Cd14 and toll like receptors regulate lipopolysaccharide induction of kgf 1 protein expression
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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

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
Introduction Induction of KGF-1 Protein Expression

  • 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.


Fibroblast growth factors

Fibroblast Growth Factors Induction of KGF-1 Protein Expression

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).


Keratinocyte growth factors
Keratinocyte Growth Factors Induction of KGF-1 Protein Expression

  • 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?


Gingival fibroblast expression of kgf 1
Gingival Fibroblast Expression of KGF-1 Induction of KGF-1 Protein Expression

  • 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


Purpose Induction of KGF-1 Protein Expression

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.


Materials and methods
Materials and Methods Induction of KGF-1 Protein Expression

  • 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.


Lps induction of kgf 1 protein expression
LPS Induction of KGF-1 Protein Expression Induction of KGF-1 Protein Expression

KGF-1 (pg/ml)

mean ±sd; n=4


Lps induction of mcd14 on gingival fibroblasts 18hr
LPS Induction of mCD14 on Gingival Fibroblasts (18hr) Induction of KGF-1 Protein Expression

Control

Western Blotting

LPS stimulation of gingival fibroblasts induced a 67% induction of expression of mCD14 in total cell extracts.

LPS-Txd


Lps induction of cd14 membrane expression
LPS Induction of CD14 Membrane Expression Induction of KGF-1 Protein 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


Signaling mechanism by which lps regulates kgf 1 expression
Signaling Mechanism by Which LPS Regulates KGF-1 Expression Induction of KGF-1 Protein 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


Gingival fibroblast expression of toll like receptors 2 and 4

FACS analysis Induction of KGF-1 Protein Expression

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.


Toll like receptor regulates the lps induction kgf 1
Toll-like Receptor Regulates the LPS Induction KGF-1 Induction of KGF-1 Protein Expression

  • 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


Conclusions
Conclusions Induction of KGF-1 Protein Expression

  • 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.


Regulation of kgf 1 expression in oral chronic inflammatory conditions
Regulation of KGF-1 Expression in Oral Chronic Inflammatory Conditions

Host Induced Expression

Micro Induced Expression


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