Estrogen-related Receptor α Regulates Osteogenic Differentiation Of Human Periodontal Ligament Stem Cells

Estrogen-related receptor α (ERRα) is the first identified orphan nuclear receptor.ERRα does not bind any known endogenous ligand. Instead, it activates gene transcriptionconstitutively in a ligand-independent manner. ERRα is capable of regulating thetranscription of genes involved in multiple cellular and physiological processes. ERRαalso regulates osteogenic differentiation of MSCs. Moreover, ERRα has been identified asa novel therapeutic target for treating osteoporosis and other bone diseases.Human periodontal ligament tissue-derived mesenchymal stem cells (PDLSCs) haverecently been used in stem cell-mediated therapies because of their multipotency,especially toward osteogenic differentiation. However, it is still unclear whether ERRα canregulate the osteogenic differentiation of PDLSCs. To investigate the role of ERRα in theosteogenic differentiation of PDLSCs in vitro, the following experiments were designedand carried out:1. The isolation and identification of PDLSCs and the expression of ERRα in PDLSCsObjective: To obtain primary PDLSCs and detect the expression of ERRα in PDLSCs.Methods:(1) We took the healthy premolars from adolescent for orthodontic reason,scraped middle1/3of the periodontal ligament tissues on the roots. Limiting dilutiontechnique was used for PDLSC cloning and purification, and colony-forming assay andCCK-8assay were performed;(2) Stem cell surface markers were detected using flowcytometry;(3) Osteogenic and adipogenic induction of PDLSCs were performed anddetermined by Alizarin red S and Oil red O staining respectively;(4) RT-PCR analysis wasused to detect ERRα mRNA expression in PDLSCs;(5) Immunocytochemistry andimmunofluorescence staining were used to detect ERRα expression and its proteinlocalization in cells. Results:(1) The cloned and purified PDLSCs were fibroblastcell-like morphology, and had the colony-forming capacity and the proliferationcharacteristics of stem cells;(2) PDLSCs expressed the surface markers of mesenchymalstem cells, and had the capacity of osteogenic and adipogenic differentiation;(3) PDLSCsexpressed ERRα mRNA and protein, and the protein was expressed both in nucleus andcytoplasm, and maily expressed in the nucleus. Conclusion: We successfully isolated andidentified PDLSCs with characteristics of mesenchymal stem cells, and ERRα was widelyexpressed in the cells.2. The effects of lentivirus-mediated ERRα RNA interference on the osteogenicdifferentiation of PDLSCsObjective: To study the effects of down-regulation of ERRα gene on PDLSC osteogenicdifferentiation. Methods:(1) We designed four pairs of miRNA oligonucleotides againstERRα gene and connected them with the vector pcDNA6.2-GW/EmGFP-miR. Then wetransient transfected the DNA of the four miRNA expression plasmid into293cells, anddetermined the interference efficiency by real-time quantitative PCR and Western blotanalysis;(2) The plasmid with the highest interference efficiency were used to buildlentiviral plasmid pLenti-miR-ERRα. Lentiviral packaging, purification and infection ofPDLSCs were carried out to generate the stable transfected cells. CCK-8analysis wasperformed to determine the cell proliferation. Real-time quantitative PCR and Western blot were performed to determine ERRα mRNA and protein expression respectively;(3)The osteogenic differentiation of lentivirus transduced PDLSCs was determined by ALPstaining, Alizarin red S staining and real-time quantitative PCR analysis of the mRNAexpression of the osteogenesis-related genes (ALP, OCN, RUNX2and OPN). Results:(1)We successfully constructed four miRNA expression plasmids, which down-regulatedERRα mRNA and protein expression. The pcDNA6.2-miR-ERRα-4, which had thehighest interference efficiency, was used to recombinant cloning, lentiviral packaging, andgenerate stably infected PDLSCs;(2) Lenti-miR-ERRα stably infected PDLSCs displayedsignificantly reduced mRNA and protein levels. CCK-8results showed that the activity ofmiR-ERRα PDLSCs cells was slightly lower than that of the blank or the negative controlgroup;(3) MiR-ERRα PDLSCs displayed significantly reduced ALP activity,mineralization ability, as well as mRNA expression of ALP, OCN, RUNX2and OPNcompared with the control group; Conclusion:(1) Lentiviral miRNA vector stablydown-regulated ERRα expression in PDLSCs;(2) Knockdown of ERRα gene reducedosteogenic differentiation of PDLSCs, suggesting that ERRα may promote osteogenicdifferentiation of PDLSCs.3. The role of ERRα coactivated by PGC-1α on the osteogenic differentiation ofPDLSCsObjective: To study the effects of activation of ERRα by its coactivator PGC-1α onosteogenic differentiation of PDLSCs. Methods:(1) The osteogenic induction of PDLSCswas under the treatment of the ERRα reverse the agonist XCT790for7d, then the ALPactivity assay, ALP and Alizarin red S staining were performed;(2) After the conditionsof the electroporation transfection of PDLSCs were optimized, PDLSCs were transfectedwith expressing plasmid of ERRα, PGC-1α, ERRα+PGC-1α or negative vector group, andreal-time quantitative PCR and Western blot analysis were performed to confirm theeffects of transfection;(3) The transfected and XCT790treated PDLSCs were induced inosteogenic medium for7d, followed by the determination of intracellular Ca2+concentration and mRNA expression of ALP, COL1A1, OCN, OPN and RUNX2usingreal-time quantitative PCR analysis. Results:(1) XCT790significantly reduced the protein levels of ERRα and PGC-1α, strikingly inhibited ALP activity within2weeks andultimately reduced mineralization of PDLSCs;(2) We successfully constructed ERRα andPGC-1α expression vector and transfected PDLSCs by electroporation. Quantitativereal-time PCR and Western blot were used to verify the expression of mRNA and protein;(3) After osteogenic induction for7days, ERRα over-expression up-regulated ALP anddown-regulate RUNX2and OPN mRNA expresion. PGC-1α over-expression significantlyup-regulated ALP, COL1A1and RUNX2and down-regulated OPN mRNA expression.ERRα+PGC-1α over-expression significantly up-regulated ALP, COL1A1and RUNX2mRNA expression, while it did not significantly affected OCN and OPN;(4) XCT790didnot change mRNA expression of ERRα, but it intensively inhibited protein level of ERRαand the mRNA and protein level PGC-1α. It significantly reduced mRNA expression ofALP, OCN and RUNX2, while significantly promoted the mRNA expression of OPN andintracellular Ca2+concentration. Conclusion: ERRα, coactivated by PGC-1α, maypromote the osteogenic differentiation of PDLSCs by up-regulate the expression of ALP,COL1A1and RUNX2.