Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts.
Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.
Takayama
Yoshiharu
Y
Functional Biomolecules Laboratory, National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901 Japan. takay@affrc.go.jp
Takahashi
Hitomi
H
Mizumachi
Koko
K
Takezawa
Toshiaki
T
D019921:Biotinylation; D015153:Blotting, Western; D019070:Cell Lineage; D002462:Cell Membrane / Q000378:metabolism; D003094:Collagen / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D016376:Oligonucleotides, Antisense; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003094:Collagen; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D007781:Lactoferrin