Type IIA procollagen containing the cysteine-rich amino propeptide is deposited in the extracellular matrix of prechondrogenic tissue and binds to TGF-beta1 and BMP-2

Abstract

Type II procollagen is expressed as two splice forms. One form, type IIB, is synthesized by chondrocytes and is the major extracellular matrix component of cartilage. The other form, type IIA, contains an additional 69 amino acid cysteine-rich domain in the NH2-propeptide and is synthesized by chondrogenic mesenchyme and perichondrium. We have hypothesized that the additional protein domain of type IIA procollagen plays a role in chondrogenesis. The present study was designed to determine the localization of the type IIA NH2-propeptide and its function during chondrogenesis. Immunofluorescence histochemistry using antibodies to three domains of the type IIA procollagen molecule was used to localize the NH2-propeptide, fibrillar domain, and COOH-propeptides of the type IIA procollagen molecule during chondrogenesis in a developing human long bone (stage XXI). Before chondrogenesis, type IIA procollagen was synthesized by chondroprogenitor cells and deposited in the extracellular matrix. Immunoelectron microscopy revealed type IIA procollagen fibrils labeled with antibodies to NH2-propeptide at approximately 70 nm interval suggesting that the NH2-propeptide remains attached to the collagen molecule in the extracellular matrix. As differentiation proceeds, the cells switch synthesis from type IIA to IIB procollagen, and the newly synthesized type IIB collagen displaces the type IIA procollagen into the interterritorial matrix. To initiate studies on the function of type IIA procollagen, binding was tested between recombinant NH2-propeptide and various growth factors known to be involved in chondrogenesis. A solid phase binding assay showed no reaction with bFGF or IGF-1, however, binding was observed with TGF-beta1 and BMP-2, both known to induce endochondral bone formation. BMP-2, but not IGF-1, coimmunoprecipitated with type IIA NH2-propeptide. Recombinant type IIA NH2-propeptide and type IIA procollagen from media coimmunoprecipitated with BMP-2 while recombinant type IIB NH2-propeptide and all other forms of type II procollagens and mature collagen did not react with BMP-2. Taken together, these results suggest that the NH2-propeptide of type IIA procollagen could function in the extracellular matrix distribution of bone morphogenetic proteins in chondrogenic tissue.

Figure 1

Diagram of alternative splicing of type IIA procollagen gene. (A) Probes were designed to detect the two splice forms of type II procollagen mRNA. A riboprobe (black bar) is specific for type IIA mRNA (+exon 2) and the oligonucleotide probe (gray bar) spans the splice junction and is therefore specific for type IIB procollagen mRNA. (B) Three antibodies were used to localize the type II procollagen domains. Triangles indicate potential epitopes for a rabbit antiserum against human recombinant exon 2, squares indicate epitopes for a rat antiserum against bovine type II collagen fibrillar domains, and circles indicate rabbit antiserum against bovine type II collagen COOH-propeptide. The specific number of epitopes for each antiserum is unknown.

Figure 2

Double labeled immunofluorescence of human fetal limb during chondrogenesis viewed by confocal laser scanning microscopy. Both type IIA procollagen NH₂-propeptide (A, red) and triple-helical domains (B, green) are localized in the ECM of condensing cells of the emerging digital rays (C). D–F show differential expression of type IIA and IIB procollagen mRNA in a 50-d human fetal limb. (D) Bright-field photograph of the hand showing the condensation of chondroprogenitor cells (CP) and chondrocytes (C). (E) Localization of type IIA procollagen mRNA by in situ hybridization to chondroprogenitor cells. (F) Expression of type IIB mRNA in a serial section. E and F are photographed with dark-field illumination. Bar, 200 μm. G–I show type IIA NH₂-propeptide (G, red) and triple-helical domains (H, green) in chondroprogenitor cells (CP), the ECM of cartilage, and the perichondrium in the growth cartilage of 50-d human fetal limb. The COOH-propeptide is immunolocalized within cells and not in the ECM throughout stages of chondrogenesis (J and L). Bar in A–C and G–L, 72 μm.

Figure 3

Double immunohistochemistry of growth cartilage in a 50-d gestation human fetal limb. A–D were reacted with antisera to type II collagen fibrillar domain (green) and type IIA procollagen NH₂-propeptide (red). In all panels, yellow or reddish-yellow color indicates colocalization. E and H were reacted with antibody to type II collagen fibrillar domain (green) and type II collagen COOH-propeptide (red). G was a negative control, and H showed the antibody to integrin β1 (red) and type II collagen fibrillar domain (green). Labels indicate nucleus (N), extracellular matrix (M), and secretory granules (SG). Arrow in B and C indicate ECM. Bars in A–D, 9.0 μm and in E–H, 5.4 μm.

Figure 3

Double immunohistochemistry of growth cartilage in a 50-d gestation human fetal limb. A–D were reacted with antisera to type II collagen fibrillar domain (green) and type IIA procollagen NH₂-propeptide (red). In all panels, yellow or reddish-yellow color indicates colocalization. E and H were reacted with antibody to type II collagen fibrillar domain (green) and type II collagen COOH-propeptide (red). G was a negative control, and H showed the antibody to integrin β1 (red) and type II collagen fibrillar domain (green). Labels indicate nucleus (N), extracellular matrix (M), and secretory granules (SG). Arrow in B and C indicate ECM. Bars in A–D, 9.0 μm and in E–H, 5.4 μm.

Figure 4

Fetal tissue undergoing chondrogenesis immunolabeled en bloc with antibody specific to the NH₂-propeptide of type IIA procollagen (A). Collagen fibrils sheared from fetal cartilage and observed following staining in PTA demonstrate a 70-nm periodicity (arrows, B). Following incubation in antibody, the fibrils are seen to label with a 70-nm periodicity (arrows, C). The identity of the label as antibody is confirmed by addition of secondary antibody–gold conjugate (D). Bars: (A) 100 nm; (B–D) 100 nm.

Figure 5

Immunoprecipitation of type IIA NH₂-propeptide– BMP-2 complex. (A) BMP-2 bound to rhIIA-GST protein can be immunoprecipitated by type IIA NH₂-propeptide antisera (lane 1). Control reactions show no immunoprecipitation with BMP-2 alone (lane 2), and immunoprecipitation of BMP-2–rhIIA-GST complex with preimmune serum (lane 3). A mixture of IGF-1 and exon 2 protein was not immunoprecipitated with type IIA NH₂-propeptide antiserum (lane 5). Lanes 4 and 6 show BMP-2 (33 kD) and IGF-1 (7 kD) alone, respectively. (B) Recombinant type IIA protein (rhIIA) bound to BMP-2 can be immunoprecipitated by BMP-2 antibody and reacted with anti-IIA + GST antiserum (lane 1), neither type II collagen NH₂-propeptide (rhIIN, exons 3–8, lane 2) nor GST (lane 3) can be immunoprecipitated. Lanes 4–6 show recombinant type IIA NH₂-propeptide (rhIIA), type II NH₂-propeptide (rhIIN) and GST were reacted with anti-IIA + GST antiserum.

Figure 6

The natural type IIA procollagen binds to BMP-2. (A) Type IIA and IIB procollagen mRNA (lane 2) were amplified by RT-PCR from C5.18 cell total RNA. Lane 1 is a pGEM DNA marker. (B) Western blotting of type II collagens. Lanes 1 and 3 show immunoreactivity with type IIA NH₂-propeptide antiserum and COOH-propeptide antiserum indicating this band is pNC type IIA procollagen (PNCIIA, lane 2). Antiserum to the fibrillar domain of type II collagen indicates the presence of multiple forms of type II collagen in the culture medium, including type IIA pNC procollagen, type IIB pNC procollagen, type II pC procollagen, and mature α chains (α1[II]). Pepsin solubilized α1(II) is shown in lane 4. (C) Type IIA procollagen (lane 1) and all type II collagens (lane 2) were immunoprecipitated by specific antisera from the labeled proteins collected from C5.18 culture medium. When recombinant BMP-2 was added to labeled proteins and immunoprecipitated with BMP-2 antibody, only type IIA procollagen was presented (lane 3), but not with normal mouse serum (lane 4).

Figure 7

BMP-2 (A) and TGF-β (B) bind specifically to rhIIA-GST (type IIA NH₂-propeptide) fusion proteins. Solid phase binding assays were performed in 96-well plates coated with 5 or 10 ng/well BMP-2 (A) or TGF-β1 (B). GST alone or rhIIA-GST fusion proteins (1–5,000 ng/well) were added. The amount of binding was detected by IIA NH₂-propeptide antiserum and secondary antibody conjugated to alkaline phosphatase. Free rhIIA fusion protein was determined by subtracting the bound rhIIA-fusion protein from total rhIIA-GST fusion protein. Scatchard analysis (inset) of the data (using Cricket Graph) resulted in a K _D of 5.23 nM for BMP-2 and 7.65 nM for TGF-β1.