[Transfection of Lipoxin A4 receptor-like protein gene enhanced the inhibitory effect of Lipoxin A4 on human lung fibroblasts proliferation induced by connective tissue growth factor].
OBJECTIVE
Lipoxin 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.


METHODS
Eukaryocytic 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).


CONCLUSIONS
Transfection 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.
Lu
Chao
C
Department of Pediatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Chen
Ji-qing
JQ
Wu
Sheng-hua
SH
Wu
Yuan-jun
YJ
Zhao
Fei
F
Pan
Xiao-qin
XQ
Fei
Li
L
Guo
Mei
M
Huang
Song-ming
SM
Guo
Xi-rong
XR
Chen
Rong-hua
RH
D055513:Connective Tissue Growth Factor / Q000037:antagonists & inhibitors*; D019938:Cyclin D1 / Q000032:analysis; D004247:DNA / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D044045:Lipoxins / Q000494:pharmacology*; D008168:Lung / Q000166:cytology / Q000187:drug effects; D044042:Receptors, Formyl Peptide / Q000235:genetics / Q000502:physiology*; D044067:Receptors, Lipoxin / Q000235:genetics / Q000502:physiology*; D050796:STAT3 Transcription Factor / Q000378:metabolism; D014162:Transfection
C105101:FPR2 protein, human; D044045:Lipoxins; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C040527:lipoxin A4; D019938:Cyclin D1; D055513:Connective Tissue Growth Factor; D004247:DNA