1. Volume 114, Issue 1, Pages 139-152 (January 1998)
Collagens in the liver extracellular matrix bind hepatocyte growth factor 
Detlef Schuppan, Monika Schmid, Rajan Somasundaram, Renate Ackermann, Martin Ruehl, Toshikazu Nakamura, Ernst–Otto Riecken 
Gastroenterology 
Volume 114, Issue 1, Pages (January 1998)
DOI: /S (98)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

2. Fig. 1
(A-H) Localization of immunoreactive HGF to the fibrillar ECM of the liver. Cryostat sections of human liver specimens: A, B, E-H, cirrhosis; C and D, normal. Liver specimens were incubated with affinity-purified rabbit antibodies to a 20-mer synthetic peptide derived from the carboxy terminus of the HGF α-chain, followed by the APAAP technique for detection. (A, C, and E) Primary antibody was applied directly to sections. (B) Antibody to HGF was preincubated with 8 μg of the peptide used for immunization (negative control). (D, F, G, and H) Sections were allowed to bind 50 ng of HGF, followed by washing, before antibody incubation. (H) was additionally digested with collagenase before immunodetection. A-H are sequential sections. (A, C, and E) Note immunolocalization of HGF in the portal interstitial matrix and in perisinusoidal cells with cytoplasmic extensions. (D and F) When sections were pretreated with HGF, interstitial immunostaining for HGF was essentially unchanged, whereas the previously negative perisinusoidal spaces became prominently labeled. (H) Collagenase treatment almost abolished HGF staining. Original magnifications: A-H, 250×.
Gastroenterology  , DOI: ( /S (98) )
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3. Fig. 2
HGF is preferentially bound by immobilized interstitial collagens. One nanogram (20,000 cpm) of 125I-labeled HGF, dissolved in 100 μL of PBS containing 100 μg of BSA, was incubated with proteins that had been immobilized on polystyrene microtiter wells at 2 μg/100 μL. Bound HGF is expressed as percent of total radioactivity remaining in the wells after three buffer washes. Shown are means and SD of independent experiments performed in triplicate (n = 6-7 in the case of the native collagens [type I-IV]; n=3-4 in the case of fibronectin, fibrinogen, laminin, collagen α-chains, and reduced and alkylated [r] collagens). Binding to uncoated polystyrene or to immobilized BSA was <2%.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

4. Fig. 2
HGF is preferentially bound by immobilized interstitial collagens. One nanogram (20,000 cpm) of 125I-labeled HGF, dissolved in 100 μL of PBS containing 100 μg of BSA, was incubated with proteins that had been immobilized on polystyrene microtiter wells at 2 μg/100 μL. Bound HGF is expressed as percent of total radioactivity remaining in the wells after three buffer washes. Shown are means and SD of independent experiments performed in triplicate (n = 6-7 in the case of the native collagens [type I-IV]; n=3-4 in the case of fibronectin, fibrinogen, laminin, collagen α-chains, and reduced and alkylated [r] collagens). Binding to uncoated polystyrene or to immobilized BSA was <2%.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

5. Fig. 3
Identity of collagen-bound HGF. Autoradiography of 125I-labeled HGF subjected to 10% SDS-PAGE before (−) and after (+) reduction of disulfides. The gel shows 125I-HGF (20,000 cpm/ng, 1 ng/lane, in 100 μL of PBS/0.05% Tween 20) before (HGF) or after incubation with 2 μg of collagen type I immobilized on a polystyrene microtiter well. After a 2-hour incubation, the supernatant was aspirated (yielding free HGF), followed by three washes with 200 μL each of PBS/0.05% Tween 20 and by boiling in 100 μL of SDS sample buffer (yielding bound HGF). Five microliters of free and bound fractions were subjected to SDS-PAGE and autoradiography. Before reduction: some aggregated material not penetrating or at the top of the running gel and dimeric HGF of 97 kilodaltons. After reduction: the HGF α-chain of 69 kilodaltons and the β-chain doublet of 30 and 32 kilodaltons. Similar results were obtained with immobilized collagens type III and VI and with the α1(I) chain. The positions of globular molecular weight markers are shown on the right.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

6. Fig. 4
HGF coprecipitates with reconstituted collagen fibrils. The 10 ng (200,000 cpm) of 125I-labeled HGF as added to increasing amounts of collagen type I (■), type VI (●), and BSA (▴) immediately after dilution of the respective stock solutions into PBS/0.05% Tween 20. Final concentrations were 0, 0.1, 1, 3, 10, and 30 μg protein in 200 μL of buffer. Precipitates that formed at room temperature overnight were collected by centrifugation, washed twice, and counted for radioactivity. Shown are the means (triplicate tubes) of a representative experiment.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

7. Fig. 5
(A-C) Denatured collagen and heparin inhibit the HGF-collagen interaction. One nanogram of 125I-labeled HGF was reacted with the inhibitors (A) fibrinogen, (B) reduced and alkylated collagen type VI (CVIr), and (C) heparin (100 μL) in polypropylene microtubes for 2 hours. Solutions were then transferred to and incubated with collagen types I, III, and VI (CI, CIII, CVI) immobilized on polystyrene microtiter wells or with uncoated plastic (PL), as described in Figure 3. Inhibitors were present in increasing concentrations. 2, 0 μg/well; ▨, 0.1 μg/well; ▩, 1 μg/well; ■, 10 μg/well. Bars, means ± SD (of three independent experiments performed in triplicate) of the radioactivity remaining in the wells after three buffer washes.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

8. Fig. 5
(A-C) Denatured collagen and heparin inhibit the HGF-collagen interaction. One nanogram of 125I-labeled HGF was reacted with the inhibitors (A) fibrinogen, (B) reduced and alkylated collagen type VI (CVIr), and (C) heparin (100 μL) in polypropylene microtubes for 2 hours. Solutions were then transferred to and incubated with collagen types I, III, and VI (CI, CIII, CVI) immobilized on polystyrene microtiter wells or with uncoated plastic (PL), as described in Figure 3. Inhibitors were present in increasing concentrations. 2, 0 μg/well; ▨, 0.1 μg/well; ▩, 1 μg/well; ■, 10 μg/well. Bars, means ± SD (of three independent experiments performed in triplicate) of the radioactivity remaining in the wells after three buffer washes.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

9. Fig. 5
(A-C) Denatured collagen and heparin inhibit the HGF-collagen interaction. One nanogram of 125I-labeled HGF was reacted with the inhibitors (A) fibrinogen, (B) reduced and alkylated collagen type VI (CVIr), and (C) heparin (100 μL) in polypropylene microtubes for 2 hours. Solutions were then transferred to and incubated with collagen types I, III, and VI (CI, CIII, CVI) immobilized on polystyrene microtiter wells or with uncoated plastic (PL), as described in Figure 3. Inhibitors were present in increasing concentrations. 2, 0 μg/well; ▨, 0.1 μg/well; ▩, 1 μg/well; ■, 10 μg/well. Bars, means ± SD (of three independent experiments performed in triplicate) of the radioactivity remaining in the wells after three buffer washes.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

10. Fig. 6
(A and B) HGF binding to collagens is not enhanced by contaminating heparin or HS. One nanogram (approximately 30,000 cpm) of 125I-labeled HGF, dissolved in 100 μL of PBS containing 100 μg of BSA, was incubated with two different preparations of collagen I (CI-P, pepsin-solubilized; CI-N, neutral salt extracted), collagen VI (CVI), BSA, or a BSA-heparin conjugate (BSA-H) that had been immobilized on polystyrene microtiter wells at 2 μg/100 μL. HGF that remained bound to the wells after preincubation of the wells with heparinase III (■) or the buffer control (2) is expressed as counts per minute. Data are means ± SD of a representative experiment performed in quadruplicate. See Materials and Methods for details.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

11. Fig. 7
(A and B) Binding of HGF to collagens follows saturation kinetics. Increasing amounts of 125I-labeled HGF in 100 μL PBS/0.05% Tween 20 were incubated with 200 ng of (A) collagen type I or (B) the α1(I) chain that were immobilized on polystyrene microtiter wells. Nonspecific binding, as determined by a 200-fold excess of unlabeled HGF, ranged between 20% and 28% and was subtracted from the values before plotting. Nonspecificity increased abruptly at concentrations of 125I-labeled HGF above 50 ng/100 μL, after apparent saturation was reached. Inserts show the respective Scatchard plots with an apparent high-affinity/low-capacity and a low-affinity/high-capacity binding site. Displacement of 1 ng of radiolabeled HGF with increasing amounts of nonlabeled HGF resulted in similar binding curves. For a summary of apparent dissociation constants and molar ratios, see insert.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

12. Fig. 8
(A and B) HGF binds to collagen chains and particular chain fragments after transfer to nitrocellulose. 125I-labeled HGF (20 ng, 400,000 cpm in 4 mL of PBS/0.05% Tween 20) was incubated with nitrocellulose replicas of human collagens and collagen fragments: blotted at 0.5 μg protein per lane from (A) 7.5% and (B) 15% SDS-PAGE. (A) Pepsin-solubilized human collagen types I, II, III, IV, V, and VI, single chains α1(I), α2(I), and reduced and alkylated collagen types IV (IVr) and VI (VIr). Arrows mark internal collagenous molecular weight standards in kilodaltons: 600, tetramers of collagen type VI; 300, trimers of collagen types I and III; 200, dimers of collagen types I and III; 120, α1(V); 105, α2(V); 100, α1(I); 95, α2(I); 90, dimers of collagen type VI; 60, P1 fragment of α1(IV); 55, α3(VI); and 50, P2 fragment of α2(IV). There is a trace of collagen type V in the collagen type VI preparation. (B) Cyanogen bromide (CB) peptides of α1(I), α2(I) and of collagen type III. On the left is shown a blot stained with Amido Black, on the right a parallel blot incubated with radiolabeled HGF. Positions of internal molecular weight markers (in kilodaltons) are as follows: 62, the dimeric α2(I) CB3-5; 45, α1(III) CB5-9; 31, α1(I) CB7, α2(I) CB3, α2(I) CB4 or α2(I) CB5; 27, α1(I) CB8; 22, α1(III) CB9;19, α1(I) CB6; 14, α1(I) CB3; 8, α1(III) CB3. Note the exclusive binding of radiolabeled HGF to peptides with the mobilities of α1(I) CB8, α1(I) CB6, the incompletely cleaved dimeric α2(I) CB3-5, α2(I) CB3 (or α2(I) CB5), α1(III) CB9 (which runs as a reactive trimer of 75 kilodaltons before reduction, not shown), and the incompletely cleaved dimeric α1(III) CB5-9.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

13. Fig. 8
(A and B) HGF binds to collagen chains and particular chain fragments after transfer to nitrocellulose. 125I-labeled HGF (20 ng, 400,000 cpm in 4 mL of PBS/0.05% Tween 20) was incubated with nitrocellulose replicas of human collagens and collagen fragments: blotted at 0.5 μg protein per lane from (A) 7.5% and (B) 15% SDS-PAGE. (A) Pepsin-solubilized human collagen types I, II, III, IV, V, and VI, single chains α1(I), α2(I), and reduced and alkylated collagen types IV (IVr) and VI (VIr). Arrows mark internal collagenous molecular weight standards in kilodaltons: 600, tetramers of collagen type VI; 300, trimers of collagen types I and III; 200, dimers of collagen types I and III; 120, α1(V); 105, α2(V); 100, α1(I); 95, α2(I); 90, dimers of collagen type VI; 60, P1 fragment of α1(IV); 55, α3(VI); and 50, P2 fragment of α2(IV). There is a trace of collagen type V in the collagen type VI preparation. (B) Cyanogen bromide (CB) peptides of α1(I), α2(I) and of collagen type III. On the left is shown a blot stained with Amido Black, on the right a parallel blot incubated with radiolabeled HGF. Positions of internal molecular weight markers (in kilodaltons) are as follows: 62, the dimeric α2(I) CB3-5; 45, α1(III) CB5-9; 31, α1(I) CB7, α2(I) CB3, α2(I) CB4 or α2(I) CB5; 27, α1(I) CB8; 22, α1(III) CB9;19, α1(I) CB6; 14, α1(I) CB3; 8, α1(III) CB3. Note the exclusive binding of radiolabeled HGF to peptides with the mobilities of α1(I) CB8, α1(I) CB6, the incompletely cleaved dimeric α2(I) CB3-5, α2(I) CB3 (or α2(I) CB5), α1(III) CB9 (which runs as a reactive trimer of 75 kilodaltons before reduction, not shown), and the incompletely cleaved dimeric α1(III) CB5-9.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

14. Fig. 9
(A-D) Collagen-bound HGF induces scattering. MDCK cells were seeded on immobilized collagen type I (coated at 2 μg/microtiter well) that had been preincubated with HGF at (A) 0, (B) 10, or (C) 50 ng/100 μL, followed by three buffer washings. (D) Two nanograms of HGF added in solution served as positive control. Cells were cultured in complete medium at a density of 2 × 103 cells/100 μL per microtiter well. After 30 hours, scattering was documented after fixation and staining of the cells by photography.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

15. Fig. 9
(A-D) Collagen-bound HGF induces scattering. MDCK cells were seeded on immobilized collagen type I (coated at 2 μg/microtiter well) that had been preincubated with HGF at (A) 0, (B) 10, or (C) 50 ng/100 μL, followed by three buffer washings. (D) Two nanograms of HGF added in solution served as positive control. Cells were cultured in complete medium at a density of 2 × 103 cells/100 μL per microtiter well. After 30 hours, scattering was documented after fixation and staining of the cells by photography.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

16. Fig. 9
(A-D) Collagen-bound HGF induces scattering. MDCK cells were seeded on immobilized collagen type I (coated at 2 μg/microtiter well) that had been preincubated with HGF at (A) 0, (B) 10, or (C) 50 ng/100 μL, followed by three buffer washings. (D) Two nanograms of HGF added in solution served as positive control. Cells were cultured in complete medium at a density of 2 × 103 cells/100 μL per microtiter well. After 30 hours, scattering was documented after fixation and staining of the cells by photography.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

17. Fig. 9
(A-D) Collagen-bound HGF induces scattering. MDCK cells were seeded on immobilized collagen type I (coated at 2 μg/microtiter well) that had been preincubated with HGF at (A) 0, (B) 10, or (C) 50 ng/100 μL, followed by three buffer washings. (D) Two nanograms of HGF added in solution served as positive control. Cells were cultured in complete medium at a density of 2 × 103 cells/100 μL per microtiter well. After 30 hours, scattering was documented after fixation and staining of the cells by photography.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

18. Fig. 10
Collagen-bound HGF stimulates hepatocyte DNA synthesis. Primary rat hepatocytes (8000 cells/microwell) were seeded on collagen-coated wells with or without preimmobilized HGF, or with HGF added in solution. DNA synthesis relative to control hepatocytes that were not exposed to HGF but otherwise treated identically was measured after addition of 1 μCi of [3H]thymidine over 24 hours. Shown are the means ± SEM of a representative experiment with five wells for each time point. 2, 0.5 ng soluble HGF; ▨, 10 ng soluble HGF;▩,  ̃1.3 ng bound HGF; ■,  ̃2.5 ng bound HGF.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions