Proteomic analysis of human periodontal ligament cells under hypoxia

Abstract

Background: The periodontal ligament is essential for homeostasis of periodontal tissue. A hypoxic milieu of the periodontal tissue is generated under periodontitis or during orthodontic treatment, which affects the periodontal and bone remodelling process. Here, we provide a comprehensive proteomic characterization of periodontal ligament cells under hypoxic conditions, aiming to reveal previously unappreciated biological changes and to help advance hypoxia-based therapeutic strategies for periodontal diseases.

Methods: Human periodontal ligament cells (hPDLCs) were characterized using immunohistochemistry (IHC) and flow cytometry (FACS). Successful hypoxia treatment of hPDLCs with 1% O₂ was confirmed by qRT-PCR. Proliferation was evaluated using an MTT assay. The proteomic expression profile under hypoxia was studied with the isobaric tags for relative and absolute quantification (iTRAQ) approach followed by protein identification and bioinformatic analysis, and western blot verification was performed.

Results: The hPDLCs were positive for vimentin, CD73 and CD105 and negative for keratin, CD34 and CD45. After hypoxia treatment, the mRNA expression of hypoxia-inducible factor 1a (HIF1a) was upregulated. The proliferation rate was elevated during the first 6 h but decreased from 6 h to 72 h. A total of 220 differentially expressed proteins were quantified in hPDLCs under hypoxia (1% O₂, 24 h), including 153 upregulated and 67 downregulated proteins, five of which were verified by western blot analysis. The Gene Ontology enriched terms included the energy metabolic process, membrane-bound organelle and vesicle, and protein binding terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated several involved pathways, including glycolysis/gluconeogenesis, biosynthesis of amino acids, the HIF-1 signalling pathway, and focal adhesion. A protein-protein interaction (PPI) network demonstrated the dominant role of autophagy over apoptosis under hypoxia.

Conclusion: The proteomic profile of hPDLCs under hypoxia was mainly related to energy metabolism, autophagy, and responses to stimuli such as adhesion and inflammation. Previously unrecognized proteins including solute carrier family proteins, heat shock proteins, ubiquitination-related enzymes, collagen and S100 family proteins are involved in adaptive response to hypoxia in hPDLCs and are thus of great research interest in future work.

Keywords: Fibroblasts; Hypoxia; Pathogenesis of periodontal disease(s); Periodontal ligament; Proteome.

Fig. 1

a Characterization of hPDLCs. Immunohistochemical staining showed the expression of vimentin in the cytoplasm (A, left), but keratin was not found (A, right). The insert shows a higher magnification of 200X. Scale bar, 100 μm. b The flow cytometry results showed that hPDLCs expressed mesenchymal stem cell-associated surface markers (CD73 and CD105) but did not express hematopoietic surface markers (CD34 and CD45). c Proliferation rates of hPDLCs. During the first 6 h, the proliferation rates of hPDLCs under hypoxia were higher than those of hPDLCs under normoxia. From 24 h to 72 h, the proliferation rates under hypoxia decreased. The values are presented as the mean ± SD (*Student’s t-test, P < 0.05). d hPDLCs under hypoxia expressed higher levels of HIF1a mRNA than control group hPDLCs

Fig. 2

Overview of iTRAQ proteomic analysis. a Hierarchical clustering of the differentially expressed proteins. The heat map demonstrates that expression patterns were altered under hypoxia. Red denotes high relative expression, and green denotes low relative expression. b The differentially expressed proteins according to fold changes (FCs) and P values are depicted with a volcano plot. Proteins with a P < 0.05 and a FC < 5/6 or >1.2 were considered to be significantly differentially expressed. Red dots denote upregulated proteins, and green dots denote downregulated proteins

Fig. 3

Enrichment analysis of GO terms for the differentially expressed proteins. The upregulated (a) and downregulated (b) protein terms in the biological process, cellular component and molecular function categories are depicted

Fig. 4

Enrichment analysis of KEGG pathways for the differentially expressed proteins. The bubble chart depicts the top 20 enriched pathways. The colour of each dot denotes the P value, and the size of each dot denotes the number of differentially expressed proteins

Fig. 5

Protein–protein interaction network. Proteins related to apoptosis and autophagy were chosen for analysis

Fig. 6

Western blot verification of the iTRAQ analysis results. The protein expression of MIF, S100A10, LDHA, and GAPDH was upregulated, while that of S100A9 was downregulated, consistent with the iTRAQ results