Insulin-like growth factor binding protein (IGFBP) proteases: Functional regulators of cell growth

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Progress in Growth Factor Research, Vol. 6. Nos. 24, pp. 273-284, 1995 Copyright © 1996 Else~er Science Ltd. All rights reserved Printed in Great Britain. 0955--2235/95 $29.00 + .00

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INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP) PROTEASES: FUNCTIONAL REGULATORS OF CELL GROWTH Roopa Rajah, Lorraine Katz, Steven Nunn, Paulo Solberg, Tara Beers and Pinchas Cohen* Department of Pediatrics, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

The IGFBP proteases were first described in pregnancy serum as a proteolytic activity against IGFBP-3. Since then, IGFBP proteases have been described in many other clinical situations, in various body fluids, and have been shown to cleave IGFBP-2 to -6 with varying specificity. The molecular nature of some of these proteases is being unraveled and three classes o f IGFBP proteases have been recognized. These include kallikreins, cathepsins and matrix metalloproteinases ( MMPs). We utilized two cellular systems to demonstrate the significance of IGFBP proteases in cellular growth regulation. In primary cultures of prostatic cells, we have shown that prostate-specific antigen (PSA ) has the ability to enhance IGF mitogenic action by reducing the effects o f lGFBPs. Similar kallikreins such as gamma nerve growth factor (NGF) share this activity. Within the prostatic milieu, we have also demonstrated epithelial production of the acid-activated IGFBP protease, cathepsin D, and its secretion into seminal plasma, as well as the serum of patients with prostate malignancy. We have also identiffed M M P s in prostatic cells and fluids. Using cultured airway smooth muscle ( A S M ) cells, we have demonstrated the synergism between IGFs and inflammatory agents in mediating A S M cell proliferation. Examination of this phenomenon revealed that these agents (e.g. leukotriene D 4 and interleukinl-fl) induce the secretion of an IGFBP protease which cleaves the IGFBPs secreted by A S M cells, allowing IGFs to stimulate proliferation. Using several methods, including immunoblotting and immunodepletion techniques, we have identified this protease as MMP-1. These two pathophysiological systems demonstrate the importance of IGFBP proteases as autocrine paracrine growth regulators. Furthermore, IGFBP proteases may be critical elements in malignant and benign proliferative diseases, including prostate cancer and the A S M hyperplasia of long-standing asthma.

Keywords: IGFBP, I G F B P proteases, PSA, y - N G F , cathepsins, MMPs.

*Correspondence to: P. Cohen at: Division of Endocrinology, Department of Pediatrics, University of Pennsylvania, Children's Hospital of Philadelphia, 409 Abrahamson Pediatric Research Building, 34th and Civic Center Blvd: Philadelphia, PA 19104, U.S.A.

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INTRODUCTION Insulin-like growth factors (IGFs) are potent mitogens that stimulate cell growth in vivo and in vitro [1-5]. IGF action is determined by the availability of free IGFs

to interact with the IGF receptors. The amount of free IGFs in a system is modulated by the levels of IGF-binding proteins (IGFBPs), which bind to IGFs with high affinity. Recently, different groups of proteases capable of cleaving IGFBPs have been shown to play a key role in modulating free IGF levels and actions. Two main phenomena which are related to the mechanism of IGFBP protease action in facilitating IGF action include: (a) IGFBP inhibition of IGF action through sequesterization of IGFs away from their receptors; and (b) cleavage of the IGFBPs into fragments with lower affinity to IGFs, thus allowing for increased receptor activation. A theoretical model of such interactions is illustrated in Fig. 1. In this review we will discuss the speculations and the validations related to these two phenomena and demonstrate that IGFBP proteases are key modulators of IGF bioavailability and bioactivity through their modification of the IGF binding proteins. CLASSIFICATION OF IGFBP PROTEASES Several groups of proteases capable of cleaving specific IGFBPs have been identified as members belonging to three molecular super families, including kallikreins [2, 6-8]. cathepsins [9-11] and matrix metalloproteinases [12-14]. The first biochemically identified IGFBP protease, prostate-specific antigen (PSA), is an IGFBP-3 protease identified in seminal plasma [15] and was characterized as a kallikrein-like serine protease. Other kalikrein-like IGFBP proteases include the ?'-nerve growth factor (?'-NGF) which shares a common domain with, and has a high DNA sequence homology to, PSA [16, 17]. A related enzyme, plasmin, has been shown to act as an IGFBP protease. The role of these kallikrein proteases as IGFBP proteases are still being investigated. Cathepsins are dipeptidylpeptide hydrolases and are intracellular acidic proteinases found in aqueous extracts of a variety of animal and human tissues. These proteases are active at acid pH ranging from pH 5.5 to pH 4, and are considered important in many physiological and pathological processes including neoplastic infiltration. The speculation is that some of these processes may be triggered by IGFBP proteolysis. The matrix metalloproteinases are peptide hydrolases which require a metal ion for their catalytic activity. This group of enzymes is inactivated by metal chelators, as well as by naturally occurring inhibitors. The first recognized IGFBP proteases in pregnancy serum were recently demonstrated to be members of the MMP family [12]. Kallikrein-like IGFBP Proteases PSA

We have studied PSA extensively in terms of its role as an IGFBP-3 protease [5, 15, 18]. Since human seminal fluid contains prostatic secretions, we postulated that the seminal plasma millieu partially reflects the in vivo extracellular prostatic environment. While IGFBP-3 is produced by primary cultures of prostate fibroblast cells (PC-F) in culture, no IGFBP-3 is detectable in seminal plasma by Western

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THEORETICAL MECHANISMS OF IGFBP PROTEASE ACTIONS IGF4/-HI IGFBP

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ligand blotting (WLB). However, radioimmunoassay for IGFBP-3 in seminal plasma demonstrated significant amounts (approximately 200 ng/ml) of IGFBP-3, a situation analogous to that observed in pregnancy serum. In order to detect IGFBP-3 protease activity in seminal plasma we employed the IGFBP protease assay. [125I]-IGFBP-3 was degraded by adding pregnancy serum, seminal plasma or PSA, but not by non-pregnant serum or prostate cell conditioned media [8]. Autoradiographs measuring IGFBP-3 protease activity demonstrated that purified PSA cleaved IGFBP-3 in neutral pH, yielding a cleavage pattern identical to that seen with seminal plasma. IGFBP-1, -2, -4 and -6 were not significantly degraded by PSA, suggesting PSA to be a potent and specific IGFBP-3 protease [15]. Within the seminal plasma milieu, we have recently shown, however, that PSA also cleaves IGFBP-5. Notably, PSA has a unique cleavage pattern different from that of pregnancy serum and other proteases. IGFBP-3 fragments were functionally analyzed by affinity labeling and WLB. Silver staining of total protein and WLB of immunoreactive fragments with an IGFBP-3 specific antiserum demonstrated several multiple fragments. N-terminal sequence analysis revealed proteolytic recognition sites for PSA in the IGFBP-3 molecule. Three of the sites were consistent with a 'kallikrein-like' enzymatic activity [5]. In addition, immunoprecipitation of the seminal plasma with a specific IGFBP-3 antibody developed in our laboratory, followed by SDS-PAGE, revealed that seminal plasma contains IGFBP-3 fragments with a molecular weight of approximately 25 kDa. These IGFBP-3 fragments are identical to the fragments generated in vitro when recombinant-glycosylated IGFBP-3 is cleaved by PSA, and subjected to the same electrophoresis methodology. Therefore we conclude that PSA acts as a specific IGFBP-3 protease in vivo. Biological action o f P S A as an IGFBP protease. In order to demonstrate a biological role for PSA as an IGFBP-3 protease, we incubated recombinant IGFBP-3 with and without PSA, and crossed-linked it to [~25I]-IGF-I or [~z5I]IGF-II in the presence or absence of increasing concentrations of each peptide. After subjection to SDS-PAGE and densitometric analysis, displacement curves for IGF-I and IGFII were generated for both intact and cleaved IGFBP-3. Cleavage of IGFBP-3 by

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PSA caused a modest decrease in the affinity of IGFBP-3 fragments for IGF-II, but the affinity of IGF-I for the PSA-derived IGFBP-3 cleavage products fell by an order of magnitude. This represent an important mechanism by which PSA can enhance IGF action, since decreased affinity of a PSA-generated IGFBP-3 fragment for IGFs will result in an increased availability of free IGF to interact with the IGF receptor. In order to test the latter theory at the prostate epithelial cell level, we performed clonal assays for prostate epithelial cell (PC-E) growth in the presence and absence of IGF, IGFBP-3 and exogenous PSA. It should be noted that PC-E in our culture system do not produce substantial amounts of any of these three proteins. However, in vivo, prostate epithelial cells produce PSA, and prostate fibroblasts (based on our in vitro data) presumably secrete IGFBP-3 and IGF-II which would be available for the epithelial cells. When compared with cells grown under serumfree conditions without insulin or IGFs for 5 days, cells grown in the presence of sub-maximally stimulating concentrations of IGFs doubled their growth rate. The addition of IGFBP-3 at concentrations of 200 ng/ml had no effect when added alone. However IGFBP-3 completely blocked the stimulatory effects of simultaneously added IGFs. The most noteworthy phenomenon observed in this experiment was the reversal of the inhibitory effects of IGFBP-3 on IGF-stimulated PC-E growth when cells were grown in the presence of PSA [16]. It appears, therefore, that PSA functions as a PC-E growth enhancer in the presence of IGFs and IGFBP-3 (as found in serum) by a mechanism directly related to its IGFBP-3 protease characteristics (Fig. 2). y-NGF Another member of the kallikrein family, y-NGF [17, 18], which shares high sequence homology with PSA, was recently characterized as IGFBP protease [8]. The potential IGFBP proteolytic activity of N G F was evaluated by incubation of ~25I-labeled IGFBPs with proteases, followed by SDS-PAGE analysis. Both glycosylated and non-glycoslyated IGFBP-3 were cleaved by N G F at a concentration of 1 ng/pl, thus proving N G F to be a more potent IGFBP protease than PSA. NGFgenerated, lower molecular weight, IGFBP-3 fragments (detected by immunoblotting and cross-linking to IGFs) had 10-fold lower affinity to IGFs than intact IGFBP-3.2.5S N G F (a and fl subunits) and other kallikreins had little or no proteolytic activity against IGFBPs, suggesting that the y-subunit of N G F is the proteolytically active component. Unlike PSA, N G F also displayed potent proteolytic activity against IGFBP-4 and -6. These data also suggest that N G F may be involved in the growth of cells by more than one mechanism. In addition to binding to its own receptors, N G F is capable of cleaving IGFBPs and thus, enhancing IGF action. This synergistic interaction between N G F and the IGF axis may have important implications for cell growth, development and repair in the brain and other tissues. IGFBP-1 and -2 appear to be relatively resistant to the activity of kallikrein proteases. Other kallikreins Additional kallikreins which we have studied, including epidermal growth factor binding protein (EGFBP), human plasma kallikrein (hPK) and renin, are relatively poor IGFBP proteases.

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100 FIGURE 2. Regulation of cell growth by IGF axis components. The effects of IGF-II (2 riM), recombinant IGFBP-3 (4 riM) and PSA (2 /IM) on prost ate epithelial cell growth, evaluated using the 4-day microtiter assay. Results are expressed as mean + S E M relative to control (cells grown in basal media); *P < 0.001 relative to control; §P < 0.001 relative to IGF stimulation but not significantly different from control.

Cathepsins as IGFBP Proteases

Cathepsins are lysosomal proteases, active only below pH 5.5, and are secreted from various normal and cancerous cells and cell lines. Cathepsins have been shown to cleave IGFBP-1 to -5 in proteolytic studies [9, 10]. The first demonstration of cathepsin as an IGFBP protease was the immuno-depletion of cathepsin D in MCF-7 conditioned media which effectively attenuated acid-activated IGFBP-3 proteolysis [19]. Later, the acid-activated IGFBP protease in LNCaP and PC3 cellconditioned media was identified as cathepsin D, based on its proteolytic activity at acidic pH optimum and immunoblotting. Furthermore, immunoadsorption of cathepsin D from the media attenuated the acid-activated IGFBP hydrolysis. [~25]IGF-I binding to prostate cancer cells was reduced in the presence of LNCaP conditioned media at neutral pH but not by acid-incubated conditioned media [10, 11]. We have shown that cathepsin D is an acid IGFBP protease in both normal and malignant prostatic epithelial cells in primary culture, but not in prostatic stromal cells. We also have demonstrated the presence of cathepsin D in seminal plasma, where it is active as an IGFBP protease [11]. The physiologically relevant role of cathepsins as IGFBP proteases is much in debate. It is suspected that an acidic microenvironment, which would allow cathepsins to remain active, may exist during tumor growth and invasion. The abnormally high release of H ÷ ions from membrane-bound proteins in cancerous cells [9] may provide an acidic environment for extracellular cathepsin action and, thereby, may permit an accelerated rate of cell growth responsible for tissue invasion. These possibilities suggest a role for cathepsin D in the regulation of cellular IGF. The role of cathepsins in the largely neutral extracellular environment is yet unclear.

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278 Matrix Metalloproteinases as IGFBP Proteases

The matrix metalloproteinase family includes a number of collagenases such as interstitial collagenase (MMP-1), gelatinase A (MMP-2), stromolysin 1 (MMP-3), and gelatinase B (MMP-9). Although MMPs may play an important pathological role in the invasion and metastasis of cancer cells [45], little is known about the role of these proteases as co-mitogens. The first suggestion of MMPs as IGFBP protease was made by the demonstration of a Zn2÷-dependent protease(s) produced by dermal fibroblasts in vitro and mouse pregnancy serum [12, 13]. These proteases were inhibitable by EDTA, and degradation of recombinant human IGFBP-3 by conditioned media was blocked by MMP-specific tissue inhibitor of metallo-proteinases (TIMP-1). Removal of MMPs-1, -2 and -3 from conditioned medium by sequential immunoaffinity and gelatin--Sepharose chromatography resulted in the complete loss of IGFBP-3-degrading proteinase activity. These studies suggest that MMPs may play a role in regulating cellular growth and proliferation via degradation of IGFBP-3, thus enhancing IGF bioavailability. We recently identified an IGFBP protease activity in airway smooth muscle (ASM) cell conditioned media and demonstrated the link between the induction of this protease by the asthma associated pro-inflammatory eicosanoid, leukotriene D 4 (LTD4), and the synergistic action of IGF-I and LTD 4 on the growth of these cells [4]. More recently we also characterized this protease to be MMP-1 by immunodepletion of this conditioned media with anti-MMP-I antibody [20]. These findings lead to the presumption that MMPs could potentially be co-mitogens by regulating IGF action. Asthma-associated airway hyperplasia has been shown to be primarily a result of smooth muscle cell proliferation. Analysis of human bronchial smooth muscle (hBSM) cell conditioned medium, by Western ligand blotting, demonstrated a marked LTD 4 induced reduction in the levels of the IGF binding proteins, predominantly IGFBP-2 and -3, which are elaborated into the conditioned media. Western ligand blotting and immunoblotting demonstrated these IGFBPs to be cleaved to lower molecular weight forms. The effect of IGFBP release into conditioned media was not associated with changes in the mRNA levels of these binding proteins. Immunodepletion of LTD 4 treated hBSM cell conditioned medium using anti-MMP1 demonstrated dose-dependent reduction of IGFBP-2 proteolysis, suggesting that MMP-1 is the primary IGFBP-2 protease. MMP-specific, naturally occurring tissue inhibitor of metalloproteinases (TIMP-1) inhibited proteolysis of IGFBPs by MMP-1. Immunoblotting the hBSM cell conditioned medium demonstrated a dosedependent increase in MMP-1 response to LTD 4 and IL-113. These observations demonstrate that MMP-1 is an IGFBP-2 protease, induced by the increased levels of eicosanoids, with a significant role in modulating IGF action in hBSM cell culture. Similar mechanisms may be applicable in vivo in conditions such as asthma, which we associate with an increase in the inflammatory cytokines which regulate MMP-1 (Fig. 3). IGFBP PROTEASES IN CLINICAL STATES Pregnancy-associated I G F B P Proteases

Pregnancy-associated IGFBP-3 protease activity, which has been shown to appear after 6-weeks gestation, is responsible for the decrease and progressive

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disappearance of intact IGFBP-3 by Western ligand blot (without a change in IGFBP-3 immunoreactivity), and the appearance of lower molecular weight fragments of IGFBP-3 with a lower affinity to IGFs [21, 22]. The IGFBP-3 protease is active in neutral pH, and its activity rises with gestation [24]. It has been demonstrated that IGFBP-2, -4 and -5 also undergo proteolysis during pregnancy [21, 23, 24], but not IGFBP-1 or -6. Both the IGFBP-3 and -5 proteases in pregnancy serum appear to be cation dependent, and their activities are decreased by serine protease inhibitors [21, 24, 25]. Recently, it has been suggested that matrix metalloproteinases represent the IGFBP-3 protease in the serum of late gestational pregnant

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rats [12]. Serum levels of MMPs rise as pregnancy progresses, while levels of tissue inhibitor of matrix metalloproteinases (TIMP) decline [26]. Plasmin has also been proposed to be the pregnancy-associated IGFBP-3 protease [27]. The breakdown of basement membrane components during trophoblast invasion involves the secretion of both MMPs and serine proteinases (plasminogen activator) [28], and the activation of trophoblast-derived MMPs appeared to be a plasmin-dependent phenomenon [29]. A number of other serine proteases including chymotrypsin, trypsin, plasma kallikrein and neutrophil elastase have been shown to activate one or more of the precursors of MMPs in proteolytic cascades [29]. Thus, it has been speculated that these proteases may play a role in facilitating trophoblastic invasion. Recently it has been demonstrated that the pregnancy serum protease may play a role in regulating IGF bioactivity, making IGFs more available to the cells [30]. Therefore, it is possible that this proteolytic activity could have an impact on placental, and consequently on fetal growth, by releasing free IGFs and allowing increased receptor binding. Other Serum 1GFBP Proteases

Proteases for IGF binding proteins have been recognized as potential modulators of IGF action in several clinical conditions. Since first identified in pregnancy serum, IGFBP-3 proteolytic activity has also been reported in the serum of severely ill patients in states of cachexia, critically ill newborns, patients with AIDS and newly diagnosed juvenile diabetics [19, 31-33]. The functional significance of IGFBP proteases in these conditions is poorly understood, but in catabolic states, IGFBP proteases appear to be an acute-phase reactant. IGFBP proteases have also been documented in children with growth hormone receptor deficiency, and it has been postulated that the proteases may participate in somatic and tissue growth by modulating IGF activity [34]. In future, IGFBP proteases in serum may serve as diagnostic markers for particular disease states. I G F B P Proteases in Urine

Children with chronic renal failure (CRF) manifest reduced linear growth together with normal or elevated levels of growth hormone, low-normal levels of IGF-I [35, 36] a substantial increase in the RIA levels of serum IGFBP-1, a smaller increase of serum IGFBP-2 and a modest increase of serum IGFBP-3 [20, 37, 38]. The increase in serum IGFBP-3 by RIA in CRF is associated with reduced levels of intact IGFBP-3 measured by techniques such as Western ligand blotting, and an increase in the levels of IGFBP-3 fragments detected by immunoblotting. The apparent cleavage of serum IGFBP-3 is associated with the presence of IGFBP-3 protease activity in the urine (but not serum) of CRF patients [39]. Two patterns of IGFBP-3 proteolysis have been found, which appear to have a unique pattern. The absence of significant serum IGFBP-3 protease activity suggests that the protease is concentrated in the urine following filtration, or that it may be produced in the kidney itself. Alterations in urinary levels of IGFBP-3 presumably affect the amount of unbound IGF available for binding to receptors within the renal tubule. Thus, these abnormalities may play a role in the pathogenesis of renal disease and in the growth abnormalities of renal failure.

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281 IGFBP Proteases in Cancer

Because of the significant autocrine/paracrine role of the IGF axis in the mediating cellular growth, it has been proposed that alterations in elements of the IGF axis may influence or promote the growth of tumors. Several neoplastic conditions are associated with altered expression of IGFs and related molecules, and various IGFBPs have been shown to be expressed by different tumors [40, 41]. Several malignancies, including prostate cancer and leukemia, have also been associated with the presence of IGFBP-3 protease activity in serum [19, 23, 42]. Prostatespecific antigen, which has been demonstrated to be an IGFBP-3 protease in seminal plasma [15], is found in abnormally high levels in the serum of patients with malignant prostate cancer, and serves as a tumor marker. IGFBP-3 proteases have also been demonstrated in the cerebrospinal fluid (CSF) of patients with leukemia and CSF tumors [43]. Proteolytic activity may play a role in normal and abnormal tissue proliferation by altering the affinity of the binding proteins for the growth factors, releasing free IGFs and allowing increased receptor binding [16]. It is not yet known whether the increased IGFBP proteases in neoplastic conditions represents the production of proteases by tumor cells or the loss of protease inhibitors. Further research will determine if these abnormalities will have a role as diagnostic markers of malignancy. THERAPEUTIC POTENTIALS OF IGFBP PROTEASE INHIBITORS It is logical to assume that IGFBP protease inhibitors may have antiproliferative potential, since they block IGFBP proteolysis, increase IGFBP levels, reduce free IGF levels, decrease IGF binding to its receptor and, thereby, may decelerate growth and proliferation. Kallikrein inhibitors such as Aprotinin, Antipain, Amastatin, PMSF and trypsin-chymotrypsin inhibitor (Bowman-Birk inhibitor, BBI) prevent proteolysis of IGFBPs by N G F with varying efficiency [8]. Other protease inhibitors such as pepstatin specifically inhibit cathepsins [9, 10]. Proteolytic activity of MMPs is inhibited by naturally occurring TIMP-1 and -2 [12, 14, 46], and synthetic proteases such as galardin and Batimastat (BB-94) [47]. Overexpression of TIMP-2 markedly reduced melanoma growth in the skin of immunodeficient mice. Our observations suggest a mechanism to account for the growth inhibiting property of TIMPs [14, 47]. Preliminary observations suggest that BBI may inhibit IGF-mediated growth of cancer cell-lines by preventing proteolytic cleavage of IGFBPs. BBI is categorized as a trypsin-chymotrypsin inhibitor, effective against various serine proteases. BBI is naturally present in vegetables and epidemiological studies have identified legumes as possible protective agents in the decreased occurrence of breast, colon and prostatic cancers in vegetarian populations [48]. BBI has been shown to suppress X-ray induced transformation and radiation/chemical-induced transformation of BALB/3T3 cells in vitro [49]. In vivo effects of BBI as a tumor suppressor require further investigation. Currently, the precise mechanism by which a vegetarian diet exerts suppressive effects is unknown. BBI and other protease inhibitors are being used in experimental studies to demonstrate the potential action of synthetic protease inhibitors on IGFBP proteolysis. The role of protease inhibitors in clinical conditions remains to be explored.

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SUMMARY The alterations of the IGF-IGFBP-IGF-R balance in the autocrine-paracrine environment of the developing neoplasia has been demonstrated to influence or promote cellular growth [2, 3, 21, 37, 49]. It is now evident that the proteolytic activity of IGFBP protease decreases the affinity of the binding proteins to IGFs and therefore plays a critical role in modulating IGF availability at the tissue level. Therefore, IGFBP proteases must be considered as potential co-mitogens and the IGFBP proteases may emerge as clinically relevant anti-proliferative agents. REFERENCES 1. Cohen P, Peehl DM, Lamson G, Rosenfeld RG. Insulin-like growth factors (IGFs), IGF receptors, and IGF-binding proteins in primary cultures of prostate epithelial cells. J Clin Endocrinol Metab. 1991;73: 401-407. 2. Cohen P, Lamson G, Okajima T, Rosenfeld R G. Transfection of the human insulin-like growth factor binding protein-3 gene into Balb/c fibroblasts inhibits cellular growth. Mol Endocrinol. 1993; 7: 380-386. 3. Cohen P, Peehl DM, Baker B, Liu F, Hintz RL, Rosenfeld RG. Insulin-like growth factor axis in prostatic stromal cells from patients with benign prostatic hyperplasia. I. Clin Endocrinol Metab. 1994; 79: 1410-1415. 4. Cohen P, Bhala A, Herrick D, Noveral J, Grunstein M. The effects of Leukotriene D 4 on the proliferation of airway smooth muscle cells are mediated by modulation of the IGF axis. Am J Physiol. (Lung), 1995. In press. 5. Fielder PJ, Gargosky SE, Vaccarello M, Wilson K, Cohen P, Diamond F, Guevarar-Ageirre J, Rosenbloom AL, Rosenfeld RG. Serum profiles of insulin-like growth factors and their binding proteins in adults with growth hormone receptor deficiency treated with insulin-like growth factorI. Acta Paediatr. 1993; 388 (Suppl.): 40--43. 6. Cohen P, Graves HCB, Peehl DM, Kamarei M, Giudice LC, Rosenfeld RG. Prostate specific antigen (PSA) is an IGF binding protein-3 (IGFBP-3) protease found in seminal plasma. J Clin Endocrinol Metab. 1991; 73: 491-497. 7. Fielder PJ, Rosenfeld RG, Cohen P. Biochemical analysis of PSA proteolyzed IGFBP-3. Growth Regul. 1994; 4: 164--72. 8. Rajah R, Bhala A, Cohen P. Murine gamma NGF is an IGFBP protease. Endocrinology (Accepted for publication March 1996). 9. Conover C, DeLeon DD. Acid-activated insulin-like growth-binding protein-3 proteolysis in normal and transformed cells. Role of cathepsin D. J Biol Chem. 1994; 2691: 7076-7080. 10. Conover C, Perry J, Tindall D. Endogenous cathepsin D-mediated hydrolysis of insulin-like growth factor-binding proteins in cultured human prostatic carcinoma cells. J Clin Endocrinol Metab. 1995; 80: 987-993. 11. Nunn S, Peehl DM, Cohen P. Cathepsin D is an acid activated IGFBP protease produced in normal and malignant epithelial cells. Endocrine Soc. 1995. 12. Fowlkes JL, Suzuki K, Nagase H, Thrailkill KM. Proteolysis of insulin-like growth factor binding protein-3 during rat pregnancy: a role for matrix metalloproteinases. Endocrinology 1994; 135: 2810-2813. 13. Fowlkes JL, Enghild JJ, Suzuki K, Nagase H. Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures. J Biol Chem. 1994; 269: 25,742-25,746. 14. Rajah R, Nunn S, Grunstein MM, Cohen P. LTD 4 induces MMPs which function as IGFBP-2 protease in airway smooth muscle. J Clin Invest. 1995. Submitted. 15. Cohen P, Graves HC, Peehl DM, Karnarei M, Giudice LC, Rosenfeld RG. Prostate-specific antigen (PSA) is an insulin-like growth factor binding protein-3 protease found in seminal plasma. J Clin Endocrinol Metab. 1992; 75: 1046-1053. 16. Cohen p, Peehl DM, Graves HCB, Rosenfeld RG. Biological effects of prostate specific antigen (PSA) as an IGF binding protein-3 (IGFBP-3) protease. J Endocrinol. 1994; 142: 407-415.

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