OBJECTIVELipoxin A(4) is formed by the metabolism of arachidonic acid. Anti-inflammatory and anti-proliferative effect of lipoxin A(4) has been shown in many human diseases. Recently, as a novel high affinity receptor for ligand lipoxin A(4), Lipoxin A(4) receptor-like protein (LRLP) has been identified. Currently close attention is paid to the important contribution of connective tissue growth factor (CTGF) in lung fibrosis. The purpose of the study was to transfect LRLP gene into human lung fibroblasts and investigate the mechanism of its enhancing antagonistic effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by connective tissue growth factor.

METHODSEukaryocytic expression vector pEGFP/LRLP which contained LRLP and green fluorescence protein fusion gene (GFP) was constructed and transfected into human lung fibroblasts (HLF). After selecting with G418, HLF/LRLP cell clone which stably expressed LRLP/GFP fusion protein was isolated and characterized by the laser scanning confocal microscope. Cultured HLF and HLF/LRLP were stimulated for 24 h with CTGF (1 microg/ml) in the presence and absence of pretreatment of Lipoxin A(4) (10.0 nmol/L) for 30 min. Inhibition of cell proliferation was determined by MTT assay. Cell cycle analysis was performed by flow cytometry. Western blot was used to detect the expression of cyclin D(1) protein. Electrophoretic mobility shift assay (EMSA) was employed to detect the DNA binding activity of STAT(3).

RESULTS(1) HLF/LRLP cell clone which stably expressed LRLP and GFP fusion protein was successfully obtained. (2) Proliferation of HLF and HLF/LRLP was induced by 1 microg/ml CTGF. Pretreatment with 10 nm Lipoxin A(4) inhibited the proliferation of HLF and HLF/LRLP. And the inhibitory rate of HLF/LRLP was significantly higher than that of HLF [(54.1 +/- 4.2)%, (21.2 +/- 3.7)%, P < 0.05]. (3) The flow cytometry analysis showed that compared with HLF, more HLF/LRLP were arrested at G(0)/G(1) phase in the presence of pretreatment of Lipoxin A(4). [(76.3 +/- 3.5)%, (60.8 +/- 2.0)%, P < 0.05]. (4) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of cyclin D(1) protein expression in HLF and HLF/LRLP. And its antagonistic effect on HLR/LRLP was stronger than that on HLF (P < 0.05). (5) Ten nmol/L Lipoxin A(4) antagonized CTGF induced increase of STAT(3) DNA binding activity, and its antagonistic effect on HLF/LRLP was more powerful than that on HLF (P < 0.05).

CONCLUSIONSTransfection of Lipoxin A(4) receptor-like protein gene enhanced the inhibitory effect of Lipoxin A(4) on human lung fibroblasts proliferation induced by CTGF. Its mechanism might be related to regulation of cyclin D(1) protein expression and STAT(3) DNA binding activity.

Connective Tissue Growth Factor ; antagonists & inhibitors ; Cyclin D1 ; analysis ; DNA ; metabolism ; Fibroblasts ; cytology ; drug effects ; Humans ; Lipoxins ; pharmacology ; Lung ; cytology ; drug effects ; Receptors, Formyl Peptide ; genetics ; physiology ; Receptors, Lipoxin ; genetics ; physiology ; STAT3 Transcription Factor ; metabolism ; Transfection

OBJECTIVETo clone and identify novel proteins binding to the death domain of the death receptor 4 (DR4).

METHODSThe yeast two-hybrid system was used for this study. Automatic sequencing was carried out for DNA sequencing. The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares, respectively. Co-immunoprecipitate method was used to confirm human formyl peptide receptor-like 1 (FPRL1) binding specifically with DR4CD (the cytoplasmic domain of DR4) in HEK293T cells.

RESULTSTwo positive clones, named as pADB1 and pADB2, were obtained. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of FPRL1 was 97%. The insert of pADB2 shared no homology with any known peptides in GeneBank. Co-immunoprecipitate analysis further confirmed that FPRL1 could bind to DR4CD in vivo specifically.

CONCLUSIONSFPRL1 may associate with DR4CD in vivo specifically. The functional studies of FPRL1 in signaling pathway mediated by TNF-related apoptosis inducing ligand (TRAIL) are in active progress in our laboratory.

Amino Acid Sequence ; Apoptosis ; Apoptosis Regulatory Proteins ; Base Sequence ; Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Humans ; Membrane Glycoproteins ; metabolism ; Molecular Sequence Data ; Protein Structure, Tertiary ; Receptors, Formyl Peptide ; metabolism ; Receptors, Lipoxin ; metabolism ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor ; genetics ; metabolism ; Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; metabolism

In this study, we observed that lysophosphatidylglycerol (LPG) completely inhibited a formyl peptide receptor like-1 (FPRL1) agonist (MMK-1)-stimulated chemotactic migration in human phagocytes, such as neutrophils and monocytes. LPG also dramatically inhibited IL-1beta production by another FPRL1 agonist serum amyloid A (SAA) in human phagocytes. However, LPG itself induced intracellular calcium increase and superoxide anion production in human phagocytes. Keeping in mind that phagocytes migration and IL-1beta production by FPRL1 are important for the induction of inflammatory response, our data suggest that LPG can be regarded as a useful material for the modulation of inflammatory response induced by FPRL1 activation.
Chemotaxis, Leukocyte/*drug effects ; Humans ; Interleukin-1beta/*biosynthesis ; Lysophospholipids/*pharmacology ; Monocytes/drug effects/immunology/metabolism/physiology ; Neutrophils/drug effects/immunology/metabolism/physiology ; Peptides/metabolism/pharmacology ; *Phagocytes/drug effects/immunology/metabolism/physiology ; Receptors, Formyl Peptide/*metabolism ; Receptors, Lipoxin/*metabolism ; Serum Amyloid A Protein/metabolism/pharmacology

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.
Animals ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Chemotaxis, Leukocyte ; Humans ; Interleukin-8/*biosynthesis ; MAP Kinase Kinase Kinases/metabolism ; Neutrophils/drug effects/*immunology ; Proto-Oncogene Proteins/metabolism ; Rats ; Receptors, Formyl Peptide/metabolism ; Receptors, Lipoxin/metabolism ; Serum Amyloid A Protein/*antagonists & inhibitors ; Signal Transduction ; Transcription, Genetic

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.
Animals ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Chemotaxis, Leukocyte ; Humans ; Interleukin-8/*biosynthesis ; MAP Kinase Kinase Kinases/metabolism ; Neutrophils/drug effects/*immunology ; Proto-Oncogene Proteins/metabolism ; Rats ; Receptors, Formyl Peptide/metabolism ; Receptors, Lipoxin/metabolism ; Serum Amyloid A Protein/*antagonists & inhibitors ; Signal Transduction ; Transcription, Genetic