Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M202335200 on April 24, 2002

J. Biol. Chem., Vol. 277, Issue 30, 26872-26878, July 26, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/30/26872    most recent
M202335200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ruehl, M.
Right arrow Articles by Schuppan, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ruehl, M.
Right arrow Articles by Schuppan, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Epithelial Mitogen Keratinocyte Growth Factor Binds to Collagens via the Consensus Sequence Glycine-Proline-Hydroxyproline*

Martin RuehlDagger, Rajan SomasundaramDagger, Ines Schoenfelder, Richard W. Farndale§, C. Graham Knight§, Monika Schmid, Renate Ackermann, Ernst Otto Riecken, Martin Zeitz, and Detlef Schuppan||

From the Department of Medicine I, Klinikum Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, 12200 Berlin, Germany, the § Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom, and the  Department of Medicine I, University of Erlangen-Nuernberg, Ulmenweg 18, 91054 Erlangen, Germany

Received for publication, March 11, 2002, and in revised form, April 22, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that mesenchymal cell-derived keratinocyte growth factor (KGF), a key stimulator of epithelial cell proliferation during wound healing, preferentially binds to collagens I, III, and VI. Binding is inhibited in a dose-dependent manner by denatured single collagen chains and collagen cyanogen bromide peptides. This interaction is saturable with dissociation constants of ~ 10-8 to 10-9 M and estimated molar ratios of up to three molecules of KGF bound to one molecule of triple helical collagen. Furthermore, collagen-bound KGF stimulated the proliferation of transformed keratinocyte or HaCaT cells. Ligand blotting of collagen-derived peptides points to a limited set of collagenous consensus sequences that bind KGF. By using synthetic collagen peptides, we defined the consensus sequence (Gly-Pro-Hyp)n as the collagen binding motif. We conclude that the preferential binding of KGF to the abundant collagens leads to a spatial pattern of bioavailable KGF that is dictated by the local organization of the collagenous extracellular matrix. The defined collagenous consensus peptide or its analogue may be useful in wound healing by increasing KGF bioactivity and thus modulating local epithelial remodeling and regeneration.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the past years, components of the extracellular matrix including collagens were shown to interact with several growth factors and cytokines, thus modulating their local availability and biological activity (1-5). We were able to demonstrate specific interactions of platelet-derived growth factor (forms AA, BB, and AB), hepatocyte growth factor (HGF),1 interleukin 2, and oncostatin M with collagens (6-9). Interestingly, the biological activity of collagen-bound platelet-derived growth factor, HGF, interleukin 2, and oncostatin M was not abolished by binding to collagens, suggesting that these abundant matrix proteins may represent an important biological reservoir for these growth factors.

Keratinocyte growth factor (KGF)/fibroblast growth factor 7 (FGF-7) is a highly specific mitogen for various epithelial cells. KGF promotes proliferation and migration and was found to induce angiogenesis and stabilize endothelial barriers (10, 11). Therefore, KGF plays an important role in cutaneous wound healing, for example, and in regeneration of gastric and intestinal epithelium after injury (10, 12-14). KGF is synthesized by various types of mesenchymal cells such as lung, dermal, or gastrointestinal fibroblasts and myofibroblasts located predominantly in the subepithelial connective tissues (10, 15, 16), but it has never been detected in epithelial cells. However, many epithelia including dermal and gastrointestinal epithelial cells express the FGF receptor 2-IIIb, the only known high affinity receptor for KGF, explaining their responsiveness to this epithelial mitogen (17-20). In vitro and in vivo studies show a beneficial or protective effect of KGF on cutaneous wound healing (10), lung injury (21), experimental colitis (22), cyclophosphamide-induced cystitis (23), and gastric wound healing (13, 16, 24, 25). In line with these findings are clinical trials with FGF-10 for wound healing and treatment of mucositis caused by cancer therapy (26-29). FGF-10, also termed KGF-2, is closely related to KGF/FGF-7 in structure (57% sequence homology) and activity and binds to the same receptor (FGF receptor 2-IIIb), underlining the therapeutic potential of this group of growth factors (30).

The known interactions of the FGFs with heparin or heparan sulfate moieties of cell membranes and extracellular proteoglycans can differentially modulate their activities. For example, heparan sulfate proteoglycans potentiate the biological activity of FGF-1 but strongly inhibit the activity of KGF/FGF-7 (31).

Here we describe the specific interaction of KGF/FGF-7 predominantly with the abundant collagens I, III, and VI and their constituent chains. We define a minimal consensus collagen binding motif for KGF, study the effect of collagen-bound KGF in cell culture, and discuss the implications of this interaction for wound healing and epithelial regeneration.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Human recombinant KGF (163 amino acids) was purchased from Biomol (carrier-free, No. 51566, Hamburg, Germany), and recombinant hepatocyte growth factor was purchased from R&D Systems (carrier-free, No. 294-HG, Wiesbaden-Nordenstadt, Germany). All other reagents were from either Merck or Sigma and were of the highest purity available. Polystyrene microtiter plates (Immulon 2, Removawells) were from Dynatech (Hamburg, Germany). Cell culture experiments were done in Falcon 96-well plates (Falcon, BD Biosciences GmbH, Heidelberg, Germany).

Native type I, III, IV, and VI collagens were isolated from human placenta or skin, and type II collagen was purified from human articular cartilage. Collagen purification and the biochemical modifications of collagen chains were performed as described previously (6-9). Cyanogen bromide (CNBr) peptides were prepared by dissolving 2 mg of single collagen chains in 1 ml of 70% formic acid at room temperature, flushing the tube for 10 min with nitrogen, and adding 2 mg of CNBr followed by a 4-h incubation at 37 °C and lyophilization (32). These peptides were purified using gel filtration and ion-exchange fast protein liquid chromatography.

The collagen mimetics H-Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly-NH2 ((GPO)10), H-Gly-Cys-Pro-(Gly-Pro-Pro)10-Gly-Cys-Pro-Gly-NH2 ((GPP)10), H-Gly-Pro-Cys-(Gly-Pro-Pro)5-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Pro)5-NH2 (GFOGER-GPP), and H-Gly-Ala-Cys-(Gly-Ala-Pro)5-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Ala-Pro)5-NH2 (GFOGER-GAP) were synthesized as described previously (33, 34). Their spontaneous assembly into triple helices was demonstrated by determining melting curves by polarimetry. At a concentration of 5 mg/ml, the midpoints of the melting curves occurred at 82.3 ± 1.4 °C for (GPO)10, 45.8 ± 0.8 °C for (GPP)10, and 44.3 ± 0.3 °C for GFOGER-GPP. The peptide GFOGER-GAP was non-helical even at 5 °C.2

Methods

Immobilization of Collagens and Collagen Peptides-- The coating of microtiter plates and calculation of coating efficiencies were performed as described previously (7, 8). Native collagens, collagen chains, and CNBr peptides were immobilized on polystyrene microtiter wells at concentrations of 2 µg/100 µl/well and 300-600 ng/100 µl/well, respectively, for binding studies and at 10-fold lower concentrations for inhibition experiments. Immobilization was done in 50 mM ammonium bicarbonate, pH 9.6, overnight at 4 °C followed by three washes with phosphate buffered saline (PBS), pH 7.4. Nonspecific binding sites were blocked with PBS containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaureate) for 1 h at room temperature for binding studies and with PBS, 0.5% bovine serum albumin (BSA) for inhibition studies. Coating efficiencies for 2 µg/well native collagens and collagen chains ranged between 21 and 48% (7, 8).

Radiolabeling and KGF Binding Assay-- KGF was radiolabeled with the [125I]Bolton-Hunter reagent (PerkinElmer Life Sciences) according to the manufacturer's recommendations. [125I]KGF was separated from free iodine by a Sepharose G25 column (PD10, Amersham Biosciences) in PBS containing 0.05% Tween 20 as described previously (7, 8). Incorporated radioactivity ranged between 20,000 and 30,000 cpm/ng [125I]KGF. The precipitation with trichloroacetic acid (10% v/v) in the presence of 200 µg of BSA/200 µl usually yielded 90-96% of protein-bound radioactivity. The purity of radiolabeled KGF was demonstrated by SDS-PAGE and autoradiography (data not shown).

For binding studies, 1-2 ng of [125I]KGF in 100 µl of PBS, 0.05% Tween 20 was added to the collagen-coated wells and incubated for 2 h at room temperature, and finally after three washes in binding buffer (PBS, 0.05% Tween 20), radioactivity bound to the collagen-coated wells was measured in a gamma -counter (Berthold, Bad Wildbach, Germany).

Ligand Blot-- For ligand blots 2 µg of collagen I, single collagen chains alpha 1(I), alpha 2(I), CNBr peptides of chain alpha 1(I), and pepsin-resistant triple helical fragments of collagens IV and VI were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The blots were blocked with PBS, 0.3% Tween 20 overnight at 4 °C, washed three times in binding buffer, and incubated with approximately 50 ng of [125I]KGF diluted in 10 ml of binding buffer (100,000 cpm/ml) for 2 h at room temperature followed by three washes with binding buffer before air-drying and autoradiography. As a control, a parallel blot was stained with Amido Black after electrophoretic transfer.

Dot Blot-- For dot blots, serial dilutions of collagen I, chain alpha 1(I), CNBr peptide alpha 1CB6, and the collagen mimetics (GPO)10, (GPP)10, GFOGER-GPP, and GFOGER-GAP were immobilized on nitrocellulose membranes at concentrations of 0.02-4 µg/dot. BSA was used as a negative control. Blocking, incubation, and autoradiography were done as described for the ligand blots.

Inhibition Experiments-- 1-2 ng of [125I]KGF and increasing concentrations (0, 0.01, 0.1, 1, and 10 µg/100 µl) of single chains of collagen types I and VI, CNBr peptides of collagen type I, collagen mimetics ((GPO)10, (GPP)10, GFOGER-GPP, and GFOGER-GAP)), high molecular weight heparin, or hepatocyte growth factor (0-200 ng/100 µl) were preincubated in a total volume of 350 µl for 2 h at room temperature in detergent-blocked polypropylene tubes. 100 µl of the mixture was then added in triplicate to microtiter wells precoated with collagen or collagen chains. After an additional 2 h of incubation and three washes with PBS, bound radioactivity was measured as described above.

Saturation Binding Experiments-- For saturation binding studies, increasing amounts of unlabeled KGF (0-300 ng) were added to 2 ng of the labeled growth factor in a final volume of 100 µl of binding buffer and incubated for 2 h at room temperature in microtiter wells, which were precoated with 200 ng/100 µl/well of native triple helical collagens. Bound [125I]KGF was determined as described above after subtraction of the radioactivity bound to BSA-coated wells, which ranged from 8 to 17%.

Influence of Osmolality on Binding of KGF to Collagens-- Collagens IV and VI, the alpha 1 and alpha 2 chains of collagen type I, and the CNBr peptides of alpha 2(I) were immobilized at 2 µg/100 µl/well on microtiter plates and incubated with 1 ng/100 µl of 125I-labeled KGF under the following conditions. Solutions were adjusted by increasing amounts of NaCl (50-1500 mmol/liter) in a buffer of 10 mmol/liter Tris-HCl, 0.05% Tween 20, pH 7.4, resulting in osmolalities between 120 and 3020 mosM. The binding of [125I]KGF to precoated collagens was performed as described for inhibition experiments.

Human Keratinocytes (HaCaTs) Biological Activity Assay-- To determine the biological activity of collagen-bound KGF, a modified KGF bioassay was used. Spontaneously immortalized HaCaTs, kindly provided by N. Fusenig (Heidelberg, Germany), were cultured in 80-cm2 flasks containing Dulbecco's modified Eagle's medium with 2 mM glutamine supplemented with penicillin (107 units/liter), streptomycin (10 mg/liter), and 10% fetal calf serum (Biochrom, Berlin, Germany) under standardized conditions (37 °C, 8% CO2) in a humidified atmosphere. The alpha 1 chain of collagen type I was coated on microtiter plates (Falcon, BD Biosciences GmbH, Heidelberg, Germany) at a concentration of 2 µg/100 µl/well (0.3 cm2) overnight at 4 °C. Wells were blocked with 2% BSA in PBS for 1 h followed by extensive washing with PBS, 0.05% Tween 20. KGF in PBS was added to the wells at increasing concentrations and incubated for 2 h at room temperature. After 2 h, unbound KGF was removed by washing with PBS, 0.05% Tween 20 followed by three washes with PBS. 100 µl of trypsinized HaCaT cells in the logarithmic growth phase (100,000 cells/ml medium) was plated on the collagen-coated wells to which different amounts of KGF had been bound. Soluble KGF added to already seeded HaCaT cells served as a positive control. Cells were then cultured for 72 h, and cell numbers were measured by a colorimetric assay, sulforhodamine B (SRB), as described previously (35).

Statistical Analysis-- Binding data are expressed as mean ± S.E. Dissociation constants and the number of binding sites obtained by saturation experiments were analyzed according to the method of Scatchard (6-9).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

KGF Binds to Native Collagens I-VI, Single Collagen Chains, and Chain Fragments-- Radiolabeled KGF bound specifically to all immobilized native and heat-denatured collagens tested. The binding to collagens I, II, and IV and single chains of collagens I, III, and IV ranged between 7 and 11% after the subtraction of nonspecifically bound radioactivity and reached 16-27% for collagens III and VI and single chains of collagen VI. KGF also bound to immobilized CNBr peptides of alpha 1(I) (in the order CB8 right-arrow CB6 right-arrow CB7 right-arrow CB3) and to the CNBr peptides of alpha 2(I) (Fig. 1).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1.   KGF binds to immobilized native collagens, to single collagen chains and to cyanogen bromide peptides of collagen I. Native (triple helical) collagens I, II, III, IV, and VI, single chains of collagens I (alpha 1, alpha 2), III, IV, and VI after reduction and alkylation (ra), and collagen CNBr peptides (CB) were immobilized on polystyrene microtiter wells followed by incubation with 1-2 ng of radiolabeled KGF. Detergent-blocked polystyrene served as control (P). After three washes, bound radioactivity was measured (expressed as percent of the initially added radioactivity). Shown are the means ± S.E. of at least five independent experiments performed in triplicate. alpha 1(I)CB and alpha 2(I)CB are the mixture of CNBr peptides of alpha 1(I); alpha 2(I) chains, CB3, CB6, CB7, and CB8 are purified CNBr peptides of alpha 1(I).

Ligand Blot-- KGF binding was confirmed by ligand blotting. As shown in Fig. 2, [125I]KGF highlighted the chains of collagens I, IV, and VI after separation by SDS-PAGE and electrophoretic transfer to nitrocellulose membrane. In comparison to protein staining, the autoradiography showed strong binding to all chains and CNBr peptides but reduced the binding to alpha 1(I)CB3 and alpha 1(VI), for example, supporting the results of the microtiter well assays.


View larger version (76K):
[in this window]
[in a new window]
  		Fig. 2.   Binding of KGF to collagens and collagen peptides after transfer to nitrocellulose membranes. Reduced and alkylated collagens I and IV, the chains of collagens I and VI, and the CNBr peptides of alpha 1(I) (2 µg/lane) were separated by SDS-PAGE and blotted to nitrocellulose membranes in duplicate. Blocking of unspecific binding sites was followed by either protein staining with Amido Black or incubation with 50 ng of [125I]KGF/10 ml for 2 h, washing, and autoradiography. Molecular masses (in kDa) of the CNBr peptides or of the pepsin-derived fragments are as follows: alpha 1(I), 98; alpha 2(I), 96; alpha 3(VI) long, 62; alpha 1(VI), 54; alpha 2(VI), 52; alpha 3(VI) short, 50; alpha 1(I)CB7, 30; alpha 1(I)CB8, 28; alpha 1(I)CB6, 20; and alpha 1(I)CB3, 14. Shown is a representative blot.

Because these results suggested common or similar binding sites on the collagens under investigation and because most of the collagen fragments bound KGF, we used minimal collagen mimetics with and without the characteristic collagenous triple helical structure. A dot blot analysis revealed strong binding to the minimal triple helical peptide (GPO)10, whereas (GPP)10 showed reduced but still detectable binding (Fig. 3). However, collagen mimetics containing an inserted sequence (GFOGER-GPP) or alanine-residues (GFOGER-GAP) that disrupt the triple helical structure did not interact with KGF. These results suggest that between 5 and 10 triplets of the structures (GPO) and (GPP) are essential for KGF binding, although binding is especially favored by having hydroxyproline in the X' position of the (GXX')n-collagenous structure.


View larger version (78K):
[in this window]
[in a new window]
  		Fig. 3.   Binding of KGF to synthetic collagen mimetics. Increasing amounts of collagen I (CI), chain alpha 1(I), peptide alpha 1(I)CB6, collagen mimetics ((GPO)10, (GPP)10, GFOGER-GPP, and GFOGER-GAP), and BSA were immobilized on nitrocellulose membranes. Unoccupied binding sites were blocked with 0.3% Tween 20 in PBS and incubated for 2 h with 50 ng of [125I]KGF/10 ml followed by three washes with PBS, air drying, and autoradiography.

Collagen Peptides and Collagen Mimetics Inhibit Binding of KGF to Immobilized Collagens-- To further prove specificity of the KGF-collagen interaction, inhibition experiments were performed. The binding of radiolabeled KGF to "immobilized" collagens, collagen chains, or collagen fragments (CNBr peptides) could be inhibited by "soluble" collagen chains (Fig. 4, A and B), CNBr peptides (Fig. 4C), or collagen mimetics (Fig. 4D). As shown in Fig. 4A, the soluble alpha 1(I) chain could strongly inhibit KGF binding to the immobilized alpha 1(I) chain with half-maximal inhibition at a 1:1 molar ratio, taking into account a coating efficiency of 40%. To further define the KGF binding sequences on the alpha 1(I) chain, alpha 1(I)CNBr fragments were used in inhibition experiments, which demonstrated primarily alpha 1(I)CB6 and alpha 1(I)CB8 as inhibitors of KGF binding to alpha 1(I) (Fig. 4C). The binding to collagen VI and the inhibition of KGF binding to immobilized collagen I chains, collagen IV, and collagen VIr/a by soluble collagen VI chains (Fig. 4B, CVI r/a) demonstrate the cross-inhibitory potential of different collagens and chains. In line with the binding assays, the collagen mimetics (GPO)10 and (GPP)10 were the best inhibitors of KGF in this setting, whereas GFOGER-GPP had a somewhat lower inhibitory potential and non-helical control GFOGER-GAP had no inhibitory potential (Fig. 4D). Similarly, when KGF was reacted with immobilized alpha 1(I)CB6, (GPO)10 was the best inhibitor (data not shown).


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4.   Single chains of collagens I and VI, alpha 1(I)CB peptides, and collagen mimetics inhibit binding of KGF to various immobilized collagens and collagen chains. 1-2 ng of [125I]KGF were preincubated with increasing amounts of the soluble alpha 1(I) chain (A), reduced and alkylated collagen VI (B), CNBr peptides of alpha 1(I) (C), or collagen mimetics ((GPO)10, (GPP)10, GFOGER-GPP, and GFOGER-GAP) (D) followed by the addition to various collagens and collagen chains immobilized at 200 ng/well for 2 h and the determination of bound radioactivity. Binding is expressed as the percentage of bound radioactivity in the presence of inhibitor relative to the bound radioactivity in the absence of inhibitor. Shown are the results representative of four experiments performed in triplicate.

Saturation Binding Studies and Estimated Affinities of the KGF-Collagen Interaction-- To determine the binding affinities of the KGF-collagen interaction, saturation binding studies were performed. Increasing amounts of unlabeled KGF were incubated with a constant amount of [125I]KGF (~1 ng = 0.04 pmol/well), reaching a saturation of 5-7 pmol of added KGF/100 µl on 200 ng/well (~0.65 pmol) of immobilized collagen types I (Mr = ~300,000) (Fig. 5A), III (Mr = ~300,000) (Fig. 5B), and VI (Mr = ~320,000) (Fig. 5C) with preestablished coating efficiencies between 30 and 40%. Scatchard analysis yielded binding sites of comparable affinity on the tested collagens with dissociation constants (KD) between 10-8 and 10-9 mol/liter. Based on these data, 1 M immobilized native interstitial collagens I or III was estimated to bind approximately 1 M KGF, microfibrillar collagen VI, and 3 M KGF.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   Binding of KGF to immobilized collagens follows saturation kinetics. Increasing amounts of unlabeled KGF were added to a constant amount (1 ng) of labeled KGF followed by incubation for 2 h with immobilized collagen I (A), collagen III (B), or collagen VI (C) immobilized at 200 ng/100 µl/well. Calculation of binding sites was performed as described under "Experimental Procedures." Dissociation constants (KD) of the KGF-collagen interactions were determined graphically by the method of Scatchard (insets), yielding low capacity/high affinity and high capacity/low affinity KD values ~10-9 and 10-8 M, respectively. The number of KGF molecules bound per collagen molecules was calculated to be between 1 and 3. Shown are the results of one of three representative experiment(s) performed in triplicate.

Partial Inhibition of the KGF-Collagen Interaction by Heparin-- KGF binding to collagens I, IV, and VI could be partly inhibited by preincubation with heparin (Fig. 6A). KGF as a heparin binding growth factor could not be displaced completely from collagen by heparin with still 50-70% KGF bound at maximal heparin concentrations (10 µg/100 µl/well). In comparison to the inhibition by collagen mimetics that left only 30% KGF bound (Fig. 4D), these data suggest collagen-binding domains on KGF that are different from the heparin binding region.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 6.   A, partial inhibition of KGF binding to collagens I, IV, and VI by heparin. Binding of KGF to different immobilized collagens was inhibited by preincubation with increasing amounts of heparin in analogy to the inhibition experiments with collagen chains (Fig. 4). Even with maximal heparin concentrations, KGF binding to collagens was inhibited only by 50%. B, competition of KGF binding to collagens by HGF. 2 ng of [125I]KGF were preincubated with increasing concentrations (0-100 ng/100 µl) of HGF and then added to collagens I, III, or VI immobilized at 200 ng/well for another 2 h followed by determination of bound radioactivity. Binding is expressed as the percentage of bound radioactivity in the presence of inhibitor relative to the bound radioactivity in the absence of inhibitor. For the different collagens, the molar excess of HGF over KGF at half-maximal inhibition (17- and 27-fold) is indicated. Shown are the results of one of three representative experiment(s) performed in triplicate. C, influence of osmolality on KGF binding to collagens. Collagens IV and VI, the collagen chains alpha 1(I) and alpha 2(I), and the CNBr peptides of alpha 2(I) were immobilized at 2 µg/well and incubated with 1 ng/100 µl [125I]KGF at increasing osmolality. Uncoated wells were used as control. After a 2-h incubation, bound radioactivity was determined.

Competition of KGF Binding to Collagens by HGF-- Because we previously showed that HGF is a collagen binding as well as a heparin binding growth factor, we investigated the competition of HGF and KGF for binding to collagens type I, III, and VI. As indicated in Fig. 6B, a 17-27-fold molar excess of HGF over KGF resulted in 50% inhibition of KGF binding to collagens I, III, and VI. Maximal inhibition (~90%) of KGF binding to collagens type I and III was achieved by a 100-120-fold molar excess of HGF over KGF, whereas only 50% of the microfilamentous collagen VI could be displaced by an even 140-fold molar excess of HGF. These results clearly point to consensus binding sites for both cytokines on interstitial collagens I and III, whereas the microfibrillar collagen VI may contain additional binding sites.

Influence of Osmolality of KGF Binding to Collagens-- Fig. 6C demonstrates that KGF binding to collagens and collagen chains can be disrupted by ionic forces. The binding was reduced to 50% at osmolalities of ~100-150 mosM for collagen IV and the alpha 1(I) chain and at 200 mosM for the alpha 2(I) chain, whereas 250-300 mosM were needed for a 50% reduction of binding to collagen VI and the CNBr peptides of alpha 2(I). Background levels for all collagens were reached at 1500 mosM.

Collagen-bound KGF Is Biologically Active-- Collagen-bound KGF induced a strong proliferative response on HaCaT keratinocytes (Fig. 7), reaching maximal stimulation when 25-30 pmol of KGF had been preincubated in alpha 1(I)-coated wells. Because ~12% preincubated KGF was bound to the alpha 1(I) chain under these conditions (Fig. 1), the biological activity of collagen-bound KGF was equivalent to the activity of the same amount of KGF in solution (Fig. 7).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 7.   Collagen-bound KGF is biologically active. The collagen chain alpha 1(I) was coated on microtiter wells at a concentration of 2 µg/100 µl/well and blocked with 1% BSA in PBS, 0.05% Tween 20. KGF in PBS was added at increasing concentrations and incubated for 2 h at room temperature. After 2 h, unbound KGF was removed by washing with PBS/Tween followed by three washes with PBS. 100 µl of trypsinized HaCaT cells in the logarithmic growth phase (100,000 cells/ml) were plated in medium on collagen-coated wells, which had been preincubated with different concentrations of KGF. In parallel, soluble KGF added to the HaCaT cells served as positive control (see inset). Cells were then cultured for 72 h, and the cell number was determined by a colorimetric cytotoxicity assay after trichloroacetic acid fixation and staining with sulforhodamine B. Shown are results of two experiments performed in triplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We demonstrated that KGF binds to immobilized collagens in the order type VI right-arrow III right-arrow I right-arrow II right-arrow IV in vitro. Furthermore, collagen-bound KGF is biologically active as shown by HaCaT proliferation assay. The interaction of KGF with native and denatured collagens could be inhibited by single collagen chains and smaller collagen chain fragments. Saturation binding experiments yielded dissociation constants of approximately 10-8 to 10-9 M, which are in the range of other growth factor collagen interactions (6-9) as well as in the range of the affinity of KGF for its receptor (10-8 to 10-9 M) (36, 37). The disruption of the interaction by an increase in osmolality shows that this binding is mediated mainly by ionic forces, which has been demonstrated for other collagen binding (7, 8). 1 M of immobilized native interstitial collagens type I and III was estimated to bind approximately 1 M KGF and microfibrillar collagen type VI, and 3 M KGF, respectively. However, this is very likely an underestimation of the available binding sites in vivo, because experiments were performed with collagens immobilized on plastic, which may limit accessibility.

Cross-inhibition and ligand blot experiments suggested collagenous consensus binding motifs for KGF. This was proven by using synthetic collagen peptides containing the sequences (GPO)10 and (GPP)10 that spontaneously form a collagen-like triple helix (33, 34). The preferred binding of KGF to (GPO)10 over (GPP)10 indicates that the hydroxyl group of hydroxyproline makes an important contribution to the interaction. The slightly weaker interaction with GFOGER-GPP, a triple helical peptide with a run of 10 GPP triplets interrupted by an integrin binding motif (34), suggests that a minimal number of "consecutive" GPP/GPO stretches is necessary for the KGF-collagen interaction. Binding does not occur to the analogous non-helical peptide GFOGER-GAP, suggesting that the native triple helical structure defined by stretches of GPP/GPO is necessary for effective binding of KGF to collagen. As shown in Fig. 2, the cyanogen bromide peptides alpha 1(I)CB6, CB7, and CB8 but not alpha 1(I)CB3 bound KGF. Upon closer inspection, the sequence of alpha 1(I)CB3 comprises only four isolated GPP or GPO triplets compared with 12, 12, and 9 triplets partly in sequence for CB6, CB7, and CB8, respectively. The peptide alpha 1(I)CB6 harbors a stretch with more than two triplets of GPP or GPO (five in sequence), whereas alpha 1(I)CB7 and alpha 1(I)CB8 contain only one or two GPP or GPO triplets. Thus, the larger number of GPP/GPO motifs in CB6, CB7, and CB8 suggests that even in the longer CNBr peptides (149, 264, 271, and 279 amino acids for peptides CB3, CB6, CB7, and CB8, respectively), a minimal number of sequential GPP or GPO triplets are required for binding of KGF.

KGF as a member of the FGF family binds also to heparin and heparan sulfate (31, 38, 39), which are involved in the interaction of KGF with its receptor FGF receptor 2-IIIb (37, 40). The strong support for heparin/heparan sulfate-independent binding of KGF to collagens apart from the proven purity of our preparations (6-9) is provided by our binding and inhibition data with the synthetic collagen mimetics, which do not contain heparan sulfate. Because heparin led to a 50% inhibition of the KGF-collagen interaction (Fig. 6A), a maximal binding to the extracellular matrix (and also the KGF receptor) appears to necessitate a combined KGF-collagen and KGF-heparin/heparan sulfate interaction. HGF, another mesenchyme-derived heparin as well as collagen binding epithelial growth factor, differs from KGF in that HGF-collagen binding is almost completely inhibited by heparin (8). Because an excess of HGF could partly displace KGF from collagen (Fig. 6B), both growth factors may have similar binding sites for heparin/heparan sulfate and collagens.

The binding and storage of biologically active KGF by the collagenous extracellular matrix as shown by our in vitro and cell culture experiments may play an important role in the local availability and activity of this growth factor. It is well documented that KGF is up-regulated in the mesenchyme-underlying areas of epithelial lesions, such as those occurring after skin injury, in the intestine, or the liver to promote epidermal, intestinal, and hepatic reepithelialization, wound healing, and regeneration (10, 12, 14, 25, 41). In support of this finding, the local application of KGF in several rodent models of gastrointestinal and lung injury (chemotherapy- and/or radiation-induced) has led to a significant reduction of mortality in KGF-treated animals (22, 42). In the intestine, this is accompanied by enhanced crypt cell proliferation and rapid reepithelization without scarring. Therefore, KGF may have therapeutic potential for the gastrointestinal tract and in a similar way for the liver, the lung, or skin (24, 27, 41). Especially in chronic lesions, e.g. from cutaneous wounds or Crohn's disease, where fibrogenesis, i.e. an up-regulation of extracellular matrix production, is observed (43), the binding of KGF to collagens may enhance (local) epithelial regeneration by its short term storage followed by release from abundant collagens, e.g. by matrix metalloproteases (MMPs). The activation of MMPs and their ability to cleave native and denatured collagens may enhance the availability of collagen-bound KGF, which as shown by our cell culture experiments, maintains its biological activity. In this context, it is of interest that KGF was shown to induce the production of MMP-1, MMP-9, and the plasminogen activator uPA in epithelial cell lines from human prostate or porcine periodontal ligament (44, 45) that may further increase its local release. Another more targeted approach to release KGF from the collagenous matrix may be the use of synthetic collagen mimetics based on the sequence (GPO)10.

In conclusion, our finding of the specific interaction of KGF with collagens via the binding motif (GPO)n opens a novel approach to enhance and modulate the local availability and activity of KGF at the site of active lesions. In vivo experiments are needed to show how far GPO-containing peptides can be used to promote epithelial wound healing in inflammatory and repair processes, such as that found in the damaged skin, the gastrointestinal tract, the liver, the lung, and other epithelial systems.

    ACKNOWLEDGEMENT

We thank Prof. N. Fusenig (Deutsches Krebs forschungs Zentrum, Heidelberg, Germany) for providing HaCaT cells.

    FOOTNOTES

* This study was supported in part by Grants Schu 646/1-10 and SFB366 C5/C10 from the Deutsche Forschungsgemeinschaft, a grant from the Interdisciplinary Center for Clinical Research by the University of Erlangen-Nuernberg, and by a program grant from the Medical Research Council (to C. G. K. and R. W. F.).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.

Dagger Both authors contributed equally to this work.

|| Recipient of a Hermann-and-Lilly-Schilling Professorship. To whom correspondence should be addressed: Medizinische Klinik I, University of Erlangen-Nürnberg Krankenhausstrasse 12, 91054 Erlangen, Germany. Tel.: 09131-853-3398/3386; Fax: 09131-853-6003; E-mail: detlef.schuppan@med1.imed.uni-erlangen.de.

Published, JBC Papers in Press, April 24, 2002, DOI 10.1074/jbc.M202335200

2 D. J. Olney, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HGF, hepatocyte growth factor; KGF, keratinocyte growth factor; FGF, fibroblast growth factor; CNBr or CB, cyanogen bromide; PBS, phosphate-buffered saline; BSA, bovine serum albumin; MMP, matrix metalloproteases; uPA, urokinase-type plasminogen activator; HaCaT, human keratinocytes; (GPO)10, (Gly-Pro-Hyp)10; (GPP)10, (Gly-Pro-Pro)10.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ruoslahti, E., and Yamaguchi, Y. (1991) Cell 64, 867-869[CrossRef][Medline] [Order article via Infotrieve]
2. Taipale, J., and Keski-Oja, J. (1997) FASEB J. 11, 51-59[Abstract]
3. Vaday, G. G., and Lider, O. (2000) J. Leukocyte Biol. 67, 149-159[Abstract]
4. Davis, G. E., Bayless, K. J., Davis, M. J., and Meininger, G. A. (2000) Am. J. Pathol. 156, 1489-1498[Abstract/Free Full Text]
5. Schuppan, D., Ruehl, M., Somasundaram, R., and Hahn, E. G. (2001) Semin. Liver Dis. 21, 351-372[CrossRef][Medline] [Order article via Infotrieve]
6. Somasundaram, R., Ruehl, M., Tiling, N., Ackermann, R., Schmid, M., Riecken, E. O., and Schuppan, D. (2000) J. Biol. Chem. 215, 38170-38175
7. Somasundaram, R., and Schuppan, D. (1996) J. Biol. Chem. 271, 26884-26891[Abstract/Free Full Text]
8. Schuppan, D., Schmid, M., Somasundaram, R., Ackermann, R., Ruehl, M., Nakamura, T., and Riecken, E. O. (1998) Gastroenterology 114, 139-152[CrossRef][Medline] [Order article via Infotrieve]
9. Somasundaram, R., Ruehl, M., Schaefer, B., Schmid, M., Ackermann, R., Riecken, E. O., Zeitz, M., and Schuppan, D. (2002) J. Biol. Chem. 277, 3242-3246[Abstract/Free Full Text]
10. Werner, S. (1998) Cytokine Growth Factor Rev. 9, 153-165[CrossRef][Medline] [Order article via Infotrieve]
11. Gillis, P., Savla, U., Volpert, O. V., Jimenez, B., Waters, C. M., Panos, R. J., and Bouck, N. P. (1999) J. Cell Sci. 112, 2049-2057[Abstract]
12. Housley, R. M., Morris, C. F., Boyle, W., Ring, B., Biltz, R., Tarpley, J. E., Aukerman, S. L., Devine, P. L., Whitehead, R. H., and Pierce, G. F. (1994) J. Clin. Invest. 94, 1764-1777[Medline] [Order article via Infotrieve]
13. Zeeh, J. M., Procaccino, F., Hoffmann, P., Aukerman, S. L., McRoberts, J. A., Soltani, S., Pierce, G. F., Lakshmanan, J., Lacey, D., and Eysselein, V. E. (1996) Gastroenterology 110, 1077-1083[CrossRef][Medline] [Order article via Infotrieve]
14. Playford, R. J., Marchbank, T., Mandir, N., Higham, A., Meeran, K., Ghatei, M. A., Bloom, S. R., and Goodlad, R. A. (1998) J. Pathol. 184, 316-322[CrossRef][Medline] [Order article via Infotrieve]
15. Rubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., and Aaronson, S. A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 802-806[Abstract/Free Full Text]
16. Finch, P. W., and Cheng, A. L. (1999) Gut 45, 848-855[Abstract/Free Full Text]
17. Tanahashi, T., Suzuki, M., Imamura, T., and Mitsui, Y. (1996) J. Biol. Chem. 271, 8221-8227[Abstract/Free Full Text]
18. Ueda, T., Sasaki, H., Kuwahara, Y., Nezu, M., Shibuya, T., Sakamoto, H., Ishii, H., Yanagihara, K., Mafune, K., Makuuchi, M., and Terada, M. (1999) Cancer Res. 59, 6080-6086[Abstract/Free Full Text]
19. Jang, J. H., Shin, K. H., and Park, J. G. (2001) Cancer Res. 61, 3541-3543[Abstract/Free Full Text]
20. La Rosa, S., Uccella, S., Erba, S., Capella, C., and Sessa, F. (2001) Appl. Immunohistochem. Mol. Morphol. 9, 319-328[CrossRef][Medline] [Order article via Infotrieve]
21. Deterding, R. R., Havill, A. M., Yano, T., Middleton, S. C., Jacoby, C. R., Shannon, J. M., Simonet, W. S., and Mason, R. J. (1997) Proc. Assoc. Am. Physicians 109, 254-268[Medline] [Order article via Infotrieve]
22. Farrell, C. L., Bready, J. V., Rex, K. L., Chen, J. N., DiPalma, C. R., Whitcomb, K. L., Yin, S., Hill, D. C., Wiemann, B., Starnes, C. O., Havill, A. M., Lu, Z. N., Aukerman, S. L., Pierce, G. F., Thomason, A., Potten, C. S., Ulich, T. R., and Lacey, D. L. (1998) Cancer Res. 58, 933-939[Abstract/Free Full Text]
23. Ulich, T. R., Whitcomb, L., Tang, W., O'Conner Tressel, P., Tarpley, J., Yi, E. S., and Lacey, D. (1997) Cancer Res. 57, 472-475[Abstract/Free Full Text]
24. Potten, C. S., O'Shea, J. A., Farrell, C. L., Rex, K., and Booth, C. (2001) Cell Growth Differ. 12, 265-275[Abstract/Free Full Text]
25. Finch, P. W., Pricolo, V., Wu, A., and Finkelstein, S. D. (1996) Gastroenterology 110, 441-451[CrossRef][Medline] [Order article via Infotrieve]
26. Igarashi, M., Finch, P. W., and Aaronson, S. A. (1998) J. Biol. Chem. 273, 13230-13235[Abstract/Free Full Text]
27. Xia, Y. P., Zhao, Y., Marcus, J., Jimenez, P. A., Ruben, S. M., Moore, P. A., Khan, F., and Mustoe, T. A. (1999) J. Pathol. 188, 431-438[CrossRef][Medline] [Order article via Infotrieve]
28. Jimenez, P. A., and Rampy, M. A. (1999) J. Surg. Res. 81, 238-242[CrossRef][Medline] [Order article via Infotrieve]
29. Han, D. S., Li, F., Holt, L., Connolly, K., Hubert, M., Miceli, R., Okoye, Z., Santiago, G., Windle, K., Wong, E., and Sartor, R. B. (2000) Am. J. Physiol. 279, G1011-G1022[Abstract/Free Full Text]
30. Haseltine, W. A. (2001) J. Am. Acad. Dermatol. 45, 473-475[CrossRef][Medline] [Order article via Infotrieve]
31. Berman, B., Ostrovsky, O., Shlissel, M., Lang, T., Regan, D., Vlodavsky, I., Ishai-Michaeli, R., and Ron, D. (1999) J. Biol. Chem. 274, 36132-36138[Abstract/Free Full Text]
32. Matsudaira, P. (1990) Methods Enzymol. 182, 602-613[Medline] [Order article via Infotrieve]
33. Knight, C. G., Morton, L. F., Onley, D. J., Peachey, A. R., Ichinohe, T., Okuma, M., Farndale, R. W., and Barnes, M. J. (1999) Cardiovasc. Res. 41, 450-457[Abstract/Free Full Text]
34. Knight, C. G., Morton, L. F., Peachey, A. R., Tuckwell, D. S., Farndale, R. W., and Barnes, M. J. (2000) J. Biol. Chem. 275, 35-40[Abstract/Free Full Text]
35. Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon, J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S., and Boyd, M. R. (1990) J. Natl. Cancer Inst. 82, 1107-1112[Abstract/Free Full Text]
36. Bottaro, D. P., Rubin, J. S., Ron, D., Finch, P. W., Florio, C., and Aaronson, S. A. (1990) J. Biol. Chem. 265, 12767-12770[Abstract/Free Full Text]
37. Hsu, Y. R., Nybo, R., Sullivan, J. K., Costigan, V., Spahr, C. S., Wong, C., Jones, M., Pentzer, A. G., Crouse, J. A., Pacifici, R. E., Lu, H. S., Morris, C. F., and Philo, J. S. (1999) Biochemistry 38, 2523-2534[CrossRef][Medline] [Order article via Infotrieve]
38. Basilico, C., and Moscatelli, D. (1992) Adv. Cancer Res. 59, 115-165[Medline] [Order article via Infotrieve]
39. Faham, S., Linhardt, R. J., and Rees, D. C. (1998) Curr. Opin. Struct. Biol. 8, 578-586[CrossRef][Medline] [Order article via Infotrieve]
40. Jang, J. H., Wang, F., and Kan, M. (1997) In Vitro Cell. Dev. Biol. 33, 819-824
41. Bajaj-Elliott, M., Breese, E., Poulsom, R., Fairclough, P. D., and MacDonald, T. T. (1997) Am. J. Pathol. 151, 1469-1476[Abstract]
42. Khan, W. B., Shui, C., Ning, S., and Knox, S. J. (1997) Radiat. Res. 148, 248-253[Medline] [Order article via Infotrieve]
43. Matthes, H., Herbst, H., Schuppan, D., Stallmach, A., Milani, S., Stein, H., and Riecken, E. O. (1992) Gastroenterology 102, 431-442[Medline] [Order article via Infotrieve]
44. Ropiquet, F., Huguenin, S., Villette, J. M., Ronfle, V., Le, Brun, G., Maitland, N. J., Cussenot, O., Fiet, J., and Berthon, P. (1999) Int. J. Cancer 82, 237-243[CrossRef][Medline] [Order article via Infotrieve]
45. Putnins, E. E., Firth, J. D., and Uitto, V. J. (1995) J. Invest. Dermatol. 104, 989-994[CrossRef][Medline] [Order article via Infotrieve]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Eur. J. Cardiothorac. Surg.Home page
J. B. Lopes, L. A.O. Dallan, S. P. Campana-Filho, L. A.F. Lisboa, P. S. Gutierrez, L. F. P. Moreira, S. A. Oliveira, and N. A.G. Stolf
Keratinocyte growth factor: a new mesothelial targeted therapy to reduce postoperative pericardial adhesions
Eur. J. Cardiothorac. Surg., February 1, 2009; 35(2): 313 - 318.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
T. Takahashi, O. Yamada, M. J Soares, and K. Hashizume
Bovine prolactin-related protein-I is anchored to the extracellular matrix through interactions with type IV collagen
J. Endocrinol., February 1, 2008; 196(2): 225 - 234.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. A. Smethurst, D. J. Onley, G. E. Jarvis, M. N. O'Connor, C. G. Knight, A. B. Herr, W. H. Ouwehand, and R. W. Farndale
Structural Basis for the Platelet-Collagen Interaction: THE SMALLEST MOTIF WITHIN COLLAGEN THAT RECOGNIZES AND ACTIVATES PLATELET GLYCOPROTEIN VI CONTAINS TWO GLYCINE-PROLINE-HYDROXYPROLINE TRIPLETS
J. Biol. Chem., January 12, 2007; 282(2): 1296 - 1304.
[Abstract] [Full Text] [PDF]

Home page
 GutHome page
W Dieterich, B Esslinger, D Trapp, E Hahn, T Huff, W Seilmeier, H Wieser, and D Schuppan
Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease
Gut, April 1, 2006; 55(4): 478 - 484.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
G. E. Davis and D. R. Senger
Endothelial Extracellular Matrix: Biosynthesis, Remodeling, and Functions During Vascular Morphogenesis and Neovessel Stabilization
Circ. Res., November 25, 2005; 97(11): 1093 - 1107.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/30/26872    most recent
M202335200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ruehl, M.
Right arrow Articles by Schuppan, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ruehl, M.
Right arrow Articles by Schuppan, D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement