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RESEARCH REPORT |
Department of Periodontology & Oral Biology, Goldman School of Dental Medicine, Boston University, 100 East Newton Street, G05, Boston, MA, 02118, USA;
*corresponding author, samar{at}bu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: periodontal ligament fibroblasts (PDLF) • gingival fibroblasts (GF) • periodontium • DNA microarray • differential gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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While some efforts have been made recently to identify and characterize the functional differences between PDLF and GF by scanning electron microscopy and subtractive hybridization (Giannopoulou and Cimasoni, 1996; Park et al., 2001), the accurate pattern of differential gene expression between PDLF and GF remain unknown. Significant strides in analysis of gene expression have been made by means of DNA microarray technology. The technique has recently been successfully used in identifying host molecular pathways by comparative analysis of the host transcriptional response to infection (Detweiler et al., 2001), gaining insights into the mechanisms that control life-span and age-related phenotypes (Ly et al., 2000), as well as delineating differences in gene expression that might account for the discordant phenotype in autoimmune diseases (Wilson et al., 2000). In the present study, we analyzed differentially expressed genes between PDLF and GF, and clustered them according to their biological functions.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Isolation
After cells became subconfluent, they were immediately subjected to lysis in solution RNA STAT-60 (TEL-TEST, Friendswood, TX, USA), and the total cellular RNA was isolated according to the manufacturer's instructions. Poly A+ RNA was purified with OligoTex mRNA isolation columns (Qiagen, Valencia, CA, USA). PDLF and GF RNA samples used for subsequent experiments were pooled from cells from three subjects for each tissue type. Equal amounts of RNA were used from each cell passage. We checked the integrity of RNA samples by evaluating total cellular RNA on denaturing agarose gel electrophoresis, and further measured the purity and quantity of RNA samples by spectrophotometry.
Microarray Construction and Image Processing
Each mRNA sample (1 µg at 50 ng/µL) was used in triplicate for cDNA array hybridization. Fluorescent-tagged nucleotides were used to label cDNAs from PDLF (Cy5-dCTP) and GF (Cy3-dCTP) mRNAs during oligo-primed reverse transcription (RT). The microarray was spotted with a collection of a cDNA library containing 9018 unique human cDNA clones (IncyteGenomics, St. Louis, MO, USA). After hybridization, the array was imaged by modified scanning confocal fluorescent microscopy.
Microarray Data Analysis
Further analysis, such as data normalization, data filtering, and pattern identification, was conducted with GEMTools 2.5 (IncyteGenomics, St. Louis, MO, USA). Two criteria were taken into account: (1) the element's signal to background ratio and (2) the area percentage representing the hybridization size of an element on the microarray. The default values are 2.5 for signal-to-background ratio and 40% area. The level of detectable differential expression is 1.75-fold, which is the minimum threshold for statistical significance (p < 0.05). The results for chip validation and significance determination study can be accessed at: http://dentalschool.bu.edu/research/lifearray/. Given that false-positive observations may still occur when the levels of differential expression are low, or the signal-to-background ratios are small (Tusher et al., 2001), we further filtered the data with a more stringent threshold (three-fold differential expression), to ensure both statistical and biological significance.
Northern Analysis
Total RNA (20 µg each) were separated by 1% agarose-formaldehyde gel electrophoresis and transferred onto Hybond-N+ nylon membrane (Amersham, Piscataway, NJ, USA). The cDNA fragments to be hybridized were generated from RT-PCR reaction with the use of gene-specific primers designed by Primer 3 software (Rozen and Skaletsky, 1998). Each probe was hybridized with the membrane for 2 hrs at 68°C. Radioactive signals were quantified with a PhosphorImager and IMAGEQUANT software (Molecular Dynamics, Sunnyvale, CA, USA). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was used as an internal control. The Student paired-sample t test was used to analyze the data, and the significance was defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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).
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). About 1.8% (163 genes) of all genes were found differentially expressed by at least three-fold. Among them, 84 gene transcripts (0.93%) were found to be comparatively more abundant in PDLF, whereas 79 genes (0.87%) were expressed at higher levels in GF (Fig. 2). To analyze the differences in expression pattern more thoroughly, we sorted all 163 of these genes according to their biological function (Table). We noticed that there were significant differences between PDLF and GF on several functional groups. First, 20 genes encoding transmembrane proteins were more abundantly expressed in PDLF compared with GF. Half of these genes have been up-regulated over five-fold, with the most significant one, milk fat globule-EGF factor 8 (MFG-E8), having a 31.5-fold differential expression. However, only 9 genes in the same functional group were up-regulated in GF, with comparatively low differential expression ratios (less than 4.5-fold). Furthermore, 14 cytoskeleton-related proteins were overexpressed in PDLF, whereas only 2 genes showed slightly higher expression in GF. On the other hand, many more cell-cycle regulation proteins (11 vs. 3) and metabolism-related proteins (14 vs. 3) were expressed at higher level in GF than in PDLF.
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). The relative amount of MFG-E8 mRNA to GAPDH was 33-fold higher in PDLF than in GF, while the relative amount of STK15 mRNA was 4.1-fold higher in GF than in PDLF (Fig. 3B), concordant with their 31.5- and 4.7-fold differential expression, respectively, observed in microarray results. The relative ratio of TPM2 to GAPDH in PDLF was 2.2-fold higher than in GF, whereas the transcript level of MRPL3 showed 1.9-fold increase in GF than in PDLF (Fig. 3B). These were comparable with the three-fold differential expression for both of the genes observed in microarray.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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It was observed that transmembrane proteins were strongly up-regulated in PDLF compared with GF (Table
). Most strikingly, MFG-E8 was markedly up-regulated in PDLF compared with GF, with a 31.5-fold differential expression. MFG-E8 has been known as a major glycoprotein of the milk fat globule membrane and may promote cell adhesion by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus (Andersen et al., 2000). Recently, an antimicrobial function has been conferred on MFG-E8 in phagosomes that participate in tissue remodeling, apoptotic cell clearance, and restriction of the spread of intracellular pathogens (Garin et al., 2001). This would substantiate the participation of PDLF in tissue remodeling, apoptosis, and restriction of pathogen spread through regulation of membrane surface proteins. Syndecan 4 (SDC4), a selectively enriched and widespread focal adhesion component and a regulator of growth factor signaling (Woods et al., 2000), was also up-regulated in PDLF, which substantiates the role of PDLF in site-specific responses to the extracellular signals. Sortilin (SORT1) was observed as another gene significantly up-regulated in PDLF, with a differential expression ratio of -17.4. Sortilin is a transmembrane type-I receptor (Mazella, 2001) that may regulate the function of cells involved in immune response and inflammation (Mazella et al., 1998). Taken together, the preferential expression of these specific cell-surface glycoproteins and proteoglycans on PDLF provides further suggestion of a role of PDLF in the regulation of inflammatory and immune responses by modulating cell-surface molecules. This could also help us understand the role of PDLF in the responses to periodontal pathogens and its ultimate contribution to wound-healing processes.
Another interesting observation in this study was the up-regulation of cytoskeleton-related proteins in PDLF compared with that in GF (14 vs. 2). Transgelin (TAGLN) is a transformation- and shape-change-sensitive protein found in fibroblasts (Lawson et al., 1997). Although its precise function is still unknown, its abundance in PDLF may contribute to the physiology and structural integrity of periodontal tissue. The expression of desmoplakin (DSP) in the PDLF has been considered to protect gap junctions in the PDLF against cell transformation caused by cell contraction, which may relate to tooth movement and repair of periodontal tissues (Yamaoka et al., 1999). Given the adaptive role of the periodontium, the presence of these specific cytoskeletal molecules suggests a central role of PDLF in the maintenance of periodontal tissue homeostasis and in tooth movement.
We also noticed that genes encoding cell-cycle regulation proteins and metabolism-related proteins tended to be more abundantly expressed in GF than in PDLF. Serine/threonine kinase 15 (STK15) is associated with centrosomes and plays a key role in mitosis (Zhou et al., 1998). The expression of STK15 mRNA was specifically observed at the G2/M phase of the cell cycle during proliferation (Kawasaki et al., 2001). CDC25B is a mitotic inducer controlling transition from the G2 to the M phase of the cell cycle (Korner et al., 2001). It has been shown that over-expression of Cdc25B enhances the proliferation of mammary epithelial cells, resulting in hyperplasia (Ma et al., 1999). Up-regulation of these genes may indicate faster progression of GF through the cell cycle and may facilitate fibroblast proliferation, an event important for tissue repair. In addition, significant up-regulation of metabolism-related genes involved in the synthesis of proteins and fatty acids and the processing of nucleic acids in GF compared with PDLF may further corroborate the enhanced GF proliferation compared with PDLF. There has been evidence that GF has a significantly greater proliferation rate (Larjava et al., 1989) than PDLF. Therefore, the increased expression of cell-cycle regulation and metabolism-related genes may partially explain enhanced GF proliferation compared with PDLF observed in periodontal wound healing (Oates et al., 2001).
Interestingly, IL-8 mRNA was found to be highly expressed in GF compared with PDLF, with a differential expression of 85.1-fold. Previous studies have shown that constitutively high levels of IL-8 are observed in cultured human gingival fibroblasts (Kent et al., 1996). Several reasons can be proposed for this observation: (i) GF is more engaged to respond to inflammatory stimuli than PDLF; (ii) neutrophil-mediated pro-inflammatory processes may be regulated in part by GF in the cytokine network of immuno-participant cells (Takashiba et al., 1992); and (iii) GF may play a role in the regulation of IL-8 production in the formation of the cytokine network (Takigawa et al., 1994). Further studies are warranted to elucidate the present role of IL-8 in GF compared with PDLF.
Recent studies of fibroblast cell lines from different donors identified an age-related difference with regard to differential gene expression (Ly et al., 2000). This concern has no bearing on our data, since we were careful in selecting our samples only from age-matched donors. However, due to the sample selection of this study, it has by no means represented the overall populations in regard to age, gender, and genetic background. Caution must be exercised in extrapolating the present data to the general population, since they reflect differential gene expression in healthy Caucasian adults (24-48 yrs old). Further studies aimed at determining whether the observations in this study can be generalized to other populations are warranted.
Due to limitations intrinsic to the array technology, currently available dot-printed chips include only a subset of human genes. Therefore, it is likely that some important genes may be missing from the set of cDNAs present on the chip. Indeed, the expression of S100A4 and CD40 was absent in the present array profile but was well-characterized by Northern analysis.
In summary, the present study compared the gene expression profile between cultured PDLF and GF by means of DNA microarray. PDLF and GF appear to display different gene expression patterns that may reflect intrinsic functional differences of the two cell populations. This differential expression of genes may well coordinate with their tissue-specific activities during inflammatory/immune responses and in the maintenance of tissue homeostasis.
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ACKNOWLEDGMENTS |
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Received November 12, 2001; Last revision February 25, 2002; Accepted April 3, 2002
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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