Division of Molecular Cardiology , Faculty of Medicine , Kyushu University , Fukuoka , Japan .
Biochem Biophys Res Commun 188 : 1198-204 ( 1992)
Abstract
Platelet derived growth factor ( PDGF ) has been shown to induce c-fos and c-myc proto-oncogenes .
In the present study , we investigated the effects of genistein , a tyrosine kinase inhibitor , and NiCl2 , a Ca2+ influx blocker , on PDGF-induced Ca2+ transient and on expression of c-fos and c-myc mRNA .
In the presence of extracellular Ca2+ , PDGF induced elevation of intracellular Ca2+ concentration ( [ Ca2+]i ) and increases in c-fos and c-myc mRNA , as detected by reverse transcription polymerase chain reaction ( RT-PCR ) and Northern hybridization .
PDGF-induced [ Ca2+]i elevation was composed of an initial transient increase ( first component ) followed by steady state elevation ( second component ) .
Genistein ( 10 microM ) blocked the 1st , but not the 2nd , component of [ Ca2+]i elevation induced by PDGF .
NiCl2 ( 1 mM ) and removal of extracellular Ca2+ inhibited the 2nd , but not the 1st , component .
In the presence of 10 microM genistein and 1 mM NiCl2 , PDGF induced c-fos and c-myc mRNA , although the [ Ca2+]i elevation could be completely blocked by these two agents .
These results indicate that elevation of [ Ca2+]i is not a prerequisite condition for PDGF to induce c-fos and/or c-myc mRNA in rat aortic smooth muscle cells in primary culture .
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Department of Internal Medicine , Yale University School of Medicine , West Haven , Connecticut .
Am J Physiol 263 : F623-36 ( 1992)
Abstract
The early growth response gene 1 ( Egr-1 ) is a member of the family of immediate early response genes .
Egr-1 encodes a nuclear phosphoprotein that binds a specific nonameric DNA sequence through three zinc-finger domains and functions as a transcriptional activator .
We tested whether the vasoactive agents platelet-derived growth factor ( PDGF ) , arginine vasopressin ( AVP ) , serotonin ( 5-HT ) , and angiotensin II ( ANG II ) induced Egr-1 mRNA in cultured rat mesangial cells ( MCs ) and investigated the role of protein kinase C ( PKC ) in mediating the induction process .
PDGF , AVP , and 5-HT induced Egr-1 mRNA within 15 min , reaching peak levels at -60 min.
After PDGF and 5-HT stimulation , Egr-1 mRNA levels returned to baseline within 4 h , whereas AVP induced a sustained increase for up to 8 h . There was a very close correlation between doses required for Egr-1 induction and induction of MC proliferation .
ANG II was a very weak MC mitogen and induced only a small increase in Egr-1 mRNA .
Comparison of control cells with cells depleted of PKC by 48 h of PMA treatment revealed that induction of Egr-1 by PDGF and 5-HT is independent of PKC .
In contrast , however , the Egr-1 response to AVP was diminished in PKC-depleted cells .
AVP induced Egr-1 mRNA 10.9-fold in control cells , compared with 7.8-fold in PKC-depleted cells .
Egr-1 mRNA after AVP stimulation remained elevated in control cells for up to 8 h but returned to baseline after 120 min in PKC-depleted cells .
Similar results were obtained using the PKC-inhibitor H-7 .
Using immunocytochemistry , PDGF and AVP were found to induce Egr-1 protein within 30 min localized to the nucleus .
We conclude that there is a strong correlation between induction of Egr-1 after stimulation with PDGF , AVP , 5-HT , and ANG II and the proliferative response elicited by these agents in MCs .
AVP induces Egr-1 by both PKC-dependent and PKC-independent pathways , whereas the effects of PDGF and 5-HT are independent of PKC .