EXPRESSION AND INSULIN-LIKE GROWTH-FACTOR DEPENDENT PROTEOLYSIS OF INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-4 ARE REGULATED BY CELL CONFLUENCE IN VASCULAR SMOOTH-MUSCLE CELLS

Insulin-like growth factor (IGF)-I is markedly induced after balloon injury in the rat aorta, where it may serve to mediate vascular repair. Because the bioavailability of IGF-I is modulated by IGF-binding proteins (IGFBPs), we examined the regulation of IGFBPs by IGFs in primary cultures of rat aortic smooth muscle cells (SMCs). Serum-deprived SMC-conditioned medium contains IGFBPs of 38 to 45 kD (only in confluent cultures), 30 kD (possibly IGFBP-2), 28 kD, and 24 kD (IGFBP-4), the latter being the most abundant. IGF-I and IGF-II but not insulin evoked a marked decrease of IGFBP-4 as early as 4 hours after treatment. IGFBP-4 mRNA abundance, however, was entirely unaffected by IGF-I for up to 48 hours. IGF-I analogues with high affinity for the IGF-I receptor and weak affinity for IGFBP paradoxically evoked a small increase in IGFBP-4, probably through a general increase in protein synthesis. IGF-I only minimally decreased IGFBP-4 content in medium of sparse cultures, whereas it completely abolished IGFBP-4 content in conditioned medium of superconfluent SMCs. IGF-I also evoked a concentration-dependent increase in the abundance of IGFBP-3 in confluent, but not sparse, SMCs without affecting IGFBP-3 mRNA. Addition of IGF-I to cell-free medium conditioned by confluent, but not by sparsely cultured, SMCs led to rapid degradation of IGFBP-4. Interestingly, IGFBP-4 mRNA was markedly induced in confluent relative to sparsely grown SMCs,in an IGF-I independent fashion. Thus, both biosynthesis and IGF-dependent proteolysis of IGFBP-4 are increased in confluent SMCs' Proteolysis was maximal at 37-degrees-C and was abrogated by EDTA and by benzamidine. Phenylmethylsulfonyl fluoride and the plasmin inhibitor bdellin had minor inhibitory activity, whereas aprotinin, angiotensin-converting enzyme inhibitors, and N-ethylmaleimide were without effect. The protease does not affect the structure of IGF-I as determined by reverse-phase high-performance liquid chromatography and size-exclusion chromatography of I-125-IGF-I incubated for up to 24 hours with SMC-conditioned medium containing IGFBP-4. In summary, SMCs elaborate a cation-dependent protease in a confluence-dependent fashion, which degrades bound IGFBP-4 and likely releases free structurally intact IGF-I, presumably to interact with the cell surface receptor and/or other IGFBPs.