Quorum-sensing molecule dihydroxy-2,3-pentanedione and its analogs as regulators of epithelial integrity
AbstractBackground and Objective
Quorum-sensing molecules regulate the behavior of bacteria within biofilms and at the same time elicit an immune response in host tissues. Our aim was to investigate the regulatory role of dihydroxy-2,3-pentanedione (DPD), the precursor of universal autoinducer-2 (AI-2), and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) in the integrity of gingival epithelial cells.Material and Methods
Human gingival keratinocytes were incubated with four concentrations (10 μmol L−1, 1 μmol L−1, 100 nmol L−1 and 10 nmol L−1) of DPD and its analogs for 24 hours. The numbers of viable cells were determined using a proliferation kit, matrix metalloproteinase (MMP)-2 and -9 activities were determined by gelatin zymography, and expression of occludin protein and occludin mRNA were determined by western blotting and RT-qPCR, respectively.Results
Increased cell proliferation was observed in gingival keratinocytes incubated with 100 nmol L−1 of butyl-DPD. MMP-9 activity was elevated in cells incubated with 10 μmol L−1 of ethyl-DPD. On the other hand, MMP-2 activity did not show any significant change when gingival keratinocytes were incubated with or without DPD or analogs. Western blot analyses demonstrated five forms (105, 61, 52.2, 44 and 37 kDa) of occludin. Incubation with 1 μmol L−1 and 100 nmol L−1 of DPD and with 10 nmol L−1 of ethyl-DPD increased dimeric (105 kDa) forms of occludin, while incubation with 100 nmol L−1 of isobutyl-DPD increased monomeric (61 kDa) forms. DPD and ethyl-DPD decreased, and 100 nmol L−1 of isobutyl-DPD and 10 nmol L−1 of butyl-DPD increased, the monomeric (52.2 kDa and 44 kDa) forms of occludin, whereas ethyl-DPD decreased and isobutyl-DPD increased, the low-molecular-weight (37 kDa) forms. According to RT-qPCR analysis, the exposure of gingival keratinocytes to 10 μmol L−1 of isobutyl-DPD up-regulated expression of occludin.Conclusion
The results indicate that isobutyl-DPD has the potential to enhance the integrity of the epithelium by stimulating the formation of occluding, without affecting the proliferation or gelatinolytic enzyme activities of the exposed cells. The modulatory effect of an AI-2 analog on the epithelial cell response is shown for the first time.