[Redox factor 1 increases proliferation of neonatal rat cardiac fibroblasts].
OBJECTIVE
To investigate the function of REF1 in the proliferation and collagen synthesis of neonatal rat cardiac fibroblasts, and the underlying mechanisms.


METHODS
Neonatal rat cardiac fibroblasts were transfected with the adenoviral vector containing rat wild type Ref1 (Ad-Ref1) or mutated Ref1 (Ad-mutRef1). The mutations resulted in Cys to Ala at amino acids 65 and 93, which eliminated the redox function of the REF1 protein. MTT was used to check the cell viability and flow cytometry was used to analyze the cell proliferation with the count of cell numbers and the percentage of cells in S phase of the cell cycle. The expressions of Ref1, collagen I (Col I) and collagen III (Col III) were determined by RT-PCR and Western blot. The translocation of REF1 was examined by fluorescence staining and revealed under fluorescence microscope. Electrophoretic mobility shift assay (EMSA) was used to check the effect of REF1 on AP1 DNA binding ability. The high glucose medium (25 mmol/L) was applied to culture cardiac fibroblasts. The effect of high glucose on AP1 DNA binding activity, the expression and translocation of REF1 were examined.


RESULTS
MTT analysis showed that Ad-Ref1 promoted the relative viability of cardiac fibroblasts (0.671+/-0.044 vs control 0.364+/-0.007, n=6, P<0.01). The percentage of cells in S phase of the cell cycle was increased significantly in the Ad-Ref1 transfected cells (16.8%+/-0.62% vs control 9.04%+/-0.43%, n=3, P<0.05), as demonstrated by flow cytometry analysis. The expressions of Col I and Col III at mRNA level were increased when cells transfected with Ad-Ref1, while Ad-mutRef1 did not show such effects. Compared with the redox-deficient mutant Ad-mutRef1 (C65/93A), EMSA results demonstrated that Ad-Ref1 resulted in a marked increase in AP1 DNA binding. We also found that the cardiac fibroblasts cultured in high glucose (25 mmol/L) medium resulted in an increase in AP1 DNA binding activity, which was similar as seen in Ad-Ref1 transfected cells. There was also an increased accumulation of nuclear REF1 protein when cells were cultured in high glucose medium, although the expressions of REF1 at both mRNA and protein levels were not affected.


CONCLUSIONS
REF1 can increase proliferation and collagen synthesis of cardiac fibroblasts, which may be related to its ability to up-regulate AP1 DNA binding.
Gen
Yan
Y
Department of Biochemistry and Molecular Biology, Peking University School of Basic Medical Sciences, Beijing 100191, China.
Li
Tao
T
Hu
Xiao-qing
XQ
Zhang
Chen-guang
CG
Chen
Ping
P
Ma
Kang-tao
KT
Zhou
Chun-yan
CY
D000818:Animals; D000831:Animals, Newborn; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D003094:Collagen / Q000096:biosynthesis; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000494:pharmacology*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D009206:Myocardium / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
D003094:Collagen; C470319:Apex1 protein, rat; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase