Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities

Abstract

Background/purpose: Peroxisome proliferator-activated receptor γ (PPARγ) is a major transcription factor of energy metabolism-associated genes, and three PPARγ isoforms have been identified in periodontal tissues and cells. When energy metabolism homeostasis is affected by PPARγ downregulation in periodontal ligament fibroblasts (PDLFs), osteo/cementogenic abilities are markedly lost. Herein, we investigated whether PPARγ agonists promote periodontal tissue regeneration, and which PPARγ isoforms and metabolic pathways are indispensable for osteo/cementogenic abilities.

Materials and methods: A PPARγ agonist was locally administered to regenerate murine periodontal tissue. The distinct functions of the PPARγ isoforms in PDLFs were assessed using an overexpression strategy. Candidate metabolic processes were searched using gene ontology analysis of PPARγ-knockdown PDLFs. In vitro differentiation assays were performed to evaluate the effects of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), two major isoprenoid intermediates.

Results: PPARγ agonists accelerated periodontal tissue regeneration. Full-length PPARγ overexpression specifically enhanced the osteo/cementogenic differentiation of PPARγ agonist-induced PDLFs. The isoprenoid metabolic process was the top-ranked downregulated metabolism-associated pathway following PPARγ knockdown; FPP and GGPP enhanced and suppressed PDLFs' differentiation, respectively. Gene expression analysis of human clinical periodontal tissues revealed that osteocalcin correlated with farnesyl pyrophosphate synthetase (FDPS), which catalyzes FPP production, but not with two FPP conversion enzymes: geranylgeranyl diphosphate synthase 1 (GGPS1) or farnesyl diphosphate farnesyltransferase 1 (FDFT1).

Conclusion: Preferable PPARγ agonistic actions depend on the full-length PPARγ isoform. FPP increased PDLFs' osteo/cementogenic abilities. Therefore, administering FPP and precisely controlling FDPS, GGPS1, and FDFT1 activities could be a novel strategy for accelerating periodontal tissue regeneration.

Keywords: Energy metabolism; Isoprenoid synthesis; Periodontal ligament fibroblasts; Periodontal tissue regeneration; Peroxisome proliferator-activated receptor γ.

Figure 1

Local administration of rosiglitazone promotes periodontal tissue regeneration in a murine model. (A) The vertical distances from the cemento-enamel junction to the alveolar bone crest at the mesial and distal roots at 14 days after ligature removal were measured and summed (n = 13). (B) Demineralized male maxilla sections treated with 0 or 1.2 mg/kg of rosiglitazone for 14 days after ligature removal were stained with Masson's trichrome. ∗P < 0.05 was considered to be a statistically significant difference from the control. n.s. = not significant. The border of the alveolar bone and periodontal ligament tissue are indicated by dash line. Scale bars correspond to 500 and 100 μm at low and high magnification, respectively. Ros = rosiglitazone.

Figure 2

Overexpression of full-length PPARγ specifically enhances troglitazone-induced osteo/cementogenic differentiation of PDLFs. (A, B) PDLF-empty, PDLF-PPARγ, PDLF-PPARγ-UBI, and PDLF-PPARγ-PDL were cultured in mineralization-inducing medium for a maximum of 12 days. ALP activities were normalized by cell numbers (A) and calcium deposition was visualized by Alizarin Red S staining (B). ∗∗∗P < 0.001 was considered a statistically significant difference compared with the PDLF-empty treated with troglitazone on day 6. n.s. = not significant, Tro = troglitazone.

Figure 3

The isoprenoid metabolic process is most severely downregulated metabolic pathway in PPARγ-knockdown PDLFs. (A) Gene ontology analysis of the downregulated genes in PDLFs transfected with siRNA for PPARG by comparing with PDLFs transfected with control siRNA. Raw RNA-seq data were obtained from NCBI's Gene Expression Omnibus (GEO) under accession number GSE178607. (B) PDLF-1 cells were cultured in mineralization-inducing medium for 3 and 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM). The gene expression levels of AKR1C3, ALDH3A2, AKR1C1, and SDC3, which were categorized in “isoprenoid metabolic process” and identified as the downregulated genes in PPARG-knockdown PDLFs, were evaluated. (C) PDLFs were cultured in mineralization-inducing medium for 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM) and FPP (20 μM) or GGPP (20 μM). ALP activities were normalized by cell numbers. (D) PDLFs were cultured in mineralization-inducing medium for 12 days in the presence of FPP (0, 10, 20 μM), and calcium deposition was visualized and quantified by Alizarin Red S staining. (E) Demineralized 1.5-month-old maxilla sections were stained without the primary antibody or with mouse IgG, α-FDPS, α-GGPS1, or α-FDFT1 antibodies, ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 indicated significantly higher expression levels compared with those in DMSO-treated PDLFs at each day point (B), non-treated PDLFs in either DMSO, troglitazone, or rosiglitazone treatment group (C), and non-treated PDLFs (D). Tro = troglitazone. Ros = rosiglitazone. P = pulp. D = dentin. Pdl = periodontal ligament tissue. Ab = alveolar bone. Scale bars correspond to 100 μm.

Figure 4

Correlation between OCN or COL1A1 and FDPS, GGPS1, or FDFT1 expression in human clinical periodontal tissue. Human clinical periodontal tissues (18 samples from different patients) were collected, and the correlative expression levels of OCN or COL1A1 with FDPS, GGPS1, or FDFT1 were examined. The high (r > 0.8 or r < −0.8) and weak correlations (0.2 < r < 0.4 or −0.4 < r < −0.2) are indicated by green and orange lines, respectively.

Supplemental Figure 1

Top-ranked terms enriched in the siPPARG-downregulated genes in PDLFs.