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RESEARCH REPORT |
Department of Periodontal Science, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8525, Japan;
* corresponding author, stakashi{at}cc.okayama-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: gene profiling • periodontal ligament fibroblasts • subtractive hybridization • in situ hybridization
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Despite the identical genomic DNA in the periodontal ligament and gingival fibroblasts, the phenotypic differences between them are obvious. The gene expression profile of each cell type has not yet been elucidated, although each cell expresses a select set of characteristic genes. A typical eukaryotic cell line contains approximately 250,000 cytoplasmic mRNAs and as many as 10,000 different kinds of mRNAs. However, there is only a 2% difference in the mRNAs present in the two closely related lineages (Hedrick et al., 1984). We speculated that human periodontal ligament fibroblasts (HPFs) and human gingival fibroblasts (HGFs) represent a difference in the gene expression profile, because they share common embryonic development from cells of the cranial neural crest (Palmer and Lubbock, 1995).
In this study, we aimed to profile the genes highly expressed by HPFs. We constructed a library, followed by subtraction of HPF cDNA from HGF cDNA. The clones isolated from the library were examined for mRNA expression levels for both PFs and GFs in vitro and in vivo. Furthermore, they were analyzed for sequence homology to known genes.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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The mRNA (5 µg) isolated from the fifth-passage cells was reverse-transcribed to first-strand cDNA by means of SuperScript II (GIBCO BRL, Grand Island, NY, USA) and oligo (dT)12–18 primer (GIBCO) at 45°C, and then blunt-ended second-strand cDNA was synthesized from the cDNA as described previously (Gubler and Hoffman, 1983).
To confirm the differences in cell types, we examined osteocalcin mRNA expression by polymerase chain-reaction (PCR) as a marker of osteoblastic characteristics (Ducy et al., 2000).
Construction of a Subtractive cDNA Library
Subtractive hybridization was carried out as described previously (Klickstein, 1992), with slight modifications. In brief, the blunt-ended cDNA from HPFs was ligated with an EcoRI adapter (Takara, Kyoto, Japan) to both ends by means of T4 DNA ligase (NEB, Beverly, MA, USA). Complementary DNA less than 400 by was removed by means of a column (Size Sep 400 Spun Columns; Pharmacia Biotech, Uppsala, Sweden). The HGF cDNA was digested with both Alu I and Rsa I (NEB) to make the blunt-ended short fragment. The HPF cDNA (40 ng) with the EcoRI site was subtracted from the blunt-ended HGF cDNA (2 µg) under the following conditions: The cDNA mixture was heated to 99°C for 5 min, cooled on ice immediately, and then kept at 37°C for 24 hrs. The remaining double-stranded cDNA with the EcoRI site was ligated into a ZAP Express Predigested Vector (Stratagene, La Jolla, CA, USA) and packaged with the use of Gigapack III Gold Packaging Extracts (Stratagene) according to the manufacturer’s instructions. The phage library was screened in the presence of 1 M isopropylthio-ß-D-galactoside and 250 mg/mL of 5-bromo-4-chloro-3-indoyl-ß-D-galactoside (Sigma Chemical, St. Louis, MO, USA). Positive phages were stocked in 1 mL of SM buffer, and some were converted to pBK-CMV phagemid for sequence analysis.
Reverse Dot-blot Hybridization
The cloned DNA was amplified by PCR with 1 µL of phage stock and M13-20 and BK Reverse primers (Stratagene). The PCR product (2 µL) was dot-blotted on a Hybond N+ membrane (Amersham Biosciences, Tokyo, Japan) and incubated with the probe at a concentration of 1 x 106 cpm/mL in the buffer as previously described (Davis et al., 1994) at 42°C for 36 hrs. After the RNAs were recovered from fifth-passage HPFs and HGFs, cDNA pools were synthesized as described above, and then labeled with [
-32P] dCTP (Amersham Biosciences) by means of the Bca Best Labeling Kit (Takara). After a final wash with 4 x SSC containing 0.1% SDS at 42°C, the hybridization signals were visualized in a Bio Imaging Analyzer (BAS 2000; FUJI, Tokyo, Japan). The signal intensity of each DNA sample was quantified with NIH Image Ver. 1.62, and normalized against that of ß-actin. After stripping the HPF probe, we performed hybridization with the HGF probe, and then quantified and normalized the signal intensity of each DNA sample as above. Clones were selected for sequence analysis and Northern analysis when signal ratios differed by more than two-fold between the two probes.
DNA Sequencing and Homology Search
The phagemid DNA was purified by means of a Plasmid Mini Kit (Qiagen, Hilden, Germany), and the sequences from both ends were partially determined by the dideoxy sequencing procedure with an Automatic 377 sequencer (Perkin-Elmer, Foster City, CA, USA). The nucleotide sequence homology was analyzed by use of the BLASTN and BLASTX homology programs through GenBank DNA databases (final searches in April, 2002). We performed the functional classification of the genes identified as previously described (Adams et al., 1993), by using the Locus Link program from the National Center for Biotechnology Information.
Northern Blot Analysis
Messenger RNA (1 µg) from the seventh-passage cells was separated on 1.0% agarose gel with formaldehyde and transferred to a Hybond N+ membrane (Amersham Biosciences). The membrane was incubated with the probe at a concentration of 5 x 105 cpm/mL in the hybridization buffer as previously described (Church and Gilbert, 1984) at 65°C for 16 hrs. After a final wash with 2 x SSC containing 0.1% SDS at 65°C, the hybridization signals were visualized and quantified as described above. DNA insert from the phagemid clone was amplified by PCR, and labeled with [-32P] dCTP by the method described above, and then used for hybridization. In addition, we collected mRNA from seventh-passage cells from two different donors and used it for Northern hybridization.
Tissue Preparation and in situ Hybridization
We took periodontal tissues from the maxillary first molars of male Wistar rats (10–12 wks old) under deep anesthesia and fixed them for 3 hrs in a solution containing 4% paraformaldehyde. The experimental protocol was carried out according to the guidelines for animal care of Okayama University Dental School. The tissues were decalcified in a 10% EDTA (pH 7.4) solution at 4°C for 4 wks and then embedded in paraffin according to standard procedures.
In situ hybridization was performed according to the method described by Noji et al.(1989), with minor modifications. Briefly, the tissue sections were deparaffinized in xylene, rehydrated, acetylated, and pre-hybridized in a solution of 2 x SSC, 50% formamide at 50°C. The PstI-HindIII gene fragment (433 bp) of a PDL-29 cDNA was subcloned into pBluescript SK(-) (Stratagene), and then sense and anti-sense riboprobes were synthesized by T3 and T7 RNA polymerases from the construct, according to the instructions supplied with the DIG RNA Labeling kit (Roche Diagnostics, Mannheim, Germany). We applied a 20-µL quantity of hybridization mixture (50% de-ionized formamide, 4 mg/mL BSA, 4 mg/mL tRNA, 10% dextran sulfate, and 2 x SSC) to each slide. The specimens were incubated for 15–17 hrs in a humid chamber at 50°C with the probe, at a concentration of 0.4 ng/µL for hybridization. The sections were washed with a solution of 50% formamide and 2 x SSC at 50°C for 1 hr, treated with 20 mg/L RNase A (Roche Diagnostics) at 37°C for 30 min in NTE buffer (0.5 M NaCl, 10 mM Tris [pH 8], 1 mM EDTA), washed further with 0.1 x SSC at 50°C for 1 hr, and then developed by means of a DIG Nucleic Acid Detection Kit (Roche Diagnostics).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Among 6 x 103 plaques from the subtractive library, 400 independent clones containing DNA inserts between 400 bp and 2400 bp were obtained and used for reverse dot-blot hybridization. Thirty-three clones were highly expressed by HPFs, and all of them had similarities with known human genes (Table).
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, 1C). A single 2.2-kb transcript was detected in both HPFs and HGFs, and the size of the transcript was consistent with that of the DNA insert in the clone. In the case of the other two donors. the PDL-29 mRNA level in HPFs was much higher than that in HGFs (Fig. 1C).
Expression of PDL-29 mRNA in vivo
By in situ hybridization on the healthy rat periodontal tissue sections with the anti-sense probe, dense signals indicating PDL-29 mRNAs were detected in both GFs and PFs (Fig. 3). However, little difference in signal intensity was seen between them in the tissue. No signal was detected in the tissue in the case of the sense probe.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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It is interesting that none of the clones had been reported as a unique gene of PFs. Recently, 15 human periodontal ligament fibroblast-specific cDNAs were isolated by subtractive hybridization between cultured HPFs and HGFs (Park et al., 2001). However, the profile of the clones isolated here is completely different from that of the cDNAs. This may be due to a difference in stringency in the subtractive hybridization. Among the clones isolated, only 3 (PDL-28, -29, -33) have been shown to be expressed in tissues or cell types (http://bodymap.ims.u-tokyo.ac.jp/). Therefore, we classified them according to known functions. The "Energy Metabolism" category contains COX assembly factor (PET112), which is involved in energy metabolism at amino acid residues (Petruzzella et al., 1998). The "Signal Transduction" and "Hormones and Cytokines" categories include D1S155E (Jeffers et al., 1990), NUDC (Miller et al., 1999), RSP-1 (Cutler et al., 1992), TMSB4 (Gondo et al., 1987), and CIN85 (Take et al., 2000). These are related to either an altered proliferative response or the developmental potential of cells. The "Transcription and Translation machinery" category includes ribosomal components and elongation factors, which may be involved in the high turnover and remodeling of matrix proteins. The "Other Metabolism" category includes the gene for annexin A2, which has been identified as a factor enhancing osteoclast formation and bone resorption (Takahashi et al., 1994). The annexin A2 and osteocalcin produced by HPFs may play roles in bone remodeling during periodontal regeneration. These functional annotations suggest that the gene expression profile is associated with a characteristic of HPFs.
From healthy human periodontal tissue, PDL-29, identified as a COX assembly factor, showed much stronger mRNA expression by PFs than that by GFs in vitro. The similarities between PFs and GFs were revealed in the other two donors. However, little difference in PDL-29 mRNA expression was seen between the two cell types in healthy rat periodontal tissue. These results suggest that HPFs express PDL-29 mRNA highly, at least in vitro. The PDL-29 expression by PFs may vary from that by GFs in the magnitude of mRNA in response to some exogenous stimuli such as growth factors or inflammatory mediators in vivo.
The PDL-29 cDNA contained a sequence in the 3'-untranslated region (3'-UTR) just before the poly (A)-tail of PET112 (Fig. 2A). An additional homology search with human genomic BLAST in the NCBI revealed that the insertion sequence was located in exon 15 of the PET112 genome (NT_022851). These findings suggest that PDL-29 is an alternative-splicing variant of the COX assembly factor. Several known cis-acting elements (Ogbourne and Antalis, 1998) are found in the insertion sequence of the cDNA (Fig. 2B). It is well-known that the promoter element in the 3'-UTR of the eukaryote gene—namely, silencers—also controls the localization, stability, and translation of its transcripts (Decker and Parker, 1995). Since the silencers identified here usually destabilize RNA, the protein production of PDL-29 may not parallel its transcription.
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ACKNOWLEDGMENTS |
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Received May 31, 2002; Last revision May 5, 2003; Accepted May 8, 2003
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REFERENCES |
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