Distribution of small integrin-binding ligand, N-linked glycoproteins (SIBLING) in the condylar cartilage of rat mandible

Abstract

The Small Integrin-Binding LIgand, N-linked Glycoprotein (SIBLING) family is one category of non-collagenous proteins closely related to osteogenesis. In this study, the authors systematically evaluated the presence and distribution of four SIBLING family members, dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (OPN), in rat mandibular condylar cartilage using protein chemistry and immunohistochemistry. For protein chemistry, SIBLING proteins in the dissected condylar cartilage were extracted with 4M guanidium-HCl, separated by ion-exchange chromatography, and analyzed by Western immunoblotting. Immunohistochemistry was employed to assess the distribution of these four SIBLING proteins in the condylar cartilage of 2-, 5- and 8-week-old rats. Results from both approaches showed that all four members are expressed in the condylar cartilage. DSPP, unlike that observed in dentin and bone, exists as a full-length form (uncleaved) in the condylar cartilage. The NH(2)-terminal fragment of DMP1 is mainly detected in the matrix of the cartilage while the COOH-terminal fragment is primarily localized in the nuclei of cells in the chondroblastic and hypertrophic layers. The data obtained in this investigation provide clues about the potential roles of these SIBLING proteins in chondrogenesis.

Fig. 1

Separation of NCPs extracted from the rat mandibular condylar cartilage. Q-Sepharose ion-exchange chromatography separated NCPs extracted from rat mandibular condylar cartilage into 120 fractions. Each fraction contained 1 ml of 6 M urea solution.

Fig. 2

Stains-All staining for chromatographic fractions 15–93 of the Gdm-HCl extract from the rat mandibular condylar cartilage. Digits at the top of the figure represent fraction numbers. The blue protein bands in fractions 48–54 migrating between the 53 kDa and 78 kDa molecular weight markers represent OPN. The blue protein bands around 80 kDa (between the 78 kDa and 114 kDa markers) in fractions 54–81 represent BSP. The identification of these Stains-All positive protein bands as OPN and BSP was confirmed by Western immunoblotting (see Fig. 3).

Fig. 3

Western immunoblotting for DSPP and its fragments, DMP1 and its fragments, BSP and OPN. (A) Western immunoblotting using anti-DSP polyclonal antibody for fractions 27–39 of Q-Sepharaose chromatography of NCPs extracted from rat condylar cartilage. Positive control (Cont): 0.3 μg of DSP isolated from rat incisor dentin; 27–39: 60 μl of sample from fractions 27-39 were treated with 2.5% of β-mercaptoethanol before loading. Long arrow indicates the migrating position of full-length DSPP; arrowhead indicates the migrating position of DSP. (B) Western immunoblotting using anti-DMP1-N-9B6.3 monoclonal antibody. Cont: 1 μg of the NH₂-terminal (37 kDa) fragment of DMP1 and the full-length form of DMP1 isolated from rat long bone. 51: 60 μl of sample from fraction 51 of Q-Sepharaose chromatography of NCPs extracted from rat condylar cartilage. Both the 37 kDa fragment (arrowhead) and full-length form of DMP1 (long arrow) are detected in the extract from the rat condylar cartilage. In comparison with the DMP1 molecular species in the extract from rat long bone (Cont in Fig. 3B), the full-length form of DMP1 is more abundant in the extract from the cartilage. (C) Western immunoblotting using anti-DMP1-C-857 polyclonal antibody. Cont: 1 μg of the COOH-terminal (57 kDa) fragment and the full-length form of DMP1 isolated from rat long bone. 51: 60 μl of sample from fraction 51 of Q-Sepharaose chromatography of NCPs extracted from rat condylar cartilage. While the full-length form of DMP1 (long arrow) was visualized, the 57 kDa fragment (arrowhead) was hardly detectable in the extract from the rat condylar cartilage. (D) Western immunoblotting using anti-BSP-10D9.3 monoclonal antibody. Cont: 1 μg of BSP isolated from rat long bone. 69: fraction 69 of Q-Sepharaose chromatography of NCPs extracted from rat condylar cartilage. Arrow indicates the migrating position of BSP. (E) Western immunoblotting using the anti-OPN polyclonal antibody. Cont: 1 μg of OPN isolated from rat long bone. 51: fraction 51 of Q-Sepharaose chromatography of NCPs extracted from rat condylar cartilage. Arrow indicates the migrating position of OPN.

Fig. 4

H&E and IHC staining for paraffin sections of the condylar cartilage from 2-, 5- and 8-week-old rats. Column 1, 2-week-old rat; column 2, 5-week-old rat; column 3, 8-week-old rat. Ar, articular layer; Pr, prechondroblastic layer; Ch, chondroblastic layer; Hy, hypertrophic layer; C-B, cartilage–bone interface. Row A, H&E staining; row B, IHC for DSPP; row C, IHC for the NH₂-terminal fragment of DMP1; row D, IHC for the COOH-terminal fragment of DMP1; row E, IHC for BSP; row F, IHC for OPN. Bar equals 100 μm in all microphotographs. DSPP was observed in the cells of all five layers and in the ECM of the articular layer and prechondroblastic layer at 2 weeks (Fig. 4B1). At 5 and 8 weeks (Figs. 4B2 and 4B3), the signal for DSPP was detected in the cells of the chondroblastic and hypertrophic layers, as well as in the ECM of the articular, prechondroblastic and chondroblastic layers. The signal for the NH₂-terminal fragment of DMP1 was mainly observed in the ECM of the articular layer and prechondroblastic layer at 2 weeks (Fig. 4C1). At 5 and 8 weeks (Figs. 4C2 and 4C3), the signal for the NH₂-terminal fragment of DMP1 was mainly observed in the cells and the ECM of the chondroblastic and hypertrophic layers. Note that the IHC staining in the 5-week-old group is weaker than in the 8-week-old group. The signal for the COOH-terminal fragment of DMP1 (Figs. 4D1-4D3) was primarily observed in the nuclei of cells of the chondroblastic and upper part of the hypertrophic layers. BSP was observed in the cells of all five layers and in the ECM of the articular layer at 2 weeks (Fig. 4E1). At 5 weeks (Fig. 4E2), BSP signal was strong in the nuclei of cells in the prechondroblastic and chondroblastic layers and in the ECM of all five layers. At 8 weeks (Fig. 4E3), the signal for BSP was mainly observed in the prechondroblastic, chondroblastic and hypertrophic layers. OPN was observed in the cells of all five layers and in the ECM of the articular layer at 2 weeks (Fig. 4F1). At 5 and 8 weeks (Figs. 4F2 and 4F3), the signal for OPN was mainly observed in the nuclei of the chondroblastic layer and in the ECM of the prechondroblastic and chondroblastic layers.

Fig. 5

Double staining immunofluorescence on the condylar cartilage from 8-week-old rats using a mixture of anti-DMP1-N-859 polyclonal and anti-DMP1-C-8G10.3 monoclonal antibodies. (A) Light microscopic IHC was performed with the anti-DMP1-N-859 polyclonal antibody. The NH₂-terminal fragment of DMP1 was prominent in the chondroblastic and hypertrophic layers (boxed). (B) A higher magnification of the boxed area in A. (C) Light microscopic IHC was performed with the anti-DMP1-C-8G10.3 monoclonal antibody. The COOH terminal fragment was observed in the nuclei of cells in the chondroblastic and hypertrophic layers (boxed). (D) A higher magnification of the boxed area in C. (E) Double staining immunofluorescence analysis showed that the NH₂-terminal fragment (red color) was mainly observed in the ECM of the chondroblastic and hypertrophic layers, or around the cell nuclei in the prechondroblastic, chondroblastic and hypertrophic layers, while the signal for the COOH terminal fragment (green color) was mainly found in the nuclei of cells in the chondroblastic and hypertrophic layers, and at the cartilage–bone interface.