Signaling pathways induced by lipoproteins derived from Mycoplasma salivarium and a synthetic lipopeptide (FSL-1) in normal human gingival fibroblasts.

Lipoproteins derived from Mycoplasma salivarium and a synthetic lipopeptide (FSL-1) activate human gingival fibroblasts to induce IL-6 production and ICAM-1 expression. Human gingival fibroblasts were treated with lipoproteins or FSL-1 and then examined for the activation of mitogen-activated protein kinases (MAPKs), ERK1/2, p38, and SAPK/JNK, and transcription factors, AP-1 and NF-kappaB. Western blotting indicated that p38 and SAPK/JNK were activated in response to the stimulators, but the activation of ERK1/2 could not be discriminated because ERK1/2 was activated in the absence of stimulators. The p38 inhibitor SB 203580 also suppressed their IL-6 production-inducing activities, whereas the ERK1/2-activating MAPK kinase (MEK1) inhibitor PD 98059 did not suppress their activities. Moreover, they were capable of inducing the activation of AP-1 and NF-KB. NF-kappaB activation was also confirmed by the phosphorylation of IkappaB-alpha. On the basis of these results, it was concluded that lipoproteins of M. salivarium and FSL-1 are capable of activating the MAPKs p38 and SAPK/JNK and the transcriptional factors AP-1 and NF-kappaB in human gingival fibroblasts.

Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease.

Bacteroides forsythus is a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared from B. forsythus cells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-kappaB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-kappaB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.

Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam(2)CGDPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentans-derived lipopeptide MALP-2 (Pam(2)CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-kappaB reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-kappaB-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.