Dataset Information

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

ABSTRACT: It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was found between DecPDL and PermPDL cells, comparative secretome analysis evidenced that one of the most stickling differences involved cell adhesion molecules, including laminin subunit gamma 1 (LAMC1) and beta 2 (LAMB2). Next, total RNA and protein extracts were obtained from fresh PDL tissues of deciduous (n = 6) and permanent (n = 6) teeth, and Western blotting and qPCR analysis were used to validate our in vitro findings. Western blot analysis confirmed that LAMC1 was increased in DecPDL fresh tissues (p<0.05). Furthermore, qPCR data analysis revealed that mRNA levels for laminin subunit beta 1 (LAMB1), beta 3 (LAMB3), LAMC1, and gamma 2 (LAMC2) were higher in DecPDL fresh tissues, whereas transcripts for LAMB2 were increased in PermPDL (p<0.05). In conclusion, the differential expression of laminin chains in DecPDL and PermPDL suggests an involvement of laminin-dependent pathways in the control of physiological differences between them.

SUBMITTER: Giovani PA

PROVIDER: S-EPMC4858149 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

Giovani Priscila A PA Salmon Cristiane R CR Martins Luciane L Paes Leme Adriana F AF Rebouças Pedro P Puppin Rontani Regina M RM Mofatto Luciana S LS Sallum Enilson A EA Nociti Francisco H FH Kantovitz Kamila R KR

PloS one 20160505 5

It has been suggested that there are histological and functional distinctions between the periodontal ligament (PDL) of deciduous (DecPDL) and permanent (PermPDL) teeth. Thus, we hypothesized that DecPDL and PermPDL display differences in the constitutive expression of genes/proteins involved with PDL homeostasis. Primary PDL cell cultures were obtained for DecPDL (n = 3) and PermPDL (n = 3) to allow us to perform label-free quantitative secretome analysis. Although a highly similar profile was ...[more]

PMID: 27149379

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Project description:Deciduous and permanent human teeth represent a model system to study ageing of mesenchymal populations. Aging is tightly connected to self-renewal and proliferation and thus, mapping potential molecular differences in these characteristics between populations constitutes an important task. Specifically designed microarray panels were used. We have detected a number of molecules that were differentially expressed in dental pulp mesenchyme from deciduous and permanent teeth extracted from young children and adults, respectively. Among the differentially regulated genes HMGA2, a stem cell-associated marker, stood out as a remarkable example with a robust expression in deciduous pulp cells. In addition to this, we discovered that several proliferation-related genes, including CDC2A and CDK4, were up-regulated in deciduous pulp cells, while matrix genes COL1A1, fibronectin and several signaling molecules, such as VEGF, FGFr-1 and IGFr-1 were up-regulated in the pulp cells from permanent teeth. Taken together, our data suggest that deciduous pulp cells are more robust in self- renewal and proliferation, whereas adult dental pulp cells are more capable of signaling and matrix synthesis. The deciduous teeth were collected from 3-12-year-old children (N=8), whereas the permanent teeth were collected from adults, 19-52 years of age (N=8). The tooth was mechanically separated into two halves to remove all soft tissue in the pulp. The pulp tissue was digested and the dissociated cells were passed through cell strainer and plated with culture medium. The cells were then passaged at 90% confluency to a 10 cm dish for expansion. RNA was extracted from passage one cells with the Qiagen RNeasy Mini Kit. Microarray analysis was performed using PIQORTM Stem Cell Microarray chip (Miltenyi Biotech), which is comprised of 942 relevant genes for stem/progenitor cells and key genes for cell differentiation, to identify gene sets that are differentially expressed between cells in the deciduous and adult teeth. A total of 1 μg RNA for each sample was used for amplification and further analysis with the PIQORTM stem cell microarray chip (Miltenyi Biotec), followed by detection with a laser scanner (Agilent Technologies). In total, 8 comparisons were performed with dataset consisting of two microarrays, each containing a total of 11 slides with signal intensities within the R/Bioconductor statistical framework. The Linear Model for Microarray Data package (Limma) was used to pre-process the raw data, perform quality controls and estimate statistical significance. Expression intensities were background-corrected using a convolution of normal and exponential distributions with an offset of 50, which were then normalized within every slide using loess normalization. We analyzed the red and green channels separately using the mixed model method and different groups were quantile-normalized separately. The correlation was computed between the two channels for the same spot. Differentially expressed genes were identified by computing the contrasts, fitting to a linear model, performing the hypothesis tests, and correcting for multiple testing by performing the false discovery rate using the Benjamini-Hochberg correction.

Project description:Background and objectivesDental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition.MethodsProtein extracts from deciduous (n = 25) and permanent (n = 12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins.ResultsA total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth.ConclusionsClear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

Dataset Information

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

Publications

Secretome Profiling of Periodontal Ligament from Deciduous and Permanent Teeth Reveals a Distinct Expression Pattern of Laminin Chains.

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