Interstitial collagens I, III, and VI sequester and modulate the multifunctional cytokine oncostatin M.

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 oncostatin M (OSM), a profibrogenic cytokine and modulator of cancer cell proliferation, specifically binds to collagen types I, III, IV, and VI, immobilized on polystyrene or nitrocellulose. Single collagen chains inhibit these interactions in a dose-dependent manner. Cross-inhibition experiments of collagen-derived peptides point to a limited set of OSM-binding collagenous consensus sequences. Furthermore, this interaction is found for OSM but not for other interleukin-6 type cytokines. OSM binding to collagens is saturable, with dissociation constants around 10(-8) m and estimated molar ratios of 1-3 molecules of OSM bound to one molecule of triple helical collagen. Furthermore, collagen-bound OSM is biologically active and able to inhibit proliferation of A375 melanoma cells. We conclude that abundant interstitial collagens dictate the spatial pattern of bioavailable OSM. This interaction could be exploited for devising collagenous peptide-antagonists that modulate OSM bioactivity in tumor growth and fibrotic disorders like rheumatoid arthritis and hepatic fibrosis.

Oncostatin M, a glycoprotein of 28 kDa, is a pleiotropic cytokine and member of the IL-6 type cytokine family. It displays significant similarities in primary and predicted secondary structures with leukemia-inhibitory factor (LIF), granulocyte colony-stimulating factor (G-CSF), IL-6, and IL-11 (15,16). Members of the IL-6 family share the ability to modulate differentiation of a variety of cell types, which carry gp130 as a common receptor subunit (17) as part of a complex family of hetero-and homodimeric receptors (18). OSM is primarily produced by activated T-cells and monocytes but is detected in other cells, such as endothelial cells and different tumor cell lines derived from meningioma, Kaposi sarcoma, keratocanthoma, and breast carcinoma (16, 19 -22). Depending on the cell type, OSM shows a variety of biological activities on cell growth and differentiation (23). Thus OSM can inhibit the growth of several tumor cell lines, e.g. murine M1 myeloid leukemic cells (24) but stimulates proliferation of fibroblasts (25), endothelial cells (26), and the intermediate cells derived from AIDS-associated Kaposi sarcoma (27). The proinflammatory potential of OSM was demonstrated after subcutaneous injection of OSM in mice, leading to classic morphological signs of inflammation at the site of injection, e.g. extravasation of leukocytes and by its ability to stimulate IL-6 production in endothelial cells in vitro (28,29). On the other hand, intravenous coinjection of OSM and lipopolysaccharide into BALB/c mice was shown to inhibit lipopolysaccharide-induced production of TNF-␣ and to reduce lethality (30), pointing to the anti-inflammatory potential of OSM in other scenarios. OSM is a profibrogenic cytokine, as demonstrated by excessive extracellular matrix deposition in a transgenic mouse model of OSM-expressing pancreatic ␤-islet cells (31) and by enhanced collagen and TIMP-1 expression in hepatic stellate cells (32), fibroblasts, and osteoblasts after addition of OSM (33,34). Furthermore, OSM was shown to play an important role in liver development and maturation (35,36).
Here, we describe the specific interaction of OSM with mainly fibrillar collagens I and III, and collagens IV and VI, and their constituent chains, which are not shared by other IL-6 type cytokines, and the biological effect of collagen-bound OSM in cell culture.  1 The abbreviations used are: ECM, extracellular matrix; BSA, bovine serum albumin; G-CSF, granulocyte colony-stimulating factor; HGF, hepatocyte growth factor; HSC, hepatic stellate cells; IL-2, interleukin-2; LIF, leukemia-inhibitory factor; OSM, oncostatin M; PBS, phosphate-buffered saline; PDGF, platelet-derived growth factor; TIMP, tissue inhibitor of matrix metalloproteinases; TNF-␣, tumor necrosis factor ␣. placenta or skin. Preparation of the pure native collagens and isolation of their respective collagen chains were performed as described before (12).

Methods
Coating of Microtiter Plates with Collagens, Single Collagen Chains, and Collagen Cyanogen Bromide Peptides-Coating of microtiter plates and calculation of coating efficiencies were performed as described (12). Briefly, native collagens and collagen chains were immobilized on polystyrene microtiter wells at concentrations of 2 g/100 l/well for binding studies, and at 200 ng/200 l/well 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. Unspecific binding sites were blocked with PBS, containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaureate), for 1 h at 4°C. Coating efficiencies for 2 g/well native collagens and collagen chains ranged between 21 and 48% (4,6).
Radiolabeling and OSM Binding Assay-OSM was radiolabeled with the 125 I-labeled Bolton-Hunter reagent (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer's recommendations. 125 I-Labeled OSM was separated from free iodine by a Sepharose G-25 column (PD 10, Amersham Biosciences, Inc., Freiburg, Germany) in PBS, containing 0.05% Tween 20 (12). Incorporated radioactivity ranged from 20,000 to 30,000 cpm/ng of OSM. Precipitation with trichloroacetic acid (10% v/v) in the presence of 200 g of BSA/200 l, usually yielded 90 -96% of protein-bound radioactivity. Purity of radiolabeled OSM was demonstrated by SDS-PAGE and autoradiography (not shown).
For binding studies 1-2 ng of [ 125 I]OSM/100 l of PBS/0.05% Tween were 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 ␥-counter (Berthold, Bad Wildbach, Germany).
Precipitation Assays-Native collagen I was used for fluid-phase binding of radiolabeled OSM. Increasing concentrations (0, 1, 5, 10, and 20 g/200 l) of native, solubilized collagen I or BSA were incubated in PBS/0.05% Tween 20, pH 7.4, in 1.5-ml polypropylene tubes (Eppendorf, Hamburg, Germany) at 37°C for 1.5 h which allowed collagen type I fibrils to form. After fibril formation 1 ng of [ 125 I]OSM was added for an additional 2 h at room temperature. After centrifugation at 10,000 rpm (12,500 ϫ g) for 15 min, the supernatant was removed and the radioactivity in the collagen/BSA/OSM precipitate was determined.
Inhibition Experiments-Because solubilized triple-helical, native collagen types I, III, and VI may rapidly form fibrils in neutral buffers, reproducible results with inhibition experiments were only obtained by using single chains from these collagens, either after heat denaturation or after reduction and alkylation. 1-2 ng of [ 125 I]OSM and 5 g/100 l of collagen chains were preincubated in a total volume of 350 l for 2 h at room temperature in detergent-blocked polypropylene tubes. 100 l of the mixture was then added in triplicate to the collagen-precoated microtiter wells. After a further 2 h of incubation and three washes with binding buffer, bound activity was measured as described above.
Saturation Binding Experiments-For saturation binding, increasing amounts of unlabeled OSM (0 -300 ng) were added to 1 ng (approximately 50,000 cpm) of [ 125 I]OSM in a final volume of 100 l of binding buffer and incubated for 2 h at room temperature in microtiter wells, which were precoated with 200 ng/100 l/well native triple-helical collagens or single collagen chains. To exclude different binding affinities of radiolabeled versus unlabeled OSM, binding experiments were also performed by using [ 125 I]OSM up to a concentration of 50 ng per well. The resultant binding curves showed a superimposable pattern (data not shown).
Dependence of OSM Binding on pH, Osmolality, and Divalent Cations-To assess the influence of pH, osmolality, or the presence of divalent cations, collagens were immobilized at 2 g/100 l/well and incubated with 1 ng/100 ml 125 I-labeled OSM under the following con-ditions: pH was adjusted between 6.0 and 9.0 by addition of 2 M NaOH or 2 M HCl to PBS/0.05% Tween 20; osmolality was adjusted by using 10 mM Tris-HCl/0.05% Tween 20, pH 7.4, containing increasing amounts of NaCl (50 -1500 mM), resulting in osmolalities between 120 and 3020 mosM; the divalent cations calcium, magnesium, manganese, or EDTA were added from stock solutions to 50 mM Tris-HCl/0.05% Tween 20 to yield final concentrations of 0.24 -15 mM, and osmolality was adjusted to 300 mosM with NaCl. Binding of [ 125 I]OSM to precoated collagen types I and VI and the ␣(1)-chain of collagen type I was performed as described for inhibition experiments.
A375 Cell Proliferation Assay-To determine the biological activity of collagen-bound OSM, a modified OSM bioassay was used (37). Briefly, the human melanoma cell line A375 (ATCC CRL-1872) was cultured in 80-cm 2 flasks containing Dulbecco's modified Eagle's medium, glutamine (2 mM), and mercaptoethanol (50 M), supplemented with penicillin (107 units/liter), streptomycin (10 mg/liter), and 5% fetal calf serum (Biochrom, Berlin, Germany) under standardized conditions (37°C, 8% CO 2 ) in a humidified atmosphere. Collagen I was coated on microtiter wells (Removawells) at a concentration of 2 g/100 l/well overnight at 4°C. Wells were blocked with BSA (1%) in PBS/0.05% Tween 20 for 1 h followed by extensive washing with PBS/Tween. OSM was added at increasing concentrations and incubated for 2 h at room temperature. After 2 h, unbound OSM was removed by washing with PBS/0.05% Tween 20, followed by three washes with PBS. 100 l of trypsinized A375 cells in the logarithmic growth phase (100,000 cells/ml of medium) was then plated on the collagen-coated wells, which had been preincubated with different concentrations of OSM. In parallel, soluble OSM added to already plated A375 cells served as a positive control. Cells were cultured for 72 h, and the cell number was assessed by a colorimetric cytotoxicity assay (sulforhodamine B-staining) (38).
Statistical Analysis-Binding data are expressed as mean Ϯ S.E. Dissociation constants and the number of binding sites obtained by saturation experiments were analyzed according to the method of Scatchard (12,39).

OSM Binds to Native Collagens I, III, IV, and VI and Single
Collagen Chains-Native triple-helical collagens I, III, IV, and VI or their heat-denatured or reduced and alkylated single chains immobilized on polystyrene microtiter wells bound between 20 and 40% of radiolabeled OSM (1-2 ng) (Fig. 1). Unspecific binding of radiolabeled OSM to blocked polystyrene wells was around 10%. Native and denatured collagens bound OSM with the order: type III Ͼ I Ͼ VI Ͼ IV. When bound [ 125 I]OSM was eluted from collagen-coated wells by boiling, then reduced with SDS-gel sample buffer and analyzed by SDS-PAGE and autoradiography, only intact OSM was identified (data not shown). Precipitation experiments confirmed the results of the solid phase assays: Fibrils of native collagen I in FIG. 1. OSM binds to immobilized native (triple helical) collagens and to single collagen chains. Native collagen (C) types I, III, IV, and VI, and single collagen chains of collagen types III, IV, and VI after reduction and alkylation (ra), as well as chains ␣1(I) and ␣2(I) were immobilized on polystyrene microtiter wells at 2 g/100 l for native collagens and collagen chains, followed by incubation with 1-2 ng of radiolabeled OSM. Detergent-blocked polystyrene served as control (p). After three washes bound radioactivity was measured and is expressed as a percentage of the initially added radioactivity. Shown are the means (ϮS.E.) of at least five independent experiments performed in triplicate.
Single Collagen Chains Inhibit Binding of OSM to Immobilized Collagens-Binding of OSM to immobilized collagen types I and III and to the monomeric constituent chains of collagen types I, III, and IV was inhibited dose dependently by single collagen chains. Collagen chains were able to block OSM binding to their respective but also to heterotypic immobilized collagens (Fig. 3), demonstrating the potential of collagen chains for cross-inhibition. Single chains of collagens I, III, and VI inhibited binding of OSM to the immobilized native triple helical collagens I and III by up to 30%, whereas these chains inhibited OSM binding to immobilized collagen chains by up to 80% (Fig. 3). Inhibition varied when different combinations of soluble collagen chains and collagenous substrates were used. Thus, 5 g/100 l ␣1(III) blocked binding of OSM to its immobilized homotypic chain, to immobilized ␣1(I) and collagen type IV chains by up to 80%, but only by 40% to the immobilized ␣2(I)-chain. Similar results were obtained when inhibitor and immobilized ligands were reversed. Thus, the ␣2-chain of collagen type I blocked OSM binding to ␣1(III) by 50%. When 20 g/100 l homotypic inhibitor were used, OSM binding was completely inhibited (Fig. 4).
Saturation Binding Studies and Estimated Affinities of the OSM-Collagen Interaction-When increasing amounts of unlabeled OSM were incubated with a constant amount of [ 125 I]OSM, binding to immobilized collagens was saturable (Fig. 5, A and B, for collagens I and VI, respectively). Saturation was reached between 6 and 10 pmol of OSM/100 l on 200 ng/well of immobilized collagen type I and between 2 and 4 pmol OSM/100 l on 200 ng/100 l of immobilized collagen type VI. Taking into account the coating efficiencies of the respective immobilized collagens (6) and the amount of OSM needed for saturation, Scatchard analysis (12, 39) yielded binding sites of comparable affinity on the tested collagens, with dissociation constants (K D ) of approximately 10 Ϫ8 M. Based on these data, 1 mol of immobilized collagen types I, III, and VI, and 1 mol of the single chains of collagen types I and VI were estimated to bind approximately three (for the native collagens), or 1-1.5 mol (for the ␣-chains of collagen type I) of OSM, respectively (not shown).
Cross Competition of OSM Binding with IL-6 Type Cytokines-In cross-inhibition experiments with IL-6 type cytokines only G-CSF and IL-6 showed a slight inhibitory effect on the binding of [ 125 I]OSM to collagen types I, III, and VI, whereas unlabeled OSM was by far the best inhibitor (Fig. 6, data for collagens I and VI not shown). The modest inhibitory effect of G-CSF is remarkable, because previous experiments suggested some binding of G-CSF but not of LIF, IL-6, or IL-11 to collagens. 2 This suggests a structural motif on OSM for ECM binding different from the gp130 receptor interactive site.
Role of pH, Osmolality, and Divalent Cations-Binding of OSM to collagen types I, III, IV, and VI and to the ␣1(I)-chain was not significantly influenced by lowering the pH to 6.0 or increasing it to 9.0, with deviations never exceeding 20% of the amount bound at pH 7.4. Similarly, calcium, magnesium, manganese, or EDTA up to a maximal concentration of 15 mM did not modify the interaction to a significant degree (results not shown). When incubations were performed with increasing concentrations of NaCl, yielding osmolalities between 120 and 3020 mosM, maximal binding of OSM to collagen types I and VI was observed at low osmolalities. Binding was reduced to 50% at approximately 200 mosM, reaching background levels at 2000 mosM, indicating a major contribution of ionic forces to the interaction between OSM and collagens (Fig. 7).
A375 Cell Proliferation Assay-Collagen-bound OSM inhibited the growth of the A375 melanoma cell line as assessed by a colorimetric cytotoxicity assay (sulforhodamine B) that correlates with cell numbers. Although collagen-bound OSM showed a half-maximal inhibitory activity when preincubated at approximately 7 ng/ml, this was already reached by only 0.3 ng of soluble OSM in parallel experiments. Taking into account that 35% OSM is bound to collagen type I under these conditions, collagen I-bound OSM is approximately 7-fold less biologically active than "free" OSM in this cell-culture setting (Fig.  8). DISCUSSION We showed that the pleiotropic and multifunctional cytokine OSM is bound by collagen types I, III, IV, and VI in vitro. Our data from inhibition and cross-inhibition experiments point to common collagenous binding sites, which are saturable and display dissociation constants of 10 Ϫ8 -10 Ϫ9 M. This range is comparable to that found for other protein-protein interactions, e.g. that between plasminogen and fibronectin (40), and is similar to that described for the interaction of the growth factors/cytokines PDGF, HGF, and IL-2 with collagens (4, 6, 12). Furthermore, these affinities are in the range of the interaction of OSM with its low affinity receptor type II (41). This suggests that OSM, PDGF, HGF, and IL-2 (4, 6, 12) harbor identical or overlapping binding sites. Furthermore, these growth factors must contain consensus as well as unique collagen binding sites.
The binding of OSM to the chains of collagens I, III, and IV points to the common collagenous motif (Gly-Pro-Hyp) x as binding partner. This is supported by the finding that other proteins of the extracellular matrix such as laminin and fibronectin did not bind OSM significantly and that the synthetic collagen peptide (Gly-Pro-Hyp) 10 is an inhibitor of the OSMcollagen interaction (data not shown).
We could show that other members of the IL-6 type family, namely LIF, G-CSF, IL-6, and IL-11 did not bind to collagens nor interfered significantly with OSM binding to collagens (Fig.  6). It is notable that, out of the IL-6 type family, OSM is the only stimulator of connective tissue deposition, as shown in an OSM-transgenic mouse model of pancreatic fibrogenesis (31). Preferential binding to (interstitial) collagens, which are the most prominent matrix components in fibrosis, with a resultant reduction in bioactivity, may serve as a (negative) feedback regulation during fibroproliferation. Thus OSM has recently been implicated in the induction of TIMP-1 (32,33,42), TIMP-3 (43), and collagen type I in dermal fibroblasts (44). Similarly, OSM up-regulates TIMP-1 in activated hepatic stellate cells (HSC), the major matrix producing cells in human liver fibrosis, whereas low levels of OSM-mRNA are found in fibrotic human liver (32). OSM might act via synergistic mechanisms to enhance fibrosis: 1) inhibition of ECM degradation by increasing the expression of TIMP-1 and hence, inhibition of matrix metalloproteinases, and 2) stimulation of collagen production. OSM enhances collagen synthesis in HSC mainly by post-transcriptional regulation (32) via stabilization of the procollagen ␣1(I) stem-loop structure (45). Our findings indicate that, in the setting of fibrosis and wound healing, collagens are not only the most abundant matrix components but may also serve to store and modulate various growth factors/cytokines, namely PDGF BB and oncostatin M, as profibrogenic factors; HGF, as a potent mitogen for hepatocytes and other epithelia; and IL-2, as a T-cell growth factor. The synergy of such proproliferative and profibrogenic factors and their accumulation in the extracellular matrix will lead to reconstitution of fibrous tissue and the epithelial-mesenchymal equilibrium but may also result in enhanced fibrosis, once matrix remodeling is chronically induced, which involves the action of proteases such as the matrix metalloproteinases. Thus a vicious circle of remodeling and matrix-bound growth factor release may maintain fibroblast proliferation and enhanced matrix synthesis.
It was shown that IL-6, which is up-regulated by OSM (25,29), is necessary for regeneration of hepatocytes (46), illuminating again the pluripotent function of OSM in the setting of fibrosis on the one hand and (epithelial) regeneration on the other. Because it was shown that oncostatin M has anti-neoplastic potential, as demonstrated in several cell lines (15) but also in an in vivo model (47), our finding that collagen-bound OSM inhibits melanoma cell proliferation (Fig. 8), makes this interaction an interesting target for local in vivo anti-neoplastic strategies.
In summary, our study demonstrates a specific binding of OSM to interstitial collagens, which may be exploited to modulate the local availability and activity of this cytokine in wound repair and inflammation such as pancreatic and liver fibrosis, and in rheumatoid arthritis, or in neoplastic diseases such as melanoma, glioma, or AIDS-related Kaposi sarcoma. OSM antagonists such as blocking antibodies were successfully tested in murine arthritis models, and OSM-antagonistic RNA aptamers are already in development (48,49). In this regard, antagonists for extracellular OSM binding sites, e.g. competitive collagenous peptides or their nonpeptidic analogues, are a further promising strategy for modification of OSM biological activity.
FIG. 8. Collagen-bound OSM is biologically active. Collagen I was coated on microtiter wells (Removawells) at a concentration of 2 g/100 l/well and blocked with 1% BSA in PBS/0.05% Tween 20. OSM was added at increasing concentrations and incubated for 2 h at room temperature. After 2 h, unbound OSM was removed by washing with PBS/Tween, followed by three washes with PBS. Then 100 l of trypsinized A375 melanoma cells in the logarithmic growth phase (100,000 cells/ml) was added. In parallel, OSM in solution was added to A375 cells on uncoated wells serving as positive control (see inset). Cells were then cultured for 72 h, and cell numbers were determined by a colorimetric assay after trichloroacetic acid fixation and staining with sulforhodamine B. Shown are results of one representative experiment (out of three) performed in triplicate.