MDCK cells secrete neutral proteases cleaving insulin-like growth factor-binding protein-2 to -6

Liliana Shalamanova, Bernd Kübler, Jens-Gerd Scharf, and Thomas Braulke

University of Hamburg, Children's Hospital, Department of Biochemistry, D-20246 Hamburg; and Division of Gastroenterology and Endocrinology, Department of Medicine, University of Göttingen, D-37075 Göttingen, Germany


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Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) may be an important mechanism to regulate IGF availability and IGF-independent functions of IGFBPs. We analyzed the secretion of IGFBP proteases in Madin-Darby canine kidney (MDCK) cells. The results showed that several specific proteases were secreted, cleaving IGFBP-2 to -6 at neutral pH. The proteolytic activity against IGFBP-6 differed at least from IGFBP-5 protease activity in its sensitivity both to IGF-II and to the hydroxamic acid-based disintegrin metalloprotease inhibitor, as well as serine protease inhibitors. During partial purification steps, the serine protease inhibitor-sensitive fraction with IGFBP-6 protease activity was separated from fractions characterized by the presence of a 30-kDa disintegrin immunoreactive band. Whereas the IGFBP-4 and -6 proteases are predominantly secreted across the basolateral membrane, the majority of IGFBPs are sorted to the apical medium from filter-grown cells. These studies indicate that the side-specific secretion of several distinct IGFBP proteases with partially overlapping IGFBP specificities may be another level in the regulation of IGF-dependent epithelial functions.

insulin-like growth factor-binding proteins and proteases; polarized sorting; disintegrin metalloprotease


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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INSULIN-LIKE GROWTH FACTORS (IGFs) I and II are single-chain polypeptides expressed in many tissues and participate in the regulation of growth and differentiation of various cell types as autocrine and/or paracrine factors. Most of the metabolic and mitogenic effects of IGFs are mediated by IGF-I or insulin receptors exhibiting tyrosine kinase activity upon ligand binding (18). The capacity of IGFs to affect cell growth and metabolism via interaction with cell surface receptors is controlled by a family of IGF-binding proteins (IGFBPs). Six distinct high-affinity IGFBPs, designated IGFBP-1 to IGFBP-6, have been characterized, differing in molecular mass, posttranslational modifications, and tissue and developmental regulated expression (10, 44). Additional components of the IGF system are IGFBP-specific proteases. Limited proteolysis of IGFBPs is believed to be the major mechanism for the release of IGFs from IGFBP · IGF complexes, generating fragments with reduced affinity for IGFs (26). Proteolytic activity at neutral pH has been detected for IGFBP-2 (15), IGFBP-3 (16, 42), IGFBP-4 (14, 22, 30), and IGFBP-5 (9, 39), which are characterized by distinct fragmentation patterns, substrate specificity, and inhibitor profile. Furthermore, several studies suggest that acidic proteases may be involved in inactivation and the regulation of the extracellular IGFBP level, presumably in the lysosomal degradation pathway (2, 3, 8, 12, 27, 36). Two IGFBP proteases, the IGF-dependent IGFBP-4 protease and the IGFBP-5 serine protease secreted by cultured fibroblasts, have recently been reported to be identical with the pregnancy-associated plasma protein A and the complement component C1s, respectively (6, 23).

IGFs, IGFBPs, and IGF receptors have been reported to contribute to processes of cell proliferation and differentiation of epithelial cells (11, 32, 46). Prerequisite for the constitution of an epithelial permeability barrier are highly polarized cells characterized by morphologically, functionally, and biochemically distinct apical and basolateral plasma membranes. The basolateral localization and secretion of IGF receptors and IGF-II, respectively, as well as differential sorting of IGFBP-2, -4, and -6 in polarized HT29-D4 colon carcinoma cells (33), demonstrate the complexity of IGF-mediated epithelial cell regulation. Because nothing is known about the synthesis of IGFBP proteases in epithelial cells, the Madin-Darby canine kidney (MDCK) cell line was used as a model to examine and characterize secreted IGFBP proteases. Here we report that MDCK cells secrete different proteases cleaving IGFBP-2 to -6. Proteases cleaving IGFBP-4 and -6 are delivered preferentially to the basolateral side.


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Materials. Sodium [125I]iodine (carrier free; specific activity 16.9 mCi/µg iodine), [35S]methionine (100 Ci/mmol), and prestained protein standard (Rainbow) were purchased from AmershamPharmaciaBiotech (Freiburg, Germany). Recombinant human (rh)IGF-II from GroPep (Adelaide, Australia) was iodinated by the chloramine T method (45) to a specific activity of 60-80 µCi/µg. Recombinant IGFBP-1 and IGFBP-2 and -5 were purchased from UBI (Lake Placid, NY) and GroPep, respectively. Nonglycosylated rhIGFBP-3 was a generous gift from Drs. A. Sommer and C. Maack (Celtrix, Santa Clara, CA). rhIGFBP-4 and -6 produced in yeast (20) were kindly provided by Dr. J. Zapf (University Hospital, Zurich, Switzerland). Recombinant human arylsulfatase A (ASA) was a kind gift from Dr. T. Dierks (University of Göttingen, Germany). The IGFBPs and ASA were iodinated using IODO-GEN (Pierce Chemical, Rockford, IL) as described (31). The hydroxamic acid-based metalloprotease inhibitor TAPI was prepared at Immunex (Seattle, WA) (29) and was a kind gift from Dr. S. Rose-John (University of Kiel, Kiel, Germany). Antibodies directed against the prodomain (rb 132), the cystein-rich domain (rb 122), and the disintegrin domain (rb 119) of human ADAM 12 S (25) were kindly provided by Dr. U. Wewer (University of Copenhagen, Copenhagen, Denmark). Peroxidase-conjugated goat anti-rabbit IgG came from Dianova (Hamburg, Germany).

Blot analysis. 125I-labeled IGF-II ligand blot analysis was performed according to Hossenlopp et al. (16), with slight modifications. Briefly, conditioned media of 0.35 ml were mixed with 0.82 ml of ice-cold 96% ethanol and kept on ice for 1 h. Precipitated probes were solubilized and separated by SDS-PAGE (12.5% acrylamide) under nonreducing conditions. After electrotransfer and blocking in 1% fish gelatin, the nitrocellulose membranes were probed with 125I-IGF-II and exposed to X-ray films (X-Omat AR, Kodak). For immunoblotting, the membranes were blocked with 5% nonfat skim milk and probed with the rb 119, 122, and 132 antisera (dilution 1:100-1:500) and anti-rabbit IgG coupled to horseradish peroxidase (dilution 1:15,000). Reactive bands were visualized by the SuperSignal enhanced chemiluminescence detection system (Pierce Chemical) and exposure to X-ray films.

Cell culture. MDCK cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum (FCS), 2 mM glutamine, 4.5 g/l (wt/vol) glucose, and penicillin/streptomycin. The MDCK cells were routinely grown on 35-mm dishes (Greiner, Germany) or, to obtain polarized cell monolayers, on 24-mm Transwell (Costar, Cambridge, MA) (0.4-µm pore size) polycarbonate filters, as described recently (5). Serum-free medium conditioned for 24-72 h in the presence or absence of IGF-II (50 nM) was obtained as described earlier (4).

Northern blot analysis. The isolation of total RNA from MDCK cells, radiolabeling by random priming of cDNA probes for human IGFBPs, and the Northern blot hybridization were carried out as described previously (35).

IGFBP protease assay. Fifty microliters of conditioned media or 5-20 µl of column fractions dialyzed against 20 mM Tris · HCl, pH 7.4, containing 10 mM NaCl, were incubated with 125I-labeled IGFBPs (5,000-10,000 cpm) at 37°C for 6-18 h. When indicated, protein inhibitors were included. After solubilization, the samples were subjected to SDS-PAGE (12.5% acrylamide) and visualized by autoradiography, as described previously (24), or by phosphorimaging (Cyclone, Packard, Meriden, CT).

Protease purification. Three hundred milliliters of conditioned MDCK medium were sequentially precipitated with 30 and 45% ammonium sulfate. The latter precipitate was dissolved in 3 ml 20 mM Tris buffer, pH 7.4, containing 10 mM NaCl (TBS) and was desalted by Sephadex G25 column chromatography (PharmaciaBiotech, 1.0 × 35 cm). Two-milliliter fractions were collected at a flow rate of 0.8 ml/min, and 50-µl aliquots were assayed for IGFBP-6 protease activity. Active fractions were pooled and loaded onto a 2-ml DEAE-Sephadex column equilibrated with TBS. The column was washed with the same buffer (1 ml/min) until absorbance (280 nm) returned to the baseline. The proteins were eluted with a stepwise gradient of 0.1, 0.25, 0.5, 0.75, and 1.0 M NaCl in 20 mM Tris buffer, pH 7.5. One-milliliter fractions were collected, dialyzed against TBS, and tested for IGFBP-6 protease activity. The protease-containing fractions were pooled (fractions 7 and 8 to pool I, fractions 9-12 to pool II, and fractions 13-15 to pool III), dialyzed against 50 mM Tris buffer, pH 7.4, and loaded onto a hydroxyapatite column (PharmaciaBiotech; 1.0 × 1.0 cm). After the column had been washed, the proteins were eluted with a two-step gradient of 150 and 300 mM KPi in 50 mM Tris buffer, pH 7.4.

Other methods. Hexosaminidase activity was determined as described earlier (40). To examine the secretion of total newly synthesized proteins, cells were labeled with [35S]methionine (80 µCi/ml) for 4.5 h. Aliquots (20%) of the apical or basolateral media were precipitated with 1 ml of ice-cold acetone at -20°C for 24 h. After centrifugation, the pellets were dried and solubilized for SDS-PAGE under reducing conditions. In parallel, aliquots of media were used to determine the total radioactivity incorporated in secreted proteins by trichloroacetic acid precipitation. The radiolabeled polypeptides visualized by autoradiography or phosphorimaging were quantified by densitometric scanning (Hewlett-Packard Scan Jet 4c/T and the Advance Image Data Analyzer programme, Raytest, Straubenhardt, Germany).


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Secretion of IGFBP proteases. When conditioned media from MDCK cells were tested for IGFBP protease activity at neutral pH using 125I-labeled rhIGFBP-1 to -6 as substrates, no fragmentation of IGFBP-1 was detected within a 6-h incubation period. During the cell-free incubation, the 125I-IGFBP-2 was cleaved into two fragments of 24 and 13.5 kDa, and the 30-kDa nonglycosylated 125I-IGFBP-3 was cleaved into four fragments of 25, 21, 15, and ~8.5 kDa (Fig. 1). 125I-IGFBP-4 was fragmented to 17- and 10-kDa products, and the 125I-IGFBP-5 was cleaved to a 22-kDa doublet, 16- and 8-kDa peptide fragments, in the presence of conditioned MDCK medium. 125I-IGFBP-6 was almost completely hydrolyzed, depending on the incubation time, with a transient appearance of 17-, 10-, and 6-kDa IGFBP-6 fragments (Fig. 1). When the unrelated 62-kDa 125I-ASA was incubated with conditioned MDCK medium for 14 h at 37°C under conditions identical to 125I-IGFBP-3, no ASA proteolysis products were detected (not shown).


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Fig. 1.   Proteolysis of insulin-like growth factor-binding proteins (IGFBPs) by conditioned media from Madin-Darby canine kidney (MDCK) cells. 125I-labeled IGFBP-1 to -6 were incubated with 50 µl of nonconditioned (1) or 72-h-conditioned media from MDCK cells (2) at 37°C and pH 7.4 for 6 h (IGFBP-1 and -5) or 12 h (IGFBP-2, -3, -4, and -6). Samples were analyzed by SDS-PAGE and autoradiography. Experiments were carried out 1-2 times with a single medium of >= 3 different MDCK cell cultures.

Characterization of IGFBP proteases secreted by MDCK cells. To characterize the IGFBP protease(s) present in media of MDCK cells in more detail, the experiments were carried out using 125I-IGFBP-4, -5, or -6 as representative substrates, as indicated. The pH optimum of proteolytic activities with 125I-IGFBP-4 and -6 as substrates was estimated between 6.5 and 7.4 (not shown). By the addition of 50 nM IGF-II to the conditioned medium from MDCK cells, neither the proteolysis of 125I-IGFBP-4 nor that of 125I- IGFBP-5 was affected, whereas the protease activity cleaving 125I-IGFBP-6 was efficiently inhibited in vitro under these conditions (Fig. 2). Time course experiments, however, showed that the final degradation of IGFBP-6 was not prevented by IGF-II, whereas the rate of degradation was prolonged (not shown). To classify the proteases catalyzing the cleavage of the IGFBPs according to their inhibitor profile, media from MDCK cells were incubated with 125I-IGFBP-5 or -6 in the presence or absence of the metalloprotease inhibitors 1,10-phenanthroline (Phe, 10 mM) and TAPI (0.1 mM) and the serine protease inhibitors 3,4-dichloroisocoumarin (0.2 mM) and aprotinin (Apr, 0.3 µM). The proteolysis of 125I-IGFBP-5 was hardly affected by the inhibitors tested with the exception of the formation of the 8-kDa IGFBP-5 fragment, which was inhibited by TAPI and aprotinin (Fig. 3). In contrast, 125I- IGFBP-6 proteolytic activity in conditioned medium was almost completely inhibited by Phe and TAPI with the exception of the formation of 4-8% of the 17-kDa fragment, whereas the serine protease inhibitors impaired the proteolysis of 125I-IGFBP-6 moderately or weakly (not shown for Apr). These data suggest that 1) different IGF-sensitive IGFBP proteases are present in the medium of MDCK cells and 2) more than one protease with a distinct inhibitor profile may be involved in the degradation of a specific IGFBP.


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Fig. 2.   Effect of insulin-like growth factor II (IGF-II) on the proteolysis of IGFBP-4, -5, and -6 in conditioned media from MDCK cells. 125I-labeled IGFBP-4, -5 and -6 were incubated with 50 µl of nonconditioned (Co) or conditioned media (72 h) for 8 h at 37°C at neutral pH. Aliquots of conditioned media were incubated with 125I-IGFBPs in the presence (+) or absence (-) of 50 nM IGF-II, followed by SDS-PAGE and autoradiography. Experiments were carried out 1-2 times with a single medium of >= 3 different MDCK cell cultures.



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Fig. 3.   Effects of protease inhibitors on IGFBP-5 and IGFBP-6 protease activity in MDCK medium. Aliquots of 48-h-conditioned MDCK cell medium (25 µl) were incubated with 125I-IGFBP-5 or -6 (5,000-9,000 counts/min) in the absence of inhibitors (-) for 10 h at 37°C. 1,10-Phenanthroline (Phe 10 mM), hydroxamic acid-based metalloprotease inhibitor (TAPI, 0.1 mM), 3,4 dichloroisocoumarin (DCI; 0.2 mM), or aprotinin (Apr; 0.3 µM) was added as indicated. Reaction products were separated by SDS-PAGE and visualized by autoradiography. As a control (Co), 125I-IGFBP-5 or -6 was incubated in nonconditioned medium under identical conditions. The autoradiogram of 1 representative experiment of 4-6, testing various protease inhibitors, is shown. The percentage of intact IGFBP-5 or -6 after the incubation was determined by densitometry and is listed below each lane.

For further characterization, the IGFBP-6 protease was partially purified. A fraction containing protease activity with 125I-IGFBP-6 as substrate was precipitated from conditioned MDCK medium with ammonium sulfate and was bound to DEAE-Sephacel. IGFBP-6 protease activity was detected in fractions eluted with 0.5 M NaCl (fractions 7 and 8) and with 1.0 M NaCl (fractions 13-15) (Fig. 4A). The active fractions were pooled (pools I and III, respectively), as well as the inactive fractions 9-11 (pool II), and were applied separately to a hydroxyapatite column. No proteolytic activity was found in the flow through, and only a weak 125I-IGFBP-6 protease activity was recovered in fractions of pool I eluted at 150 mM KPi (I-1). The loss of IGFBP-6 protease activity might be due to the dialysis of the fractions eluted or the separation of a cofactor required for proteolysis. Strong IGFBP-6 proteolytic activity was found in fractions of pool III also eluted at 150 mM KPi (III-1), but not in fractions eluted at 300 mM KPi (III-2) (Fig. 4B). Because the IGFBP-6 protease activity in conditioned media was inhibited by the disintegrin metalloprotease-specific inhibitor TAPI, the fractions eluted from the hydroxyapatite column were tested by immunoblotting with rb 119 antibodies directed against the disintegrin domain of a disintegrin and metalloprotease (ADAM) 12. Under denaturating conditions, a single immunoreactive band at 30 kDa was found exclusively in I-1 and I-2 but not in the pool III-eluted fractions (Fig. 4C). When the fivefold concentrated I-1 and I-2 fractions were analyzed by SDS-PAGE and silver staining, in addition to the major BSA band, several weak bands of 88, 105, 40, and 34 kDa were detected, but no polypeptide of 30 kDa (not shown). In the initial fraction used for purification, a second disintegrin immunoreactive band at 35 kDa was detected. ADAM 12 S is the only soluble member of this protease family known so far that is composed of four domains forming an ~68-kDa mature glycosylated protease after cleavage of the 25-kDa prodomain (25). To examine whether the 30-kDa disintegrin immunoreactive band is a fragment of ADAM 12 S, aliquots of the fraction applied on the DEAE-column (AD) and of the fractions eluted with low salt concentration (I) were immunoblotted with antibodies against the disintegrin domain (rb 119), the cysteine-rich domain (rb 122), and the prodomain (rb 132) of human ADAM 12 S. The staining pattern of the two fractions with the three antibodies shown in Fig. 5 is partially consistent with the expected sizes of full-length ADAM-12 S (~92 kDa) or the mature protease (~65 kDa). All polypeptide bands of smaller molecular mass indicate the presence of truncated fragments. Because these fragments were found in fractions that lost IGFBP-6 protease activity, it is likely that they present inactive protease polypeptides. Thus the 30-kDa immunoreactive band may consist of the disintegrin domain (~10 kDa), the cysteine-rich domain (~9 kDa), and at least 11 kDa of the metalloprotease domain. Larger molecular mass complexes of ADAM 12 S with other proteins, as detected with the rb 122 antibody, have been described (25). Because the cross-reactivity of the anti-human ADAM 12 S antibodies with the polypeptides secreted by MDCK cells is unclear, the significance of the data is speculative.


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Fig. 4.   Partial purification of IGFBP-6 protease. A: ammonium sulfate precipitates of conditioned medium from MDCK cells were dissolved, dialyzed, and applied on a DEAE-Sephacel column. Bound proteins were eluted by a stepwise NaCl gradient (top). Dialyzed aliquots of every second fraction were tested for IGFBP-6 proteolytic activity, followed by SDS-PAGE and autoradiography (bottom). Underlined fractions were pooled separately (pools I, II, and III). B: after dialysis, pools I, II, and III were applied separately to an hydroxyapatite column. Bound proteins were eluted at 150 mM (1) or 300 mM (2) KPi. Dialyzed aliquots of these fractions were incubated with 125I-IGFBP-6 for 6 h at 37°C and analyzed by SDS-PAGE and autoradiography. Co, control incubation of 125I-IGFBP-6 with buffer. AD, aliquots of applied sample on DEAE-Sephacel. C: aliquots of the same fractions shown in B, eluted from hydroxyapatite column, were solubilized under nonreducing conditions and analyzed by SDS-PAGE and anti-disintegrin immunoblotting with the rb 119 antibody. Elution of proteins from the DEAE- or hydroxyapatite column in a second purification protocol with linear NaCl or KPi gradients, respectively, did not result in better separation of IGFBP-6 protease activities.



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Fig. 5.   Immunoblot analysis of MDCK medium fractions with antibodies against different ADAM 12 S domains. Aliquots of MDCK medium applied on the DEAE-ion exchange column (AD) and of the fraction eluted from the column with 0.35 M NaCl (I) were separated by SDS-PAGE under nonreducing conditions, transferred to nitrocellulose, and analyzed with antibodies against the disintegrin domain (rb 119), the cysteine-rich domain (rb 122), and the prodomain (rb 132) of ADAM 12 S. Immunoreactive polypeptides were visualized by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence.

When the IGFBP-6 protease activity was measured in the III-1 fraction in the presence and absence of protease inhibitors, the proteolytic activity was inhibited by neither Phe nor by TAPI but was almost completely blocked by the serine protease inhibitors DCI and Apr (Fig. 6), indicating the separation of the IGFBP-6 metalloprotease from the serine protease. Of interest, after incubation of 125I-IGFBP-6 with the fraction III-1, a prominent fragment of 10 kDa was formed compared with the continuing proteolytic cleavage in the unfractionated medium.


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Fig. 6.   Effects of protease inhibitors on partially purified IGFBP-6 protease-containing fraction. Aliquots of the partially purified fraction III-1 eluted from hydroxyapatite column (see Fig. 5) were incubated with 125I-IGFBP-6 for 8 h at 37°C in the presence or absence (-) of Phe (10 mM), TAPI (0.1 mM), DCI (0.2 mM), or Apr (0.3 µM). Reaction products were separated by SDS-PAGE and visualized by phosphorimaging. Densitometric evaluation revealed the percentage of remaining intact IGFBP-6 given below each lane. The experiment was carried out twice with similar results.

Polarized sorting of IGFBP-6 protease. To analyze whether newly synthesized IGFBP proteases are selectively directed to the apical or basolateral membrane in polarized MDCK cells, apical and basolateral media conditioned for 24 h were collected from cells grown on filter inserts 4 days after reaching confluence. Incubation of 125I-IGFBP-4 with apical medium revealed that 64% of the IGFBP-4 remained intact compared with only 28% of the 125I-IGFBP-4 after incubation in the basolateral medium (Fig. 7). Similarly, the majority of IGFBP-6 proteolytic activity was found in the same basolateral medium (Fig. 7), whereas 54% of the IGFBP-6 remained intact in the apical medium. These data indicate that proteases cleaving both IGFBP-4 and -6 are secreted preferentially to the basolateral side. For comparison, filter-grown MDCK cells labeled with [35S]methionine for 4.5 h secrete more newly synthesized proteins to the apical than to the basolateral medium (~1.8-fold) as measured by TCA-insoluble radioactivity. The composition and the ratio of labeled proteins secreted into the apical and basolateral media were different and polypeptide dependent (Fig. 8A). Thus, when beta -hexosaminidase activity was determined, a 2.6-fold (range 2.3-3.1, n = 6) higher activity was measured in the apical than in the basolateral medium (Fig. 8B). In addition, conditioned media from the apical and basolateral sides were analyzed by ligand blotting with 125I-labeled IGF-II. In both media, one band with an estimated molecular mass of 28 kDa and a prominent band at 25 kDa were detected, showing an approximate 4.2-fold higher abundance in the apical (range 3.9-4.7; estimated by densitometry) than in the basolateral medium (Fig. 8C). These data indicate that the presence of proteases cleaving IGFBP-4 and -6 as substrate in the basolateral medium is specific and not due to loading differences. The identity of the 28- and 25-kDa IGFBPs could not be determined, because none of the tested antibodies raised against various human or rodent IGFBPs cross-reacted with canine IGFBPs in immunoblots, and no hybridization of RNA from MDCK cells with any of the cDNAs probes specific for human IGFBP-1 to -6 was observed (not shown).


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Fig. 7.   Polarized secretion of IGFBP proteases. Medium was collected from the apical (a) or basolateral (b) side of filter-grown MDCK cells conditioned for 24 h. Aliquots of the media (50 µl) were tested for protease activity against 125I-IGFBP-4 and 125I-IGFBP-6, followed by SDS-PAGE and autoradiography. As a control (Co), 125I-IGFBP-4 or -6 was incubated in nonconditioned medium under identical conditions. Autoradiograms of 1 representative experiment of 2 (IGFBP-4) or 4 (IGFBP-6) are shown. Aliquots contain 12.2 (a) and 4.9 mU/ml (b) beta -hexosaminidase activity secreted within 24 h.



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Fig. 8.   Polarized secretion of proteins. A: filter-grown MDCK cells were labeled with [35S]methionine ([35S]Met) for 4.5 h at 37°C. Twenty percent each of the apical (a) and basolateral (b) media, corresponding to 18,000 and 10,000 counts/min TCA-insoluble radioactivity, respectively, were precipitated with acetone, separated by SDS-PAGE (10%), and visualized by phosphorimaging. This experiment was repeated twice with identical results. *Distinct proteins secreted into apical medium; left-arrow , proteins preferentially secreted to one side. B: beta -hexosaminidase activity was measured in duplicates in apical and basolateral media from 2 cell cultures in parallel. The experiment was repeated 3 times with MDCK cells after different passages, resulting in variations of the absolute activity values but with a reproducible ratio of activities in the apical and basolateral media. Enzyme activity in the apical medium is given as a percentage (means ± SD) of activity in the basolateral medium (5-18 mU/ml and 24 h). C: media from filter-grown MDCK cells were collected after 24 and 48 h from the apical (lanes 1 and 3) or basolateral side (lanes 2 and 4) and analyzed by SDS-PAGE (12.5% acrylamide), 125I-IGF-II ligand blot, and autoradiography. Positions of molecular mass marker proteins are indicated. A representative blot of 6 independent experiments is shown.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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The present study shows that MDCK cells secrete neutral proteases that cleave IGFBPs either to fragments of defined sizes, like IGFBP-2, -4, and -5, or to small peptides like IGFBP-6, hardly detectable under the conditions used. Inhibitor studies and data from the partial purification of the proteases using IGFBP-5 and -6 as substrates indicate that at least two classes of proteases, metallo- and serine proteases, are secreted by MDCK cells with different substrate specificities. The IGFBP proteases in the conditioned medium separated by ion exchange chromatography into two fractions are inhibited either by serine protease inhibitors or by metalloprotease inhibitors such as 1,10-phenanthroline and the hydroxamic acid-based inhibitor TAPI. The latter has been shown to be a potential inhibitor of tumor necrosis factor-alpha -converting enzyme (TACE), a member of the disintegrin metalloproteases (1, 29). The presence of several immunoreactive polypeptides, including a 30-kDa protein band in TAPI-sensitive IGFBP-6 protease fractions detected with specific antibodies directed against the disintegrin and cysteine-rich domain of ADAM 12 S, suggests the involvement of disintegrin metalloproteases in IGFBP-6 proteolysis. These metalloproteases have been reported to contribute to the proteolysis of IGFBP-3, -4, and -5 in human pregnancy serum and in cleavage of IGFBP-3 by placental trophoblasts (17, 22). Recently, the direct interaction with IGFBP-3 and the proteolysis of IGFBP-3 by recombinant ADAM 12 S have been shown (38). Whether disintegrin metalloproteases are involved in the proteolysis of IGFBPs in MDCK cells remains to be demonstrated and requires the sequencing of the 30-kDa immunoreactive polypeptide and the analysis of the ADAM expression pattern. The observed protective effect of IGF-II on the proteolysis of IGFBP-6 may be due to the preferential binding of IGF-II to IGFBP-6 compared with IGFBP-4 or -5, resulting in conformational changes that prolong the degradation rate rather than a direct effect on the protease.

Whereas the identity of proteases cleaving IGFBP-6 is still unknown and remains to be determined, IGFBP-6 proteolytic activity was also detected in acidified conditioned media from NIH-3T3 and HaCaT human keratinocytes (7, 27). However, the pH optimum, the complete degradation, and the inhibitor profile suggest that different acid-activated proteases, including the lysosomal protease cathepsin D, might be involved in IGFBP-6 proteolysis, either directly or indirectly by activation of IGFBP-6 proteases. Thus, it is rather unlikely that these acid-activated proteases play a role in regulation of the extracellular IGFBP-6 level. Data from mouse fibroblasts partially deficient for several of the lysosomal enzymes indicate that the acid-activated proteases may play a role in degradation of endocytosed IGFBP-3 (3).

The data presented here demonstrate that at least the proteases cleaving IGFBP-4 and -6 are secreted preferentially to the basolateral medium of polarized MDCK cells. In contrast, the activity of the lysosomal beta -hexosaminidase and the abundance of IGFBPs in the apical medium are ~2.6 and 4 times higher, respectively, than in the basolateral medium. It has been reported that, in the human colon adenocarcinoma cell line Caco-2, a majority of mannose 6-phosphate-containing lysosomal enzymes accumulate in the apical medium (41). This polarized distribution results from selective receptor-mediated uptake of lysosomal enzymes from the basolateral surface, followed by transcytotic delivery to the apical side (41). It is still unclear whether the low IGFBP level in basolateral medium results from increased proteolysis. The addition of serine protease inhibitors or TAPI increases, if anything, the abundance of IGFBPs in the apical rather than in the basolateral medium (unpublished results); however, the stability of the TAPI in hydrous solution at 37°C is low, and long-term toxic effects on cultured cells cannot be excluded (S. Rose-John, personal communication). Recently, Remacle-Bonnet et al. (33) reported on preferential sorting of IGFBP-2 and -4 to the basolateral side, whereas IGFBP-6 is primarily delivered to the apical surface of polarized enterocyte-like HT29-D4 human colonic carcinoma cells. However, the IGFBP protease activities are not determined in the media of HT29-D4 cells, and the molecular mechanism underlying the distinct pattern in IGFBP abundance in media from polarized cells is not known. It is believed that the transport of newly synthesized proteins to the basolateral side represents a default pathway for exocytosis, whereas the sorting to the apical side is signal dependent, occurring in the trans-Golgi network of MDCK cells (28, 34). Sorting signals for directing proteins in polarized cells to the apical or basolateral surface are best characterized for membrane proteins. For MDCK cells, it has been well documented that glycosylphosphatidylinositol-anchored proteins are specifically targeted to the apical plasma membrane (24). On the other hand, tyrosine and di-leucine-containing signals have been identified in the cytoplasmic domains of membrane proteins, which mediate sorting to the basolateral cell surface (19) and might be followed by a subsequent transcytotic delivery to the apical membrane. Furthermore, both N-linked and O-linked glycosylations of proteins, as well as linear glycosaminoglycan chains, appear to contain apical sorting information (21, 37, 43) recognized by molecules with lectin activity (13). However, there is evidence that the sorting mechanism for individual signals may vary considerably among different epithelial cell types (19). This might explain the discrepancies between IGFBP expression in the media of polarized MDCK cells reported in this study and expression in intestinal HT29-D4 cell media (33).

We propose that a majority of proteases cleaving IGFBP-4 and -6 in epithelial MDCK cells are delivered to the basolateral side and might be involved in release of IGFs by proteolysis of exogenous IGFBP · IGF complexes derived from the cellular environment, e.g., mesenchymal cells. Basolateral IGFBP proteolysis is a prerequisite to guarantee the access of IGFs to cell surface IGF receptors. A small percentage of IGFBP proteases might be delivered to the apical side via an indirect pathway, which might control the steady-state polarized distribution of IGFBPs. In studies with epithelial MDBK and Caco-2 cell lines, differences in the synthesis and secretion of distinct IGFBPs and in their mode of regulation have been reported, which might be necessary for the maintenance of the proliferative state and/or the initiation differentiation of these cells (11, 46). Further studies are required to identify the molecular mechanism of selective IGFBP sorting and to evaluate the physiological significance in polarized MDCK cells.


    ACKNOWLEDGEMENTS

This work was supported by Deutsche Forschungsgemeinschaft SFB 402 A6 (B. Kübler and T. Braulke), SFB 402 A5 (J.-G. Scharf), and Graduiertenkolleg 336 (L. Shalamanova).


    FOOTNOTES

Address for reprint requests and other correspondence: T. Braulke, Univ. of Hamburg, Children's Hospital---Biochemistry, Martinistr. 52, D-20246 Hamburg, Germany (E-mail: braulke{at}uke.uni-hamburg.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 November 2000; accepted in final form 16 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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