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. Y Imai, … , C Rees, D R Clemmons J Clin Invest. 1997;100(10):2596-2605. https://doi.org/10.1172/JCI119803. Research Article IGFs are pleiotrophic mitogens for porcine smooth muscle cells (pSMC) in culture. The effects of IGFs on cells are modulated by various insulin-like growth factor-binding proteins (IGFBP). IGFBP-5 is synthesized by pSMC and binds to the extracellular matrix. However, IGFBP-5 is also secreted into conditioned medium of cultured cells and is cleaved into fragments by a concomitantly produced protease. These fragments have reduced affinity for the IGFs and cleavage makes it difficult to assess the role of intact IGFBP-5. To study the consequence of accumulation of intact IGFBP-5 in medium, we determined the cleavage site in IGFBP-5 and prepared a protease resistant mutant. Amino acid sequencing of purified IGFBP-5 fragments suggested Arg138-Arg139 as the primary cleavage site. Arg138-Arg139-->Asn138-Asn139 mutations were introduced to create protease-resistant IGFBP-5, which has the same affinity for IGF-I as the native protein. This mutant IGFBP-5 remained 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 actions. The significance of inhibitory effects of the protease resistant IGFBP-5 was further demonstrated […] Find the latest version: https://jci.me/119803/pdf 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 Yumi Imai,* Walker H. Busby, Jr.,* Christine E. Smith,‡ Jane B. Clarke,* Aaron J. Garmong,* Gayle D. Horwitz,* Catherine Rees,* and David R. Clemmons* *Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7170; and ‡Searle/Monsanto Inc., St. Louis, Missouri 63196 Abstract IGFBP-5. In conclusion, the accumulation of protease-resis- tant IGFBP-5 in the medium was inhibitory to IGF-I ac- IGFs are pleiotrophic mitogens for porcine smooth muscle tions on pSMC. This suggests that proteolysis can prevent cells (pSMC) in culture. The effects of IGFs on cells are IGFBP-5 from acting as an inhibitor of IGF-I–stimulated modulated by various insulin-like growth factor–binding effects and that it serves as an important mechanism for regu- proteins (IGFBP). IGFBP-5 is synthesized by pSMC and lating cellular responsiveness to IGF-I. (J. Clin. Invest. binds to the extracellular matrix. However, IGFBP-5 is also 1997. 100:2596–2605.) Key words: Somatomedin-C • serine secreted into conditioned medium of cultured cells and is protease • atherosclerosis • smooth muscle cell migration • cleaved into fragments by a concomitantly produced pro- IGF-I receptor tease. These fragments have reduced affinity for the IGFs and cleavage makes it difficult to assess the role of intact Introduction IGFBP-5. To study the consequence of accumulation of in- tact IGFBP-5 in medium, we determined the cleavage site IGF-I is a pleotypic mitogen for smooth muscle cells (SMC)1 in IGFBP-5 and prepared a protease resistant mutant. (1). Rat, porcine, and human smooth muscle cells have been Amino acid sequencing of purified IGFBP-5 fragments shown to respond to IGF-I with increased DNA synthesis (1– suggested Arg138- Arg139 as the primary cleavage site. Arg138- 3), cell growth (1, 3, 4), cellular migration (5), and protein syn- Arg139→Asn138-Asn139 mutations were introduced to create thesis (6), as well as increases of specific extracellular matrix protease-resistant IGFBP-5, which has the same affinity for (ECM) proteins such as, plasminogen activator inhibitor-1 (7). IGF-I as the native protein. This mutant IGFBP-5 re- These cells possess abundant IGF receptors (8) and transfec- mained intact even after 24 h of incubation and it inhibited tion with antisense oligonucleotides that inhibit receptor syn- several IGF-I actions when added to pSMC culture me- thesis (9) or incubation with anti–IGF-I antisera (10) results in dium. The mutant IGFBP-5 (500 ng/ml) decreased IGF-I attenuation of their DNA synthesis responses in the basal state stimulated cellular DNA synthesis by 84%, protein synthe- and of their responses to growth factors contained in serum sis by 77%, and it inhibited IGF-I stimulated migration of (9). Therefore, IGF-I appears to be a potent SMC mitogen pSMC by 77%. It also inhibited IGF-I stimulation of IRS-1 when added alone and a potent comitogen for other growth phosphorylation. In contrast, the same amount of native factors as well as an agent that is capable of stimulating extra- IGFBP-5 did not inhibit IGF-I actions. The significance of cellular matrix protein secretion. Both processes are thought inhibitory effects of the protease resistant IGFBP-5 was fur- to be important in establishing and maintaining neointima for- ther demonstrated in pSMC transfected with mutant or na- mation. tive IGFBP-5 cDNAs. The mutant IGFBP-5 accumulated In addition to synthesis of IGF-I, these cells also synthesize in culture medium of transfected cells, while native IGFBP-5 and secrete three forms of insulin-like growth factor binding was degraded into fragments. PSMC overexpressing the proteins (IGFBP-2, IGFBP-4, and IGFBP-5) (11, 12). While mutant IGFBP-5 also responded poorly to IGF-I compared substantial concentrations of intact IGFBP-2 are present in with mock transfected cells. IGF-I (5 ng/ml) increased smooth muscle cell–conditioned medium, IGFBP-4 and -5 are [35S]methionine incorporation into control cells by 36% much less abundant (11). IGFBP-4 has been shown to attenu- above the basal level, but it did not significantly change ate the actions of IGF-I (11). IGFBP-2 has been shown to both (4%) in pSMC cultures that were producing the mutant attenuate (13) and enhance IGF-I actions (14). Although it is a potent inhibitor of smooth muscle cell migration under certain conditions (5), it has been shown to enhance the effects of IGF-I on pSMC DNA synthesis (14). This may be partly due Address correspondence to David R. Clemmons, Chief, Division of to its ability to bind to proteoglycans in the extracellular Endocrinology, CB #7170, 6111 Thurston Bowles Bldg., The Univer- matrix when sufficient IGF-I is present in the microenviron- sity of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7170. ment (15). Phone: 919-966-4735; FAX: 919-966-6025. IGFBP-5 actions on smooth muscle cells have not been Received for publication 14 March 1997 and accepted in revised studied in detail, however, IGF-I–stimulated DNA synthesis in form 29 August 1997. J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: CHO, Chinese hamster ovary; 0021-9738/97/11/2596/10 $2.00 ECM, extracellular matrix; IGFBP, IGF-binding protein; IRS-1, insu- Volume 100, Number 10, November 1997, 2596–2605 lin receptor substrate-1; PI 3-kinase, phosphoinositide 3Ј-kinase; PPP, http://www.jci.org platelet-poor plasma; pSMC, porcine smooth muscle cells. 2596 Imai et al. fibroblasts is enhanced when the concentration of IGFBP-5 in 16 h of recirculation, the retained intact IGFBP-5 and IGFBP-5 frag- the extracellular matrix is increased (16). Porcine smooth mus- ments were eluted with 0.5 M acetic acid. Fractions containing immu- cle cells (pSMC) synthesize abundant amounts of IGFBP-5, noreactive IGFBP-5 and IGFBP-5 fragments were determined by im- however, no intact IGFBP-5 can be detected in their condi- munoblotting, then loaded onto a reverse phase HPLC C4 column tioned medium (11). This is due to the concomitant secretion (Vydac, Hespenia, CA) equilibrated with 0.4% triflouroacetic acid and H O. Elution was accomplished with a 0–60% CH CN gradient of an IGFBP-5 protease that cleaves IGFBP-5 into two frag- 2 3 over 1 h. The fractions were screened by immunoblotting and those ments which have very low affinities for IGF-I. Since the accu- containing IGFBP-5 fragments were concentrated by lyophilization mulation of IGFBP-5 in the medium in an intact form, as com- then subjected to amino acid sequence analysis. pared with ECM, may result in markedly different effects on Amino acid sequence analysis. Automated Edman degradation IGF-I actions, this protease has the potential to regulate the chemistry was used to determine the NH2-terminal protein sequence amount of intact IGFBP-5 in the pericellular environment and of the fractions containing proteolytic fragments. An Applied Biosys- thereby modulate IGF-I actions. These studies were under- tems Inc. model 470 gas-phase sequencer (Foster City, CA) was used taken to determine the cleavage site in IGFBP-5 and use in for the degradations using the standard sequencer cycle, 0.03 RPTH vitro mutagenesis to prepare a protease resistant form of (18). The sequentially released PTH amino acid derivatives were IGFBP-5 to determine the effects of increasing the concentra- identified by reverse phase HPLC analysis in an on-line fashion using an Applied Biosystems model 120A PTH Analyzer fitted with a tion of intact IGFBP-5 in the conditioned medium on IGF-I Brownlee 2.1-mm inner diameter PTH C-18 column.