Overexpression of protein kinase G using adenovirus inhibits cyclin E transcription and mesangial cell cycle.
The cGMP-cGMP-dependent protein kinase (protein kinase G) system plays an important role in the pathogenesis of mesangial proliferative glomerulonephritis. However, the molecular mechanisms of the inhibitory effects of the cGMP-protein kinase G system in the cell cycle progression of mesangial cells are not well known. To determine the inhibitory pathway of cGMP-protein kinase G in cultured mesangial cells, we investigated the effects of cGMP- and adenovirus-mediated overexpression of protein kinase G on the promoter activities of cyclin E, cyclin D1, and cyclin A. 8-Bromo-cGMP (8-BrcGMP) and overexpression of protein kinase G reduced [(3)H]thymidine uptake, reduced the numbers of cells in S and G(2)/M phases, and decreased the phosphorylation of retinoblastoma (Rb) protein. 8-BrcGMP (10(-3) M), protein kinase G adenovirus (Ad-cGKIbeta; 10(10) plaque-forming units/ml), atrial natriuretic peptide (ANP), and C-type natriuretic peptide (CNP) inhibited the promoter activity of cyclin E to 49, 57, 77, and 78%, respectively. On the other hand, the promoter activities of cyclin D1 and cyclin A were not changed significantly. In Western blot analysis, 8-BrcGMP, Ad-cGKIbeta, ANP, and CNP also inhibited cyclin E protein expression dose and time dependently. The p44/p42 mitogen-activated protein kinase (MAPK) kinase 1-p44/p42 MAPK had no effect on cyclin E promoter activities, and the cGMP-protein kinase G pathway did not change MAPK activity. In conclusion, our findings suggest that the reduction of the cyclin E promoter activity that downregulates G(1)/S transition plays a dominant role in the cGMP- and protein kinase G-induced inhibition of mesangial cell proliferation.
Hanada
S
S
Second Department of Internal Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-chome, Bunkyo-ku, Tokyo 113-8519, Japan.
Terada
Y
Y
Inoshita
S
S
Sasaki
S
S
Lohmann
S M
SM
Smolenski
A
A
Marumo
F
F
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002452:Cell Count; D002453:Cell Cycle / Q000502:physiology; D006152:Cyclic GMP / Q000096:biosynthesis; D017869:Cyclic GMP-Dependent Protein Kinases / Q000096:biosynthesis*; D019927:Cyclin E / Q000096:biosynthesis* / Q000235:genetics; D005434:Flow Cytometry; D017930:Genes, Reporter; D005822:Genetic Vectors; D005920:Glomerular Mesangium / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D008156:Luciferases; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013936:Thymidine / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D019927:Cyclin E; D012333:RNA, Messenger; D008156:Luciferases; D017869:Cyclic GMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D006152:Cyclic GMP; D013936:Thymidine