Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA 18:1 controls immediate‐early GF gene transcription of inflammatory cytokines, and of their receptors and regulators. Vitamin D3 is a key hormone in decreasing inflammation, and has been shown by Nastri et al. (2018) to decrease pro‐inflammatory and increase anti‐inflammatory cytokine production by P. gingivalis‐ and S. pyogenes‐infected human GF. Therefore, we hypothesized that LPA would also modulate transcription of its receptor, VDR, since chronic periodontal disease (PD) is an inflammatory condition in which we have reported that LPA is elevated in human gingival crevicular fluid and saliva (Bathena et al., 2011). After GF treatment with 1 × 10−5 M LPA 18:1 for 2h or 8h, mRNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analyses [n=3; each n= pooled mRNA derived from 3 healthy young donors; (9 total donors; the study conformed to the Declaration of Helsinki guidelines, was approved by the Creighton University IRB, and informed written consent was obtained from all donors)] revealed that LPA consistently and significantly down‐regulated the VDR vs. control (≤ −2.0 ± 0.5‐fold at 2h, and −3.1 ± 1.1 fold at 8h, respectively). Of great interest, the receptor cubilin (CUBN), which has been shown in other systems to bind vitamin D binding protein (DBP), was also down‐regulated in groups 1 (−2.0) and 2 (−4.1) at 8h; it may be involved in the secretion of vitamin D by GF, as postulated by Liu et al., (2012). LPA treatment also significantly affected transcription of the cytochromes (CYP) CYP27A1 [−3.0 ± 0.6 (2h); −3.2 ± 1.4 (8h)] and CYP27B1 (induction or repression varied by group) which is involved in the production of metabolically active metabolites and which catalyzes the hydroxylation at the C‐1α position of 25(OH)D3 and 24R, 25(OH)2D3, respectively. These results for the VDR (minimum level of significance, p < 1×10−7) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by regulating it, along with other key receptors and enzymes in the vitamin D pathway.Support or Funding InformationHealth Future Foundation (D.R.C).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ABSTRACTAimsOur laboratory has found that lysophosphatidic acid (LPA) and its cognate receptors [LPARs, (LPA1–6)] expressed by human gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) play key roles in oral fibroblast homeostasis and are implicated in the inflammation seen in periodontal disease. We have reported that PDLF express LPA1 and LPA3; however, information on the gross topographic distribution of LPARs in the periodontal ligament (PDL) was lacking, and therefore, we developed a simple method forin situlabeling of LPARs in the PDL of extracted teeth.Materials and methodsSectioning or grinding thin sections of demineralized or native teeth and periodontium have long been the standard methodologies used to assess biomarker distribution in the PDL; however, we modified traditional immunohistochemical labeling and used whole teeth with fixed, solvent permeabilized PDLs.ResultsLPA1 and LPA3 were specifically labeled in the PDL and could be visualized at both the macroand micro-levels.ConclusionThis technique effectively labeled LPARs, and it can serve as a basis for thein situvisualization of other biomolecules expressed in the PDL.Clinical SignificanceThe ability to observe PDL LPAR distribution at the macro-level complements the microscopic data, and it is useful for detecting and documenting molecular changes in the PDL/PDLF that were brought about by age, experimental treatments, or pathologies like periodontal disease.How to cite this articleCerutis DR, Headen KV, Ogunleye AO, Williams DE. A High-resolution Immunohistochemical Method for studying Receptor Expression on the Periodontal Ligament of Whole-mount Human Tooth Roots. Int J Experiment Dent Sci 2016;5(2):99-103.

We have found that lysophosphatidic acid (LPA) controls the healing and [Ca2+]i responses of human gingival fibroblasts via the LPA1 and LPA3 receptor subtypes, and that LPA3 may modulate their inflammatory responses. We have used immunofluorescence to detect LPA receptor (LPAR) distribution in human gingivae, but that technique does not afford visualization of the state of collagen fiber organization. We hypothesized that using the second harmonic generation (SHG) technique would enable detection of changes in the appearance, collagen fibril density, and fibril organizational structure of human gingiva, and that this technique would complement visualizing LPAR expression by confocal microscopy. Pieces of intact, paraformaldehyde‐fixed, methanol:acetone (1:1) de‐lipidated attached gingivae [~1.25 mm thick (from IRB‐approved, consented donors) normal or with moderate‐severe periodontal disease] were used. Confocal analysis using anti‐LPA1 and anti–LPA3 antibodies confirmed that LPA3 is primarily found in the epithelium, and that nuclear LPA1 expression in the sub‐epithelial connective tissue is greatly diminished in periodontal disease. SHG analysis clearly showed the normal collagen structure and subsequent reduction in the abundance and organization in the collagen fibrils of periodontal gingivae. This multidisciplinary study shows that SHG effectively complements confocal microscopy as a tool to help understand periodontal changes at the level of receptors and collagen organization.

BACKGROUND:The small bioactive lipid lysophosphatidic acid (LPA) plays critical roles in both normal physiology and inflammation in many systems. However, its actions are just beginning to be defined in oral biology and pathophysiology.METHODS:Microarray analysis was used to test the hypothesis that human gingival fibroblasts (GFs) would show significant changes in wound-healing and inflammation-related gene transcripts in response to a major human salivary and gingival crevicular fluid LPA species, 18:1, and that they would express transcript for the major LPA-producing enzyme autotaxin. The microarray results were validated for three highly relevant upregulated inflammatory transcripts using quantitative reverse transcription-polymerase chain reaction (QRT-PCR). Liquid chromatography-tandem mass spectrometry was used to assay time-dependent LPA species production by GFs.RESULTS:LPA 18:1 significantly regulated 20 GF novel and 27 known genes linked to the control of inflammation (P ≤0.01). QRT-PCR validation of interleukin (IL)-8, IL-11, and suppressor of cytokine signaling 2 (SOCS2) messenger RNAs confirmed statistically significant differences from control (P ≤0.05). Autotaxin transcript was present, and GFs were found to produce multiple LPA species in a time-dependent manner.CONCLUSIONS:The upregulation of transcripts for known GF proinflammatory (IL-6, IL-8) and anti-inflammatory (IL-11) ILs, along with SOCS2, shows that LPA transiently regulates a complex set of GF genes critical to periodontal wound healing and inflammation. These results implicate LPA exerting actions on GFs that are compatible with functioning as a mediator in oral fibroblast biology and inflammatory responses. Therefore, LPA may potentially modulate/regulate periodontal inflammation.

Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA controls immediate‐early gene transcription of inflammatory cytokines, and their receptors and regulators in GF; therefore, we hypothesized that LPA would modulate transcription of the isozymes SPHK1 and/or SPHK2, which make sphingosine‐1‐phosphate (S1P). S1P is under much investigation as a major regulator in immunity and inflammation. Critically, S1P is a primary controller of human neutrophil responses (such as chemotaxis, degranulation, and oxidative burst), making it highly relevant to the inflammation seen in periodontal disease. After GF treatment with 1 × 10−5 M 18:1 LPA for 2h or 8h, RNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analysis revealed that LPA consistently and significantly upregulated SPHK1 versus control (3.9 ± 0.5 and 4.7± 0.7 fold at 2h and 8h, respectively). Of great interest, the proton‐sensing receptor GPR68/OGR1 was highly induced: 7.3 ± 4.0 fold (2h) and 18.8 ± 12.8 fold (8h). LPA1–3 can all dimerize with GPR68 /OGR1, which is linked to COX‐2 and inflammatory cytokine induction in other systems. QRT‐PCR was performed to validate the microarray results, using 3 biologic replicates for four periodontal disease‐relevant up‐regulated genes at the 8h time point. These results (level of significance, p < 0.01) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by inducing S1P production and through LPA1–3 hetero‐dimerizing with GPR68. Support: Health Future Foundation (D.R.C).

We have shown that lysophosphatidic acid (LPA) is an essential regulator of human oral fibroblast biology, and that gingival fibroblasts (GF) express the LPA receptors (LPARs) LPA1–5. Therefore, we hypothesized that LPA controls multiple crucial regulatory pathways in GF. The profile of immediate‐early genes induced by LPA in GF was determined. After treatment with 1 × 10−5 M LPA for 2h or 8h, RNA extraction was done. Agilent Whole Human Genome Oligonucleotide Microarray analysis identified a complex array of significantly regulated mRNAs (> than, or < than 2‐fold of control): 2,609 (2h) and 2,348 (8h). Some of the salient Gene Ontology Biological Processes regulated include cellular energetics, cellular motility, cell‐matrix adhesion, cell‐cell adhesion, cellular calcium (and ion) homeostasis, reactive oxygen species metabolic processes, cell‐cell signaling, cell surface receptors and associated signal transduction pathways, stress pathways, immune regulation and antigen presentation, and defense response to bacteria. LPA treatment also significantly downregulated the expression of LPA4. These results provide concrete evidence that LPA is a mediator of key importance for oral fibroblasts, with the potential to exert complex actions during periodontal inflammation and wound healing. Support: Health Future Foundation (D.R.C).

The purpose of this study was to investigate the effect that practice patterns and other variables had on graduates' level of satisfaction with dental school preparation and satisfaction with several career experiences and to determine if any gender-based differences occurred in these findings. Dentists who graduated from Creighton University School of Dentistry between 1985 and 2005 were surveyed regarding demographic factors, practice characteristics, satisfaction with educational preparation, and satisfaction with practice experiences. The response data were analyzed using Spearman's rho, multiple regression analysis, and the Pearson product moment. As a result of this analysis, no statistically significant differences were found between male and female dentists who graduated from Creighton University between 1985 and 2005 with respect to satisfaction with academic preparation and postgraduation practice experiences. The findings indicate that gender is not associated with graduates' level of satisfaction with their academic preparation while in dental school and their practice experiences.

BACKGROUND:We showed that the pluripotent platelet growth factor and mediator lysophosphatidic acid (LPA) controls key regenerative responses of human gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PDLFs) and positively modulates their responses to platelet-derived growth factor (PDGF). This study determined which LPA receptor (LPAR) subtype(s) LPA signals through to stimulate mitogenic extracellular signal-regulated kinase (ERK) 1/2 signaling and chemotaxis and to elicit intracellular Ca(2+) increases in GFs and PDLFs because many healing responses are calcium-dependent.METHODS:Activation of mitogen-activated protein kinase was determined using Western blotting with an antibody to phosphorylated ERK1/2. Migration responses were measured using a microchemotaxis chamber. GF and PDLF intracellular Ca(2+) mobilization responses to multiple LPA species and LPAR subtype-specific agonists were measured by using a cell-permeable fluorescent Ca(2+) indicator dye.RESULTS:LPA stimulated ERK1/2 phosphorylation via LPA(1)(-3). For GFs, LPA(1) preferentially elicited chemotaxis, and LPA(1-3) for PDLFs, as confirmed using subtype-specific agonists. Elevation of intracellular calcium seems to be mediated through LPA(1) and LPA(3), with little, if any, contribution from LPA(2).CONCLUSIONS:To the best of our knowledge, this study provides the first evidence that LPA signals through specific LPAR subtypes to stimulate human oral fibroblast regenerative responses. These data, in conjunction with our previous findings showing that LPA modulates GF and PDLF responses to PDGF, suggest that LPA is a factor of emerging importance to oral wound healing.