Copyright ©The Histochemical Society, Inc.

Intracellular Distribution of Desmoplakin in Human Odontoblasts

Yoshihiko Sawa, Shin-ichiro Kuroshima, Yuji Yamaoka and Shigemitsu Yoshida

Department of Oral Functional Anatomy, Graduate School of Dental Medicine, Hokkaido University, Kita-ku, Sapporo, Japan

Correspondence to: Yoshihiko Sawa, Department of Oral Functional Anatomy, Graduate School of Dental Medicine, Hokkaido University, N13 W7, Kita-ku, Sapporo 060-8586, Japan. E-mail: sawa{at}den.hokudai.ac.jp


    Summary
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 Materials and Methods
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 Literature Cited
 
Coexpression of desmosomal proteins and vimentin has been reported in a specific mesenchymal phenotype. This study investigated the expression of vimentin-binding desmosomal proteins in human dental pulp fibroblasts (DPF) and odontoblasts. The dental pulp has no cells expressing desmocollin (DSC) 1-3, desmoglein (DSG) 1-3, junction plakoglobin (JUP), or desmoplakin (DPK) 1 and 2 except for odontoblasts expressing DPK. A confocal image by laser-scanning microscopy demonstrated the diffuse distribution of DPK in the cytoplasm throughout the odontoblast processes. In culture, the mRNA expression of JUP and DPK1, but not DSC1-3 and DSG1-3, was detected in all DPF clones tested and also in odontoblast-like cells (OB) expressing osteocalcin and dentin sialophosphoprotein mRNAs established in the differentiation medium. The DPF having the potential to differentiate into OB expressed vimentin, but not DPK before culturing in the differentiation medium, whereas OB expressed vimentin-binding DPK1. These results suggest that DPF usually expresses DPK1 mRNA, and that the DPK1 production and the bonding of vimentin to DPK1 occur in DPF with the differentiation into odontoblasts.

(J Histochem Cytochem 53:1099–1108, 2005)

Key Words: odontoblasts • desmoplakin • vimentin


    Introduction
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DESMOSOMAL PLAQUE PROTEINS bind to the intermediate-sized filament cytokeratin in epithelial cells or desmin in cardiac muscle cells. Furthermore, the intermediate-sized filament vimentin is attached to desmosomal plaque in human meningiomal and arachnoidal cells (Kartenbeck et al. 1984Go) and in human notochord cells during the early stages of embryonic development (Lehtonen et al. 1995Go; Saraga-Babic et al. 2002Go). It has been suggested that the coexpression of desmosomal plaque proteins and vimentin independent of desmosome formation may characterize the differentiation of the specific cell populations as a predominantly epithelial phenotype or a more mesenchymal one. On the other hand, the reciprocal instructive interactions between stomodeal ectoderm and cranial neural crest–derived ectomesenchyme cells result in the differentiation of ameloblasts, of odontoblasts and cementoblasts, and of tooth formation (Cobourne and Sharpe 2003Go; Lisi et al. 2003Go). We have previously reported the expression of desmosomal proteins in human periodontal ligament fibroblasts (Yamaoka et al. 1999Go). In the dental pulp, the "desmosome-like" junctions have been reported between adjacent odontoblasts and between odontoblasts and neighboring dental pulp fibroblasts (DPF). It has been speculated that the desmosome-like junctions could be immature desmosomes because of morphological peculiarities, different from the typical desmosomes in epithelial cells (Sasaki et al. 1982Go; Iguchi et al. 1984Go; Calle et al. 1985Go; Holland 1987Go). It is thought that desmosomal plaque proteins bind with the intermediate-sized filament vimentin because odontoblasts are the mesenchymal phenotype; however, whether odontoblasts have the ability to produce desmosomal proteins and whether the proteins attach to vimentin in the cells have not been directly addressed. This study investigated the coexpression of desmosomal proteins and vimentin in human odontoblasts and DPF.


    Materials and Methods
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 Materials and Methods
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Subjects
Human dental pulp from teeth extracted for orthodontic reasons and gingival tissue were obtained from 18- to 28-year-old individuals having normal premolars and third molars with complete root formation (n=20). All procedures were performed with appropriate informed consent, and the protocol was approved by the Institutional Review Board on the Use of Human Subjects of Hokkaido University Graduate School of Dental Medicine. Immediately after extraction the teeth were divided parallel to the tooth axis by chisel, and the pulp tissue was extracted from the pulp cavity with tweezers. The coronal dental pulp and gingival tissue were cut into frozen sections in a cryostat, or used to establish cultured DPF.

Cell Culture
The dental pulp tissue extracted from the pulp cavity was treated with PBS (pH 7.2) containing 0.02% trypsin, 0.1% EDTA, and 0.01% EGTA at 37C for 30 min to obtain cells of the odontoblast and subodontoblast layers. After the treatment, dental pulp was removed and isolated cells were cultured in DMEM (Gibco Life Technologies Inc.; Grand Island, NY): containing 10% FBS, penicillin G (100 units/ml), streptomycin (100 mg/ml), and amphotericin B (0.25 mg/ml) at 37C in an atmosphere containing 5% CO2, changing the medium and monitoring growth every 2 days. After a confluent monolayer of migrating cells had formed (~3–7 days), the harvested cell suspension was diluted in a 96-well microtitration plate to establish near single clones. The cells forming a confluent monolayer in one well were divided into two wells of a six-well plate and subcultured in DMEM or in the differentiation medium: osteoblast basal medium containing 0.2 mM ascorbic acid and 15% FBS (Clonetics Corporation; Walkersville, MD), which was supplemented with 0.05 ng/ml transforming growth factor-ß1 (Techne Corporation; Minneapolis, MN), 0.5 mM hydrocortisone (Sigma-Aldrich Corporation; St Louis, MO), and 10 mM ß-glycerophosphate (Sigma) (Lecka-Czernik et al. 1999Go; Allard et al. 2000Go; Unda et al. 2001Go). Clones at the third passage of 106 cells were used to test the osteocalcin (OC) and dentin sialophosphoprotein (DSPP) mRNA expression by RT-PCR and were examined in immunoprecipitation with anti-desmoplakins (DPKs). Normal human foreskin keratinocytes (PHK16-0b; Japanese Cancer Resources Bank, Osaka, Japan) cultured in a keratinocyte basal medium (Cambrex Bio Science Walkersville, Inc.; Walkersville, MD) were also used as controls for the expression of desmosomal proteins and vimentin.

RT-PCR
The extraction of total RNA from DPF cultured in DMEM or in the differentiation medium was achieved with a QIAshredder column and RNeasy kit (Qiagen Inc.; Tokyo, Japan). The RT was performed on 30 ng of total RNA, followed by 35 cycles of PCR for amplification with 50 pM of primer sets for ß-actin, for alkaline phosphatase (ALP), osteonectin (ON), OC, and DSPP as reported (Papagerakis et al. 2002Go), and for desmosomal proteins in which the specificities had been confirmed by the manufacturer (Sigma-Genosys Ltd.; Cambridge, UK) (Table 1). The PCR products were separated on 2% agarose gel (NuSieve, FMC; Rockland, ME) and visualized by Syber Green I (Takara Shuzo Co. Ltd.; Tokyo, Japan).


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Table 1

Sequence of primers

 
Immunostaining
Frozen 8-µm serial tissue sections were cut in a cryostat and fixed in 30% acetone-10 mM PBS (pH 7.2) for 10 min at room temperature, followed by the treatment with first antibodies for 8 hr at 4C. The examination of the expression of desmosomal cadherin; desmocollins (DSCs) and desmogleins (DSGs) in dental pulp cells used a mixture (anti-DSCs) of equal volumes of monoclonal antibodies to human DSC1, DSC2, and DSC3, and a mixture (anti-DSGs) of equal volumes of monoclonal antibodies to human DSG1, DSG2, and DSG3, with the ingredients purchased from Progen Biotechnik (Heidelberg, Germany) and diluted in PBS (100 ng/ml) before use. The examination of the expression of desmosomal plaque proteins: junction plakoglobin (JUP) and DPKs in dental pulp cells used an anti-human JUP (anti-JUP, Progen) and a commercial cocktail of monclonal antibodies to bovine DPK1 and DPK2 (anti-DPKs, Progen). The expression of plakin family proteins was tested by monclonal antibodies to human plectin (anti-plectin, MedSystems Diagnostics GmbH; Vienna, Austria), and to envoplakin (anti-envoplakin, Santa Cruz Biotechnology Inc.; Santa Cruz, CA). The expression of vimentin was tested by a monoclonal antibody to human vimentin (anti-vimentin, DakoCytomation Co. Ltd.; Copenhagen, Denmark). These antibodies were diluted in PBS (100 ng/ml) before use. The sections exposed to the first antibodies were treated with 100 ng/ml of an Alexa 488-conjugated anti-mouse IgG (Molecular Probes Inc.; Eugene, OR) at 25C for 30 min and examined by fluorescence microscopy and laser-scanning microscopy (Axiovert 135M, Carl Zeiss; Jena, Germany) with a x63 oil planapochromatic lens (numerical aperture x1.4). Serial confocal fluorescence images of 10 optical slices on the Z-axis were acquired at 500-nm intervals within a depth of 4 to 9 µm below the cut surface. The digital images were processed using Adobe Photoshop, version 5.0 (Adobe Systems Incorporated; San Jose, CA) and three-dimensional images were re-constructed using a Zeiss LSM Image Browser version 3.0 (Carl Zeiss).

Immunoprecipitation and Western Blot
The DPF monolayer (106 cells) was solubilized in 1 ml of cell lysis buffer (50 mM HEPES [pH 7.3], 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 100 mM NaF, 10 mM Na pyrophosphate, 1% Triton X, and 5% glycerol). The lysate of whole cell protein (2 mg/ml) was centrifuged at 12,500 x g for 20 min at 4C, and 10 µg of anti-DPKs (Progen) and 30 µl of Protein G–agarose beads (Roche Diagnostics GmbH; Mannheim, Germany) were added to 1 ml of the supernatant. After gently shaking the mixture at 4C for 12 hr, the beads were mixed in 0.1 ml of sample buffer and centrifuged. The supernatant was loaded on 15% polyacrylamide gel by electrophoresis, and the separated proteins were transferred onto a PVDF membrane (Invitrogen Life Technologies; Carlsbad, CA). Immunoblots with 10 ng/ml of anti-DPKs (Progen), of peroxidase-conjugated goat anti-mouse IgG (Amersham Biosciences; Buckinghamshire, UK), and with 10 ng/ml of peroxidase-conjugated anti-vimentin (DakoCytomation) were performed on the separated proteins immunoprecipitated with anti-DPKs (Progen). Reaction products were visualized by chemiluminescence substrate (ECL plus Western Blotting Detection System; Amersham) on X-ray film.


    Results
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 Materials and Methods
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 Literature Cited
 
Immunohistochemical Analysis of the Expression of Desmosomal and Plakin Family Proteins in the Dental Pulp Tissue
In the gingival tissue, the epithelia at the cell–cell border were immunostained by anti-DSCs, anti-DSGs, anti-JUP, and anti-DPKs, but no gingival fibroblasts of the lamina propria were stained. In the dental pulp tissue tested, there were no cells immunostained by anti-DSCs, anti-DSGs, anti-JUP, or anti-DPKs except for odontoblasts, which reacted with only anti-DPKs (Figure 1 and Figure 2). The whole of the dental pulp tissue, including DPF and odontoblasts, was immunostained by anti-plectin, whereas no pulp cells reacted with anti-envoplakin (Figure 2). Reaction products with anti-DPKs were wholly observed in the cell bodies throughout the processes of odontoblasts by fluorescence microscopy. A confocal fluorescence image of a 500-nm optical slice by laser scanning microscopy and the reconstructed three-dimensional image demonstrated a diffuse distribution of reaction products with anti-DPKs in the cytoplasm throughout the odontoblast processes of odontoblasts (Figure 3).



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Figure 1

Expression of desmosomal proteins in coronal dental pulp. Sections were stained with hematoxylin and eosin (H-E) or immunostained and visualized by green fluorescence. In the gingival tissue, the epithelia but not gingival fibroblasts of the lamina propria were immunostained by anti-desmocollins (DSCs), anti-desmogleins (DSG), anti-junction plakoglobin (JUP), and anti-desmoplakins (DPKs). The reaction products with the antibodies were observed at the cell–cell border at similar intensities. In the dental pulp tissue, there were no cells immunostained by anti-DSCs, anti-DSGs, anti-JUP, or anti-DPKs; odontoblasts immunostained with only anti-DPKs (arrowhead). Bar = 100 µm.

 


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Figure 2

Expression of plakins in coronal dental pulp. (a) Hematoxylin and eosin. (b) Desmoplakins (DPKs). (c) Plectin. (d) Envoplakin. Only odontoblasts were immunostained with anti-DPKs (arrow). The whole of the pulp tissue including odontoblasts were immunostained by anti-plectin, whereas no pulp cells were immunostained with anti-envoplakin. There is a cross-reaction of anti-envoplakin to the edge of the section; the middle area of the odontoblast cytoplasm separated from the dentin at the odontoblast-predentin interface. Bar = 50 µm.

 


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Figure 3

The desmoplakin (DPK) distribution in odontoblast processes of coronal dental pulp. (a) Examination by fluorescence microscopy. Reaction products with anti-DPKs were observed in the cell bodies throughout the processes of odontoblasts (arrowheads). (b) Confocal fluorescence image of a 500-nm optical slice at 8 µm below the cut surface by laser-scanning microscopy. The three-dimensional images of 10 optical slices were reconstructed at the red line on the X-axis (red-highlighted box at bottom) and at the green line on the Y-axis (green-highlighted box to the right). The images demonstrate the diffuse distribution of reaction products with anti-DPKs in the whole of the cytoplasm throughout the processes of odontoblasts and the absence of reaction products in the nuclei. Bar: a,b = 10 µm.

 
Analysis of the Expression of DPK in Cultured Dental Pulp Cells
In this study, all DPF clones established (n=22) showed the expressions of ALP and ON mRNAs, but no OC and DSPP mRNAs in culture in DMEM before culturing in the differentiation medium. After culturing in the differentiation medium, the DPF clones where ALP and ON mRNAs but no OC and DSPP mRNAs had been detected were determined as DPF without the potential to differentiate into odontoblast-like cells (OB), and the clones where ALP, ON, OC, and DSPP mRNAs had been detected were used in the cell experiments (n=8) as DPF with the potential to differentiate into OB (Buchaille et al. 2000Go; Papagerakis et al. 2002Go) (Figure 4A). In all of the DPF and OB clones established, the region common in two DPK splice variants, DPK1 and DPK2, was detected at a similar intensity level (Figure 4A). The mRNAs of DSC1-3, DSG1-3, JUP, the region common in DPK1 and 2, and the DPK1-unique region covering codon G3862TA to GAG5658 were detected in PHK16-0b. In all of the DPF and OB clones tested, no PCR products for mRNAs of DSC1-3 and DSG1-3 were detected, whereas the products for JUP mRNA, the region common in DPK1 and DPK2 messages, and the DPK1-unique region were detected (Figure 4B).



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Figure 4

Gene expression of odontogenic and desmosomal proteins in cultured dental pulp cells. (a) The dental pulp fibroblast (DPF) clones that showed the expressions of alkaline phosphatase (ALP) and osteonectin (ON) mRNAs, but no osteocalcin (OC) and dentin sialophosphoprotein (DSPP) mRNAs in culture in DMEM before culturing in the differentiation medium (Lane 1). The expression of ALP, ON, OC, and DSPP mRNAs after culturing in the differentiation medium (Lane 2) was determined as DPF having the potential to differentiate into OB. In the DPF and OB, the region common in two desmoplakin (DPK) splice variants, DPK1 and DPK2 (DPKc), was detected at a similar intensity level. (b) The mRNAs of desmocollin (DSC)1-3, DSG1-3, junction plakoglobin (JUP), DPKc, and the DPK1-unique region covering codon G3862TA to GAG5658 (DPK1a, 1b) were detected in PHK16-0b. The mRNAs of DPKc, DPK1a, DPK1b, and JUP, but none of DSC1-3 and DSG1-3 were detected in the DPF before culturing in the differentiation medium in lane 1 in (a), which has the potential to differentiate into OB, and detected in OB in Lane 2 in (a): the dental pulp fibroblasts cultured in the differentiation medium.

 
In the immunohistochemical analysis, the expression of DPK but no vimentin was observed in PHK16-0b. The DPF clones having the potential to differentiate into OB showed the expression of vimentin but no DPK before culturing in the differentiation medium, whereas they showed the expression of vimentin and DPK after culturing in the differentiation medium in all clones tested (Figure 5).



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Figure 5

Production of desmoplakin (DPK) and vimentin in cultured dental pulp cells. The immunostained cells were visualized by green fluorescence. The PHK16-0b was immunostained by anti-DPKs but not by anti-vimentin. The dental pulp fibroblasts (DPF) before culturing in the differentiation medium, having the potential to differentiate into odontoblast-like cells (OB), was not immunostained by anti-DPKs, but stained by anti-vimentin. The OB, the DPF cultured in the differentiation medium, was immunostained by anti-DPKs and anti-vimentin. Bar = 100 µm.

 
In the Western blot analysis with anti-DPKs and anti-vimentin for the immunoprecipitated proteins with anti-DPKs in whole-cell lysate, the reaction with anti-DPKs at the molecular weight of DPK1 (250 kDa) but not with anti-vimentin at the molecular weight (57 kDa) was detected in PHK16-0b. In the DPF clones having the potential to differentiate into OB, no reaction with anti-DPKs or with anti-vimentin was detected before culturing in the differentiation medium, whereas reactions with anti-DPKs and anti-vimentin were detected at molecular weights of DPK1 and vimentin after culturing in the differentiation medium in all OB clones tested (Figure 6).



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Figure 6

Coexpression of desmoplakin and vimentin in cultured dental pulp cells. Western blot analysis with anti-desmoplakin (DPKs) and anti-vimentin was performed on immunoprecipitated products with anti-DPKs in whole cell lysate. In PHK16-0b, the reaction with anti-DPKs was detected at the molecular weight of DPK1 (250 kDa), whereas with anti-vimentin, it was not detected at the molecular weight of vimentin (57 kDa). In the dental pulp fibroblasts, before culturing in the differentiation medium and having the potential to differentiate into odontoblast-like cells (OB), no reaction with anti-DPKs or with anti-vimentin was detected. In OB cultured in the differentiation medium, reactions with anti-DPKs and anti-vimentin were detected at the molecular weights of DPK1 and vimentin.

 

    Discussion
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 Materials and Methods
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 Literature Cited
 
The Expression of Desmosomal Proteins in Dental Pulp Tissue
The formation of an adhesive core with the desmosomal cadherins DSC and DSG is indispensable to the desmosome construction (Garrod et al. 2002Go). Desmosomes have been shown to be present in the odontoblast layer using freeze-fracture replicas (Goldberg et al. 1981Go). However, we did not detect DSC1-3 and DSG1-3 in the dental pulp tissue sections or their messages in cultured DPF and OB (Figure 1 and Figure 4). Therefore, it may be that odontoblasts do not form typical desmosomes (Sasaki et al. 1982Go; Iguchi et al. 1984Go; Calle et al. 1985Go; Holland 1987Go). The JUP, where DSCs and DSGs bind, was also not detected; however, a reaction with anti-DPKs was only observed in the odontoblast layer (Figure 1 and Figure 2). DPK is a member of the plakin family defined by the presence of a plakin repeat domain that binds to the intermediate-sized filaments. DPK has the plakin repeat domain-A absent in plectin and envoplakin, which are members of the plakin family and have a plakin domain common to plectin and envoplakin (Leung et al. 2002Go; Fontao et al. 2003Go). Plectin is a major cytoplasmic component in almost all somatic cells and forms the side arms of the intermediate filaments. Envoplakin is a constituent of the differentiated epidermal keratinocyte envelope (DiColandrea et al. 2000Go; Leung et al. 2002Go). In the examination for the immunoreaction with anti-plectin and anti-envoplakin to evaluate the specificity of anti-DPKs, the whole pulp tissue was immunostained with anti-plectin, and none of the pulp cells reacted with anti-envoplakin; with anti-DPKs, the reaction was only in the odontoblast layer, suggesting that the expression of DPKs is specific to odontoblasts (Figure 2). We have previously suggested a possible function of desmosomal proteins including DSC and DSG to maintain gap junctions in human periodontal ligament fibroblasts (Yamaoka et al. 1999Go). The function of DPK may be different from the desmosomal proteins in the periodontal ligament fibroblasts because odontoblasts expressed DPK only. In all dental pulp tissue tested, the three-dimensional image analysis showed a diffuse distribution of the reaction products with anti-DPKs in the whole cytoplasm throughout the processes, except for in the nuclei of odontoblasts (Figure 3). The DPKs are large proteins with a molecular mass of at least 215 kDa, which play an essential role in anchoring the intermediate-sized filaments to the cell membrane through its COOH-terminal plakin repeat domain; this affects the cytoskeletal strength (Huen et al. 2002Go; Leung et al. 2002Go; Fontao et al. 2003Go). DPK may contribute to maintaining the strength of odontoblast processes by forming complexes with the intermediate-sized filaments.

The Expression of mRNA of Plaque Proteins in Cultured Dental Pulp Cells
Odontoblasts continuously secrete dentin sialoprotein and dentin phosphoprotein that are encoded by a single gene, DSPP. The OC is expressed only during the later stages of odontoblast differentiation, and ALP and ON are expressed in odontoblasts and in many other cell types (Buchaille et al. 2000Go; Papagerakis et al. 2002Go). In this study, all DPF clones established expressed ALP and ON mRNAs in DMEM, and the clones expressing OC and DSPP mRNAs after culturing in the differentiation medium were selected as DPF having the potential to differentiate into OB (Figure 4A). The desmoplakin gene encodes two alternatively spliced forms, DPK1 and DPK2, where a 599 residue within the DPK1 coiled–coiled rod domain is missing (Green et al. 1990Go; Gallicano et al. 1998Go; North et al. 1999Go). In all DPF clones, including the DPF without the potential to differentiate into OB and in OB after culturing in the differentiation medium the region common in DPK1 and DPK2 mRNAs, the DPK1 unique region (Val1195 to Glu1793) and JUP, but not DSC1-3 and DSG1-3, were detected (Figure 4). In PHK16-0b, the mRNAs of DSC1-3, DSG1-3, JUP, the region common in DPK1 and 2, and the DPK1-unique region were detected. Desmosomes are the most common type of adhesive intercellular junction in vertebrate epithelia and in the cell lines. Desmosomes are composed of the adhesive core with desmosomal cadherins, DSCs and DSGs, which mediate intercellular adhesion, and the desmosomal plaques, JUP and DPK, which are cytoplasmic membrane–associated proteins (Garrod et al. 2002Go). The detection of the gene expression on all desmosomal proteins tested in PHK16-0b suggests that the analysis was successful (Figure 4B). These results may suggest that odontoblasts have the ability to produce DPK1 and JUP, but not desmosomal cadherins.

Coexpression of DPK and Vimentin in Cultured Dental Pulp Cells
The expression of DPK, but not vimentin, specific to mesenchymal cells was confirmed in PHK16-0b. The DPF having the potential to differentiate into OB expressed vimentin but no DPK before culturing in the differentiation medium, whereas OB expressed vimentin and DPK (Figure 5). On the immunoprecipitated proteins with anti-DPKs, DPK1, but no vimentin, was detected in PHK16-0b. In the DPF having the potential to differentiate into OB neither vimentin nor DPK were detected before culturing in the differentiation medium, whereas vimentin and DPK1 were detected in OB (Figure 6). This suggests that the DPK gene is generally expressed but degrades without translation in DPF, and that DPK1 is coexpressed with vimentin in OB. It has been reported that desmin-binding DPK1 in Purkinje fiber cells, and vimentin-binding DPK1 in meningeal cells and the follicular dendritic cells function as the cytoskeleton (Angst et al. 1990Go; Virata et al. 1992Go; Gallicano et al. 2001Go; Huen et al. 2002Go; Leung et al. 2002Go). The results suggest that the cytoplasmic vimentin-binding DPK1 may play a role as cytoskeletal molecules in human odontoblasts.


    Acknowledgments
 
This work was supported by a grant-in-aid for Exploratory Research from the Ministry of Education, Science, Sports, and Culture of Japan (No. 16659497).


    Footnotes
 
Received for publication September 13, 2004; accepted March 9, 2005


    Literature Cited
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 Summary
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 Materials and Methods
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 Literature Cited
 

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