Effects of PI3-k/Akt short hairpin RNA on proliferation, fibronectin production and synthesis of thrombospondin-1 and transforming growth factor-beta1 in glomerular mesangial cells induced by sublytic C5b-9 complexes.
OBJECTIVE
To explore proliferation of glomerular mesangial cells (GMC) and secretion of extracellular matrix (fibronectin induced by sublytic C5b-9 complexes), and then ascertain the role of phosphatidylinositol 3-kinase (PI3-k)/Akt signal pathway in these processes, by using small hairpin RNAs.


METHODS
The expression of cyclin D(2), (3)H-thymidine into DNA and production of fibronectin including thrombospondin-1 and transforming growth factor-beta(1) in the GMCs stimulated by sublytic C5b-9 or transfected with expression vectors of PI3-k and Akt short hairpin RNA or LY294002 (PI3-k inhibitor) were measured by Real-time quantitative polymerase chain reaction (PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and (3)H-thymidine incorporation ((3)H-TdR), respectively.


RESULTS
The expression of cyclin D(2), (3)H-thymidine into DNA and fibronectin in the GMCs stimulated by sublytic C5b-9 could all be increased, and the elevations of these parameters mentioned above were also markedly reduced in the GMCs transfected with vectors of PI3-k and Akt short hairpin RNA or LY294002, respectively.


CONCLUSIONS
These data indicate that sublytic C5b-9 can promote proliferation of GMCs and secretion of fibronectin as well as synthesis of thrombospondin-1 and transforming growth factor-beta(1). The PI3-k/Akt signal pathway in these reactions, mediated by sublytic C5b-9 complexes, may play at least a partial role.
Gao
L
L
Department of Microbiology and Immunology, Nanjing Medical University, Nanjing, China.
Zhang
Y
Y
Qiu
W
W
Xu
W
W
Feng
X
X
Ren
J
J
Jiang
X
X
Wang
H
H
Zhao
D
D
Wang
Y
Y
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D050776:Complement C5b / Q000378:metabolism*; D003186:Complement C9 / Q000378:metabolism*; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000096:biosynthesis*; D005920:Glomerular Mesangium / Q000201:enzymology / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D051381:Rats; D019700:Thrombospondin 1 / Q000096:biosynthesis*; D053773:Transforming Growth Factor beta1 / Q000096:biosynthesis*
D003186:Complement C9; D017931:DNA Primers; D005353:Fibronectins; D019700:Thrombospondin 1; D053773:Transforming Growth Factor beta1; D012313:RNA; D050776:Complement C5b; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt