Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin‐6 expression through multiple mitogen‐activated protein kinase pathways in periodontal ligament fibroblasts

Abstract

Actinobacillus actinomycetemcomitans plays a major role in the pathogenesis of aggressive periodontitis. Lipopolysaccharide (LPS) derived from A. actinomycetemcomitans is a key factor in inflammatory cytokine generation within periodontal tissues. In this study, we identify major mitogen-activated protein kinase (MAPK) signaling pathways induced by A. actinomycetemcomitans LPS, Escherichia coli LPS and interleukin-1beta (IL-1beta) in a murine periodontal ligament (mPDL) fibroblast cell line. Immunoblot analysis was used to assess the phosphorylated forms of p38, extracellular-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) MAPK following stimulation with A. actinomycetemcomitans LPS, E. coli LPS and IL-1beta. IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction, while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA). We utilized biochemical inhibitors of p38, ERK and JNK MAPK to identify the MAPK signaling pathways needed for IL-6 expression. Additional use of stable mPDL cell lines containing dominant negative mutant constructs of MAPK kinase-3 and -6 (MKK-3/6) and p38 null mutant mouse embryonic fibroblast (MEF) cells were used to substantiate the biochemical inhibitor data. Blocking p38 MAPK with SB203580 reduced the induction of IL-6 mRNA by A. actinomycetemcomitans LPS, E. coli LPS and IL-1beta by >70%, >95% and approximately 60%, respectively. IL-6 ELISA indicated that blocking p38 MAPK reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1beta by approximately 60%, approximately 50% and approximately 70%, respectively. All MAPK inhibitors significantly reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1beta whereas only p38 inhibitors consistently reduced the A. actinomycetemcomitans LPS, E. coli LPS and IL-1beta induction of IL-6 mRNA steady-state levels. The contribution of p38 MAPK LPS-induced IL-6 expression was confirmed using MKK-3/6 dominant negative stable mPDL cell lines. Wild-type and p38alpha(-/-) MEF cells provided additional evidence to support the role of p38alpha MAPK in A. actinomycetemcomitans LPS-stimulated IL-6. Our results indicate that induction of IL-6 by E. coli LPS, IL-1beta and A. actinomycetemcomitans LPS requires signaling through MKK-3-p38alpha ERK, JNK and p38 MAPK in mPDL cells.

Inhibitory effects of orally administrated liposomal bovine lactoferrin on the LPS-induced osteoclastogenesis

“…Although E. coli LPS used to provoke in vivo periodontal tissue damage is not periodontal pathogen, it has the same kind of responses as A.a. LPS. 32 In addition, periodontal tissue destruction caused by topical application of E. coli LPS into the rat gingival sulcus is well established. [7][8][9][10][11] In our animal model, typical B-cell lesion-like human chronic periodontitis did not develop.…”

Endogenous heat shock protein GroEL of A. actinomycetemcomitans preferentially targets primary human CD8+ T cells

“…A. actinomycetemcomitans is a gram-negative, facultative, anaerobic, nonspore-forming, and nonmotile coccobacillus. Previous investigations have suggested that A. actinomycetemcomitans produces a variety of virulence factors including the leukotoxin (Ltx) (Lally et al, 1997;Korostoff et al, 1998), cytolethal distending toxin (Cdt) (Shenker et al, 1999(Shenker et al, , 2001, and lipopolysaccharide (Lps) (Kiley and Holt, 1980;Patil et al, 2006). A. actinomycetemcomitans also expresses heat shock protein GroEL (Hsp64) (Koga et al, 1993).…”

Inhibitory Effect of Panduratin A on c-Jun N-Terminal Kinase and Activator Protein-1 Signaling Involved in Porphyromonas gingivalis Supernatant-Stimulated Matrix Metalloproteinase-9 Expression in Human Oral Epidermoid Cells

“…8) Together these transcription factors control the activation of gene promoters containing AP-1, nuclear factor-kappa B (NF-kB), and C/EBP (ccAAT enhances binding protein) elements to control the expression of target genes resulting inflammatory responses including MMPs. 9) A number of strategies have been designed to block the excess production of MMPs by periodontal host cells to treat periodontitis. Inhibition of MMPs via blocking enzymatic activity offers a straightforward therapeutic target in periodontal disease therapy.…”