Serum transcriptionally regulates Na(+)-K(+)-ATPase gene expression in vascular smooth muscle cells.
The present study was designed to examine the effects of serum on Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit gene expression in cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas. Addition of 10% serum to VSMC for 24 h increased Na(+)-K(+)-ATPase activity 1.5-fold and alpha 1- and beta 1-subunit protein levels 1.9-fold. Serum (10%) caused a 3.5-fold increase in alpha 1-mRNA levels and a 6.7-fold increase in beta 1-mRNA levels, with peak elevations at 12 h. The protein synthesis inhibitor cycloheximide abolished serum-mediated beta 1-mRNA induction but did not affect serum-mediated alpha 1-mRNA induction. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) significantly reduced serum-mediated beta 1-mRNA induction but had no effect on serum-mediated alpha 1-mRNA induction. Transfection experiments with the 5'-flanking sequences of the alpha 1- or beta 1-subunit genes linked to the luciferase reporter gene revealed that 10% serum caused 2.8- and 6.5-fold increases in luciferase activity, respectively. Among growth factors, only basic fibroblast growth factor (FGF) enhanced luciferase activities for the alpha 1- and beta 1-subunit genes. We conclude that 1) serum stimulates alpha 1- and beta 1-mRNA expression, alpha 1- and beta 1-subunit protein accumulation, and Na(+)-K(+)-ATPase activity; 2) serum-mediated beta 1-mRNA induction partly requires de novo synthesis of intermediate regulatory proteins and activation of PKC and TK, whereas serum-mediated alpha 1-mRNA induction occurs through PKC- and TK-independent mechanisms; 3) the 5'-flanking regions of the alpha 1- and beta 1-subunit genes are serum responsive; and 4) FGF mimics stimulatory effects of serum on promoter activities for the alpha 1- and beta 1-subunit genes.
Nemoto
J
J
Department of Nephrology, Jichi Medical School, Tochigi, Japan.
Muto
S
S
Ohtaka
A
A
Kawakami
K
K
Asano
Y
Y
D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000201:enzymology*; D001769:Blood; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D003470:Culture Media; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004790:Enzyme Induction; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006133:Growth Substances / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000096:biosynthesis*; D013936:Thymidine / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects
D003470:Culture Media; D006133:Growth Substances; D007328:Insulin; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D005346:Fibroblast Growth Factors; D004247:DNA; D003513:Cycloheximide; D000254:Sodium-Potassium-Exchanging ATPase; D013936:Thymidine