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CD14 and Toll-like Receptors Regulate Lipopolysaccharide Induction of KGF-1 Protein Expression

CD14 and Toll-like Receptors Regulate Lipopolysaccharide Induction of KGF-1 Protein Expression. EE Putnins*, Sanaie AR, Firth JD, Wu Q. Faculty of Dentistry The University of British Columbia Vancouver, B.C., CANADA. Introduction.

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CD14 and Toll-like Receptors Regulate Lipopolysaccharide Induction of KGF-1 Protein Expression

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  1. CD14 and Toll-like Receptors Regulate Lipopolysaccharide Induction of KGF-1 Protein Expression EE Putnins*, Sanaie AR, Firth JD, Wu Q. Faculty of Dentistry The University of British Columbia Vancouver, B.C., CANADA

  2. Introduction • Abnormal epithelial cell proliferation is one aspect of PDD onset and progression. • Understanding the regulators of early disease onset may explain why some individuals are resistant/susceptible to disease. • If the epithelial barrier is perturbed then LPS from Gram negative microorganisms is likely to stimulate gingival fibroblasts.

  3. Fibroblast Growth Factors Interested in paracrine mediated epithelial specific growth factors. Growth factors that are secreted by fibroblasts and specifically stimulate epithelial cell proliferation and migration. Keratinocyte Growth Factors-1 (FGF-7) and -2 (FGF-10).

  4. Keratinocyte Growth Factors • Mitogenic for a wide variety of epithelial cells. • Epithelial cells express the appropriate receptor (FGFR2-iiib) • Increased expression of KGF-1 in chronic inflammatory conditions (e.g. Ulcerative Colitis/Crohn’s disease, Psoriasis). What about Periodontal disease?

  5. Gingival Fibroblast Expression of KGF-1 • Gingival fibroblasts express KGF-1 and 2. • KGF-1 expression was induced by: • Serum • IL-1, IL-6 and TNF-a • KGF-1 expression induced by LPS purified from: • P. gingivalis (ATCC 33211) • E. coli (O55:B5) 2.4 kb 1.7 1.3 kb Sanaie AR, Firth JD, Uitto V-J, and Putnins EE (2002) J Periodont Res., 37(1):66-74

  6. Purpose Examine the signaling pathway that regulates LPS induction of KGF-1 expression. Hypothesis LPS induction of KGF-1 protein expression is mediated through a CD14 and Toll-like receptor signaling pathway.

  7. Materials and Methods • Gingival fibroblasts were isolated from tissue discarded during crown lengthening procedures. • Commercially prepared E.coli LPS (O55:B5-Sigma) • KGF-1 protein expression into conditioned media analyzed with sandwich ELISA. • CD14 expression was analyzed with: • Western blotting • Immunohistochemistry • FACS • Toll-like receptors 2 and 4 expression was analyzed using FACS analysis. • Functional analysis was done using specific blocking antibodies.

  8. LPS Induction of KGF-1 Protein Expression KGF-1 (pg/ml) mean ±sd; n=4

  9. LPS Induction of mCD14 on Gingival Fibroblasts (18hr) Control Western Blotting LPS stimulation of gingival fibroblasts induced a 67% induction of expression of mCD14 in total cell extracts. LPS-Txd

  10. LPS Induction of CD14 Membrane Expression A B • Induction of CD14 Expression (M1+M2). • C) control • D) 1hr (39% increase) • E) 3hr (121% increase) • F) 24hr (18% increase) • Significant increase in the number of gingival fibroblasts expressing high CD14 levels (M2 peak). C D E F

  11. Signaling Mechanism by Which LPS Regulates KGF-1 Expression • Preincubation of fibroblasts with CD14 blocking antibody inhibits LPS induction of KGF-1 protein expression. • CD14 is involved in LPS signaling but it lacks a intracellular signaling tail. KGF-1 (pg/ml) mean ±sd; n=4

  12. FACS analysis Significant decrease in cell surface expression of Toll-like receptors 2 and 4 in LPS-treated samples. Gingival Fibroblast Expression of Toll-like Receptors 2 and 4 Relative Change in Receptor Exp.

  13. Toll-like Receptor Regulates the LPS Induction KGF-1 • Preincubation of cells with Toll-like receptor-2 and -4 blocking antibodies negated LPS induction of KGF-1 protein expression. KGF-1 (pg/ml) mean ±sd; n=4

  14. Conclusions • LPS, in a concentration dependant manner, significantly induced KGF-1 protein expression. • CD14. • Increase in total and cell membrane expression of CD14. • CD14 is involved in the regulation of KGF-1 expression. • Toll-like receptors 2 and 4 • Both are expressed on gingival fibroblasts • Rapid decrease in cell membrane expression of both receptors is consistent with a receptor mediated internalization pathway. • Functioning of both receptors are required for LPS induction of KGF-1 expression.

  15. Regulation of KGF-1 Expression in Oral Chronic Inflammatory Conditions Host Induced Expression Micro Induced Expression

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