Inhibitory effect of MAP kinase antisense oligonucleotide on angiotensin II-induced c-myc gene expression and proliferation of rat cardiac fibroblast.
OBJECTIVE
To investigate the inhibitory effect of down-regulating mitogen activated protein kinase (MAPK) on c-myc gene expression and further on cardiac fibroblast proliferation.


METHODS
Cultured neonatal rat cardiac fibroblasts was pretreated with a phosphorothioate-protected 17-mer antisense MAPK oligodeoxynucleotide (ODN) directed against the initiation of translation sites of the p42 and p44 MAPK isoforms by liposomal transfection. A 17-mer sense and mismatch sequence MAPK ODN were used as controls. After liposomal transfecting, cells were exposed to angiotensin II (Ang II) 10 nmol.L-1 for 5 min and then harvested in lysis buffer. MAPK activity was measured by Western blot and P-81 phosphocellulose filter paper method by using [gamma-32P]ATP and myelin basic protein as substrates. c-myc mRNA expression stimulated by Ang II for 30 min was measured by Northern blot. DNA synthesis and collagen protein synthesis induced by Ang II for 24 h were measured by [3H]thymidine incorporation and [3H]Proline incorporation, respectively.


RESULTS
Antisense ODN 0.2 mumol.L-1 reduced Ang II-induced MAPK activities by 72%, MAPK protein expression by 80%, and suppressed c-myc mRNA expression by 97%, respectively. [3H]thymidine incorporation and [3H]proline incorporation in Ang II-induced cardiac fibroblast were inhibited by 59% and 58%, respectively.


CONCLUSIONS
A 17-mer MAPK antisense oligonucleotide directed againsts the initiation of translation sites of MAPK could specifically inhibit Ang II-stimulated cultured neonatal rat cardiac fibroblast proliferation through down-regulating MAPK activity and further depleting c-myc mRNA expression.
Laboratory of Cardiovascular Physiology, Hunan Medical University, Changsha 410078, China. dingboxj@public.cs.hn.cn
B
B
Ding
S L
SL
Huang
S Q
SQ
Zhang
Y X
YX
Li
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology; D015870:Gene Expression; D016259:Genes, myc; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats
D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D000804:Angiotensin II; D020928:Mitogen-Activated Protein Kinases