[Effects of raf-1 gene overexpression on proliferative phenotype of rat mesangial cells].
OBJECTIVE
To investigate the regulating effects of raf-1 gene overexpression on phenotypic characteristics related proliferation in rat mesangial cells (MC).


METHODS
Raf-1 gene was stably transfected and overexpressed in rat MC. Growth rate, cell cycle and activity of mitogen-activated protein kinases including ERK and JNK were compared between transfected and normal cells by cell counting, FACS and specific substrate phosphorylation.


RESULTS
The growth rate of raf-1 transfected cells was normal from the 1st to 3rd days, but slower on the 5th day. At the 7th day, the cell number in raf-1-transfected clones was much less than in controls (22.0 +/- 2.5) x 10(5) vs (33.4 +/- 2.7) x 10(5), (P < 0.05). Most of raf-1 transfected cells stayed in the G0/G1 stage in cell cycle (81.3 +/- 3.7)% vs (62.3 +/- 2.4)%, (P < 0.01); the percentage of cells in S stage was decreased markedly (8.3 +/- 0.9)% vs (27.0 +/- 1.5)%, (P < 0.01). ERK activity was elevated (214 +/- 16) pmol.mg-1.min-1 vs (82 +/- 9) pmol.mg-1.min-1, (P < 0.05), but JNK activity was inhibited by about 50% in raf-1 overexpressed cells compared to controls.


CONCLUSIONS
Raf-1 is a critical intracellular signal for regulating proliferative phenotype in rat MC. Its constitutive overexpression leads these cells to be silent indicating arrest cell cycle, which may be mediated by regulating protein in cell cycle and intracellular JNK activation.
Li
B
B
Institute of Nephrology, Beijing Medical University, Beijing, 100034.
Li
X
X
D000818:Animals; D002453:Cell Cycle; D002455:Cell Division; D002478:Cells, Cultured; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000166:cytology / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D015398:Signal Transduction
D012333:RNA, Messenger; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases