Lipase-a single-nucleotide polymorphism rs143793106 is associated with increased risk of aggressive periodontitis by negative influence on the cytodifferentiation of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Aggressive periodontitis (AgP) is characterized by general health and rapid destruction of periodontal tissue. The familial aggregation of this disease highlights the involvement of genetic factors in its pathogeny. We conducted a genome-wide association study (GWAS) to identify AgP-related genes in a Japanese population, and the lipid metabolism-related gene, lipase-a, lysosomal acid type (LIPA), was suggested as an AgP candidate gene. However, there is no report about the expression and function(s) of LIPA in periodontal tissue. Hence, we studied the involvement of how LIPA and its single-nucleotide polymorphism (SNP) rs143793106 in AgP by functional analyses of LIPA and its SNP in human periodontal ligament (HPDL) cells. MATERIALS AND METHODS: GWAS was performed using the genome database of Japanese AgP patients, and the GWAS result was confirmed using Sanger sequencing. We examined the mRNA expression level of LIPA and the protein expression level of the encoded protein lysosomal acid lipase (LAL) in periodontium-composing cells using conventional and real-time polymerase chain reaction (PCR) and western blotting, respectively. Lentiviral vectors expressing LIPA wild-type (LIPA WT) and LIPA SNP rs143793106 (LIPA mut) were transfected into HPDL cells. Western blotting was performed to confirm the transfection. LAL activity of transfected HPDL cells was determined using the lysosomal acid lipase activity assay. Transfected HPDL cells were cultured in mineralization medium. During the cytodifferentiation of transfected HPDL cells, mRNA expression of calcification-related genes, alkaline phosphatase (ALPase) activity and calcified nodule formation were assessed using real-time PCR, ALPase assay, and alizarin red staining, respectively. RESULTS: The GWAS study identified 11 AgP-related candidate genes, including LIPA SNP rs143793106. The minor allele frequency of LIPA SNP rs143793106 in AgP patients was higher than that in healthy subjects. LIPA mRNA and LAL protein were expressed in HPDL cells; furthermore, they upregulated the cytodifferentiation of HPDL cells. LAL activity was lower in LIPA SNP-transfected HPDL cells during cytodifferentiation than that in LIPA WT-transfected HPDL cells. In addition, ALPase activity, calcified nodule formation, and calcification-related gene expression levels were lower during cytodifferentiation in LIPA SNP-transfected HPDL cells than those in LIPA WT-transfected HPDL cells. CONCLUSION: LIPA, identified as an AgP-related gene in a Japanese population, is expressed in HPDL cells and is involved in regulating cytodifferentiation of HPDL cells. LIPA SNP rs143793106 suppressed cytodifferentiation of HPDL cells by decreasing LAL activity, thereby contributing to the development of AgP.

Fibroblast growth factor-2 inhibits CD40-mediated periodontal inflammation.

Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.

Identification of genetic risk factors of aggressive periodontitis using genomewide association studies in association with those of chronic periodontitis.

To identify the genetic risk factors for aggressive periodontitis (AgP), it is important to understand the progression and pathogenesis of AgP. The purpose of this review was to summarize the genetic risk factors for AgP identified through a case-control genomewide association study (GWAS) and replication study. The initial studies to identify novel AgP risk factors were potentially biased because they relied on previous studies. To overcome this kind of issue, an unbiased GWAS strategy was introduced to identify genetic risk factors for various diseases. Currently, three genes glycosyltransferase 6 domain containing 1 (GLT6D1), defensin α1 and α3 (DEFA1A3), and sialic acid-binding Ig-like lectin 5 (SIGLEC5) that reach the threshold for genomewide significance have been identified as genetic risk factors for AgP through a case-control GWAS.

Autophagy facilitates type I collagen synthesis in periodontal ligament cells.

Autophagy is a lysosomal protein degradation system in which the cell self-digests its intracellular protein components and organelles. Defects in autophagy contribute to the pathogenesis of age-related chronic diseases, such as myocardial infarction and rheumatoid arthritis, through defects in the extracellular matrix (ECM). However, little is known about autophagy in periodontal diseases characterised by the breakdown of periodontal tissue. Tooth-supportive periodontal ligament (PDL) tissue contains PDL cells that produce various ECM proteins such as collagen to maintain homeostasis in periodontal tissue. In this study, we aimed to clarify the physiological role of autophagy in periodontal tissue. We found that autophagy regulated type I collagen synthesis by elimination of misfolded proteins in human PDL (HPDL) cells. Inhibition of autophagy by E-64d and pepstatin A (PSA) or siATG5 treatment suppressed collagen production in HPDL cells at mRNA and protein levels. Immunoelectron microscopy revealed collagen fragments in autolysosomes. Accumulation of misfolded collagen in HPDL cells was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. E-64d and PSA treatment suppressed and rapamycin treatment accelerated the hard tissue-forming ability of HPDL cells. Our findings suggest that autophagy is a crucial regulatory process that facilitates type I collagen synthesis and partly regulates osteoblastic differentiation of PDL cells.

A Putative Association of a Single Nucleotide Polymorphism in GPR126 with Aggressive Periodontitis in a Japanese Population.

Periodontitis is an inflammatory disease causing loss of tooth-supporting periodontal tissue. Disease susceptibility to the rapidly progressive form of periodontitis, aggressive periodontitis (AgP), appears to be influenced by genetic risk factors. To identify these in a Japanese population, we performed whole exome sequencing of 41 unrelated generalized or localized AgP patients. We found that AgP is putatively associated with single nucleotide polymorphism (SNP) rs536714306 in the G-protein coupled receptor 126 gene, GPR126 [c.3086 G>A (p.Arg1029Gln)]. Since GPR126 activates the cAMP/PKA signaling pathway, we performed cAMP ELISA analysis of cAMP concentrations, and found that rs536714306 impaired the signal transactivation of GPR126. Moreover, transfection of human periodontal ligament (HPDL) cells with wild-type or mutant GPR126 containing rs536714306 showed that wild-type GPR126 significantly increased the mRNA expression of bone sialoprotein, osteopontin, and Runx2 genes, while mutant GPR126 had no effect on the expression of these calcification-related genes. The increase in expression of these genes was through the GPR126-induced increase of bone morphogenic protein-2, inhibitor of DNA binding (ID) 2, and ID4 expression. These data indicate that GPR126 might be important in maintaining the homeostasis of periodontal ligament tissues through regulating the cytodifferentiation of HPDL cells. The GPR126 SNP rs536714306 negatively influences this homeostasis, leading to the development of AgP, suggesting that it is a candidate genetic risk factor for AgP in the Japanese population.

Expression pattern of zinc-finger transcription factor Odd-skipped related 2 in murine development and neonatal stage.

The Odd-skipped gene, first identified as a Drosophila pair-rule zinc-finger transcription factor, plays an important role in Drosophila development. The mammalian homolog, Odd-skipped related 2 (Osr2), regulates limb, tooth, and kidney development in mouse embryos. However, the detailed expression pattern of Osr2 during neonatal development remains unclear. In this study, we investigated Osr2 expression patterns in mouse neonatal and embryo tissues using qPCR and in situ hybridization methods. First, we examined the tissue distribution of Osr2 by qPCR, and found it to be highly expressed in the uterus and moderately in the testes, small intestine, and prostate. That expression was also found in eye, kidney, placenta, lung, thymus, lymph node, stomach, and skeletal muscle tissues, and in all embryonic stages. On the other hand, Osr2 was not expressed in brain, heart, liver, or spleen samples. Next, we examined the tissue localization of Osr2 using in situ hybridization. Osr2 was found in the craniofacial region on E13.5, with notable expression in dental germ mesenchyme as well as the renal corpuscle on E17.5. As for neonatal tissues, Osr2 was expressed in the dental papilla, dental follicle, Harderian gland, nasal bone, eyelid dermis, synovial joint, and tibial subcutis. Our findings suggest that Osr2 functions in reproductive system organs, such as the uterus, testes, prostate, placenta, and ovaries. Furthermore, based on its expression in kidney, Harderian gland, eyelid dermis, and tibial subcutis tissues, this transcription factor may be involved in hormone synthesis and function. Together, our results demonstrate the role of Osr2 in postnatal development and embryogenesis.