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Expression Profiling of Periodontal Ligament Cells Stimulated with Enamel Matrix Proteins in vitro: A Model for Tissue RegenerationDepartment of Periodontology, Eastman Dental Institute, University College London, 256 Gray’s Inn Road, London WC1X 8LD, UK; Correspondence: * corresponding author, i.olsen{at}eastman.ucl.ac.uk
Several studies have examined the role of enamel matrix proteins in root formation and periodontal regeneration, although most of these have focused on a few specific genes which had previously been implicated. However, recent advances in expressional profiling have made it possible to examine the range of genetic responses involved in these processes. In the present experiments, we have therefore utilized this technique to determine the effects of enamel matrix proteins on the gene activities of periodontal ligament cells in vitro. Such cells were found to have an elevated level of RNA synthesis compared with control cells. Moreover, hybridization of the cDNA prepared from this RNA to gene array filters showed that there was differential expression of 121 genes, most of which had not previously been associated with periodontal regeneration. Some of these selective changes in gene activity might thus reflect the fundamental events that underlie periodontal development.
Key Words: periodontium • regeneration • gene arrays • enamel matrix • in vitro
Regeneration of periodontal ligament (PDL) and alveolar bone lost as a result of periodontal disease has been a major objective of periodontal therapy. A variety of treatment modalities has been applied with considerable success, with bone grafting and guided tissue regeneration being widely used procedures. However, both the extent and predictability of outcomes have been limited, and there is still widespread interest in the development of new therapeutic strategies for promoting more effective periodontal regeneration. Biological mediators have recently received considerable attention, with a variety of growth and differentiation factors being locally applied to periodontal wounds to promote regeneration of periodontal defects. Among these, much attention has been devoted to specific morphogens that are thought to be critical mediators of tooth and periodontal development. Enamel matrix proteins (EM) consist mainly of amelogenins, a group of proline-rich hydrophobic peptides (Eastoe, 1964) formed from a single gene by alternative splicing and post-translational modifications (Fincham et al., 1994). There is increasing evidence that Hertwig’s root sheath cells secrete EM proteins during root formation, and that these are involved in the formation of acellular cementum during tooth development (Slavkin, 1976; Hammarström et al., 1997). Experimental models and human clinical trials have also indicated that amelogenins are effective in inducing regeneration of the PDL, root cementum, and alveolar bone (Hammarström et al., 1997; Heijl et al., 1997). In an effort to optimize clinical efficacy and to improve understanding of the molecular mechanisms underlying both development and regeneration, several investigators, by in vitro studies, have attempted to clarify the mode of action of EM. Initial investigations have focused on the study of specific cell functions associated with the regenerative response: cell recruitment, proliferation, and differentiation into mature PDL cells and osteoblasts (Tokiyasu et al., 2000; Van der Pauw et al., 2000; Haase and Bartold, 2001; Jiang et al., 2001; Lyngstadaas, 2001). Such studies have evaluated a limited number of known "markers" based on previous investigations, providing confirmation that some genes known to be involved in wound-healing processes are activated as a result of PDL exposure to EM—for example, the PDL response mediated through an increase in intracellular cAMP (Lyngstadaas et al., 2001). During both development and wound healing, external stimuli lead to rapid changes in the catalogue of genes that are expressed, in the proteins that are produced, and eventually in the cellular phenotype. Describing the profile of early gene expression after exposure to external stimuli is thus fundamental to the understanding and possible modification of these responses (Heller et al., 1997; Khodarev et al., 1999). Until recently, Differential Display (DD) reverse-transcription/polymerase chain-reaction (RT-PCR) (Liang and Pardee, 1992) was used to examine the differences in gene expression profiles between and among different cell types (Liang et al., 1992) and in the same type of cell exposed to different stimuli (Francia et al., 1996; Furumura et al., 1998; Hooper et al., 2001). However, technical difficulties associated with the simultaneous study of multiple gene products and the labor-intensive identification of the expressed gene products have hampered progress in the use of this technique. These difficulties can now be overcome by the use of cDNA arrays that allow for the simultaneous evaluation of hundreds of genes associated with specific cell functions (Granjeaud et al., 1999) and is at present the method of choice for expression profiling (Hooper et al., 2001; Xynos et al., 2001). This technique has the advantage of simplicity, high throughput capacity, and reduction of the down-stream analysis involved in conventional DD. The present study has, therefore, used gridded cDNA arrays to elucidate some of the changes in gene expression in PDL cells exposed to EM, to clarify the molecular basis of periodontal regeneration and root development.
PDL Tissue and Cell Culture PDL tissue was obtained from premolar teeth extracted for orthodontic reasons from one healthy individual (Caucasian male, 25 yrs of age), after informed consent was obtained in accordance with a protocol reviewed and approved by the Joint Research and Ethics Committee of the Eastman Dental Institute/Hospital. PDL cells were obtained from the tissue after 2 to 4 wks of culture as previously described (Parkar et al., 1996; Kuru et al., 1998). They were incubated at 37°C in 5% CO2 in air in alpha-Minimum Essential Medium (  -MEM) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 25 µg/mL fungizone, 2 mM L-glutamine (GIBCO Life Technologies, Paisley, UK), and 10% fetal calf serum (FCS) (PAA Laboratories, Linz, Austria). Trypsin-EDTA (GIBCO) was used to detach the adherent monolayer of cells, which was subcultured and used between the third and fifth passages. To determine the ability of the PDL cultures to form mineralized nodules in vitro, we grew the PDL cells to confluence in six-well plates as described above and supplemented the medium with 50 µg/mL of ascorbic acid and 10 mM β-glycerophosphate. Von Kossa staining was carried out after 3, 4, 5, and 6 wks of culture. The cells used in all the experiments described below were derived from this same patient.
DNA, RNA, and Protein Synthesis
Cell Exposure to EM
RNA Extraction
Expression Profiling A 5-µg quantity of the extracted total cellular RNA was used to produce cDNA probes labeled with 32P-dCTP (Amersham Pharmacia Biotech). The labeled probes were then hybridized to the AtlasTM gene array filters at 68°C (Xynos et al., 2001). The filters were then washed according to the manufacturer’s instructions [once with 2 x standard saline-citrate (SSC), 1% SDS; twice with 0.1 x SSC, 0.5% SDS]. The filters were exposed to autoradiographic film overnight and for 3 days at -70°C. The autoradiographs were scanned with the use of an Alpha Imager 1200 and AlphaEase version 5.5 image capture software (Flowgen, Ashby de la Zouch, UK), and saved for later analysis.
Data Analysis To assess the reliability and repeatability of the methods, we performed the expression profiling experiments twice, each time point in duplicate and each set of RNA labeled and hybridized twice. Comparison of the individual replicate filters showed that there were no differences between them which were above the threshold limits set as described above (data not shown). For the final analysis, the replicate images were therefore merged to produce a composite image.
The early-passage cultures of PDL cells used in these experiments all produced mineralized nodules by 3 wks of culture in vitro, as assessed by von Kossa staining (data not shown). The Fig.  shows the effects of EM on macromolecular synthesis by these cells. In both the control and EM-treated cultures, the incorporation of 3H thymidine increased progressively at a similar rate until approximately 10 hrs, after which DNA synthesis remained constant (Fig., A). Protein synthesis also increased to the same extent in both types of cultures, although maximal 3H amino acid incorporation was not observed until after 15 hrs (Fig., C). In marked contrast, the results in Fig. B show that the incorporation of 3H uridine far more rapidly increased in the EM-treated cultures and by 6 hrs was nearly 150% greater than in the non-treated control cultures, which increased only slowly over a period of 24 hrs. Thus, RNA synthesis in the PDL cells was elevated within the first 6 hrs of incubation in the presence of EM, whereas there was no differential effect of EM on either protein or DNA synthesis, even at 24 hrs.
Using the AtlasImageTM software settings as described in MATERIALS & METHODS, we obtained evidence that there were differences in the expression of 121 genes by PDL cells cultured in the presence of EM, of a total of 198 genes detected on the hybridized filters. In Table 1 , these 121 differentially expressed genes have been assigned to 7 groups based on the functional activity of the corresponding protein. Although there is some overlap in the functions of several of the gene products, each group contains between 14 and 22 different genes, nearly every gene having a unique response to the presence of EM protein in the culture medium. Among individual genes, fibronectin, bone proteoglycan II, MMP 14, and BIGH3 were markedly up-regulated at 2 hrs, while two DNA-associated proteins were particularly strongly down-regulated by EM, even after this short period of exposure. By 24 hrs, the calmodulin 1, integrin beta 8, AXL tyrosine protein kinase receptor, arachidonate 5-lipoxygenase, and cytokeratin 12 genes were additionally up-regulated. Substantial numbers of individual genes were also no longer detected at this later time, generally those expressed at only very low levels at 2 hrs. Three cell-cycle proteins showed a greater than 10-fold difference between 24 and 2 hrs, while one DNA-binding protein increased more than 50-fold in the presence of EM.
There were also several genes of interest not included in Table 1  , since they did not reach either the two-fold level or the intensity limits used in the analysis. Some of these genes, which were positive at lower thresholds, have previously been identified in other studies of single gene changes induced by EM. Thus, transforming growth factor beta was up-regulated at 2 hrs but was not found to be expressed at 24 hrs (Van der Pauw et al., 2000; Lyngstadaas et al., 2001). Several other growth mediators were also found to be elevated by culture in the presence of EM protein, including connective tissue growth factor (CTGF), vascular endothelial growth factor receptor 1, and platelet-derived growth factor receptor alpha (data not shown).
The designation ’ON’ in Table 1
The present study has shown that the addition of EM protein to PDL cells in vitro elicited a substantial increase in RNA synthesis, without affecting DNA and protein. This differential effect on RNA synthesis was found to occur within the first 6 hrs and persisted at least until 24 hrs, at which time the overall levels of protein and DNA synthesis were still the same in the presence and absence of EM. Although the present study did not examine the effects of more prolonged exposure, previous studies have shown that protein and DNA synthesis were also significantly elevated after 48 hrs of incubation in the presence of EM (Van der Pauw et al., 2000; Lyngstadaas et al., 2001). The majority of the new ’early’ RNA production which we detected is undoubtedly ribosomal and would be expected to lead eventually to a greater capacity of the cells for protein synthesis. Nevertheless, the DD RT-PCR technique with cDNA filter arrays clearly demonstrated that there were also major qualitative changes in mRNA expression which were induced by EM protein. This procedure could thus provide an extensive profile of the complex sequence of changes in gene expression that accompany the development and regeneration of the periodontium and which are, at present, only poorly understood. Moreover, the use of cDNA array technology can also reveal genes which have been identified in other wound-healing responses but have not been implicated specifically in periodontal regeneration as well as gene responses to other forms of periodontal therapy. The cancer gene array used in the present experiments is comprised of more than 1000 oncogenes, growth factors and their receptors, transcription factors, and cell cycle proteins. It is therefore not unexpected that approximately 20% of these were found to be expressed by early-passage PDL cells, with more than half (121 genes) being differentially affected by EM. Of these, it was notable that only one transcription factor, the fos-related antigen 1(FRA1), which has been shown to have an important role in osteoclast differentiation and can reverse osteopetrosis in Fos knockout mice (Matsuo et al., 2000), was up-regulated at 2 hrs or even after longer incubation. However, a most striking feature of the effects of EM on PDL gene expression was the rapid, persistent, and strong up-regulation of many surface-receptor-ECM genes. Thus, after 2 hrs of exposure to EM protein, 15 of the 22 differentially expressed genes in this group were elevated, including the MMPs 1, 3, 11, and 14, which are involved in wound healing and ECM remodeling processes. The fibronectin, collagens 6 and 16, tenascin, integrin beta-8, and bone proteoglycan genes were also up-regulated, suggesting that the ECM plays a fundamental and perhaps an essential regulatory role in PDL regeneration induced by EM protein, even at an early stage. Notably, the most strongly down-regulated genes were among the DNA-chromatin group, perhaps reflecting an EM-induced program of changes in gene activity directed away from DNA replication/repair and toward more rapid differentiation of bone and other precursor cells present in the PDL. Independent assays of the expression of specific genes, for example, by Western and Northern blotting, are clearly required to corroborate the array results we have obtained before definitive biological conclusions can be drawn. Thus, while our results do not establish unequivocally that one or a small group of genes is essential in EM-induced PDL regeneration, the technique of expression profiling has nevertheless highlighted a range of multiple gene activities that warrant further analysis. This would help to clarify the fundamental molecular events underlying this process and might also identify previously unsuspected gene targets for clinical therapy.
This work was supported by an educational grant from Biora AB, Malmö, Sweden, and by the Eastman Periodontal Research Fund. Received for publication January 29, 2002. Revision received August 6, 2002. Accepted for publication September 10, 2002.
Journal of Dental Research, Vol. 81, No. 11,
776-783 (2002)
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