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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 6 1863-1868
Copyright © 1997 by The Endocrine Society

Experimental Studies

Proteolysis of Insulin-Like Growth Factor-Binding Protein-3 by Human Skin Keratinocytes in Culture in Comparison to that in Skin Interstitial Fluid: The Role and Regulation of Components of the Plasmin System1

Su Xu, Paul Savage, John L. Burton, Jane Sansom and Jeffrey M. P. Holly

Departments of Surgery (S.X., P.S., J.M.P.H.) and Medicine (S.X., J.L.B., J.S.), University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom BS2 8HW

Address all correspondence and requests for reprints to: Dr. Jeffrey M. P. Holly, Department of Surgery, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom BS2 8HW.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Proteolysis of insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is an important determinant of IGF action on cells. We have investigated this in a human skin keratinocyte cell line HaCaT. Although these cells did not normally produce an active IGFBP-3 protease, addition of plasminogen resulted in a dose-dependent proteolysis of endogenous and exogenous IGFBP-3, producing fragments similar to those cleaved by skin interstitial fluid, but different from those generated by plasmin. Protease inhibitor profiles suggested the enzyme in the conditioned medium to be a calcium-dependent serine protease.

Exogenous IGFBP-3 either inhibited or slightly stimulated IGF-I-induced cell proliferation when it was coincubated or preincubated with the cells, respectively. Both effects were attenuated in the presence of plasminogen.

Preincubation of cells with IGF-I or long R3 IGF-I divergently changed plasminogen activator inhibitor-1 and -2 secretion, but only IGF-I blocked IGFBP-3 proteolysis. Such inhibition was also observed in a cell-free protease assay. IGF-I, however, had no effect on plasmin-induced IGFBP-3 degradation.

Together, these data indicate that an IGFBP-3 protease similar to that in skin interstitial fluid is generated in plasminogen-treated HaCaT cells, and it attenuates the effects of IGFBP-3 on IGF action. IGF-I, probably by coupling with IGFBP-3, can protect it from the action of this protease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN-LIKE growth factor I (IGF-I) and IGF-II are potent mitogens for many cell types, such as skin keratinocytes (1, 2), and their function is believed to be modulated by a family of their own binding proteins (IGFBPs) (3). A general mechanism for regulating the bioavailability of IGF in both biological fluids and at the cellular level appears to be proteolysis of IGFBPs induced by various proteases. Although a number of enzymes might be involved in such proteolysis, the identity of a specific protease(s) has not been established.

In previous studies, we have shown IGFBP-3 protease activity in skin interstitial fluid from normal skin and uninvolved skin of psoriasis, and that such activity is inhibited in involved psoriatic skin area (4, 5). IGFBP messenger ribonucleic acids are present in skin epidermis (6), and keratinocytes are capable of producing IGFBPs, including IGFBP-2, -3, -4, and -6 and possibly IGFBP-5, with IGFBP-3 being the major form (7, 8, 9). Skin keratinocytes also produce plasminogen activators (PAs), primarily urokinase-type (uPA) (10) and PA inhibitors (PAI-1 and -2) (11), the principle components of the plasminogen/plasmin system, which has recently been implicated in proteolysis of IGFBPs, particularly IGFBP-3 (12, 13, 14, 15, 16).

We have used a human skin keratinocyte cell line, HaCaT, to study the IGF-IGFBP system and their possible regulatory mechanisms. HaCaT cells do not normally produce IGFBP-3 protease as we have found in the pilot study. In the present report, we have shown that the addition of plasminogen can induce IGFBP-3 proteolysis in these cells, and the property of the generated protease resembles that in skin interstitial fluid but differs from that of plasmin. Fragmentation of IGFBP-3 attenuates its effects, both inhibitory and stimulatory, on IGF-induced cell growth. IGF-I not only divergently regulates the levels of PAI-1 and PAI-2, but also down-regulates the IGFBP-3 proteolysis. The latter was probably mediated by binding of IGF-I to IGFBP-3, which protects IGFBP-3 from the action of the plasmin-activated protease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Recombinant human IGF-I and IGF-II, and long R3 IGF-I were obtained from GroPep (Adelaide, Australia). Recombinant human nonglycosylated IGFBP-3 (ngIGFBP-3) were provided by Dr. C. A. Maack of Celtrix Pharmaceuticals (Santa Clara, CA). Plasminogen, of human plasma origin (SA, 11 casein U/mg plasminogen) was purchased from Boehringer Mannheim (East Sussex, UK). Tissue inhibitor metalloprotease-1 (TIMP-1) was a gift from Dr. A. Docherty (Celltech, Slough, UK).

Cell culture

A spontaneously immortalized human keratinocyte cell line, HaCaT (17), was provided by Prof. N. E. Fusenig (German Cancer Research Center, Heidelberg, Germany). Cells at passages 33–36 were cultured in DMEM supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FCS at 37 C in a water-saturated atmosphere of 5% CO2 in air. Cells subcultured in 96-well plates were maintained in the same medium until approximately 90% confluency (3 days). The culture medium was discarded, and cells were preincubated in DMEM supplemented with 0.1% BSA [henceforth referred to as serum-free medium (SFM)] for 24 h. For IGFBP analysis and PAI measurement, the medium was then changed to fresh SFM without or with recombinant human IGF-I or long R3 IGF-I at various concentrations, and plasminogen was then added 24 h later. The conditioned medium was harvested after further 24 h and frozen at -20 C until analyzed. For proliferation assay, after 24-h incubation in SFM, cells were changed to fresh SFM containing various concentrations of ngIGFBP-3 without or with 0.5 µg/mL plasminogen. IGF-I (25 ng/mL) was added either concomitantly (coincubation with IGFBP-3) or 24 h later (preincubation with IGFBP-3). The incubation continued for an additional 48 h, and cell growth was determined using the dimethylthiazol-diphenyltetrazolium bromide (MTT) method as described previously (18).

Western ligand blotting and immunoblotting

Western ligand blotting and immunoblotting were performed as described previously (4).

Measurement of protease activity

IGFBP-3 protease activity was measured as previously described (4). Conditioned medium and plasmin at various concentrations were incubated at 37 C with [125I]ngIGFBP-3 in 50 mmol/L phosphate buffer for 24 h. In protease inhibition experiments, various protease inhibitors (final concentrations: ethylenediamine tetraacetate (EDTA), 50 mmol/L; aprotinin, 1 mg/mL; TIMP-1, 150 µg/mL) were also added before incubation. The samples were then subjected to 12.5% SDS-PAGE under nonreducing conditions. In cell-free protease assay, IGF-I or long R3 IGF-I at a final concentration of 25 ng/mL was added to the medium collected from plasminogen-treated cells together with [125I]ngIGFBP-3. The mixture was incubated and fractioned on SDS-PAGE.

Quantification of PAI protein

The PAI-1 and PAI-2 concentrations were determined by enzyme-linked immunosorbent assays (ELISAs; Tintelize, Biopool, Umea, Sweden).

Statistics

Results are presented as the mean ± SE, and comparisons were analyzed using Dunnett’s test, with significance defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Conditioned medium from HaCaT cells cultured in SFM for 48 h was assessed for IGFBP production by Western ligand and immunoblotting (Fig. 1Go, lane 2). Apart from a 42/39-kDa doublet of IGFBP-3 and a 24-kDa band migrating at the same position as serum IGFBP-4, as previously described (8), the medium from control culture contained a 34-kDa IGFBP-2 band as revealed by immunoblotting (data not shown). In addition, there was a diffuse band between 28–33 kDa, which might contain IGFBP-6 as it has been purified from HaCaT cell medium (9). Immunoblotting confirmed the 42/39-kDa form as intact IGFBP-3. Little or no 29-kDa band, the major proteolytic fragment of IGFBP-3 present in normal serum (Fig. 1bGo, lane 1), was observed in conditioned medium from control cells, indicating no proteolysis of endogenous IGFBP-3 by proteases produced by the cells.



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  		Figure 1. Proteolysis of IGFBP-3 in HaCaT cell culture by plasminogen treatment. Conditioned media from control cultures (lane 2) and from cells treated with various concentrations of plasminogen for 24 h were analyzed by Western ligand blotting (a) and immunoblotting (b). A sample from a normal serum pool (NS) was used as control.

 
To test the ability of plasmin to hydrolyze IGFBP-3 produced by keratinocytes, plasminogen was added to cell cultures, where it then was activated into plasmin by keratinocyte-produced plasminogen activators. Treatment with plasminogen at the concentration of 0.05–0.5 µg/mL for 24 h resulted in a dose-dependent reduction of the 42/39-kDa intact form of IGFBP-3 in Western ligand blotting (Fig. 1aGo, lanes 3–5). Immunoblotting further revealed that the density of a 29-kDa band increased accordingly. This indicated the proteolysis of IGFBP-3 by a protease generated in the medium. An approximately 21-kDa band of IGFBP-3 fragment was also found to be increased (Fig. 1bGo, lanes 3–5). Other IGFBPs were unchanged at these concentrations of plasminogen, but their degradation did occur when the dose of plasminogen exceeded 1 µg/mL (data not shown).

Our previous study has shown that a IGFBP-3 protease with high activity is present in normal skin interstitial fluid. This enzyme cleaves [125I]ngIGFBP-3 in a fashion similar to pregnancy serum (4). In the present study we compared the cleavage of [125I]ngIGFBP-3 induced by the conditioned medium from plasminogen-treated cells and by skin interstitial fluid. The conditioned medium degraded intact 30-kDa [125I]ngIGFBP-3, and this was plasminogen dose dependent (Fig. 2Go, lanes 6–8). The main fragments produced were approximately 21 and 15 kDa, which were of similar sizes to those generated by pregnancy serum or skin interstitial fluid (Fig. 2Go, lanes 2 and 4, respectively).



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  		Figure 2. Degradation of [125I]ngIGFBP-3 by conditioned medium from plasminogen-treated cells and plasmin. Medium from control cultures (lane 5) or from plasminogen-treated cells (lanes 6–8) or plasmin (lanes 9–11) was incubated with [125I]ngIGFBP-3 at 37 C for 24 h before being subjected to SDS-PAGE. Samples of third trimester pregnancy serum (PS) and normal skin interstitial fluid (SIF) were used for comparison.

 
The cleavage pattern of ngIGFBP-3 generated by plasminogen-conditioned medium was also compared to that resulting from pure plasmin. Plasmin of 0.05–0.5 µg/mL resulted in complete IGFBP-3 degradation (not shown). At much lower doses (0.2–20 ng/mL), plasmin degraded ngIGFBP-3 at an extent comparable to that in plasminogen-conditioned medium, but generated different fragments at approximately 15, 11, and 7.5 kDa (Fig. 2Go, lanes 9–11). In the protease inhibition study (Fig. 3Go), both plasmin and IGFBP-3 protease in the conditioned medium were markedly inhibited by the serine protease inhibitor aprotinin. However, the protease in the conditioned medium, distinct from plasmin, could be partially inhibited by EDTA. TIMP-1, a specific inhibitor for metalloproteases, had no effect.



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  		Figure 3. Effects of protease inhibitors on [125I]ngIGFBP-3 degradation by plasmin or plasminogen-conditioned medium. Plasmin or conditioned medium was incubated with [125I]ngIGFBP-3 in the absence (lanes 2 and 5) or presence of various protease inhibitors (lanes 3, 4, and 6–8) at 37 C for 24 h before being subjected to SDS-PAGE.

 
The biological significance of IGFBP-3 proteolysis was determined by MTT assay. Skin keratinocytes possess IGF-I receptors, although they appear not to produce IGF-I (19). In this study, IGF-I of 25 ng/mL stimulated HaCaT cell proliferation to approximately 130%. Proteolysis of endogenous IGFBP-3 resulting from the addition of plasminogen did not significantly alter the cellular responsiveness to IGF-I (data not shown). Thus, we further tested the effect of exogenously added IGFBP-3 (Fig. 4Go). The presence of ngIGFBP-3 during the coincubation with IGF-I resulted in a dose-dependent inhibition of IGF-I-stimulated cell proliferation. In contrast, pretreatment of the cells with ngIGFBP-3 for 24 h slightly enhanced IGF-I-induced stimulation, with the maximal effect at 100 ng/mL (nearly equamolar to 25 ng/mL IGF-I). In the presence of 0.5 µg/mL plasminogen, both enhancing and inhibiting effects of IGFBP-3 were neutralized.



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  		Figure 4. Attenuation by plasminogen of the effects of IGFBP-3 on IGF-I-induced HaCaT cell proliferation. ngIGFBP-3 without or with plasminogen were added to the cells concomitantly with or 24 h before the addition of IGF-I. The incubation continued for an additional 48 h before the measurement of cell proliferation using MTT method. Data represent the mean ± SEM of triplicate experiments. * and **, P < 0.05 and P < 0.01, respectively, compared to controls.

 
To investigate whether IGF-I could affect IGFBP-3 proteolysis in plasminogen-conditioned medium, HaCaT cells were pretreated with IGF-I for 24 h before the addition of plasminogen. In the presence of 25 ng/mL IGF-I, the decrease in IGFBP-3 was prevented, as shown by Western ligand blotting in Fig. 5aGo. Immunoblotting demonstrated that this was due to the blockage of IGFBP-3 fragmentation (Fig. 5bGo). Little or no fragmentation was induced by plasminogen at 0.05 µg/mL (Fig. 5bGo, lane 2), and the proteolytic effect of plasminogen at 0.1–0.5 µg/mL was also greatly attenuated (Fig. 5bGo, lanes 3 and 4).



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  		Figure 5. Effects of IGF-I and long R3 IGF-I on IGFBP-3 proteolysis in plasminogen-treated cells. HaCaT cells were preincubated with IGF-I (a and b) or long R3 IGF-I (c and d) for 24 h before the addition of plasminogen. Conditioned media were analyzed by Western ligand blotting (a and c) and immunoblotting (b and d).

 
We also examined the effect of long R3 IGF-I on IGFBP-3 proteolysis in HaCaT cells. This IGF-I analog retains high affinity for the IGF receptor, but has markedly decreased affinity for IGFBPs (20). As shown in Fig. 5Go, c and d, no apparent inhibition by long R3 IGF-I of the IGFBP-3 proteolysis in plasminogen-conditioned medium was observed.

The inhibition by IGF-I of the endogenous IGFBP-3 proteolysis was further confirmed by the IGFBP-3 protease assay. As shown in Fig. 6Go, the medium from plasminogen-treated cells preincubated with IGF-I blocked the degradation of [125I]ngIGFBP-3 in a manner dose dependent on IGF-I (lanes 3, 5, 7, and 9). In contrast, preincubation of long R3 IGF-I had no inhibitory effect on [125I]ngIGFBP-3 proteolysis (lanes 4, 6, 8, and 10).



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  		Figure 6. Comparison of the effects of IGF-I and long R3 IGF-I on plasmin-induced [125I]ngIGFBP-3 proteolysis. HaCaT cells were incubated with various concentrations of IGF-I or long R3 IGF-I for 24 h before 0.5 µg/mL plasminogen was added. Conditioned media were collected and incubated with [125I]ngIGFBP-3 at 37 C for 24 h before being subjected to SDS-PAGE.

 
The conversion of plasminogen to active plasmin by PA can be inhibited by PAI-1 and -2. To evaluate whether the different effects of IGF-I and long R3 IGF-I on IGFBP-3 proteolysis were related to PAI production in HaCaT cells, PAI-1 and PAI-2 in conditioned medium from control, IGF-I-treated, or long R3 IGF-I-treated cultures were measured by ELISA. As shown in Fig. 7aGo, both IGF-I and long R3 IGF-I at concentrations of 5–100 ng/mL resulted in a significant elevation of PAI-1 secretion. At 25 and 100 ng/mL, they increased PA-1 by approximately 1.6-fold. In contrast, both peptides inhibited PAI-2 production (Fig. 7bGo). At 25 and 100 ng/mL, they reduced PAI-2 by approximately 40%. These results indicated that IGF-I and long R3 IGF-I exerted a similar effect on regulating the production of PAIs.



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  		Figure 7. Dose-dependent effects of IGF-I and long R3 IGF-I on PAI-1 (a) and PAI-2 (b) levels in HaCaT-conditioned medium. Cultured HaCaT cells were incubated with various concentrations of IGF-I or long R3 IGF-I for 48 h. The PAI levels in the conditioned medium from three separate experiments were determined using ELISAs. Data represent the mean ± SEM of triplicate experiments. * and **, P < 0.05 and P < 0.01, respectively, compared to controls.

 
The data described above thus indicated that the inhibitory effect of IGF-I on IGFBP-3 proteolysis appeared not to be mediated through its receptors, although IGF-I did regulate PAI levels. To confirm this, conditioned medium was collected from the cells incubated with 0.1 µg/mL plasminogen for 24 h. IGF-I or long R3 IGF-I at a final concentration of 25 ng/mL was added to this cell-free medium and incubated with [125I]ngIGFBP-3 at 37 C for 24 h. As shown in Fig. 8Go, [125I]ngIGFBP-3 remained as the 30-kDa intact form after being incubated with the conditioned medium in the presence of IGF-I (lane 3), whereas apparent loss of the intact [125I]ngIGFBP-3 was observed with long R3 IGF-I treatment (lane 4). However, IGF-I up to 100 ng/mL had no effect on 20 ng/mL plasmin-induced [125I]ngIGFBP-3 degradation (lanes 6–8).



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  		Figure 8. Effects of IGF-I on cell-free IGFBP-3 proteolysis by conditioned medium and plasmin. Conditioned medium collected from plasminogen-treated HaCaT cells was incubated with [125I]ngIGFBP-3 in the presence of IGF-I (lane 3) or long R3 IGF-I (lane 4). For comparison, plasmin was incubated with [125I]ngIGFBP-3 in the presence of various concentrations of IGF-I. Samples were at 37 C for 24 h and then analyzed by SDS-PAGE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
IGFBP-3 proteases have been classified as serine proteases in most reports but in some circumstances, prostate-specific antigen, metalloproteinases, and cathepsin D might also cleave IGFBP-3 (21, 22, 23). Plasmin, a serine protease with wide distribution and broad spectrum specificity, has recently been implicated in IGFBP-3 proteolysis (12, 13, 14, 15, 16). In this study, we have demonstrated that exogenous plasminogen, which can be activated into plasmin by cell-derived PAs, dose dependently cleaves IGFBP-3. IGFBP-3 was much more sensitive to the enzyme than other IGFBPs. Similar susceptibility of IGFBPs to plasmin has been found in human osteosarcoma cells (13). Interestingly, in this study plasmin degraded [125I]ngIGFBP-3 into fragments of different sizes from those produced by plasminogen-conditioned medium, which suggested that in the latter there might be another protease(s) involved. This was supported by the protease inhibition profile. The protease in the conditioned medium could be inhibited not only by serine protease inhibitor but also by EDTA, which indicated different properties from those of plasmin. These data suggest that the generated IGFBP-3 protease in plasminogen-conditioned medium is a calcium-dependent serine protease. Alternatively, as plasmin is capable of activating other proteases, such as matrix metalloprotease, the proteolysis of IGFBP-3 in the conditioned medium might result from the actions of several proteases. However, that the proteolytic activity was not inhibited by TIMP-1 appears to have excluded matrix metalloproteases. The identity of this IGFBP-3 protease remains to be determined.

The proteolytic fragments of endogenous IGFBP-3 and [125I]ngIGFBP-3 resulting from the addition of plasminogen in keratinocyte-derived HaCaT cells are of similar sizes to those found in skin interstitial fluid and pregnancy serum (4), indicating that the proteases in the conditioned medium, skin interstitial fluid, and pregnancy serum might share the common cleavage sites of IGFBP-3. As plasminogen/plasmin is present in the basal layer of human epidermis (24), it raises the possibility that this protease(s) might contribute to the IGFBP-3 proteolysis in skin interstitial fluid. There are some discrepancies between the IGFBP-3 protease in plasminogen-conditioned medium and that in biological fluids. For example, the former, at high concentrations, can cleave other IGFBPs in addition to IGFBP-3, whereas the latter appears to be IGFBP-3 specific. It is, however, possible that the concentration of plasminogen/plasmin in biological fluids may fall into the range that only provokes IGFBP-3 proteolysis. Further investigation is needed to establish the physiological relevance of plasmin or plasmin-activated protease(s) as an IGFBP-3 protease.

Both inhibitory and stimulatory effects of IGFBP-3 in the regulation of IGF action on cell growth have been observed (25). Inhibition appears to result from soluble IGFBP-3 sequestering IGF-I and preventing receptor interaction (26). Proteolytic processing of IGFBP-3 may thus serve as a regulatory mechanism to increase the activity of the IGFs (27). In the current study, the inhibitory effect of exogenous IGFBP-3 coincubated with IGF-I was reversed by its proteolysis. Similarly, it has been reported that plasmin-induced endogenous IGFBP-3 degradation is able to stimulate prostate carcinoma cell proliferation (16). The mechanism for the potentiation effect of IGFBP-3 remains more obscure and may correlate with targeting the action of IGF-I via IGFBP-3 cell association, protecting IGF-I from degradation by proteases, or inhibiting IGF-I-induced receptor down-regulation and cellular desensitization (28, 29). It is thus possible that in the presence of plasminogen during the preincubation period, IGFBP-3 cell association is prevented by its proteolysis, and therefore, the potentiating effect on the action of IGF-I is attenuated. Although the potentiating effect itself may result from IGFBP-3 processing to fragments (28), the protease responsible for this cell surface processing seems to be different from the IGFBP-3 protease in HaCaT cell medium, as the former could not been inhibited by aprotinin or EDTA (28).

IGF-I has been shown to inhibit the plasmin-induced and plasmin-like protease-induced IGFBP-3 proteolysis in human osteosarcoma cells and porcine ovarian granulosa cells, respectively (13, 14). In the former, the inhibition was believed to be mediated by depressing the activity of uPA (13). It was, however, not known whether this decrease in activity resulted from a decrease in uPA production and/or an increase in the level of its inhibitor PAIs. In this study, we showed that IGF-I also dose dependently blocked the IGFBP-3 proteolysis in plasminogen-treated HaCaT cells. Intriguingly, IGF-I exerted a divergent effect on the PAIs concentrations: increasing the PAI-1 level, but decreasing the PAI-2 level. Although the net effect of PAIs on the conversion of plasminogen into plasmin was not determined, the changes in PAIs did not contribute to the IGF-I inhibitory effect on IGFBP-3 degradation in the present experimental model, because long R3 IGF-I, which regulated both PAIs in a comparable manner to IGF-I, was not able to inhibit IGFBP-3 proteolysis.

In porcine ovarian granulosa cells, long R3 IGF-I seemed to act as the native IGF-I to inhibit the plasmin-like protease-induced IGFBP-3 proteolysis (14). In the present study the effects of long R3 IGF-I on PAI production were comparable to those of IGF-I, suggesting that they are mediated through cell membrane receptors, as one expects. However, long R3 IGF-I failed to block both endogenous IGFBP-3 and exogenous [125I]ngIGFBP-3 degradation. Such distinction from IGF-I clearly indicates that ligand-membrane receptor interaction is not required for the inhibitory action of IGF-I and, thus, appears to imply that this action relies upon the affinity of IGF-I to its binding protein. A similar mechanism has been postulated in Hs578T human breast cancer cells, in which the inhibitory effect of IGF-I on IGFBP-3 degradation by a protease (undefined) was believed to be due to the formation of an IGF-IGFBP complex (30). Our present data, however, could not rule out the possibility that the nonreceptor-mediated action of IGF-I might result from IGF-I binding to the protease.

Taken together, our data demonstrate a possible feedback loop, i.e. IGFBP-3 can either stimulate or inhibit the action of IGF-I on HaCaT cells, the presence of IGFBP-3 protease attenuated both effects of IGFBP-3, and IGF-I itself is able to block IGFBP-3 proteolysis. Thus, the protease modulates IGF availability from IGFBP-3 and, in turn, modulates the activity of this protease. In skin interstitial fluid taken from psoriatic skin lesions, the IGF-II level is significantly elevated and IGFBP-3 protease activity is greatly reduced due to a locally produced, unidentified inhibitor(s) (5). In the same fluid, the PAI-1 level was significantly higher and the PAI-2 level was lower than those in skin interstitial fluid taken from the uninvolved skin (31). Based on our data, it is tempting to speculate that the rise in IGF-II could not only be responsible for the altered PAI levels, but either directly or indirectly participate in the inhibition of IGFBP-3 proteolysis in psoriatic skin lesions. Further studies are needed to define the role of IGFs in regulating the proteolytic degradation of their binding proteins under physiological and pathological conditions.


   Footnotes
 
1 This work was supported by the Bristol and Avon Dermatology Research Trust and the Medical Research Council. Back

Received August 22, 1996.

Revised December 11, 1996.

Revised February 20, 1997.

Accepted March 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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