© 1995 American Heart Association, Inc.
Articles |
Inhibition of Vascular Smooth Muscle Cell Growth Through Antisense Transcription of a Rat Insulin-Like Growth Factor I Receptor cDNA
From the Division of Cardiology, Department of Medicine, Emory University, Atlanta, Ga.
Abstract Insulin-like growth factor I (IGF I) is an
autocrine/paracrine growth factor that is produced in multiple tissues
and is essential for normal developmental growth. Its effects are
mediated by activation of a membrane-bound tyrosine kinase receptor,
IGF IR. On the basis of the partial rat IGF IR 
-chain cDNA sequence
previously reported, we cloned cDNA encoding the full-length rat IGF
IR. The deduced amino acid sequence predicts a 1370–amino acid
receptor precursor, which includes signal sequence, a 707–amino acid
-chain, a 4-Arg cleavage site, and a 629–amino acid ß-chain.
Overall, similarity to human IGF IR is 89% and 98% at the nucleotide
and amino acid levels, respectively. Antisense IGF IR expression
constructs in vectors incorporating Epstein-Barr virus replicative
signals and the cytomegalovirus promoter/enhancer or the inducible
human metallothionein IIa promoter/enhancer were assembled and stably
transfected into cultured rat aortic smooth muscle cells. Clone CA9
(constitutively expressing abundant antisense IGF IR transcripts),
clones MA5 and MA7 (expressing antisense IGF IR transcripts inducibly),
and clones ME8 and ME10 (expressing vector alone) were characterized.
There was a 57% reduction in IGF IR mRNA levels in clone CA9 after
confluence compared with clone ME10. This resulted in a 51% decrease
in IGF I binding sites in clone CA9, without a change in binding
affinity (Kd), and a 55% and 57% reduction in
DNA synthesis rates, basally and in response to 10 ng/mL IGF I,
respectively. Clones MA5/MA7 similarly showed a 54% reduction in IGF
IR number after confluence following exposure to 100 µmol/L
ZnSO4 and a 44% and 58% reduction in DNA synthesis,
basally and in response to 10 ng/mL IGF I, respectively. Growth curves
indicated that proliferation of clone CA9 in the presence of 10% serum
was reduced by 60% compared with clone ME10. Thus, cloning of cDNA
encoding the full-length rat IGF IR indicates that this receptor is
highly conserved. Antisense targeting of this receptor in vascular
smooth muscle cells (VSMCs) demonstrates that a decrease in IGF IR
density results in marked inhibition of VSMC proliferation. These
findings indicate an important role for this ligand-receptor system in
regulating VSMC growth. Specifically, they suggest that modulation of
VSMC IGF IR density may be an important mechanism whereby growth of
these cells is controlled.
Key Words: insulin-like growth factor I • molecular cloning • antisense RNA • transfection • cellular proliferation
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