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RESEARCH REPORT |
1 Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials and Hospital Dentistry, UCLA School of Dentistry, Box 951668, CHS B3-087, Los Angeles, CA 90095-1668, USA; and
2 King Faisal Specialist Hospital & Research Center, Department of Dentistry, Jeddah, Saudi Arabia;
*corresponding author, ichiron{at}dent.ucla.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) and 253-(wit 3.0 ß) amino-acid-long peptides with the characteristics of an intracellular molecule. Analysis of these data may provide new clues to the molecular mechanism of edentulous mucosa formation.
KEY WORDS: wound-healing • tooth extraction • oral mucosa, gingiva • differential gene expression • wound-inducible transcript • wit 3.0.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Wound-healing involves a complex set of cellular and molecular events targeted toward the restoration of the structural integrity of damaged tissue (Selliseth and Selvig, 1995; Speyer et al., 1996; Lalani et al., 1998). As a result of tissue damage, cytokines such as interleukin 6 (IL-6), IL-1, epidermal growth factor (EGF), transforming growth factor (TGF), and tumor necrosis factor- (TNF-) recruit phagocytic cells to the wounded site to clear away the damaged tissue, and regulate tissue repair by the epithelial and fibroblastic cells (Li et al., 1999; Nakamura and Nishida, 1999; Rozlog et al., 1999). Within the wound, the fibroblastic cells develop a temporary system of contractile surface microfilaments, which resemble smooth muscle and express high-level -smooth-muscle actin. These myofibroblasts become aligned and pull the wound margins together (Desmouliere, 1995; Thomas et al., 1995). Abnormal connective tissue is characteristically different from the original tissue (Clark et al., 1997; Schor et al., 1996), and is often formed as the result of the myofibroblastic activity in the extended wound (Phan, 1996; Cass et al., 1997; Muchaneta-Kubara and El Nahas, 1997).
During the tooth extraction wound-healing process, the chronological restoration of epithelial and connective tissues is thought to follow the pattern of the general soft-tissue wound-healing event. In 1967, Carlsson et al. described how epithelialization of the wound surface occurs parallel to reparative processes of connective tissue, which may lead to a wide range of structure and physiological characteristics of edentulous oral mucosa formation. It was reported that one group of individuals had alveolar ridges covered with thick resilient mucous membrane containing dense collagenous connective tissue and hyper-keratinized epithelium, while another group had thin atrophic membranes with less dense collagenous connective tissue and hypo-keratinized epithelium (Krajicek et al., 1984a,b). It is conceivable that edentulous oral mucosa generation is associated with a site- and/or a tissue-specific wound-healing process.
Despite the fact that tooth extraction is so prevalent, detailed literature on the wound-healing process leading to the formation of edentulous mucosa is surprisingly scarce. We hypothesized that the distinct features of edentulous mucosa are due, in part, to specific differential gene expression in response to tooth extraction wound-healing. Our study objectives were to identify and characterize candidate genes that are differentially induced in wounded oral mucosal tissue following tooth extraction. This report describes the isolation of a cDNA encoding a 3.0-kb-long mRNA, which may differentially participate in the oral mucosa wound-healing process.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Differential Display Polymerase Chain-reaction (DDPCR)
We performed differential display to compare the Day 4 edentulous mucosa and untreated gingival total RNA samples. One µg of total RNA was used for reverse transcription, followed by polymerase chain-reaction, with 24 combinations of three 3' anchor primers and eight 5' primers (Genhunter Corp., Nashville, TN, USA). Cycling parameters were: 94°C for 30 sec, 40°C for 2 min, 72°C for 30 sec for 40 cycles, followed by a final extension cycle of 72°C for 5 min. The [-35S]- or [-33P]-labeled amplified DDPCR products were then separated on 6% sequencing gels. The DNA sequencing gel was blotted onto a piece of Whatman 3M paper and dried without methanol/acetic acid fixation. For four days, the dried gel was subjected to autoradiography with Kodak BioMAX film (Eastman Kodak, Rochester, NY, USA). To reduce the chance of false-positive results, we designed stringent selection criteria. Selection of the cDNA candidate bands required:
The selected DDPCR bands were harvested from the blot, re-amplified, and then cloned into an Escherichia coli vector by means of a TA cloning system (Invitrogen, Carlsbad, CA, USA). Double-stranded plasmid DNA was sequenced by a chain termination method.
RNA Transfer Blot Analysis
From the Day 4 experiments on rats, 20 µg of total RNA samples of edentulous mucosa and the untreated gingiva were size-fractionated on 0.8% denaturing agarose gels containing formaldehyde and formamide, and then transferred to nitrocellulose filters. The RNA transfer blots were hybridized with radiolabeled candidate DDPCR products. For a housekeeping gene standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used.
Full-length cDNA Cloning
We used a Rapid Amplification of cDNA Ends (RACE) protocol to obtain the 5' side-overlapping cDNAs from one of the candidate clones, by means of commercially available protocols (Marathon cDNA Amplification and Smart Race cDNA Amplification kit, Clontech, Palo Alto, CA, USA). One µg total RNA of edentulous mucosa was converted to double-stranded cDNA, which was then modified with a universal adapter at the 5' and 3' terminals. The PCR amplification reactions were performed with a combination of the universal primer and sequence specific primers: 5'-CTGCCTCATCTCAACTGTGTAGCCT-3' or 5'-GCGCTCCTGTTCCTTGCAACCCTGCTG-3'. The PCR products were analyzed on 1% agarose gels and confirmed by DNA transfer blot hybridization. The amplified cDNA was cloned and sequenced. The obtained DNA sequences were analyzed and compared with the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/).
In situ Hybridization
In 10 additional rats, molar extraction from the right-side maxilla was performed according to the method previously described. Maxillary alveolar tissue containing Day 4 and Day 7 post-extraction edentulous mucosal samples were harvested after being fixed by perfusion in 4% paraformaldehyde in phosphate-buffered saline (PBS). Specimens were decalcified for 7 days in Cal-Ex (Fischer Scientific, Fair Lawn, NJ, USA) and embedded in paraffin. The mounted 8-µm sections from Day 4 and Day 7 post-extraction specimens were de-paraffinized and washed twice in DEPC-phosphate-buffered saline (PBS) (pH 7.4) for 5 min, followed by a single wash with DEPC-PBS containing 100 mM glycine and 0.3% Triton X-100, before incubation in Tris HCl-EDTA (TE) buffer containing 10 µL RNase-free proteinase K. The sections were fixed with 4% paraformaldehyde in PBS, washed for 5 min with DEPC-PBS, incubated for 10 min with 0.1 M triethanolamine buffer and 0.25% acetic anhydride, then incubated at 37°C for 10 min with pre-hybridization buffer containing 4X SSC with 50% v/v formamide (20X SSC = 17.5% sodium chloride and 8.82% sodium citrate, pH 7.0). Sense and antisense probes were prepared by means of a Digoxigenin (DIG) RNA labeling kit (Boehringer Mannheim, Indianapolis, IN, USA). The sections were incubated overnight at 42°C in a humidified chamber with the hybridization buffer containing 40% formamide, 10% dextran sulfate, 1X Denhardt's, 4X SSC, 10 mM dithiothreitol, 1 mg/mL yeast tRNA, 1 mg/mL denatured salmon sperm DNA, and 10 ng labeled RNA probe. The hybridized slides were washed at 37°C for 15 min with 2X SSC and 1X SSC, consecutively. The sections were then incubated for 30 min at 37°C in NTE buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, pH 8.0) containing 20 µg/mL RNase A, and washed for 30 min with 0.1X SSC. Color detection was performed with nitroblue tetrazolium (NBT). Some sections were stained with hematoxylin and eosin, then examined under a light microscope.
Reverse-transcription Polymerase Chain-reaction (RT-PCR) Examination on Fibroblasts
From 4 rats, the Day 7 edentulous mucosa and the untreated gingiva were harvested. The fibroblasts were then isolated according to the method of Layman and Diedrich (1987). The harvested tissues were resected to produce pieces of tissue 1 mm3 and washed in three changes of PBS. Tissue sections were then placed separately on the 100-mm culture plate containing Dulbecco's Modified Eagle Medium (Gibco BRL, Grand Island, NY, USA), supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, 100 µg/mL of streptomycin and 0.25 µg/mL of amphotericin B. Fibroblasts grown from these tissues were passed 4 to 8 times. Total RNA of the cultured fibroblasts derived from rat edentulous mucosa and the untreated gingiva was separately isolated. For a comparison, total RNAs of human oral keratinocytes and oral mucosa fibroblasts were obtained according to the protocol approved by UCLA's IRB (#G98-05-046-04).
The expression pattern of the selected candidate gene in these rat and human cells was examined by RT-PCR with the use of primers flanking an open reading frame area of the selected candidate gene, CS13up 5'-TCAGCTCTTCCTCAGACTCAGGTCACTG-3', and CS13dw 5'-ATGAGCTGCACCATTGAGAAGGCATTGC-3'. A pair of primers targeting rat beta actin was used as a housekeeping gene control. RT-PCR was performed at 94°C for 1 min followed by 25 cycles at 94°C for 1 min, 72°C for 1 min, 72°C for 1 min, and extension at 72°C for 7 min. The resulting PCR products were separated by electrophoresis in 2% agarose gel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The edentulous mucosa exhibited 8 specific DDPCR bands that were not seen in the untreated gingiva. These DDPCR products specific for edentulous mucosa were selected for re-amplification, then directly cloned and sequenced. In the following transfer blot analysis, DDPCR clone AA1 was strongly hybridized to a Day 4 edentulous mucosa mRNA species of approximately 3.0 kb (Fig. 2A). We named this differentially expressed 3.0-kb mRNA wound inducible transcript 3.0 (wit 3.0), and selected it for further characterization. AA1 contained a 410-bp-long cDNA insert with a typical poly (A) structure. Using a 5' RACE protocol, we cloned two overlapping cDNAs, CS13 (1980 bp) and CS312 (843 bp) (Fig. 2B). CS13 and CS312 revealed an open reading frame encoding a deduced peptide 215 amino acids long.
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). The epithelial cells showed active proliferation and migration toward the extraction socket, whereas the connective tissue was infiltrated with inflammatory cells, and exhibited a dense fibrosis-like extracellular matrix (Fig. 2D). In situ hybridization with antisense probe revealed that a high level of wit 3.0 mRNA was expressed by fibroblasts in the connective tissue adjacent to the wounded site, where dense fibrosis-like connective tissue was found (Fig. 2E), whereas sense probe did not reveal any positive signal (Fig. 2F). The labeling with the wit 3.0 probe was not found in the untreated gingival connective tissue (data not shown). RT-PCR analysis of the isolated cells from edentulous mucosa also supported that the wound-associated fibroblastic cells are the cellular source of wit 3.0 expression, whereas cultured fibroblasts from untreated gingiva showed minimum expression of wit 3.0 (Fig. 2G). We also tested the expression of wit 3.0 in human keratinocytes and oral mucosa fibroblasts, both of which showed minimum expression (Fig. 2G).
We noted that the primer set flanking the deduced open reading frame of wit 3.0 unexpectedly gave rise to two PCR products (Fig. 2F). Both RT-PCR products were cloned and sequenced. The shorter RT-PCR product (645 bp) encoded wit 3.0, whereas the longer RT-PCR product (764 bp) encoded a nucleotide sequence identical to wit 3.0 with a 114-bp insertion in the middle. We concluded that these RT-PCR products must represent isoforms of wit 3.0, and thus we named them wit 3.0 and wit 3.0 ß, respectively.
The nucleotide sequences of the overlapping cDNAs (AA1, CS13, and CS312) account for 2745 bp, representing the complete sequence of wit 3.0 (Fig. 3). Nucleotides and the deduced peptide sequences of the open reading frames were submitted to the GenBank for a sequence search. In part, wit 3.0 was matched with HSPC 123 derived from a human CD34+ stem cell cDNA library (Accession No. AF161472), cDNA DKFZp564o1 Homo sapiens mRNA derived from brain tissue (Accession No. AL117608), and cDNA FLJ10672 Homo sapiens mRNA derived from teratocarcinoma cell line (Accession No. AK001534). The nucleotide similarities in the matched region were 89%, 49%, and 84.6%, respectively, limited to the open reading frame region (Figs. 4A, 4B). When the deduced peptide of wit 3.0 ß was submitted for the GenBank search, it matched at 99% with FLJ10672 (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Based on the strong differential hybridization in RNA transfer blot analysis, an mRNA species recognized by the DDPCR clone AA1 was selected for further study. Three overlapping cDNAs (AA1, CS13, and CS312) (Fig. 2B) represent the full-length sequence of wit 3.0, which appeared to encode two hypothetical peptides of 215 amino acids (wit 3.0 ) and 253 amino acids (wit 3.0 ß) (Fig. 4). We speculate that these hypothetical peptides may be generated from either an alternative splicing process of the wit 3.0 gene or from a closely related gene family. Further studies such as genomic DNA cloning are necessary for this question to be answered.
The translated protein sequence of wit 3.0 ß exhibited a very high homology with the translated protein of FLJ1006 (Fig. 4), and thus it is highly likely that wit 3.0 ß represents the rat version of human FLJ1006. To date, wit 3.0 does not completely match the sequences in the GenBank. Because the sequence similarity with human cDNAs HSPC 123, DKFZp564o1, and FLJ1006 was limited to the open reading frame region of wit 3.0 and ß, we postulate that this open reading frame of wit 3.0 is likely to be translated as an active peptide. We estimate that the wit 3.0 and wit 3.0 ß proteins have molecular weights of approximately 25 kDa and 31 kDa, with isoelectric points (pI) of 5.17 and 5.11, respectively. From the Kyte-Doolittle analysis, we learn that both proteins are hydrophilic molecules without a typical signal peptide sequence, indicating that they are not secreted proteins.
The proposed short open reading frame of wit 3.0 will leave an unusually long 3' untranslated region of over 2000 bases. Three human cDNA clones (HSPC123, DKFZp564o1, and FLJ10672) similarly contain long 3' untranslated regions. The biological function of the 3' untranslated region is gradually being revealed. The long 3' untranslated region of transferring receptor mRNA participates in the peptide translation by binding to cytosolic aconitase (Hentze et al., 1987; Casey et al., 1988). Histone mRNA also contains a long 3' untranslated region which appears to support its stability in conjunction with the cell's DNA synthesis activity (Marzluff, 1992). The 3' untranslated region may also regulate the polarized location of mRNAs within the cytoplasma (Kislauskis and Singer, 1992). It is unclear whether the long 3' untranslated region of wit 3.0 bears any biological functions.
During the wound-healing process following tooth extraction, the initial restoration of epithelial integrity is complete within 4 to 7 days (Kapur and Shklar, 1963). Active connective tissue remodeling is accompanied by increased type I collagen expression. Adaptation of the oral mucosa tissue within the tooth extraction area often results in the generation of distinct epithelial and connective tissue (Ross and Benditt, 1965; Wirthlin et al., 1984). Wounding involves the temporal expression of potent cytokines, which may induce an unreported phenotypic change to fibroblastic cells. Induction of wit 3.0 gene transcription in wounded oral mucosa is a novel observation in this study, which may be one of the phenotype adaptation events of the gingival fibroblasts in the tooth extraction wound. To date, the expression of wit 3.0 in other wound-healing sites has not been addressed. The cDNA HSPC123 and FLJ10672 exhibiting the significant sequence similarity to wit 3.0 are isolated from human CD34+ stem cell and teratocarcinoma cDNA libraries, respectively. Recently, the role of circulating stem cells in damage repair has been reported in many different tissues (Blau et al., 2001). Although no evidence is available, it is tempting to speculate that wit 3.0 expressing fibroblastic cells participating in the tooth extraction wound-healing process may include these colonized stem cells.
In summary, we present the wit 3.0 transcripts that are differentially expressed in edentulous oral mucosa. Although the function of wit 3.0 is unknown, the distinct expression pattern of wit 3.0 may suggest a potential role in oral wound-healing.
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ACKNOWLEDGMENTS |
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Received September 5, 2001; Last revision February 5, 2002; Accepted February 13, 2002
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