PROTEASE-RESISTANT FORM OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-5 IS AN INHIBITOR OF INSULIN-LIKE GROWTH-FACTOR-I ACTIONS ON PORCINE SMOOTH-MUSCLE CELLS IN CULTURE

IGFs are pleiotrophic mitogens for porcine smooth muscle cells (pSMC) in culture, The effects of IGFs on cells are modulated by various insu lin-like growth factor-binding proteins (IGFBP), IGFBP-5 is synthesize d by pSMC and binds to the extracellular matrix, However, IGFBP-5 is a lso secreted into conditioned medium of cultured cells and is cleaved into fragments by a concomitantly produced protease. These fragments h ave reduced affinity for the IGFs and cleavage makes it difficult to a ssess the role of intact IGFBP-5. To study the consequence of accumula tion of intact IGFBP-5 in medium, we determined the cleavage site in I GFBP-5 and prepared a protease resistant mutant, Amino acid sequencing of purified IGFBP-5 fragments suggested Arg(138)-Arg(139) as the prim ary cleavage site, Arg(138)- Arg(139)-->Asn(138)-Asn(139) mutations we re introduced to create protease-resistant IGFBP-5, which has the same affinity for IGF-I as the native protein, This mutant IGFBP-5 remaine d intact even after 24 h of incubation and it inhibited several IGF-I actions when added to pSMC culture medium, The mutant IGFBP-5 (500 ng/ ml) decreased IGF-I stimulated cellular DNA synthesis by 84%, protein synthesis by 77%, and it inhibited IGF-I stimulated migration of pSMC by 77%, It also inhibited IGF-I stimulation of IRS-1 phosphorylation, In contrast, the same amount of native IGFBP-5 did not inhibit IGF-I a ctions, The significance of inhibitory effects of the protease resista nt IGFBP-5 was further demonstrated in pSMC transfected with mutant or native IGFBP-5 cDNAs, The mutant IGFBP-5 accumulated in culture mediu m of transfected cells, while native IGFBP-5 was degraded into fragmen ts, PSMC overexpressing the mutant IGFBP-5 also responded poorly to IG F-I compared with mock transfected cells, IGF-I (5 ng/ml) increased [S -35]methionine incorporation into control cells by 36% above the basal level, but it did not significantly change (4%) in pSMC cultures that were producing the mutant IGFBP-5, In conclusion, the accumulation of protease-resistant IGFBP-5 in the medium was inhibitory to IGF-I acti ons on pSMC, This suggests that proteolysis can prevent IGFBP-5 from a cting as an inhibitor of IGF-I-stimulated effects and that it serves a s an important mechanism for regulating cellular responsiveness to IGF -I.