INHIBITION OF VASCULAR SMOOTH-MUSCLE CELL-GROWTH THROUGH ANTISENSE TRANSCRIPTION OF A RAT INSULIN-LIKE GROWTH-FACTOR-I RECEPTOR CDNA

Insulin-like growth factor I (IGF I) is an autocrine/paracrine growth factor that is produced in multiple tissues and is essential for norma l developmental growth. Its effects are mediated by activation of a me mbrane-bound tyrosine kinase receptor, IGF IR. On the basis of the par tial rat IGF IR alpha-chain cDNA sequence previously reported, we clon ed cDNA encoding the full-length rat IGF IR. The deduced amino acid se quence predicts a 1370-amino acid receptor precursor, which includes s ignal sequence, a 707-amino acid alpha-chain, a 4-Arg cleavage site, a nd a 629-amino acid beta-chain. Overall, similarity to human IGF IR is 89% and 98% at the nucleotide and amino acid levels, respectively. An tisense IGF IR expression constructs in vectors incorporating Epstein- Barr virus replicative signals and the cytomegalovirus promoted-enhanc er or the inducible human metallothionein IIa promoterienhancer were a ssembled 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 transcr ipts inducibly), and clones ME8 and ME10 (expressing vector alone) wer e characterized. There was a 57% reduction in IGF IR mRNA levels in cl one 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 (K-d), and a 55% and 57% reduction in DNA synthesis r ates, basally and in response to 10 ng/mL IGF I, respectively. Clones MA5/MA7 similarly showed a 54% reduction in IGF IR number after conflu ence following exposure to 100 mu mol/L ZnSO4 and a 44% and 58% reduct ion in DNA synthesis, basally and in response to 10 ng/mL IGF I, respe ctively. Growth curves indicated that proliferation of clone CA9 in th e presence of 10% serum was reduced by 60% compared with clone ME10. T hus, cloning of cDNA encoding the full-length rat IGF IR indicates tha t this receptor is highly conserved. Antisense targeting of this recep tor in vascular smooth muscle cells (VSMCs) demonstrates that a decrea se in IGF IR density results in marked inhibition of VSMC proliferatio n. These findings indicate an important role for this ligand-receptor system in regulating VSMC growth. Specifically, they suggest that modu lation of VSMC IGF IR density may be an important mechanism whereby gr owth of these cells is controlled.