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Isolation and Expression of FIP-2 in Wounded Pulp of the Rat
M. Oyama,
F. Myokai,
T. Ohira,
N. Shiomi,
K. Yamashiro,
H. Arai,
F. Nishimura and
S. Takashiba*
Department of Pathophysiology-Periodontal Science, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama, 700-8525, Japan;
Correspondence: * corresponding author,stakashi{at}cc.okayama-u.ac.jp
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ABSTRACT
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Pulpal wound healing followed by cavity preparation may involve reactionary or reparative dentinogenesis in relation to the cavity position; however, little is known about the molecular responses. We aimed to isolate and analyze genes induced or suppressed in the wounded pulp to identify molecular processes involved in the pulp responses to injury. Twenty-three cDNAs were isolated by cDNA subtraction between healthy and wounded pulp of rats. By library screening, we identified rat 14.7K-interacting protein (rFIP)-2A and B genes homologous to human FIP-2, being involved in regulating membrane trafficking and cellular morphogenesis. RT-PCR analysis showed induction for only rFIP-2B in the wounded pulp. In situ hybridization analysis revealed that both rFIP-2s were expressed strongly in condensing mesenchymal cells of the palatal process and surrounding Meckel’s cartilage, but not in intramembranous chondrogenic cells. Thus, up-regulated rFIP-2B expression may play a role in regulating membrane trafficking or cellular morphogenesis of these embryonic and wounded pulpal cells.
Key Words: FIP-2 • wounded dental pulp tissue • subtractive hybridization • in situ hybridization
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INTRODUCTION
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Close regulation of odontoblast differentiation and subsequent secretory activity is critical for dentinogenesis during both embryogenesis and tissue repair (Smith and Lesot, 2001). After injury to the mature tooth, the fate of the odontoblast can vary according to the intensity of the injury. Milder injury can result in up-regulation of functional activity, leading to focal secretion of a reactionary dentin matrix, while greater injury can lead to odontoblast cell death. The regulation of odontoblast death after cavity preparation may be important for reparative dentinogenesis, because dentinogenesis in the damaged pulp may start after the elimination of apoptotic cells (Kitamura et al., 2001). In general, reparative dentinogenesis is observed 1 wk after a moderate wound, such as the formation of a cavity whose depth is half the thickness of the dentin, whereas the induction of apoptosis and the elimination of apoptotic cells are observed within 3 or 4 days after wounding occurred (Sveen and Hawes, 1968; Taylor and Byers, 1990; Kitamura et al., 2001). Many genes may be expressed differentially in the pulpal healing process; however, little is known about these genes.
In this study, we assumed that specific genes would be up-regulated or down-regulated in the pulpal healing following an experimental wound. We aimed to isolate wound-inductive (WIN) and wound-suppressive (WSP) cDNA and to analyze their mRNA expression to identify molecular processes involved in the rat pulp responses to injury.
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MATERIALS & METHODS
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Mechanical Wound and Tissue Preparation
The experimental protocol was approved by the Okayama University Dental School Review Board for animal care (no. 1-005-117). Twenty Wistar rats (male, 10–12 wks old), each weighing from 300 to 350 g, were used. The rats were deeply anesthetized with an intraperitoneal injection of 5% sodium pentobarbital (Nembutal, Dianippon Pharmaceutical Co., Suita, Japan) at a dose of 30 mg/kg, and a cavity depth of half the thickness of the dentin was prepared in the maxillary first molar (Fig. 1Aa ). The cavities were left exposed to the oral environment for 1 wk, and then the teeth were extracted with the animals under the anesthesia mentioned above. For a control, a maxillary first molar without a cavity was extracted from the opposite side of the mouth in the same rat.
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Figure 1. Histology of pulp, detection of cDNAs, and reverse Northern analysis. (A) Histology of pulp tissues. The wounded and healthy teeth were fixed with PBS containing 4% paraformaldehyde, demineralized with 10% EDTA for 4 wks, and embedded in paraffin. Hematoxylin and eosin staining was performed on serial sections 7 µm thick. The pulps 1 wk after cavity preparation (a,b) and healthy pulp (c,d) are shown. Pulpal cells underneath the cavity are indicated by the arrow, and neither reparative dentin formation nor apparent disruption of the odontoblast layer is observed (a,b). Bar equals 300 µm.
(B) Procedure of subtractive hybridization. The target and driver single-stranded (ss) cDNAs bound to the beads were synthesized from the total RNA (100 ng) isolated from the wounded and healthy pulp tissues. The target complementary sscDNA (c-sscDNA) was synthesized from the target sscDNA-beads with an EcoRI-dT primer (5'-GGCGAATTCTGCAGTTTTTTTTTTTTTT-3'). After auto-subtraction, the target c-sscDNA was subtracted twice from the driver sscDNA beads. The subtracted c-sscDNA was amplified by polymerase chain-reaction (PCR) with use of the EcoRI-dT primer, and was displayed on a 3% agarose gel. The procedure is described in detail in the Appendix.
(C) Display of amplified cDNAs. The wounded pulp cDNA subtracted from the healthy pulp cDNA was used as WIN cDNA, while the healthy pulp cDNA subtracted from the wounded pulp cDNA was used as WSP cDNA. The subtracted cDNA was amplified, and the product underwent gel electrophoresis. Lanes: 1, wound-inductive (WIN) cDNA; 2, wound-suppressive (WSP) cDNA; M, 100-bp DNA ladder.
(D) Messenger RNA expression of genes in pulp tissues. The cDNAs (WIN-2, 6, 10, 11, 13, WSP-1, 2, 6, 7, GAPDH) underwent electrophoresis on an agarose gel (Ag). They were then transferred to a membrane, and hybridized with the probe from wounded pulp tissue (Wo) and healthy pulp tissue (He). Relative signal intensity (each cDNA/GAPDH) is shown in the upper panel. *WIN-11 was used as a probe for screening of the cDNA library. Two independent hybridizations were performed, and a typical result is shown.
(E) WIN-11 mRNA in adult rat tissues. WIN-11 mRNA was detected in the tissues shown by an arrow in the upper panel, while β-actin mRNA was detected in the tissues shown by an arrow in the lower panel. Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis. Three independent hybridizations were performed, and a typical result is shown.
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To avoid contamination of the gingival tissue and periodontal ligaments, we broke coronal dentin using dental forceps, and, using a dental curette, recovered pulp tissue from the chamber for further analysis.
Reciprocal Subtractive Hybridization, Cloning, and Homology Search
Subtractive hybridization and cloning were performed as described by Myokai et al.(2003), and as described in detail in the Appendix. Total RNA was isolated from the pulp tissue by the acid guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). The target and driver single-stranded (ss) cDNAs bound to the beads were synthesized from total RNAs of both wounded and healthy pulp (Fig. 1B ). The complementary (c)-sscDNA was synthesized from the sscDNA beads. The target cDNA was subtracted and amplified by polymerase chain-reaction (PCR), and cloned into the EcoRI site of a pUC118 plasmid vector (Takara, Otsu, Japan). The plasmid containing a cDNA insert longer than 250 bp was prepared with the use of Qiagen Plasmid Miniprep Kits (Qiagen, Hilden, Germany), and sequenced by the dideoxy sequencing procedure (Sanger et al., 1977) in an Automatic 377 sequencer (Perkin-Elmer, Foster City, CA, USA). A nucleotide homology search was performed in the Rat Genome Database (http://www.rgd.mcw.edu/), the Rat Genome Assembly (http://www.hgsc.bcm.tmc.edu/), and both BLASTN and BLAST EST homology programs in GenBank DNA databases (final searches in October 30, 2003).
Reverse Northern and Northern Hybridization
Reverse Northern hybridization was performed as described previously (Ohira et al., 2004) and in the Appendix. Briefly, the cDNAs isolated by subtraction were subjected to gel electrophoresis in duplicate, then transferred to nylon membranes. Total RNAs from the wounded and healthy pulps were reverse-transcribed with the use of oligo (dT)12–18 primer, and labeled with [ -32P] dCTP. The membranes were hybridized with the probe, and the signals were visualized in a BioImaging Analyzer (BAS 2000; FUJI, Tokyo, Japan). The signal intensity of each cDNA was quantified with the use of NIH Image (Ver. 1.62), and normalized against that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Tissue distribution of mRNA for the target gene was analyzed by Northern hybridization with adult rat multiple-tissue RNA blots in triplicate (Cat. 7764-1; Clontech, Palo Alto, CA, USA). The [-32P] dCTP-labeled probe was prepared from a cDNA fragment displaying the greatest difference in mRNA expression between the wounded and healthy pulps. The blots were finally washed with 1 x SSC containing 0.1% SDS at 50°C for 30 min. The hybridization signals were visualized in the BioImaging Analyzer.
Library Screening, Rapid Amplification of cDNA Ends, and Structural Analysis
The probe for Northern analysis was used for the following hybridizations. Five x 105 plaques from a rat liver  ZAP cDNA library (Cat. No. 937507; Stratagene, La Jolla, CA, USA) were screened. Positives were fully sequenced from both 5' and 3' ends.
To obtain a full-length cDNA, we performed rapid amplification of cDNA ends (RACE) using a rat liver Marathon-Ready cDNA kit (Cat. 7471-1; Clontech) according to the manufacturer’s instructions. Gene-specific primers (GSPs) were designed as described previously (Chenchik et al., 1996). The GSPs and arbitrary primers (APs) are shown in the Appendix. PCR products were cloned into pT7 Blue T-Vector (Novagen, Madison, WI, USA), and sequenced.
The deduced amino acid sequence for the full-length cDNA was analyzed by the SMART program (http://smart.embl-heidelberg.de/), MOTIF (http://motif.genome.jp/), and PROSITE SCAN (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=%20/NPSA/npsa%20_proscan.html).
Semi-quantitative Analysis of mRNA
The mRNA accumulation in the wounded and healthy pulps was examined by reverse transcription (RT)-PCR. The PCR mixture (50 µL) contained specific primers designed for the genes isolated as described above or for rat β-actin. An independent amplification was performed in duplicate at 36, 38, and 40 cycles. The cDNA (1 ng) prepared from the wounded or healthy pulp was added to all PCR mixtures. After the PCR product (5 µL) underwent electrophoresis on a 2% gel, the intensity of the signal was quantified with NIH Image and normalized against that of β-actin.
Tissue Preparation and in situ Hybridization
The procedures of tissue preparation and in situ hybridization are described in detail in the Appendix. Face and oral cavity tissues were taken from neonatal Wistar rats under deep anesthesia, fixed with 4% paraformaldehyde, and serial sections were made.
In situ hybridization was performed according to a method described previously (Myokai et al., 2004). In brief, the neonatal rat tissue sections and rat embryo sections (Cat. 69159-4; Novagen) were pre-treated and hybridized with either anti-sense or sense riboprobe. The hybridization signal was detected by autoradiography. The plasmids containing the coding region of the target gene were linearized, and they were used as templates to synthesize either anti-sense or sense riboprobes. The riboprobes were prepared with the use of T7 or SP6 polymerases with [-35S] UTP, and the unincorporated labeled nucleotides were removed.
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RESULTS
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Histology of Pulp Tissues and Clones Isolated
Neither reparative dentin formation nor apparent disruption of the odontoblast layer was seen 1 wk after the cavity was prepared (Fig. 1A). An inflammatory cell infiltrate, such as neutrophils or lymphocytes, was not observed in relation to the cavity position.
Both WIN and WSP cDNAs were amplified by PCR, and displayed on a gel (Fig. 1C). Forty WIN clones and 30 WSP clones were isolated, and then 23 clones containing cDNA fragments longer than 250 bp were sequenced. They were identified as 13 individual sequence types containing 2 unknown and 6 known genes, and 5 expressed sequence tags (ESTs) (Table).
Messenger RNA Accumulation of Genes Identified
Reverse Northern hybridization was used to detect 9 kinds of mRNA among 13 kinds of genes in wounded and healthy pulp tissues (Fig. 1D), while little or no signal was detected in the other genes (data not shown). WIN-11 and WSP-6 differed in mRNA expression between the wounded and healthy pulp tissues, with WIN-11 displaying the greatest difference. WIN-11 was expressed in almost all of the tissues tested, and was expressed strongly in the heart, brain, and liver (Fig. 1E).
Genes Isolated by cDNA Library Screening and RACE
We isolated the pX105-2 by screening the cDNA library using the WIN-11 probe (Fig. 2A). Clone #1 was obtained by RACE with AP 1, AP 2, GSP 1, and GSP 2. Nucleotide sequences of WIN-11, pX105-2, and the clone overlapped significantly. Additional RACE with AP 1, AP 2, and either GSP 3 or GSP 4 gave 2 clones (#2 and #3), and clones overlapped. The results indicated that 2 genes were isolated; however, they shared the same nucleotide sequence in the 3' region. Moreover, both rat genes had significant homology with human 14.7K-interacting protein (hFIP)-2, and contained a common zinc-finger domain (Fig. 2B). However, hFIP-2 has 2 putative leucine-zipper domains, while the 2 rat genes had 1, and rat (r)FIP-2A was missing a putative basic-leucine zipper motif, whereas both rFIP-2B and hFIP-2 were not.
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Figure 2. Identification and structure of 2 FIP-2s and their expression. (A) Clones obtained by cDNA library screening and RACE studies. WIN-11 (0.35 kb), pX105-2 (1.8 kb), and clone #1 (2.2 kb) significantly overlapped; therefore, they have been submitted to DDJB and assigned accession no. AB050777. Clones #2 (0.9 kb) and #3 (3.0 kb) were completely overlapped; therefore, they have been submitted to DDJB and assigned accession no. AB069907. *WIN-11 displaying the greatest difference in mRNA expression between the wounded and healthy pulp tissues by reverse Northern analysis;  , common region in rFIP-2A and rFIP-2B;  , region of rFIP-2B different from that of rFIP-2A.  , cDNA containing the specific region for rFIP-2A or rFIP-2B was cloned and used as a template for riboprobes of in situ hybridization. The procedure is described in detail in the Appendix. (B) Structure of rFIP-2s. Deduced amino acid sequences of rFIP-2s and hFIP-2 are shown. The signal peptide domain is boxed in green, the putative leucine-zipper domains are boxed in black, and the zinc-finger domain is boxed in blue. The putative bZIP motif is boxed in red. *Same amino acid as the sequence above; –, missing amino acid. (C) Expression of rFIP-2 mRNA. Complementary DNA was amplified by PCR with the primers as described below. The PCR products for rFIP-2A, B, and β-actin underwent electrophoresis on a gel. The specific primers were designed: rFIP-2A sense, 5'-TGCCCAGCCAGCCTCCTACC-3'; rFIP-2B sense, 5'-ATCTCTGTGGCCGGACCTGTTACC-3'; and rFIP-2A and rFIP-2B antisense, 5'-CCACTTCGATTCCCACACTC-3'. For an internal control, specific primers for rat β-actin were designed: sense, 5'-TTGTAACCAACTGGGACGATATGG-3'; and antisense, 5'-GATCTTGATCTTCATGGTGCTAGG-3'. Two independent PCRs were performed, and typical results are shown. Lane M, 100-bp DNA ladder. Relative signal intensity (each mRNA from/β-actin mRNA) is shown in the lower panel. , healthy pulp; , wounded pulp.
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Messenger RNA Expression of rFIP-2s
The RT-PCR analysis with different numbers of cycles allowed for the quantification of the target mRNA in the pulp (Fig. 2C). Up-regulation of mRNA expression by wounding was detected only for rFIP-2B, although mRNA for both FIP-2A and B was detected in the wounded and healthy pulp tissues.
Expression of rFIP-2s in the Rat Embryo
The rFIP-2A and B mRNAs were detected in condensing mesenchymal cells of the palatal process and surrounding Meckel’s cartilage during rat embryogenesis, whereas they were absent in the intramembranous chondrogenic cells (Fig. 3B). However, no or very weak mRNA expression for rFIP-2A and B was found in the tooth germ at cap and early crown stages (Fig. 4, in the Appendix).
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Figure 3. Expression of the 2 rFIP-2s during rat embryogenesis. (A) Histology of the rat embryo at 17 days. Hematoxylin and eosin staining was performed on the sagittal section from the rat embryo. Bar equals 200 µm. mx, maxilla; md, mandible; tg, tongue. (B) In situ hybridization in rat embryo at 17 days. Using rFIP-2A or B anti-sense riboprobe, we observed strong signals for the rFIP-2A and B mRNA in condensing mesenchymal cells of the palatal process (a,b,c,d) and surrounding Meckel’s cartilage (e,f,g,h), while they are absent in the neighboring chondrogenic cells (a to h). The results are shown as bright- (a,c,e,g) and dark-field (b,d,f,h) views. The hybridization signals of rFIP-2A (a,b,e,f) and rFIP-2B (c,d,g,h) were detected by autoradiography at 1 wk, and similar results were obtained after three-week exposure (data not shown). No significant signal was detected on any sections in the case of sense riboprobe for rFIP-2A or B (data not shown). Bar equals 50 µm.
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DISCUSSION
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Sixteen WIN and 7 WSP cDNAs were isolated from wounded pulp tissues, and were identified as 13 individual sequence types containing 6 known and 2 unknown cDNAs, and 5 ESTs (Table). Of the 16 WIN cDNAs, 8 were identified as cytochrome c, which is the terminal enzyme of the mitochondrial respiratory chain and regulates both electron transfer and energy transduction. The frequent isolation of cytochrome c may reflect a marked response to the wound in pulpal cells. Cathepsin B (catB), a lysosomal cysteine protease, has recently been implicated in apoptosis. The role of catB in the TNF--triggered apoptotic cascade has been examined in hepatocytes from catB–/– and catB+/+ mice, and apoptosis has been shown to be diminished in the catB–/– cells, suggesting that TNF--induced apoptosis was dependent, in part, upon catB activity (Guicciardi et al., 2000). Therefore, catB isolated here may be involved in TNF--induced apoptosis in pulpal cells after cavity preparation. Of the 7 WSP cDNAs, 4 were identified as known cDNAs: laminin  2, type I collagen, and 2 ribosomal proteins. At the cap stage of tooth development in mice, strong staining for the laminin 2 chain has been shown in the basement membrane in contact with enamel knot cells (Kieffer-Combeau et al., 2001). However, no significant signal for the laminin 2 chain was detected in either wounded or healthy pulp tissues by reverse Northern hybridization (data not shown). Therefore, the expression of laminin 2 may be low-level or limited locally in adult rat pulp tissues. Type I collagen distributes in skin, bone, gingiva, periodontal ligament, cementum, and most connective tissues, and is abundant in pulpal cells and odontoblasts in erupted teeth (Garcia et al., 2003). Interestingly, WSP-6 (pro- 2 collagen I) showed increased expression in the wounded pulp tissues (Fig. 1D). Since type I collagen is composed of 2 1 chains and 1 2 chain, increased expression of pro--2 collagen I may have an influence on type I collagen synthesis in the wounded pulp. There is no report of the involvement of ribosomal protein in the apoptosis of cells or in dentinogenesis. In spite of the elimination of genomic DNA with our procedure, WIN-5 had high homology with the mitochondrial genome. The extensive amplification of the subtracted cDNA by PCR may account for the detection of trace amounts of small genomic DNA fragments.
The relationships between genes from different genomes are naturally represented as a system of homologous families that include both orthologs and paralogs. Orthologs are proteins from different species that evolved by vertical descent, and typically retain the same function as the original. In contrast, paralogs are proteins from within a given species that are derived from gene duplication, and new functions may evolve that are related to the original (http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/Orthology.html). Here, 2 rFIP-2s were identified: rFIP-2B was structurally homologous to hFIP-2; however, rFIP-2A in the 5' region had no significant similarity with known genes, while rFIP-2A in the C terminus was homologous to hFIP-2 (Fig. 2B). The findings imply that rFIP-2B is orthologous to hFIP-2, while rFIP-2A is paralogous to rFIP-2B.
FIP-2 was originally identified as a novel human tumor necrosis factor -inducible cellular protein interacting with an adenovirus 14.7-kDa protein, and it is involved in the complex regulation of apoptosis; however, by itself it did not cause cell death (Li et al., 1998). Hattula and Peränen (2000) have reported that the amino-terminal region of FIP-2 interacts with the activated form of Rab8, while the carboxy-terminal region of FIP-2 binds to Huntingtin. Moreover, co-expressed FIP-2 and Huntingtin enhance the recruitment of Huntingtin to Rab8-positive vesicular structures, and, similarly, FIP-2 promotes cell polarization to Rab8, suggesting that FIP-2, together with Huntingtin and Rab8, is part of a protein network that regulates membrane trafficking and cellular morphogenesis (Hattula and Peränen, 2000). Sahlender et al.(2005) reported the role of FIP-2 in Golgi ribbon formation and exocytosis after our latest revision. In this study, in situ hybridization analysis revealed that rFIP-2A and B were expressed strongly in condensing mesenchymal cells of the palatal process and surrounding Meckel’s cartilage, but not in the intramembranous chondrogenic cells (Fig. 3B). Moreover, the expression of rFIP-2B was up-regulated, although both rFIP-2A and B were expressed in the wounded pulp tissues (Fig. 2C). These findings suggest that up-regulated rFIP-2B expression plays a role in regulating the membrane trafficking or cellular morphogenesis of these embryonic mesenchymal cells and the wounded pulpal cells.
The dentin-pulp complex shows a broad spectrum of responses to caries, which represents a summation of injury, defense, and repair events. The complex interplay among these events will be important in determining the fate of the dentin-pulp complex (Smith, 2002). In this study, the cavities were left exposed to the oral environment; however, no apparent reparative dentin formation was observed in relation to the cavity position (Fig. 1A). In general, reparative dentinogenesis occurs following pulp damage, similar to our experimental pulp wound (Sveen and Hawes, 1968; Taylor and Byers, 1990; Kitamura et al., 2001). It is currently unknown why no apparent reparative dentin formation occurred in our model. However, the high level of rFIP-2B expression may be involved in differentiation or proliferation of the wounded pulpal cells, because it expressed strongly in the condensing mesenchymal cells during palatal and mandibular development (Fig. 3B).
In conclusion, rFIP-2A and B were identified, following reciprocal subtraction, both being structurally homologous to hFIP-2, regulating membrane trafficking and cellular morphogenesis. The rFIP-2B mRNA was up-regulated in the wounded pulp and expressed strongly in condensing mesenchymal cells of the palatal process and surrounding Meckel’s cartilage during rat embryogenesis. These results suggest that up-regulated rFIP-2B expression plays a role in regulating the membrane trafficking or cellular morphogenesis of these embryonic mesenchymal cells and wounded pulpal cells.
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ACKNOWLEDGMENTS
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This study was supported by a Grant-in-Aid for Scientific Research B (No. 14370710 to ST), a Grant-in-Aid for Scientific Research B (No. 17390562 to FN), a Grant-in-Aid for Scientific Research C (No. 17591991 to FM), and a Grant-in-Aid for Young Scientists B (No. 15791103 to MO) from the Japan Society for the Promotion of Science, and by the Kobayashi Magobe Memorial Foundation, the Ryobi Teien Foundation, and the Inamori Foundation (FM), and the Suzuken Memorial Foundation (ST).
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FOOTNOTES
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A supplemental appendix to this article is published electronically only at http://www.dentalresearch.org.
Received for publication November 14, 2003.
Revision received May 13, 2005.
Accepted for publication May 31, 2005.
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REFERENCES
|
|---|
- Chenchik A, Moqadam F, Siebert P (1996). A new method for full-length cDNA cloning by PCR. In: A laboratory guide to RNA: isolation, analysis, and synthesis. Krieg PA, editor. New York: Wiley-Liss, Inc., pp. 273–321.
- Chomczynski P, Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.[Medline]
[Order article via Infotrieve]
- Garcia JM, Martins MD, Jaeger RG, Marques MM (2003). Immunolocalization of bone extracellular matrix proteins (type I collagen, osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells. Int Endod J 36:404–410.[Medline]
[Order article via Infotrieve]
- Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, et al. (2000). Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest 106:1127–1137.[Medline]
[Order article via Infotrieve]
- Hattula K, Peränen J (2000). FIP-2, a coiled-coil protein, links Huntingtin to Rab8 and modulates cellular morphogenesis. Curr Biol 10:1603–1606.[CrossRef][Medline]
[Order article via Infotrieve]
- Kieffer-Combeau S, Meyer JM, Lesot H (2001). Cell-matrix interactions and cell-cell junctions during epithelial histo-morphogenesis in the developing mouse incisor. Int J Dev Biol 45:733–742.[Medline]
[Order article via Infotrieve]
- Kitamura C, Kimura K, Nakayama T, Toyoshima K, Terashita M (2001). Primary and secondary induction of apoptosis in odontoblasts after cavity preparation of rat molars. J Dent Res 80:1530–1534.
- Li Y, Kang J, Horwitz MS (1998). Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor alpha-inducible cellular protein containing leucine zipper domains. Mol Cell Biol 18:1601–1610.[Abstract/Free Full Text]
- Myokai F, Oyama M, Nishimura F, Ohira T, Yamamoto T, Arai H, et al. (2003). Unique genes induced by mechanical stress in periodontal ligament cells. J Periodontal Res 38:255–261.[Medline]
[Order article via Infotrieve]
- Myokai F, Koyama E, Nishikawa K, Noji S, Murayama Y, Taniguchi S (2004). Aspects of interleukin-8 gene expression by gingival and dermal fibroblasts stimulated with interleukin-1beta or tumour necrosis factor alpha. J Int Acad Periodontol 6:21–28.[Medline]
[Order article via Infotrieve]
- Ohira T, Myokai F, Shiomi N, Yamashiro K, Yamamoto T, Murayama Y, et al. (2004). Identification of genes differentially regulated in rat alveolar bone wound healing by subtractive hybridization. J Dent Res 87:546–551.
- Sahlender DA, Roberts RC, Arden SD, Spudich G, Taylor MJ, Luzio JP, et al. (2005). Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis. J Cell Biol 169:285–295.[Abstract/Free Full Text]
- Sanger F, Nicklen S, Coulson AR (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467.[Abstract/Free Full Text]
- Smith AJ (2002). Pulpal responses to caries and dental repair. Caries Res 36:223–232.[Medline]
[Order article via Infotrieve]
- Smith AJ, Lesot H (2001). Induction and regulation of crown dentinogenesis: embryonic events as a template for dental tissue repair? Crit Rev Oral Biol Med 12:425–437.[Abstract/Free Full Text]
- Sveen OB, Hawes RR (1968). Differentiation of new odontoblasts and dentine bridge formation in rat molar teeth after tooth grinding. Arch Oral Biol 13:1399–1409.[CrossRef][Medline]
[Order article via Infotrieve]
- Taylor PE, Byers MR (1990). An immunocytochemical study of the morphological reaction of nerves containing calcitonin gene-related peptide to microabscess formation and healing in rat molars. Arch Oral Biol 35:629–638.[Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 84, No. 9,
842-847 (2005)
DOI: 10.1177/154405910508400912
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ABSTRACT
INTRODUCTION