Defensins HNP1 and HBD2 stimulation of wound-associated responses in human conjunctival fibroblasts.
OBJECTIVE
To study the responses of human conjunctival fibroblasts (HCFs) to stimulation by human neutrophil defensin 1 (HNP1) and beta defensin2 (HBD2).


METHODS
Defensin-stimulated gene expression in primary cultures of HCFs was analyzed by real-time PCR after exposure to various concentrations of HNP1 or HBD2. Gene and protein expression for selected collagens, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were determined by real-time PCR and ELISA analysis. Activation of p42/44 mitogen-activated protein (MAP) and Akt was analyzed by Western blot.


RESULTS
HCFs did not express significant levels of the genes for HNP1 or HBD1-3. However, HNP1 and HBD2 stimulated HCF proliferation, the activation of p42/44 MAP kinase, and Akt kinase in a dose-dependent manner. HNP1 and HBD2 were not found to be chemotoxic for HCFs. It was demonstrated with the use of U0126 and wortmannin that the activation of p42/44 MAP kinase and Akt was responsible for the increased HCF proliferation observed under HNP1 and HBD2 stimulation. HNP1 stimulated the expression of the genes for collagen I, III, VI, and VIII. In addition, it reduced the secretion of collagen I protein but increased its intracellular retention. HNP1 and HBD2 upregulated the transcription and translation of MMP1. Small increases were observed in MMP14 gene expression after HNP1 stimulation and MMP2 gene expression after HBD2 stimulation.


CONCLUSIONS
The results of this study suggest that HNP1 and HBD2 have a potential role in the biosynthetic and tissue remodeling responses of conjunctival fibroblasts.
Li
Jing
J
Singapore Eye Research Institute, Singapore.
Raghunath
Michael
M
Tan
Donald
D
Lareu
Ricky R
RR
Chen
Zhencheng
Z
Beuerman
Roger W
RW
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D003094:Collagen / Q000378:metabolism*; D003228:Conjunctiva / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008875:Middle Aged; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism*; D015854:Up-Regulation; D014945:Wound Healing / Q000187:drug effects*; D023084:alpha-Defensins / Q000494:pharmacology*; D023083:beta-Defensins / Q000494:pharmacology*
C106641:DEFB4A protein, human; D004791:Enzyme Inhibitors; D019714:Tissue Inhibitor of Metalloproteinases; D023084:alpha-Defensins; D023083:beta-Defensins; C406413:human neutrophil peptide 1; D003094:Collagen; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020782:Matrix Metalloproteinases