matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases

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Progress in Growth Factor Research, Vol. 6. Nos. 2~l ., pp. 255-263, 1995 Copyright © 1996 Elsevier Science Ltd. All rights reserved

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MATRIX METALLOPROTEINASES AS INSULIN- LIKE GROWTH FACTOR BINDING PROTEIN-

DEGRADING PROTEINASES

John L. Fowlkes, *t Kathryn M. Thrailkill, t Delila M. Serra, t Ko Suzuki~ and Hideaki Nagase:l:

tDepartments of Pediatrics, Duke University Medical Center, Durham, NC 27710, U.S.A. ~Department of Biochemistry and Molecular Biology, University of Kansas Medical Center,

Kansas City, KS 66160, U.S.A.

Insulin-like growth factor (IGF) bioavailability is modulated by specific IGFBPs, six o f which are known (IGFBPs 1 4 ) . Since IGFBPs have equal or higher affinity for IGFs than do IGF receptors, it is believed that degradation of IGFBPs by IGFBP- degrading proteinases is an important step in regulating IGF bioactivity. Recent studies from our laboratory have demonstrated that at least two IGFBPs, i.e. IGFBP- 3 and -5, are degraded under physiologic conditions by matrix metalloproteinases ( MMPs). In vitro, we have demonstrated that IGFBP-3 is degraded in human dermal

fibroblast cultures by MMPs using a variety o f techniques, including proteinase inhi- bition with a specific inhibitor o f MMPs, i.e. tissue inhibitor o f metalloproteinases (TIMP-1), immunoabsorption with specific antisera to human MMPs and a unique method developed in our laboratory, IGFBP-3 substrate zymography. Using similar methods, we have also demonstrated that MMPs, along with an unidentified 97-kDa proteinase, degrade IGFBP-5 in murine osteoblast cultures. In rat pregnancy serum, we have shown that degradation of lGFBP-3 is associated with M M P activity present in the serum, which likely arises from the placental compartment. Analysis o f the cleavage products o f IGFBP-3 produced by MMPs 1, 2 and 3 reveals that MMPs cleave exclusively within the non-homologous, mid-region o f the molecule. Together, these studies suggest that MMPs, beyond their previously described roles as extracellular matrix degrading enzymes, may also exert effects on cellular growth and differentiation via degradation of lGFBPs.

Keywords: Insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), matrix metalloproteinase (MMP), stromelysin, interstitial collagnease, gelatinase.

*Correspondence to: J.L. Fowlkes at: Department of Pediatric Endocrinology, Division of Endocrinology, Box 3080, Duke University Medical Center, Durham, NC 27710, U.S.A.

Acknowledgements--We wish to thank Dr Christophere Maack (Celtrix Pharmaceuticals, Santa Clara, CA, U.S.A.) for providing rhlGFBP-3 and IGFBP-3 antisera and Dr Michael Kiefer (Chiron, Corp., Emeryville, CA, U.S.A.) for providing rhlGFBP-5. This work was supported by National Institutes of Health Grants AR39189 (to H.N.) and DK02276 (to J.L.F.), and a March of Dimes Basil O'Connor Starter Scholar Research Award (#5-FY93-0953) to J.L.F. and a Duke Children's Telethon Grant (to K.M.T.).

255

256 J. L. Fowlkes et al.

INTRODUCTION

Insulin-like growth factors (IGF-I and IGF-II) are ubiquitous growth-promoting peptides which exert both mitogenic and/or differentiation-inducing effects on numerous cells and tissues [1, 2]. For IGFs to exert their effects at the cell surface, they first must bind specific, high affinity cell-surface receptors. However, the interaction of IGFs with cell-surface receptors is modulated in a complex manner by a group of soluble IGF-binding proteins (IGFBPs), six of which have been purified and cloned [1, 2]. To date, several studies have demonstrated that all six IGFBPs have similar or higher affinities for IGF-I and IGF-II than does the type-I IGF receptor [2], suggesting that disruption of the IGF-IGFBP complex is a probable prerequisite to IGF-IGF-receptor interaction. We and others have provided evidence that this can be accomplished by limited proteolysis of the carrier IGFBP.

Limited proteolysis has been described for IGFBPs 2-5, and various proteinases have been postulated to function as IGFBP-degrading proteinases [1, 2]. Our labo- ratories have recently reported that matrix metalloproteinases (MMPs) function as IGFBP-degrading proteinases, both in vitro and in vivo [3-5]. Although this family of zinc-dependent proteinases is generally believed to be important in the degradation and turnover of extracellular matrix molecules, both in physiologic and in pathologic conditions [6-8], our studies suggest a new role for these enzymes as regulators of IGF bioavailability and bioactivity.

MMPS AS IGFBP-DEGRADING PROTEINASES I N VITRO

IGFBP-3

In human dermal fibroblasts, we have demonstrated that both endogenous and exogenous IGFBP-3 is degraded into several immunoreactive fragments by cell-free conditioned media [9]. These studies revealed that the IGFBP-3-degrading proteinases produced by dermal fibroblasts were zinc-dependent [9]. Based on these initial findings, studies were undertaken to identify the metalloproteinases which degrade IGFBP-3.

To characterize the IGFBP-3-degrading metalloproteinases produced by human fibroblasts, we developed and employed IGFBP-3-substrate zymography [4]. This method allows for the determination of the M r of IGFBP-degrading proteinases; used in combination with proteinase inhibitors, IGFBP-3-substrate zymography can also be used to classify proteinases. Figure 1 demonstrates that several distinct IGFBP-3-degrading proteinases (M r 52,000-72,000) were detected in human fibroblast conditioned media (lane 1), suggesting that more than one IGFBP-3-degrading proteinase was produced by human fibroblasts. The addition of EDTA (10 mM) to the incubation inhibited all of the IGFBP-3-degrading proteinases (lane 2), demonstrating that all detected IGFBP-3-degrading proteinases were bivalent, cation-dependent enzymes. Since the identified proteinases were cation-dependent and their molecular masses were similar to those of previously described gelatin- degrading proteinases, i.e. gelatinases [5, 10], fibroblast conditioned media was also analyzed by gelatin-substrate zymography (lane 3). This method demonstrated that human fibroblast conditioned media contained several gelatinases with identical

MMPs as IGFBP-degrading Proteinases

M lxlO ~1

257

97

69

46

30

- - + - - E D T A

1 2 3 FIGURE 1. Characterization of lGFBP-3-degrading and gelatin-degrading proteinase activity in human fibroblast conditioned media. Samples of human fibroblast conditioned media (50 #1) were analyzed by IGFBP-3-snbstrate zymography in the agsence (lane 1) or the presence (lane 2) of 10 mM EDTA as described in Ref. 141. Dark bands represent the IGFBP-3-degrading proteinase activity in the sample. Human fibroblast conditioned media (50 #1) was also analyzed by gelatin-substrate zymography. Areas of lysis (clear zones) represent gelatin-degrading proteinase activity in the sample (lane 3). Molecular weight markers are indicated on the left.

molecular masses to those that degraded rhlGFBP-3 (compare lanes 3 and 1, respectively). These were the first data suggesting that gelatinases might function as IGFBP-degrading proteinases. Further studies demonstrated that incubation of human fibroblast conditioned medium with the organomercurial 4- aminophenylmercuric acetate (APMA) resulted in a shift in M r of the IGFBP-3- degrading proteinases to several lower molecular weight forms (~ 18-46 kDa). This is consistent with previous studies showing that organomercurials such as APMA convert zymogen forms of MMPs (proMMPs) to lower molecular weight, enzy- matically active species [8].

To confirm the presence of MMPs in human fibroblast conditioned media, the media were immunoblotted with specific antisera generated against human MMP-1 (interstitial collagenase; Fig. 2, lane 1), human MMP-2 (gelatinase A; lane 2), MMP-3 (stromelysin 1; lane 3) or a combination of all three antisera (lane 4). All three proMMPs were detected at their expected molecular masses (proMMP-1, 52/55 kDa; proMMP-2, 72 kDa; and proMMP-3, 57/60 kDa [7, 8]), which were identical in size to IGFBP-3-degrading proteinases detected by IGFBP-3-substrate zymography (lane 5). Moreover, when tissue inhibitor of metalloproteinases (TIMP-1), a specific glycoprotein inhibitor of all MMPs [7, 8], was added to cell- free fibroblast conditioned media containing exogenous rhlGFBP-3, degradation of intact rhlGFBP-3 was inhibited by >80%. These data strongly suggested that

258

Mr(x 1 0"31

J. L. Fowlkes et al.

97.

69 . i~ I . . . . .

46.

30.q

1 2 3 4 5 FIGURE 2. Demonstration that human fibroblast conditioned media contains MMPs. Human fibroblast conditioned medium was immunobintted with specific antisera generated against MMP-1 (lane 1), MMP-2 (lane 2), MMP-3 (lane 3) or a combination of all three antisera (lane 4) as described in REf. [41. For comparison, human fibrohlast conditioned medium was also analyzed by IGFBP-3-substrate zymography (lane 5). Molecular weight markers are indicated on the left.

MMPs were involved in IGFBP-3 degradation in human fibroblast cultures. Further support for this hypothesis was provided when MMP-1, MMP-2 and MMP-3 were sequentially removed from fibroblast conditioned media by immunoabsorption and gelatin-Sepharose chromatography. Removal of MMPs from fibroblast conditioned media eliminated IGFBP-3-degrading proteinase activity in conditioned media as detected by IGFBP-3-substrate zymography, while re-addition of MMP-1, -2 and -3 to MMP-depleted conditioned media restored IGFBP-3-degrading activity. Together, these data demonstrate that the IGFBP-3-degrading metalloproteinases present in fibroblast conditioned media are MMPs, and that MMPs are the princi- ple IGFBP-3-degrading proteinases produced by human fibroblasts.

IGFBP-5

Studies from our laboratory have shown that IGFBP-5 is uniquely regulated in MC3T3-E1 osteoblasts throughout a 30-day period of proliferation and differentiation: IGFBP-5 is first detectable in conditioned media of replicating pre-osteoblasts (day 5); IGFBP-5 concentrations then peak between culture days 8-12, and decline to almost undetectable levels thereafter, despite persistence of IGFBP-5 mRNA as late as day 28 [11]. These observations suggested that post- translational mechanisms might be involved in the regulation of IGFBP-5 levels. To determine if IGFBP-5 proteolysis contributed to the disappearance of IGFBP-5 in conditioned media of mature osteoblast cultures, MC3T3-E1 cells were cultured for 30 days and conditioned media from serial time points were examined for their

M M P s as IGFBP-degrading Proteinases 259

ability to degrade [125I]IGFBP-5 [5]. We found that [1:5I]rhlGFBP-5 was degraded by proteinase activity present in conditioned media, and that activity increased in a time-dependent fashion as cultures matured. IGFBP-5-degrading proteinase activity was inhibited by chelating agents, and it was variably inhibited by certain serine proteinase inhibitors. Because bone cells in culture produce MMPs, we hypothe- sized that MMPs produced by MC3T3-E1 osteoblasts accounted for the cation- dependent, IGFBP-5-degrading proteinase activity. Indeed, the addition of TIMP-1 to MC3T3-E1 conditioned media inhibited >85% of IGFBP-5 proteolytic activity, suggesting that MMPs were primary proteinases involved in the degradation of IGFBP-5. Furthermore, using [125I]rhlGFBP-5-substrate zymography, IGFBP-5 degrading proteinases were identified in a broad band with M r 52,000-72,000 and in a more narrow band at M r 97,000 (Fig. 3, lane 1). The activity of all proteinases detected by [125I]rhlGFBP-5-substrate zymography increased throughout differentiation, consistent with our previous findings. The 52-72 kDa proteinases were inhibited by EDTA (lane 2), demonstrating that they were cation-depending proteinases, and were inhibited by TIMP-1 (lane 4), confirming their identities as members of the MMP family. In contrast, the 97-kDa proteinase was partially inhibited by PMSF (lane 3), but not by EDTA or TIMP-1, suggesting that the 97-kDa proteinase may be a serine proteinase, but not an MMP.

To clarify the identities of the MMPs involved in IGFBP-5 degradation, MC3T3- E1 conditioned media were immunoprecipitated with antisera to human MMP-1, MMP-2, MMP-3 or MMP-9. Only antisera to human MMP-1 and human MMP- 2 immunoprecipitated IGFBP-5 degrading proteinases with M r of 52 kDa and 69/72 kDa, respectively. Consistent with these data, conditioned media from MC3T3-E1 cells contained proteins which were detected by immunoblotting with antisera to human MMP-1 and human MMP-2, and MMP-2 immunoreactivity, in particular, increased significantly throughout differentiation. Together, these studies suggest that murine MMPs antigenically related to human MMP-1 and MMP-2 are involved in IGFBP-5 degradation in MC3T3-EI cultures.

Mr(xl0 -3)

97.4--,

69.0-*

46.0-* . . . . . Control -+ TA +PMSF +TIMP-I

FIGURE 3. Detection and characterization of IGFBP-5-degrading proteinase activity by IJ2SI]rhiGFBP-5 - substrate zymography. MC3T3-E1 conditioned media (50/A) were analyzed by 112Sl]rhlGFBP-5-substrate zymography (see Ref. [10]) in the absence (control) or in the presence of the proteinase inhibitors 10 mM EDTA (lane 2), 10 mM P M S F (lane 3), or 10/zg/ml TIMP-1 (lane 4). Lytic areas represent IGFBP-5- degrading proteinases.


MMPS AS IGFBP-DEGRADING PROTEINASES IN VIVO

During the later stages of pregnancy in the rodent and human, IGFBP-3 under- goes limited proteolysis [12-16]; this process yields IGFBP-3 fragments which bind IGFs with significantly lower affinity than does intact IGFBP-3 [16]. Through proteolysis of IGFBP-3, free IGF-I concentrations increase in human pregnancy serum [16]. In the pregnant rat, proteolysis of IGFBP-3 is associated with increased intravascular IGF-I clearance [12] and enhanced redistribution of serum IGFs to maternal tissues and the conceptus [17].

Previous studies have demonstrated that the IGFBP-3-degrading proteinase activity present in rat and human pregnancy serum is inhibited by chelating agents. Because the serum concentrations of MMP-1, MMP-2 and MMP-9 increase in maternal serum throughout pregnancy [18-20] and because their naturally occur- ring inhibitor, TIMP-1, decreases concurrently [21], we speculated that MMPs might be involved in the limited proteolysis of IGFBP-3 during pregnancy [3]. As demonstrated in Fig. 4, TIMP-1, when added to rat pregnancy serum (lane 3), inhibited [125I]rhlGFBP-3 proteolysis by >95%. In addition, purified human MMPs, principally MMP-1 and MMP-3, degraded IGFBP-3 in solution, and MMP-3, in particular, produced a pattern of IGFBP-3 degradation products identical in size to fragments produced by pregnancy serum. Furthermore, the combined addition of anti-human MMP-1 IgG and anti-human MMP-3 IgG blocked >95% of the degradation of [~25I]rhlGFBP-3 by rat pregnancy serum. Taken together, these data demonstrate that MMPs are involved in IGFBP-3 proteolysis in the serum of late gestation rats, and suggest that MMP-1 and MMP-3 are the principal MMPs

A B

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F I G U R E 4. Inhibi t ion o f IGFBP-3-degrading protease activity in rat pregnancy scram by T I M P - 1 . (A)

[ I ~ I ] r h l G F B P - 3 was incubated with buffer (lane 1) or rat pregnancy serum without (lane 2) or with (lane 3) T I M P - I as described in Ref. Pl - (B) Three separate experiments were analyzed for the effect of T I M P -

1 to inhibit the degradation [t25IIrhlGFBP-3. Bars correlate with lanes 1, 2 and 3 as outlined above. * P < 0.0001.

MMPs as IGFBP-degrading Proteinases 261

involved. Some investigators have characterized IGFBP-3 proteinase activity as a cation-dependent, serine proteinase; our results are consistent with these findings, since serine proteinases such as chymotrypsin, trypsin, plasmin, plasma kallikrein and neutrophil elastase have all been shown to activate one or more proMMPs [8].

CONCLUSIONS

These studies demonstrate that MMPs function as IGFBP-degrading proteinases both in vivo and in vitro under physiologic conditions. The demonstration that MMPs degrade IGFBPs broadens the range of substrates described for these extracellular proteinases [6-8]. While the physiologic ramifications of IGFBP-degradation by MMPs is not entirely clear, MMPs have been implicated in a variety of processes such as tumor cell invasion, morphogenesis, trophoblast invasion, carti- lage and bone repair and turnover, wound healing and angiogenesis [16]. Since IGFs also participate in many of these same processes, changes in MMP production and/or secretion may alter IGF bioavailability due to their regulation of IGFBP degradation. In vivo, such alterations are present in pregnancy (as described above); however, increased MMP production has been reported in several other disorders in which IGFBP-3 proteolysis has also been reported, including brain tumors [22, 23], breast cancer [2, 19] and prostate cancer [24-26].

It is presently unclear how MMPs effect IGF bioavailability at the cellular level; however, previous studies have demonstrated that cytokines, such as bFGF and TGF-fl~, bind to extracellular matrix, where they can be released via proteolysis to exert their effects at the cell surface. Since IGFBP-3 and -5 have been shown to associate with extracellular matrix [1], IGFs bound to matrix-associated IGFBPs may be released by MMPs to interact with cell surface receptors during periods of cellular proliferation. Our data also demonstrate that MMPs-1, -2 and -3 cleave IGFBP-3 exclusively within the mid-region of the molecule [4]; thus, another conse- quence of MMP-mediated IGFBP proteolysis may be to cleave IGFBPs into fragments which possess unique functions. In particular, N-terminal fragments of IGFBP-3 and IGFBP-5 have been shown to enhance IGF-action in bone cells [1]. Together, these studies suggest that an important interplay exists between MMPs and the IGF-IGFBP axis and that under the appropriate conditions, MMPs may serve to release stores of IGFs, allowing for a rapid cellular response to IGFs, which requires no new synthesis of these peptides.

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24. Cohen P, Graves CBG, Peehl DM, Kamarei M, Guidice LC, Rosenfeld RG. Prostate specific antigen is an IGF binding protein-3 (IGFBP-3) protease found in seminal plasma. J Clin Endocrinol Metab. 1992; 75: 1046-1053.

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M M P s as IGFBP-degrading Proteinases 263

26. Kanety H, Madjar Y, Dagan Y, Levi J, Papa MZ, Pariente C, Goldwasser B, Karasik A. Serum insulin-like growth factor binding protein-2 (IGFBP-2) is increased and IGFBP-3 is decreased in patients with prostate cancer: correlation with serum prostate-specific antigen. J Clin Endocrinol Metab. 1993; 77: 229-233.

matrix metalloproteinases as insulin-like growth factor binding protein-degrading proteinases

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