Yasuo Fukumoto

Angiotensin II induces expression of the c-fos gene through protein kinase C activation and calcium ion mobilization in cultured vascular smooth muscle cells☆

Incubation of the serum-deprived cultures of rat vascular smooth muscle cells with angiotensin II, a potent vasoconstrictor, caused a rapid and transient increase in the c-fos mRNA level. The doses of this agonist necessary for the increase in the c-fos mRNA level coincided with those for the phospholipase C-mediated hydrolysis of phosphoinositides. Moreover, protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate and Ca2+-ionophore A23187 increased the c-fos mRNA level in an additive manner. These results suggest that angiotensin II induces expression of the c-fos gene through the activation of protein kinase C and Ca2+ mobilization in cultured vascular smooth muscle cells.

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet‐derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c‐fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane‐permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c‐fos mRNA, and this action was mimicked by 8‐bromo‐cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c‐fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.

Antiproliferative action of protein kinase C in cultured rabbit aortic smooth muscle cells.

In rabbit aortic smooth muscle cells (SMC), protein kinase C-activating 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the whole blood serum (WBS)-induced DNA synthesis. The inhibitory action of TPA was mimicked by another protein kinase C-activating phorbol ester, phorbol-12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol-12,13- didecanoate known to be inactive for this enzyme. Prolonged treatment of the cells with PDBu caused the down-regulation of protein kinase C. In these cells, WBS still induced DNA synthesis but the inhibitory action of TPA was abolished. DNA synthesis started at 18 h and reached a maximal level 24 h after the addition of WBS. TPA inhibited the WBS-induced DNA synthesis even when added 12 h after the addition of WBS. These results suggest that protein kinase C has an antiproliferative action in rabbit aortic SMC and that this action is attributed to the inhibition of the progression from the late G1 into S phase of the cell cycle. TPA also inhibited the phospholipase C-mediated hydrolysis of phosphoinositides which was induced by WBS within several minutes, but the relevance of this effect on the antiproliferative action of TPA is uncertain.

Two types of protein kinase C with different functions in cultured rabbit aortic smooth muscle cells

Incubation of cultured rabbit aortic smooth muscle cells (SMC) with phorbol-12, 13-dibutyrate (PDBu) for 48 h caused the down-regulation of protein kinase C (PKC) to the level of 30-40% of that in the control cells. The proliferative and antiproliferative actions of PKC were abolished in parallel with the loss of the down-regulation-sensitive component of PKC, but the inhibitory actions in the whole blood serum (WBS)-induced phospholipase C (PLC) reactions and intracellular Ca2+ mobilization were not affected. Immunoblot analysis with specific monoclonal antibodies against three PKC isozymes (type I, II and III) revealed that only the type III isozyme was detected in rabbit aortic SMC and that this isozyme completely disappeared after the incubation with PDBu. These results indicate that the type III isozyme is responsible for the proliferative and antiproliferative actions and suggest that the unidentified isozyme(s) is involved in the inhibitory actions in the WBS-induced PLC reactions and intracellular Ca2+ mobilization in rabbit aortic SMC.

Inhibition of DNA synthesis by phorbol esters through protein kinase C in cultured rabbit aortic smooth muscle cells

In cultured rabbit aortic smooth muscle cells (SMC), 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) induced DNA synthesis in the presence of plasma‐derived serum to a small extent, but inhibited markedly the rabbit whole blood serum (WBS)‐, platelet‐derived growth factor (PDGF)‐ and epidermal growth factor‐induced DNA synthesis. Phorbol‐12,13‐dibutyrate (PDBu) mimicked this antiproliferative action of TPA, but 4α‐phorbol‐12, 13‐didecanoate was inactive in this capacity. Prolonged treatment of the cells with PDBu caused the partial down‐regulation of protein kinase C. In these protein kinase C‐reduced cells, WBS still induced DNA synthesis, but TPA did not inhibit the WBS‐induced DNA synthesis. We have previously shown that protein kinase C is involved at least partially in the PDGF‐induced DNA synthesis in rabbit aortic SMC. The present results together with this earlier observation suggest that protein kinase C has not only a proliferative but also an antiproliferative action in rabbit aortic SMC.

Independent inhibition of DNA synthesis by protein kinase C, cyclic AMP and interferon α/β in rabbit aortic smooth muscle cells

Summary In quiescent cultures of rabbit aortic smooth muscle cells, whole blood serum-induced DNA synthesis was inhibited markedly by protein kinase C-activating 12- O -tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13-dibutyrate (PDBu), cyclic AMP-derivatives, such as dibutyryl cyclic AMP (Bt 2 cAMP) and 8-bromo-cyclic AMP, and interferon α/β. Neither TPA nor interferon α/β elevated the cellular cyclic AMP level. Neither Bt 2 cAMP nor interferon α/β induced the phospholipase C-mediated hydrolysis of phosphoinositides. The down-regulation of protein kinase C by prolonged treatment with PDBu abolished the antiproliferative action of TPA but did not affect that of Bt 2 cAMP or interferon α/β. TPA and Bt 2 cAMP inhibited the serum-induced DNA synthesis when added within 12 h after the addition of the serum, while interferon α/β was active only when added within 6 h. These results suggest that there are at least three independent signaling systems, protein kinase C- and cyclic AMP-mediated systems and an unidentified system for interferon α/β, which are involved in the antiproliferative mechanisms in rabbit aortic smooth muscle cells.

Comparison of the mode of action of ras p21 with those of protein kinases A and C in the stimulation of gene expression in NIH/3T3 cells

To compare the mode of action of ras p21 with those of protein kinases A and C in the regulation of gene expression in NIH/3T3 cells, we investigated the transcriptional activity of various enhancer/promoters and enhancer motifs in the cells transfected with the c‐Ha‐ras val12 complementary DNA (cDNA). The results indicate that the c‐Ha‐ras val12 protein stimulates the enhancer/promoters of the c‐fos gene, the metallothionein IIA gene, the simian virus 40 (SV40) virus genome and the Rous sarcoma (RS) virus genome, and the serum‐response element and the 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐response element in a manner independent of protein kinases A and C in NIH/3T3 cells.