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.

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.

LPA is a pleiotropic G protein‐coupled lipid mediator involved in many homeostatic and patho‐physiologic aspects of cellular regulation. It binds to LPA1‐6 receptor (LPAR ) subtypes. It is co‐released by activated platelets together with platelet‐derived growth factor (PDGF). We have published that human gingival (GF) and periodontal ligament (PDLF) fibroblasts express LPA1‐3, and that LPA positively regulates their responses to PDGF. In this study, we tested the hypotheses that 1) LPA binds to additional LPAR subtypes on GF and PDLF and 2) PDGF regulates plasma membrane expression of LPARs. Using immunohistochemistry (IHC) with LPAR subtype‐specific antibodies (Abs) we probed for LPA1‐5. Determination of cell‐surface expression of LPARs was done by flow cytometry (FC) with these Abs. Cells were grown in 100‐mm dishes and serum‐starved prior to measuring LPARs by FC. We found that GF and PDLF also express LPA4 and LPA5. PDGF‐BB at10 ng/ml modulated the cell‐surface expression of several LPARs between 2 and 6h post‐treatment. From these results, we conclude that 1) multiple LPAR subtypes modulate the responses of GF and PDLF to LPA, and 2) PDGF‐BB exerts complex regulation of LPARs in these cells. Support: NIH/NIDCR 1 R15 DE016855‐03 (D.R.C).

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).

Aims Our 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 for in situ labeling of LPARs in the PDL of extracted teeth. Materials and methods Sectioning 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. Results LPA1 and LPA3 were specifically labeled in the PDL and could be visualized at both the macroand micro-levels. Conclusion This technique effectively labeled LPARs, and it can serve as a basis for the in situ visualization of other biomolecules expressed in the PDL. Clinical Significance The 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 article Cerutis 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.