were embedded in paraffin and sectioned. The sections were deparaffinized with xylene. Endogenous peroxidase was blocked by incubation of the sections in 0.3% hydrogen peroxide in absolute methanol at room temperature for 30 min. After serial dilutions of ethanol in water, the sections were washed in 0.01 M PBS. Then, 10% normal goat serum (HISTOFINE, Nichirei Co. Ltd, Tokyo, Japan) was applied at room temperature for 30 min to prevent non-specific binding of antibodies. The antibodies used were anti-rat PKC ßI and ßII rabbit IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and biotin-labeled anti-rabbit IgG antibody was used as the secondary antibody. For each antibody tested, sections were washed in PBS 3X each, incubated for 1 hr with the secondary antibody, and washed in PBS 3X each. The sections were then incubated with 3,3'-diaminobenzidine tetrahydrochloride (DAKO, Japan Co., Ltd, Kyoto, Japan) for 1–2 min, rinsed with tap water, counterstained with hematoxylin, and mounted.

Laser Microdissection
Jaws of embryonic rats were dissected, embedded in TissueTek OCT medium (Sakura Finetechnical Co. Ltd, Tokyo, Japan), and then frozen in liquid nitrogen. The tissues were sectioned at 8 µm in a cryostat. The frozen sections were placed on glass slides which had been pre-treated at 200°C for 8 hrs for inactivation of ribonuclease (RNase). The frozen sections were placed at room temperature for 1–3 min and fixed in 100% methanol for 3 min. After being washed with RNase-free water, sections were stained with 1% toluidine blue and air-dried. The target areas (inner enamel epithelium, outer enamel epithelium, cells of stellate reticulum surrounded by the inner enamel epithelium, outer enamel epithelium, and dental papilla) in the sections were microdissected with a Laser Microbeam System (P.A.L.M, Bernried, Germany). Each population was estimated to be > 98% homogeneous, as determined by microscopic visualization of the recovered cells (Emmert-Buck et al., 1996).

RNA Extraction from Microdissected Samples
Total RNA was independently extracted from each population of laser-microdissected cells. Briefly, the microdissected cells within the cap were covered with 200 µL buffer solution, 4 M guanidine thiocyanate, 25 mM sodium citrate, and 0.5% sarcosyl, and the cap was placed on the tube and vortexed. After the addition of 20 µL of 2 M sodium acetate, 220 µL of water-saturated phenol, and 60 µL of chloroform-isoamyl alcohol, the tube was centrifuged at 12,000 x g at 4°C for 30 min to separate the aqueous and organic phases. The aqueous layer was transferred to a new tube. Two µL of glycogen and 200 µL of isopropanol were added and centrifuged at 12,000 x g at 4°C for 30 min. The supernatant was removed, and the pellet was washed by 70% ethanol, centrifuged, and air-dried. The total RNA was re-suspended in RNase-free water.

Real-time RT-PCR
The mRNA expression levels of PKC ßI, PKC ßII, and VEGF, normalized to that of GAPDH, were determined by real-time RT-PCR, with use of the ABI Gene AMP 5700 and ABI Sequence Detection System software. RT-PCR was performed with a QuantiTect SYBR Green RT-PCR kit (QIAGEN, Tokyo, Japan). Each 30-µL reaction mixture contained 15 µL of 2 x QuantiTect SYBR Green RT-PCR Master Mix, 1 µL of sense primer (1 µM), 1 µL of antisense primer (1 µM), 0.3 µL of QuantiTect RT Mix, 2 µL of template RNA, and RNase-free water. The conditions of RT-PCR were as follows: reverse transcription at 50°C for 30 min; PCR, initial denaturation at 95°C for 15 min, 45 cycles at 94°C for 15 sec, 55°C for 30 sec, and 72°C for 45 sec. The sequences of the PCR primer pairs that were used for each gene are as follows: rat GAPDH (product size, 66 bp), 5'-AAG TAT GAT GAC ATC AAG AAG GTG GT-3', 5'-AGC CCA GGA TGC CCT TTA GT-3'; protein kinase C ßI (product size, 62 bp), 5'-TTC GTC ACG TTC TCC TGC C-3', 5'-TTT GCT CCG TGG GTC ATC A-3'; protein kinase C ßII (product size, 61 bp), 5'-ATT CGT CAC GTT CTC CTG CC-3', 5'-TTG CTC CGT GGG TCA TCA-3'; amelogenin (product size, 68 bp), 5'-GCC CCC CAG CAA CCA-3', 5'-TGT TGG GTT GGA GTC ATG-3' GA; and vascular endothelial growth factor (product size, 194 bp), 5'-GTC CAA TTG AGA CCC TGG TG-3', 5'-CTA TGT GCT GGC TTT GGT GA-3'. All primers were selected from 2 different exons with at least 1 intervening intron. Standard curves were generated with the use of serial dilutions (1–0.0001) of known quantities of rat brain mRNA. We calculated the relative expression level by dividing the signal intensity of each gene by that of GAPDH. All values are expressed as the mean ± SEM, and statistical comparisons were made between different target areas (n = 5; the dissections and RT-PCR reactions were all repeated 5x per target area), with Student’s t test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunohistochemical Analysis
Immunohistochemical analyses of PKC ßI and ßII expression in the rat dental germ in the cap stage, early bell stage, and late bell stage were performed. PKC ßI and ßII were expressed in the inner enamel epithelium at the early bell stage (Figs. 1bGo, 1eGo) and the late bell stage (Figs. 1cGo, 1fGo). In particular, there were cells with strong PKC ßI and ßII expression in the cervical loop (near the epithelial sheath) of the inner enamel epithelium in the early bell stage and the late bell stage. Cells in the inner enamel epithelium, except for the region of the cervical loop, expressed PKC ßI and ßII faintly in the early bell stage and the late bell stage.



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  		Figure 1. Immunohistochemical analysis of PKC ßI (a,b,c) and ßII (d,e,f) expression in the rat tooth germ at the cap (a,d), early bell (b,e), and late bell (c,f) stages (scale bars, 100 µm). At the cap stage, no immunoreactivity for PKC ßI was seen (a). At the early bell stage, the inner enamel epithelium in the cervical loop shows strong immunoreactivity for PKC ßI (b'). At the late bell stage, the inner enamel epithelium in the cervical loop in the dental papilla shows weak immunoreactivity for PKC ßI (c'). At the cap stage, no immunoreactivity for PKC ßII can be seen (d). At the early bell stage, the inner enamel epithelium in the cervical loop shows strong immunoreactivity for PKC ßII (e'). At the late bell stage, the inner enamel epithelium in the cervical loop shows weak immunoreactivity for PKC ßII (f').

 
Microdissection and Real-time RT-PCR Analysis
Using the laser microdissection method, we dissected the inner and outer enamel epithelia, stellate reticulum, and dental papilla from rat dental germs in the cap stage, early bell stage, or late bell stage, and recovered the dissected cells. RNA was extracted from the dissected cells, and RT-PCR for PKC ßI, PKC ßII, amelogenin, and VEGF was carried out. Amelogenin was used as a marker of inner enamel epithelium differentiation to ameloblasts.

In the cap stage, there were low levels of expression of PKC ßI, PKC ßII, and amelogenin in the dental germ (Fig. 2cGo). A low level of VEGF expression was seen in the outer enamel epithelium, stellate reticulum, inner enamel epithelium, and dental papilla in the cap stage (Fig. 2dGo).



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  		Figure 2. Cells of the tooth germ at the cap stage before (a) and after (b) laser microdissection (scale bars, 100 µm). The white areas in Fig. 2b are areas that had been microdissected. Comparison of the levels of mRNA expression of PKC ßI, PKC ßII, amelogenin (c), and VEGF (d) in the outer enamel epithelium, stellate reticulum, inner enamel epithelium, and dental papilla, with the use of real-time RT-PCR. Data are the mean ± SEM from 5 experiments. PKC ßI and PKC ßII expression levels are not appreciably up-regulated in any of the four areas.

 
In the early bell stage, PKC ßI and ßII, along with amelogenin, were strongly expressed in the inner enamel epithelium (P < 0.05). In contrast, the expression levels of PKC ßI and ßII in the stellate reticulum and dental papilla were not appreciably high in the early bell stage (Fig. 3cGo). However, there was appreciably increased VEGF expression in the inner enamel epithelium (P < 0.05; Fig. 3dGo).



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  		Figure 3. Cells of the tooth germ at the early bell stage before (a) and after (b) laser microdissection (scale bars, 100 µm). The white areas in Fig. 3b are areas that had been microdissected. Comparison of the levels of mRNA expression of PKC ßI, PKC ßII, amelogenin (c), and VEGF (d), with the use of real-time RT-PCR analysis. Data are the mean ± SEM of 5 experiments. PKC ßI, PKC ßII, VEGF, and amelogenin expression are appreciably up-regulated in the inner enamel epithelium (P < 0.05).

 
In the late bell stage, the expression levels of PKC ßI, PKC ßII, and amelogenin in the inner enamel epithelium were slightly lower compared with that in the early bell stage (Fig. 4cGo). The expression levels of PKC ßI and PKC ßII tended to be slightly higher in the inner enamel epithelium and dental papilla than those in the outer enamel epithelium and stellate reticulum (Fig. 4cGo). The level of VEGF expression was highest in the inner enamel epithelium compared with those in the outer enamel epithelium, stellate reticulum, and dental papilla (P < 0.05).



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  		Figure 4. Cells of the tooth germ at the late bell stage before (a) and after (b) laser microdissection (scale bars, 500 µm). The white areas in Fig. 4b are areas that had been microdissected. Comparison of the levels of mRNA expression of PKC ßI, PKC ßII, amelogenin (c), and VEGF (d), with the use of real-time RT-PCR analysis. Data are the mean ± SEM of 5 experiments. The expression levels of PKC ßI, PKC ßII, VEGF, and amelogenin were reduced in the inner enamel epithelium from the early bell stage; however, they remained higher than in other areas of the dental germ.

 
Expression of amelogenin in the neighboring connective tissue or skeletal muscle was not detected throughout the stages of odontogenesis.

These results indicated that the expression levels of PKC ßI and ßII in the inner enamel epithelium showed behavior similar to that of the expression levels of VEGF and amelogenin from cap through late bell stages.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The basement membrane, which separates the enamel organ and dental papilla in the dental germ near the dental neck arcuation (near the epithelial sheath) in the early bell stage, disappears in the late bell stage, and odontoblast differentiation occurs by direct interaction of the cells of the inner enamel epithelium and dental papilla (Ten Cate, 1994). It has been suggested that type IV collagenases [matrix metalloproteinase (MMP)-2, MMP-9] are involved in the degradation of the basement membrane during tooth morphogenesis (Randall and Hall, 2002). MMP-9 expression is regulated by NF-{kappa}B (Sato and Seiki, 1993). PKC ßI and ßII are specifically required to activate NF-{kappa}B (Su et al., 2002). Moreover, it has been demonstrated that PKC ß is directly associated with the induction of expression of MMP-9 (Xie et al., 1998). This indicates that PKC ßI and ßII transmit an important signal, leading to formation of the enamel, and this occurs following odontoblast differentiation after the controlled expression of MMP.

We determined that the expression levels of PKC ßI and PKC ßII isoforms and VEGF correlate with the expression level of amelogenin in the inner enamel epithelium from cap through late bell stages of odontogenesis. This indicates that PKC ßI and PKC ßII isoforms are closely associated with initiating the differentiation of the inner enamel epithelium during early and late bell stages.

In analysis of the expression level of VEGF from the early bell stage to the late bell stage, the increased expression level of VEGF in the inner enamel epithelium correlated with the increased expression levels of PKC ßI and ßII and amelogenin in this tissue. In vascular endothelial cells, VEGF activates PKC ßI and ßII, and VEGF activates MAP kinase through the activation of Raf-1 and MEK, leading to cell proliferation. It has been reported that VEGF also affects the expression levels of PKC ßI and ßII (Takahashi et al., 1999). These reports indicate that VEGF mainly affects the inner enamel epithelium by an autocrine mechanism, and that VEGF seems to control the differentiation of the inner enamel epithelium to ameloblasts by signal transduction to the nucleus through PKC ßI and ßII.

VEGF is an important factor that induces angiogenesis and is related to PKC ßI and ßII (Mustonen and Alitalo, 1995). In the present analysis, VEGF expression was detected not only in the inner enamel epithelium but also in the stellate reticulum, dental papilla, and outer enamel epithelium of the dental germ. The location with the highest VEGF expression was the inner enamel epithelium, and it is speculated that VEGF plays a large role in the differentiation of the inner enamel epithelium.

The distribution of blood vessels in the initial stage of tooth development is important for differentiation of the dental germ (Ten Cate, 1994). Many blood vessels are in the dental sac along the circumference of the dental germ in the cap stage. These vessels invade the dental papilla. Furthermore, blood vessels gather in the presumptive area of tooth root formation. Expression of VEGF in the stellate reticulum and outer enamel epithelium may serve to regulate angiogenesis in the dental sac along the circumference of the enamel organ.

In conclusion, we studied the temporal transition of expression of PKC ßI, ßII, VEGF, and amelogenin throughout the stages of odontogenesis. Our results indicate that PKC ßI and PKC ßII isoforms and VEGF are closely associated with the differentiation of the inner enamel epithelium.


   ACKNOWLEDGMENTS
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

Received March 5, 2004; Last revision December 23, 2004; Accepted December 23, 2004


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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