Interaction between cartilage oligomeric matrix protein and extracellular matrix protein 1 mediates endochondral bone growth

Abstract

In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 colocalize in the growth plates invivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth.

Fig. 1. Binding of COMP to ECM1 in yeast

Yeast two-hybrid assay to test the interaction of proteins fused to the VP16 AD and proteins fused to the Gal4 DBD. Each pair of plasmids, as indicated, encoding proteins fused to VP16 (below the line) in the vector pPC86 (i.e., pPC86-c-jun, pPC86-ECM1, and pPC86-Rb) and those encoding proteins fused to Gal4 (above the line) in the vector pDBleu (i.e., pDB-c-fos, pDB-COMP, and pDB-lamin) were cotransfected into yeast strain MAV203. Yeast transformants were selected on SD-leu⁻/trp⁻ plates and tested for β-galactosidase activity (panel A), for growth on plates lacking histidine and uracil and containing 3AT (panel B, SD-leu⁻/trp⁻/his⁻/ura⁻/3AT⁺). The known interaction between c-Jun and c-Fos was used as a positive control, and the lack of interaction between Rb and lamin was used as a negative control.

Fig. 2. COMP binds to ECM1 in vitro and in vivo

A and B, COMP associates with ECM1 in vitro. A, Expression of GST-fused EGF domain of COMP. Samples of affinity-purified GST and GST-EGF, as indicated, were examined by SDS-PAGE and Coomassie Blue staining. B, GST pulldown assay. Purified GST (lane 1) or GST-EGF fusion protein (lane 2) were conjugated to the glutathione-sepharose beads and incubated with ECM1-conditioned medium. Proteins trapped by the EGF domain of COMP fused to GST were examined by immunoblotting with anti-ECM1 antibodies. ECM1-conditioned medium (lane 3) was used as a positive control. C, Solid-phase assay. Various amounts of recombinant ECM1 protein, as indicated, were immobilized on 96-well microtiter plates. After blocking, COMP was added to each well, and bound protein from the liquid phase was detected using monoclonal antibodies against COMP. D and E, COMP interacts with ECM1 in vivo. Co-IP assay. Cell extracts prepared from human primary chondrocytes were incubated with anti-ECM1 (lane 1) anti-COMP (lane 2) or control IgG (lane 3) followed by protein A-agarose. The immunoprecipitated protein complexes were examined by immunoblotting with anti-COMP (D) and anti-ECM1 (E) antibodies.

Fig. 3. ECM1 selectively binds to the EGF-like and C-terminal domain of COMP

A, schematic structure of COMP constructs used to map those domains (N-terminal, EGF-like, type III, and C-terminal domain) that bind to ECM1. The presence or absence of binding between COMP domains and ECM1 is indicated as plus or minus sign, respectively. B, β-galactosidase activity was used to test the interaction between the ECM1 and one of four domains of COMP. Three independent yeast transformants for each pair of plasmids were transferred onto a nitrocellulose membrane, and β-galactosidase activity was determined. The known lack of interaction between Rb and lamin served as a negative control. C, Purified Flag-tagged COMP fragment, as indicated, were separated on SDS-PAGE and visualized by Coomassie blue staining. D, Solid-phase assay. Comparable amounts of purified COMP fragments, as indicated in panel C, were immobilized on 96-well microtiter plates. After blocking, recombinant ECM1 was added to each well, and bound protein from the liquid phase was detected using antibodies against ECM1.

Fig 4. C-terminus of ECM1 is required and sufficient for interaction with COMP

A, Schematic representation of various ECM1a deletion mutants fused to either GST or His tag. The amino acid residue numbers are indicated. The predicted molecular weights (kDa) of each recombinant fusion fragment are shown on the right. B~E, Pull down assay. Purified ECM1 mutants fused to GST (B) and His-tagged ECM1 mutants (D) were separated on SDS-PAGE and visualized by Coomassie blue staining. These purified proteins were conjugated to either Glutathione-sepharose beads (C) or Pro-bond beads (E) and incubated with recombinant COMP. After washing, the bound proteins were immunoblotted using anti-COMP antibody. GST fused ECM1 mutants in panel B with expected sizes are indicated with “star”. Molecular weights are indicated on the left column.

Fig. 5. Immunohistochemistry of COMP and ECM1 in the section of long bone from a 18.5-day-old mouse embryo

A, B, C, Low-power microphotograph of a section stained with preimmuno serum (A), anti-COMP (B) or anti-ECM1 (C) polyclonal antibody (brown) and counterstained with methyl green (blue). D, E, F, High-power microphotograph of sections in A, B, C. S, resting chondrocytes; P, proliferating chondrocytes; H, hypertrophic chondrocytes; M, bone metaphysis.arrow indicates the signal.

Fig. 6. EGF domain of COMP disturbs the interaction between ECM1 and COMP

A, B, Characterization of anti-COMP monoclonal antibodies using Western blotting assay. Bacteria extracts bearing GST, GST-EGF or GST-type III, as well as the recombinant COMP or COMP conditioned medium, were subjected to 10% SDS-PAGE and detected with either 2114B3-EGF (A) or 2130E2–type III (B) monoclonal antibody. C, D, Co-IP assay. Cell extracts prepared from 293 cells cotransfected with ECM1 and COMP expression plasmids supplemented with recombinant EGF domain of COMP were incubated with anti-ECM1 (lane 1), 2114B3-EGF that recognized EGF domain of COMP (lane 2), 2130E2–type III that bound to the type III domain of COMP (lane 3), or control HA probe (serving as a control IgG (lane 4), followed by protein A-agarose. The immunoprecipitated protein complexes were examined by immunoblotting with anti-ECM1 (Rb469) (C) and anti-COMP (D) antibodies.

Fig. 7. COMP overcomes ECM1-mediated inhibition of endochondral bone growth

Metatarsals were explanted from fetal mice and cultured in the presence of stimuli in 6-tuple. After 6 days, the explants were fixed and processed for either Alizarin red/Alcian blue staining or histochemical analysis for Safranin O staining as described in “Experimental Procedures.” A, Representative photograph (a–e) and Safraninin O staining (f–j) of an explanted metatarsal after 6 days of culture in the presence of vehicle (a, f), 250 ng/mL ECM1a (b, g), 250 ng/mL COMP (c, h), 250 ng/mL ECM1a plus 250 ng/mL COMP in the absence (d, i), or presence of 250 ng/mL purified EGF domain of COMP (e, j). Straight line represents the total length (T) and the broken line indicates the zone of mineralized cartilage (M). B, Percentage increase in T (top) and M (bottom) length of metatarsal bones after 6 cultures for 6 days. (Percentage of increase = [length at day 6 − length at day 0] / length at day 0). *Significantly different from control (p < .05).