Enhanced Activity of Transforming Growth Factor β1 (TGF-β1) Bound to Cartilage Oligomeric Matrix Protein^*

Recombinant Human COMP and TGF-β1

Human COMP cDNA in the pQE mammalian expression vector (Qiagen) was stably transfected into human 293T cells (American Type Culture Collection), which were cultured in serum-free medium. Recombinant human COMP was purified from the cell culture medium to near homogeneity by nickel-nitrilotriacetic acid column affinity chromatograph. An N-terminally truncated COMP construct (COMP-ΔN) consisting of the TSP3 domains and the C-terminal domain (amino acids 269–757 of human COMP, shown in Fig. 4A) was similarly expressed and purified from 293T cell culture supernatants. The truncated COMP is missing the pentamerizing domain and EGF repeats and is secreted as a monomeric protein with an apparent molecular mass of ∼80 kDa. Human TGF-β1 was purchased from PeproTech (Rocky Hill, NJ) in a carrier-free formulation. Its activity was confirmed in pellet culture chondrogenesis assays of human bone marrow-derived stem cells (BMSCs). Recombinant human BMP-7 was a generous gift from Dr. David Rueger (Stryker Biotech, Hopkinton, MA) to A. Hari Reddi. Human BMP-2 and BMP-4 were purchased from R&D Systems (Minneapolis, MN).

Solid-phase ELISA Binding Assays

TGF-β1 was coated onto ELISA plates at 0.5 or 1 μg/ml in 50 mm sodium carbonate (pH 9.8). In certain experiments, COMP constructs were similarly coated at 1 μg/ml. Unbound sites were blocked with PBS containing 1% heat-denatured BSA and 0.05% Tween 20 at pH 7.30. COMP protein was diluted in Tris-buffered saline with 1% heat-denatured BSA and 0.05% Tween 20 at pH 7.35 and allowed to bind overnight. COMP concentrations ranged between 0.06 and 20 μg/ml, which corresponds to 140 pm to 46 nm, using a calculated molecular mass of 433 kDa for the COMP pentamer (27). For lower pH experiments, PIPES buffer was used instead of Tris. Where indicated, the divalent cations magnesium, calcium, and manganese were added as chloride salts throughout all binding, washing, and incubation steps. Where indicated, COMP was preincubated with 2 μm retinoic acid or vitamin D, diluted at least 1:5000 from Me₂SO stock. Bound COMP was detected with rabbit anti-COMP polyclonal antibody generated in-house. TGF-β1 was detected with monoclonal antibody MAB240 (R&D Systems), and BMP-7 with monoclonal antibody MAB3541. In all cases, detection of the primary antibody was performed with a peroxidase-conjugated anti-rabbit secondary antibody. For colorimetric detection, Turbo 3,3′5,5′-tetramethylbenzidine substrate (Pierce) was added for 2 min, the reaction was stopped with 2 m sulfuric acid, and absorbance was measured at 450 nm on a 96-well microplate reader. For COMP binding to immobilized TGF-β1, the background of COMP binding to immobilized BSA was subtracted. Assays were performed in triplicate and reproduced.

Negative Staining/Electron Microscopy

TGF-β1 was labeled for negative staining with colloidal thiocyanate gold and allowed to form complexes with COMP by incubation for 30 min at room temperature in Tris-buffered saline (pH 7.4) with 10 mm manganese chloride. Complexes between TGF-β and COMP were analyzed by negative staining and electron microscopy as described previously (28). The specimens were finally examined in a JEOL 1200EX transmission electron microscope operated at 60 kV.

Dual Polarization Interferometry (DPI)

DPI analysis was performed using an AnaLight4D workstation (Farfield Group, Manchester, United Kingdom), with COMP immobilized onto His-tagged AnaChip Plus chips. Soluble TGF-β1 was bound to the immobilized COMP at the indicated concentrations, and nonspecific sites were blocked with digested casein. Binding was performed in buffer containing 25 mm PIPES (pH 7.2), 150 mm NaCl, and no manganese. Data collection and analysis were performed using the AnaLight software suite and AnaLight Explorer, respectively. The DPI instrument provides absolute measurements of the change in the thickness and density of the molecular protein layer, which enabled a calculation of the mass of the immobilized COMP as TGF-β was added. These measurements are directly related to the structure and function of COMP and its interactions with TGF-β in close proximity to the measurement surface and provide the ability to determine binding constants and stoichiometry of binding.

Luciferase Assays for Transcriptional Activation

To test the effect of COMP on TGF-β-dependent transcriptional activation, we assayed the activation of a TGF-β-responsive promoter that had been stably transfected into a mink lung epithelial cell line (plasminogen activator inhibitor-1/luciferase cells kindly provided by Dr. Rifkin at New York University) (29). First, we determined that the mid-range of the TGF-β response was 4.6 ng/ml using our batch of TGF-β1 (data not shown). Next, a constant amount of TGF-β1 (4.6 ng/ml) was mixed with increasing concentrations of COMP in a chemically defined low protein medium (Opti-MEM supplemented with 50 μm manganese chloride). After 30 h, luciferase activity was assayed in the cell lysate using the Dual-Luciferase assay reporter system (Promega) with readings normalized to cell seeding density.

Quantitative RT-PCR

Three strains of human BMSCs were obtained from different donors and maintained as stem cells in α-minimal essential medium supplemented with 10% FBS and 5 ng/ml FGF2. Cells at passage 2 were transduced with lentiviral constructs to express either human full-length COMP or GFP, and untransduced BMSCs were used as a control. COMP overexpression was confirmed by Western blotting of 20 μl of cell culture supernatant with anti-COMP antibody. Cells were seeded in 6-well dishes at 2 × 10⁴ cells/well, allowed to attach in α-minimal essential medium with 10% FBS, and then treated with TGF-β1 for 48 h. Total RNA was isolated (RNeasy micro reagents, Qiagen). The mRNA expression of the TGF-β-responsive gene TSP1 was quantified with TaqMan probe Hs00962908_m1 (Applied Biosystems) and TaqMan Fast Universal PCR reagents with a 7900HT Fast thermal cycler (Applied Biosystems). The relative expression of TSP1 was normalized to the level of 18 S RNA in each sample, and the -fold change in TSP1 expression between samples was calculated using the 2^ΔΔCt method.

Docking Simulation

Docking simulation was performed as described previously (30, 31) using AutoDock3 (32) and ADT (33). We performed 50 dockings of the COMP/TGF-β1 interaction, each one starting with a random initial position and orientation of TGF-β1 (Protein Data Bank code 3KFD, fragment A) with respect to the COMP C-terminal domain (amino acids 527–757; code 3FBY, fragment A).

Statistical Analysis

Experimental analysis was performed in triplicate, with key experiments repeated at least three times. Error bars represent S.D. Unless noted otherwise, statistical comparisons were made using JMP software (SAS Software, Cary, NC) and two-sided t tests with significance set at p < 0.05.

Binding of COMP to TGF-β and BMP Ligands

The TGF-β superfamily of proteins in humans includes three TGF-β isoforms and the BMPs. To determine whether COMP binds to members of the TGF-β family of ligands, we performed solid-phase binding assays using soluble COMP and immobilized TGF-β1, BMP-2, BMP-4, and BMP-7. We found that soluble COMP bound to immobilized TGF-β1, BMP-2, BMP-4, and BMP-7 (Fig. 1A). To confirm the specificity of the binding interactions, we repeated these assays with immobilized COMP and soluble TGF-β1 and BMP-7 (Fig. 1, B and C). We found that the binding interactions were reproducible when performed with either COMP or BMP-7/TGF-β in the soluble phase. These results demonstrate that COMP binds to TGF-β1, BMP-2, BMP-4, and BMP-7. In fact, COMP bound to every member of the TGF-β superfamily we tested. In the remainder of this study, we focused on the binding interaction between COMP and TGF-β1 in more detail.

Soluble COMP bound to immobilized TGF-β1 in a dose-dependent and saturable manner in the presence of calcium chloride (Fig. 1D). This was reproducible in the absence of calcium chloride (data not shown). Analysis of the binding curve showed that half-maximal binding occurred at ∼1.4 μg of COMP binding to 1 μg of TGF-β1, which very roughly corresponds to 14 molecules of TGF-β1 bound to each pentameric COMP protein, and suggests that multiple TGF-β1 molecules interact with a single molecule of COMP.

pH Optimum of COMP/TGF-β1 Binding

We next determined the pH optimum at which COMP binds to TGF-β1. Solid-phase ELISAs were performed as described above but at varying pH values. When the pH of the binding buffers was adjusted between 6.5 and 7.5 in 0.25 increments, the maximal amount of TGF-β1 binding was found at pH 6.75 (Fig. 2A). The binding dropped off rapidly at pH 7.25 and higher.

Effect of Cofactors on COMP/TGF-β1 Interaction

The hydrophobic core formed by the COMP pentamerizing domains binds to hydrophobic molecules such as retinol and vitamin D. We used solid-phase binding assays to test whether the presence of these hydrophobic molecules affects COMP binding to TGF-β. We did not observe any change in the COMP/TGF-β1 interaction, even in the presence of the relatively high concentrations of retinol and vitamin D used in the assays (Fig. 2B).

In addition to binding hydrophobic compounds in its pentamerizing domain, there are up to 30 cation-binding sites in COMP, many of which are occupied by calcium ions (9). We therefore tested whether different cations or EDTA would affect the binding interaction between soluble COMP and immobilized TGF-β1 using ELISAs. A constant amount of COMP was allowed to bind the immobilized TGF-β1 in the presence of increasing concentrations of calcium or other divalent cations (calcium, magnesium, manganese, or EDTA). We found that manganese caused a dose-dependent increase in the amount of COMP bound to TGF-β1 (Fig. 2C). However, the binding of COMP to TGF-β1 was not affected by the addition of either calcium or magnesium to 40 mm. Surprisingly, EDTA up to 40 mm also did not affect the binding interaction (Fig. 2C). To further characterize the effect of manganese on the COMP/TGF-β interaction, increasing amounts of soluble COMP were bound to immobilized TGF-β1 in the presence or absence of 10 mm manganese. We observed a shift in the binding curve, indicating that higher affinity binding interactions occur in the presence of manganese (Fig. 2D).

Additional TGF-β1-binding Sites with Manganese

The observation that manganese increased the base-line binding of COMP to TGF-β1 raised the question of whether the presence of manganese unmasks additional binding sites for TGF-β in each COMP monomer. Alternatively, manganese could simply enhance the affinity of a single TGF-β-binding site. To discern between these possibilities, we obtained negative stained electron micrographs of gold-labeled TGF-β1 bound to COMP in the presence and absence of manganese. In the absence of manganese, TGF-β1 bound only to the CTD globule of COMP (Fig. 3, left panel). In the presence of 10 mm manganese, TGF-β1 bound to an additional site closer to the N terminus of COMP (Fig. 3, right panel). In the presence of manganese, TGF-β1 occupied the N-terminal site, the CTD-binding site, or both sites on a single arm of the pentameric COMP protein. We also observed that TGF-β simultaneously bound to multiple arms of a single COMP pentamer. These results support the hypothesis that two TGF-β-binding sites exist on each COMP polypeptide and that manganese is a required cofactor for one of these sites.

Manganese-dependent TGF-β Binding of COMP-ΔN

From the electron micrographs, the manganese-dependent TGF-β1-binding site could be located within either the EGF-like domains or the TSP3 repeats of COMP. To address this question, we generated and purified an N-terminally truncated COMP protein (COMP-ΔN) in which the secretion signal peptide was fused to C-terminal amino acids 269–757. The COMP-ΔN construct contains all TSP3 repeats and the COMP CTD but it is missing the pentamerizing domain and the four EGF domains (Fig. 4A). We found that soluble TGF-β1 bound to immobilized COMP-ΔN in a dose-dependent manner as measured by ELISA in the absence of manganese (Fig. 4B). Similarly, soluble COMP-ΔN bound to immobilized TGF-β1 in a dose-dependent manner (Fig. 4C). We tested whether the binding of COMP-ΔN to immobilized TGF-β1 was dependent on the presence of manganese. Although strong binding was observed in the presence of manganese, we did not detect binding in its absence in this particular assay.

Electron micrographs of TGF-β1 interacting with COMP-ΔN paralleled the observations made with full-length COMP. TGF-β1 bound to the CTD in a manganese-independent manner (Fig. 5, left panel), whereas binding to the more N-terminal region was observed only in the presence of manganese (right panel). Based on these electron micrographs, the manganese-dependent binding site is located within the TSP3 repeats of COMP. Taken together, these observations confirm that the CTD of COMP contains a manganese-independent TGF-β-binding site and that an additional manganese-dependent site is located within the TSP3 repeats. TGF-β1 bound to the CTD, the TSP3 repeats, or both sites on a single COMP-ΔN peptide.

Quantitative Measurement of Stoichiometry

A more quantitative analysis of binding stoichiometry was obtained using DPI performed on an AnaLight4D DPI instrument. This instrument provides a quantitative measurement of the change in the mass of immobilized COMP protein as TGF-β1 is added in solution. The number of TGF-β1 molecules bound to each COMP molecule, as well as the binding affinity of the interaction, can be calculated from these data. A binding stoichiometry of 11.5 molecules of TGF-β1 per COMP pentamer was determined, with the dissociation constant for the interaction roughly estimated to be in the nm range (Fig. 6). This stoichiometry measurement is in agreement with the ELISA estimate presented above. Using the same methodology for the monomeric COMP-ΔN, the stoichiometry measurement for TGF-β is 0.7, which suggests a 1:1 binding ratio for the COMP monomer and TGF-β1 in the absence of manganese.

COMP·TGF-β1 Complex Enhances Cellular Response to TGF-β1

Finally, we wanted to determine the effect of COMP on TGF-β1-dependent transcriptional activity. Our first approach was to use an established cell line with a TGF-β-dependent luciferase reporter. We used the plasminogen activator inhibitor-1/luciferase assay developed by Dr. Rifkin's laboratory, in which a TGF-β-responsive promoter (plasminogen activator inhibitor-1) drives the expression of firefly luciferase (29). This TGF-β-responsive construct was stably transfected into mink lung epithelial cells (29). In this system, we determined that 4.6 ng/ml TGF-β1 consistently resulted in a mid-range luciferase response in 30 h (data not shown). To test the effect of COMP on TGF-β1-dependent transcription, we preincubated 4.6 ng/ml TGF-β1 with increasing amounts of COMP, and then the COMP·TGF-β1 complex was added to the cells as described above. We observed that the COMP·TGF-β1 complex elicited a greater transcriptional response than the same amount of unbound TGF-β1. The magnitude of the increased response was dependent on the amount of COMP used (Fig. 7A). COMP alone did not activate TGF-β-dependent transcription in this assay. These results demonstrate that COMP can enhance the cellular response to TGF-β1 in a cell line using a luciferase-based measure of transcription.

To confirm that COMP enhances the cellular response to TGF-β1 in primary human cells using endogenous gene activation, we tested the expression of TSP1 mRNA, a known TGF-β-responsive gene. COMP was overexpressed in BMSCs isolated from three individual donors, and the transcription of endogenous TSP1 was measured in response to TGF-β. Untransduced or GFP-transduced cells were used as a control. We found that COMP overexpression enhanced the transcriptional response to all doses of TGF-β (Fig. 7B). These cells did not express detectable levels of endogenous COMP (Fig. 7B, inset). Taken together, these results demonstrate that COMP enhances the response to TGF-β in cell lines using a luciferase reporter and in primary cells using an endogenous TGF-β-responsive mRNA.