Department of Medicine, University of Melbourne, Heidelberg, Victoria 3084, Australia
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ABSTRACT |
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The insulin-like growth factor (IGF) system plays an important role in skin. HaCaT human keratinocytes proliferate in response to IGFs and synthesize IGF-binding protein-3 (IGFBP-3). Recently, IGFBP-6 was also identified by NH2-terminal sequencing, but it has not been identified by Western ligand blotting. In the present study, IGFBP-6 was detected in HaCaT-conditioned medium by use of immunoblotting and Western ligand blotting with 125I-labeled IGF-II. Proteolytic activity against IGFBPs, an important mechanism for regulation of their activity, was then studied. An acid-activated, cathepsin D-like protease that cleaved both IGFBP-6 and IGFBP-3 was detected. Although proteolysis did not substantially reduce the size of immunoreactive IGFBP-6, it greatly reduced the ability of IGFBP-6 to bind 125I-IGF-II as determined by Western ligand blotting and solution assay. HaCaT keratinocytes do not express IGF-I mRNA, but IGF-II mRNA and protein expression was detected. These observations suggest the possibility of an autocrine IGF-II loop that is regulated by the relative expression of IGF-II, IGFBP-3, and IGFBP-6, and IGFBP proteases in these keratinocytes, although demonstration of this loop requires further study.
insulin-like growth factor; binding protein; cathepsin D; autocrine growth factor
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INTRODUCTION |
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INSULIN-LIKE GROWTH FACTORS (IGF) I and II are potent mitogens that induce both proliferation and differentiation in many tissues. They act predominantly through the IGF-I receptor, and their actions are modulated by a family of at least six IGF-binding proteins (IGFBPs) (3, 16). IGF actions can be either potentiated or inhibited by IGF binding to IGFBPs. The IGFBPs differ in their affinities for IGFs, tissue expression, modulation of IGF activity, and susceptibility to proteolysis. IGFBP-6 differs from other IGFBPs because it has a 30- to 100-fold preferential binding affinity for IGF-II over IGF-I (2, 3, 16). It is O-glycosylated (4) and inhibits IGF-II actions to a far greater extent than IGFBPs of IGF-I (1, 21, 34).
IGFBPs can be degraded by proteases that either act specifically on an individual IGFBP or proteolyze many IGFBPs. For example, a protease secreted by human fibroblasts cleaves IGFBP-5 but not IGFBP-1, -2, -3, and -4 (26), whereas mouse 7S nerve growth factor cleaves most IGFBPs, including IGFBP-3, -4, -5, and -6 (29). The proposed role of IGFBP proteases is to release IGFs from IGFBP-containing complexes, thereby making them available for binding to IGF receptors (11).
The IGF system is involved in epidermal growth and development. This is evident from IGF-I knockout mice, in which the skin is thin and translucent because of inhibition of epidermal cell proliferation (22). In vivo, IGF-I is synthesized by melanocytes and fibroblasts but not keratinocytes (38). Nevertheless, IGF-I is a mitogen for keratinocytes (38). Keratinocytes proliferate in response to IGF-II in vivo (40). In situ hybridization of human skin identified IGFBP-3 mRNA in the basal layer of the epidermis and IGFBP-2 and IGFBP-4 in sebaceous glands and eccrine sweat glands (6). IGFBP-4 mRNA and IGFBP-5 mRNA were localized throughout the dermis, but IGFBP-1 mRNA and IGFBP-6 mRNA were not identified in human skin with this technique (6). However, IGFBP-6 mRNA was abundant in epidermal layers of human fetal skin (14).
The expression of IGFBPs has been studied previously in cultured primary human epidermal keratinocytes (25). Radioimmunoassay and Northern analysis showed that the expression of IGFBPs by these cells is dependent on their passage number, with IGFBP-6 mRNA present in early passages. The HaCaT cell line is an immortalized cell line with the characteristics of human basal epidermal keratinocytes (7). IGFBP-3 is the predominant IGFBP produced by HaCaT cells as determined by Western ligand blotting with 125I-labeled IGF-I, and this IGFBP inhibits IGF-I-induced proliferation of these cells (41). More recently, IGFBP-6 was identified by NH2-terminal sequencing as an autocrine growth inhibitor of these cells (18), although this IGFBP has not been identified by Western ligand blotting or immunoblotting. It has not been determined whether HaCaT cells synthesize IGFs. The aims of this study were therefore to further characterize IGFBP-6 and IGF expression in HaCaT keratinocytes.
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MATERIALS AND METHODS |
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Cells.
HaCaT human keratinocytes were kindly provided by Prof. N. E. Fusenig
(German Cancer Research Centre, Heidelberg, Germany) (7). Cells were
grown to confluence in Dulbecco's modified essential medium
(DMEM)-10% FCS and then changed to serum-free DMEM-0.05% BSA (Sigma,
St. Louis, MO) for 72 h, following which conditioned media were
collected, centrifuged to remove any cellular debris, and stored at
20°C before analysis. Cells appeared morphologically normal,
and few floating cells were found at the end of 72-h incubations.
Western ligand blotting.
HaCaT-conditioned media (1 ml) were concentrated by adding 2.33 ml of
cold ethanol, incubated at 20°C for 20 h and centrifuged at
11,000 g for 15 min at 4°C. The
resulting pellet was air-dried and reconstituted in nonreducing Laemmli
sample buffer. Proteins were separated by SDS-12% PAGE and transferred
to a nitrocellulose membrane. The membrane was blocked with 1% BSA,
probed with 125I-labeled IGF-II
for 18 h at 4°C, washed, and exposed to X-ray film (Biomax, Kodak,
Coburg, Australia) for 24 h.
Immunoblotting. The nitrocellulose membrane used for Western ligand blotting (WLB) was stripped with 150 mM NaCl-10 mM Tris · HCl (pH 7.4)-0.05% NaN3-3% Nonidet P-40 for 1 h at room temperature. The membrane was then blocked with 5% nonfat skim milk and probed with a polyclonal antiserum against human IGFBP-6 (kindly provided by Dr. J. Martin, University of Sydney, St. Leonards, Australia). Signal was detected using enhanced chemiluminescence (SuperSignal, Pierce, Rockford, IL) and exposure to X-ray film for 10 min.
RT-PCR. Total RNA from HaCaT cells was extracted using Trizol (Life Technologies, Mulgrave, Australia), which is based on the phenol-guanidine isothiocyanate method. Total RNA (1 µg) was reverse transcribed for 2 h using AMV reverse transcriptase (Boehringer Mannheim, Box Hill, Australia). For PCR amplification of IGFBP-6, primers hybridizing to regions corresponding to the start (5'-AATGGATCCCGGTGCCCAGGCTGCGGGCAA-3') and stop codons (5'-TTTTCTAGAGAATTCTTAGCCGCTACTCCCAGTGGGGCAGGA-3') were used (1). Amplification was performed over 40 cycles using an FTS-960 thermal sequencer (Corbett Research, Mortlake, Australia). An initial denaturation step at 95°C for 5 min was followed by five cycles, consisting of denaturation at 95°C, annealing at 62°C, and extension at 72°C, each for 30 s. The subsequent 35 cycles differed only in having a lower annealing temperature of 56°C. A final extension step was carried out at 72°C for 5 min. To amplify the IGF-I cDNA, primers used were 5'-TCTTGAAGGTGAAGATGCACACCA-3' and 5'-AGCGAGCTGACTTGGCAGGCTTGA-3', hybridizing to nucleotides 201-223 and 501-524 of the IGF-I cDNA, respectively (36). To amplify the IGF-II cDNA, primers used were 5'-ATGGGAATCCCAATGGGGAA-3' and 5'-CTTGCCCACGGGGTATCTGG-3', hybridizing to nucleotides 220-239 and 547-564 of the human IGF-II cDNA, respectively (15). The PCR conditions for IGF-I and IGF-II were identical to those for IGFBP-6, except that the first five cycles were carried out at an annealing temperature of 70°C and the subsequent 35 cycles were performed at an annealing temperature of 65°C. PCR products were identified on 1.5% agarose gels stained with ethidium bromide. The expected sizes for the IGFBP-6, IGF-I, and IGF-II cDNAs were 668 bp, 305 bp, and 345 bp, respectively. The positive control for IGF-I RT-PCR was total RNA from human adrenal gland.
Southern blotting. Membranes were prehybridized at 42°C for 60 min in Rapid-Hyb buffer (Amersham, Castle Hill, Australia). End-labeled internal oligonucleotide probes used were 5'-GCGGGCGCTGCTGCTCGGCC-3', hybridizing to nucleotides 282-301 of the IGFBP-6 cDNA, and 5'-GCTGCGGAAACAGCACTC-3', hybridizing to nucleotides 433-450 of the IGF-II cDNA (15). Membranes were hybridized for 90 min at 42°C. The membranes were washed three times for 10 min each at 42°C. The first 2 washes were in 2× standard sodium citrate (SSC)-0.1% SDS, and the final wash was in 0.1× SSC-0.1% SDS. Membranes were subsequently exposed to X-ray film for 10-60 min.
Expression and purification of recombinant glycosylated human
IGFBP-6 in Chinese hamster ovary cells.
Chinese hamster ovary cells were stably transfected with the plasmid
vector encoding human IGFBP-6, phBP6-E3 (1), using Lipofectamine (Life
Technologies). Clones were selected with -MEM-10% FCS containing
400 µg/ml geneticin. Colonies producing the highest levels of
recombinant human IGFBP-6, as determined by charcoal adsorption assay
and WLB with 125I-IGF-II, were
selected and recloned. A single clone was then expanded for production.
IGFBP-6 was purified from conditioned media by IGF-II affinity
chromatography, followed by wheat germ agglutinin lectin chromatography
to remove nonglycosylated hamster IGFBP-4. Glycosylated proteins that
contain glucosamine and/or sialic acid monosaccharides,
including IGFBP-6, were eluted with 0.3 M
N-acetylglucosamine, followed by
reverse-phase fast performance liquid chromatography (ProRPC 5/10,
Pharmacia, Boronia, Australia) with a 16-40% acetonitrile
gradient in 0.1% trifluoroacetic acid, as previously described (1).
Iodination of IGFBP-6. IGFBP-6 (2 µg) was iodinated to a specific activity of 36 µCi/µg by use of chloramine T. Iodinated IGFBP-6 was separated from free Na125I by gel filtration using a P6-DG desalting column (Bio-Rad, Richmond, CA).
Acid activation and proteolysis of IGFBP-6. The pH of medium conditioned by HaCaT cells was adjusted with 2 M HCl to pH 3, pH 4, pH 5, pH 6, and pH 7. IGFBP-6 (200 ng) or 125I-IGFBP-6 [5 × 104 counts/min (cpm)] were incubated for 20 h at 37°C with 60 µl of conditioned media. IGFBP-6 samples were assayed by charcoal adsorption assay (as detailed below), WLB, and immunoblotting. 125I-IGFBP-6 samples were analyzed by SDS-15% PAGE and exposed to X-ray film for 24 h. Similar experiments were performed with media from HaCaT cells pretreated with IGF-I or IGF-II (100 ng/ml) for 96 h.
Time-course analysis of 125I-IGFBP-6 cleavage. 125I-IGFBP-6 (5 × 104 cpm) was incubated with 60 µl of acidified HaCaT conditioned media (pH 4) for 4-20 h at 37°C. Aliquots were subjected to SDS-15% PAGE, and the gel was dried and then exposed to X-ray film for 24 h.
Characterization of proteolytic activity. HaCaT-conditioned media (pH 4) were incubated with 125I-IGFBP-6 (5 × 104 cpm) and the following protease inhibitors for 20 h at 37°C: ethylenediaminetetraacetic acid (EDTA) (1 mM), aprotinin (2 µg/ml), benzamidine (1 mM), leupeptin (1 µg/ml), pepstatin A (100 µg/ml), phenylmethylsulfonyl fluoride (PMSF; 1 mM), and trans-epoxysuccinyl-leucylamido-(4-guanidino)butane (E-64; 5 ng/ml). Samples were analyzed as described in Acid activation and proteolysis of IGFBP-6.
Charcoal adsorption assay. IGFBP-6 (200 ng) was incubated with HaCaT-conditioned media at pH 4 or pH 7, as indicated in Acid activation and proteolysis of IGFBP-6. Volumes of media corresponding to 0.01, 0.1, and 1 ng (0.02, 0.2, and 2 µl, respectively) of IGFBP-6 were then assayed as previously described (4). Briefly, samples were incubated with 125I-IGF-II (2 × 104 cpm) for 18 h at 4°C in 0.1 M NaPO4 (pH 7.4)-0.1% BSA-0.02% NaN3 in a total volume of 400 µl. Bound and free IGFs were then separated using albumin-coated charcoal followed by centrifugation at 1,300 g (20 min, 4°C). Radioactivity in the supernatant was counted. Specific binding was determined by subtraction of nonspecific binding, as assessed by binding in the absence of IGFBP (~7%), from total binding. Within each experiment, samples were assayed in duplicate. Results are shown as means ± SD of two experiments.
IGF-II immunoblotting. HaCaT-conditioned media (20 µl) and IGF-II standards (500, 100, 50, and 0 pg in unconditioned medium without serum) were applied to a 0.2-µm nitrocellulose membrane in a slot-blot apparatus under vacuum. The membrane was dried for 10 min at 37°C and then blocked for 1 h at room temperature with 5% nonfat skim milk. The membrane was probed with a monoclonal antibody (10 µg/ml) against human IGF-II (kindly provided by Prof. Nishikawa, Kanazawa Medical University, Ishikawa, Japan). Signal was detected using enhanced chemiluminescence and exposure to X-ray film for 10 min. This assay allows the detection of IGF-II without interference by IGFBPs (13).
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RESULTS |
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IGFBP-6 is synthesized by HaCaT keratinocytes.
Although the synthesis of IGFBP-6 by HaCaT keratinocytes has been
previously identified by amino acid sequencing, studies using WLB have
not identified this binding protein, possibly because 125I-IGF-I, which binds IGFBP-6
poorly, was used as tracer (23, 41). However, WLB of media conditioned
by HaCaT cells by use of
125I-IGF-II revealed two bands
with apparent molecular masses of ~30 kDa and 40 kDa (Fig.
1A).
The single band at 30 kDa was recognized by a polyclonal antiserum to
human IGFBP-6 (Fig. 1B). In
contrast, the 40-kDa band was not recognized by the antiserum to
IGFBP-6 and most likely represents IGFBP-3, which HaCaT cells are known to synthesize (41).
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HaCaT keratinocytes express an acid-activated protease for IGFBPs.
Cell-free conditioned media from HaCaT cells were acidified to pH
3-7. IGFBP-6 (200 ng) was added for 20 h at 37°C and then analyzed by Western ligand blotting. The band corresponding to IGFBP-6
was markedly decreased at pH 4, but not at other pHs tested (Fig.
3A),
whereas the band corresponding to endogenous IGFBP-3 was absent at pH
4. Despite the fact that the IGFBP-6 signal was markedly decreased on
the Western ligand blot, immunoblotting demonstrated that IGFBP-6 was
still present and its apparent molecular mass was not significantly
decreased at pH 4 under nonreducing conditions (Fig.
3B). This surprising observation was
seen consistently in seven independent experiments. The decreased
IGF-II binding was confirmed by a solution binding assay at pH 7.4 after incubation of IGFBP-6 with acidified and nonacidified conditioned
media (Fig. 3C). When
HaCaT-conditioned media were acidified (pH 4), specific binding of
125I-IGF-II was reduced to 4.3 ± 5.1% compared with 29.9 ± 1.2% after incubation with
HaCaT-conditioned media at pH 7. IGF-II binding was unaffected when
IGFBP-6 was incubated with unconditioned medium at pH 4 or pH 7, excluding an effect of pH alone.
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The acid-activated protease expressed by HaCaT cells is inhibited by
pepstatin A.
To characterize the acid-activated proteolytic activity, cell-free
conditioned media (pH 4) were incubated with
125I-IGFBP-6 and protease
inhibitors. Pepstatin A, an aspartic protease inhibitor, completely
abolished the proteolytic activity (Fig. 5,
lane 7). In contrast, serine
protease inhibitors (aprotinin, benzamidine, leupeptin, and PMSF) had
no effect on proteolytic activity (Fig. 5, lanes
4-6 and 8).
EDTA, an inhibitor of metalloproteases, also had no effect on protease
activity (Fig. 5, lane 3). E-64, an
inhibitor of cathepsin B, did not affect protease activity (result not
shown). These results are characteristic of cathepsin D-like activity.
Pretreatment of HaCaT cells for 96 h with IGF-I or IGF-II (100 ng/ml
for 96 h) had no effect on proteolytic activity (results not shown).
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HaCaT keratinocytes synthesize IGF-II but not IGF-I.
Previous studies have indicated that keratinocytes do not synthesize
IGF-I in vivo (38), but IGF expression by HaCaT cells has not been
studied. RT-PCR of total RNA from HaCaT cells with primers specific for
IGF-II demonstrated a single band at the expected size of 345 bp (Fig.
6A,
lane 2), and its identity was confirmed by Southern blotting with an internal oligonucleotide probe
(Fig. 6B, lane
4). Slot-blot immunoblot analysis of media conditioned by HaCaT cells confirmed the presence of IGF-II protein (Fig. 7, lane
5). In contrast, IGF-I mRNA was not expressed by HaCaT cells as determined by RT-PCR (Fig.
6C, lane
7).
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DISCUSSION |
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IGFBP-6 has previously been identified by amino-terminal sequencing as a growth inhibitor present in HaCaT-conditioned media (18). In the present study, we have confirmed the presence of IGFBP-6 in media conditioned by HaCaT human keratinocytes by use of WLB and immunoblotting. In a study by Xu et al. (42), a 30-kDa IGFBP was identified in HaCaT-conditioned media by WLB by use of a combination of 125I-IGF-I and 125I-IGF-II, but this was thought on the basis of immunoblotting to be IGFBP-3; however, the presence of IGFBP-6 was not excluded in that study. IGFBP-6 may not have been identified in other studies by WLB (25, 41), because 125I-IGF-I was used rather than 125I-IGF-II, and IGFBP-6 is not readily detected using the former tracer (23). The present study also identified the presence of an acid-activated protease for IGFBPs with activity against IGFBP-6. This protease was identified as cathepsin D-like on the basis of its pH specificity and inhibition by pepstatin A. Consistent with this notion, the protease also cleaved endogenous IGFBP-3, which is susceptible to proteolysis by cathepsin D (9, 12). Finally, this study shows that HaCaT keratinocytes synthesize IGF-II but not IGF-I.
There was an apparent discrepancy between the results obtained after incubation of acidified HaCaT medium with 125I-IGFBP-6, where low-molecular-weight fragments were observed, and noniodinated IGFBP-6, where no smaller fragments were observed by immunoblotting. One possible explanation for this discrepancy is that the amount of 125I-IGFBP-6 (~1 ng) added was much lower than noniodinated IGFBP-6 (200 ng), resulting in a higher protease-to-IGFBP-6 ratio in the former experiments, so that proteolysis may have been more complete. Alternatively, the small proteolytic fragments may not have been immunoreactive with the antiserum used.
Despite the fact that there was no or little discernible reduction in size of immunoreactive IGFBP-6 after incubation with acidified HaCaT medium, IGF-II binding was substantially reduced. This implies either that cleavage of a small peptide from the carboxy- or amino-terminal domain of IGFBP-6 is sufficient to interfere significantly with IGF binding, or that additional cleavage occurred at sites that abrogate IGF binding without disrupting disulfide bonds. We have previously observed that cleavage of IGFBP-6 by chymotrypsin, resulting in a ~2- to 3-kDa reduction in apparent molecular mass, is also sufficient to abolish IGF-II binding (unpublished observations). The effect of IGFBP-6 proteolysis on IGF-II binding is consistent with previous studies showing that proteolysis of IGFBPs in general decreases their binding affinity for IGFs, thereby increasing the availability of the latter to target cells (11). Alternatively, proteolysis of IGFBP-6 may have resulted in a specific binding defect, rendering the IGFBP incapable of binding iodo-IGFs but not native IGFs, as has been described for IGFBP-3 proteolyzed by pregnant serum (37).
The three known groups of IGFBP proteases include kallikreins, cathepsins (acid-activated and neutral), and matrix metalloproteases. Previously, two other proteases for IGFBP-6 have been described. Murine 7S nerve growth factor, a member of the kallikrein family, cleaves IGFBP-6 as well as IGFBP-3, IGFBP-4, and IGFBP-5 at high concentration (29). Acid-activated IGFBP-6 proteolytic activity was also found in media conditioned by a NIH-3T3 cell line (10). Although this activity was acid-activated, it was inhibited to varying extents by a number of protease inhibitors, including pepstatin, leupeptin, aprotinin, E-64, and chymostatin, suggesting that a cascade of proteases may be involved. In contrast, proteolytic activity in the present study was only inhibited by pepstatin A. Furthermore, in contrast to the present study, the proteolytic activity in media conditioned by NIH-3T3 cells was inhibited by pretreatment of cells with IGF-II and resulted in an IGFBP-6 fragment of ~17 kDa. For all these reasons, the proteolytic activity described in the present study differs from that secreted by NIH-3T3 cells.
Cathepsin D is a protease that undergoes pH-dependent, intramolecular proteolytic autoactivation (31) and is able to proteolyze a wider variety of proteins, including osteocalcin, hemoglobin, cartilage proteoglycans, myofibrillar proteins (28), and (of particular relevance to the present study) IGF-II (9). Although cathepsin D is primarily an intracellular lysosomal enzyme (31), 2-20% of total procathepsin D is secreted into conditioned media by normal cells, whereas cancer cells secrete up to 60% (8). Increased secretion of this enzyme has been reported in breast cancer cells and may be correlated with metastatic potency (5, 32).
Cathepsin D is expressed in skin and may have a role in the spread of squamous cell carcinomas. A predominantly intermediate, inactivated form of cathepsin D is abundant in the basal and spinous layers of normal skin (20), and it may have a role in cornification. Up to 70% of primary squamous cell carcinomas express mature, active cathepsin D, especially in the outer part of the tumor mass, suggesting that this protease may be involved in local invasion (19, 20). This contention is further supported by the observation that 100% of secondary squamous cell carcinoma deposits express cathepsin D (19). The extracellular role of secreted cathepsin D has been questioned because of the requirement for an acidic environment. However, activation of cathepsin D may result from enzymatic cleavage as well as autoactivation at low pH (30), which might allow the involvement of cathepsin D in proteolysis of extracellular matrix.
Cathepsin D was initially reported to cleave IGFBP-3 (12). Subsequently, Claussen et al. (9) showed that IGFBPs 1-5, but not IGFBP-6, were proteolyzed by purified cathepsin D. However, these studies were performed with recombinant IGFBP-6 expressed in yeast, whereas the preparation used in the present study was expressed in mammalian cells. Yeast-derived IGFBP-6 is not glycosylated to the same extent as mammalian-derived IGFBP-6 (21), and the carbohydrate chains are likely to differ from those in mammalian glycoproteins. For example, yeast and mammalian-derived Fc epsilon RII receptors have distinct and different sites of O-glycosylation (17). Because glycosylation may alter the extent and sites of proteolysis by cathepsin D, as demonstrated for IGFBP-3 (9), this may explain the difference between the findings in that paper and in the present study. Alternatively, the protease identified in the present study may not be cathepsin D but an as-yet-unidentified protease with similar pH and protease inhibition specificities.
It has been reported that IGFBP-6 is an autocrine growth inhibitor of HaCaT keratinocytes (18). However, proliferation assays in that study were performed in the presence of serum, which is a potential source of exogenous IGFs to which HaCaT keratinocytes are responsive (41). The present study demonstrated that these cells synthesize IGF-II and not IGF-I. A likely explanation for the inhibition of proliferation by IGFBP-6 is therefore inhibition of autocrine IGF-II effects, because IGFBP-6 inhibits IGF-II actions in other cell lines such as osteoblasts and myoblasts (1, 21, 34).
It has been postulated that cathepsin D facilitates tumor metastasis by degrading extracellular matrix, thereby allowing mobilization of tumor cells. The findings of the present study suggest an additional possibility, whereby proteolysis of IGFBP-6 by cathepsin D releases free IGF-II, which has been implicated as an autocrine growth factor in many tumor types (39), from inactive IGFBP/IGF complexes. Epidermal cells are responsive to IGF-II in vivo, as evidenced by keratinocyte hyperplasia in mice overexpressing IGF-II in skin under the influence of a keratin promoter (40). Furthermore, an increased incidence of squamous cell carcinomas was observed after 18 mo in mice overexpressing IGF-II (33).
In summary, we have demonstrated that HaCaT keratinocytes synthesize IGF-II, IGFBP-6, and a cathepsin D-like protease with activity against this binding protein. Previous studies have shown that these cells also synthesize IGFBP-3 (41) and possess an active plasmin system that is capable of proteolyzing that IGFBP (42). These cells synthesize IGF-II and proliferate in response to exogenous IGFs (41), indicating that IGF-II acts potentially as an autocrine growth factor. IGFBP-6 is a relatively specific inhibitor of IGF-II, whereas IGFBP-3 binds IGF-I and IGF-II with approximately equal affinities (24) and may inhibit the effects of both of these ligands. Both IGFBPs may therefore inhibit the autocrine IGF-II activity, whereas IGFBP-3 may additionally inhibit the effects of exogenous IGF-I such as that synthesized by melanocytes and dermal fibroblasts in vivo (38). The presence of these components of the IGF system provides the potential for precise regulation of IGF activity in skin, which may be important for the maintenance of physiological function and the prevention of proliferative disorders.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the iodination of IGFBP-6 and IGF-II by David Casley.
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FOOTNOTES |
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This work was supported by grants from the National Health and Medical Research Council of Australia and the Austin Hospital Medical Research Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests: L. Bach, Univ. of Melbourne, Dept. of Medicine, Austin & Repatriation Medical Centre (Austin Campus), Studley Rd., Heidelberg, Victoria 3084, Australia.
Received 23 July 1998; accepted in final form 11 November 1998.
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