Distribution of small integrin-binding ligand, N-linked glycoproteins (SIBLING) in the articular cartilage of the rat femoral head

Abstract

The small integrin-binding ligand, N-linked glycoprotein (SIBLING) family is closely related to osteogenesis. Until recently, little was known about their existence in articular cartilage. In this study, we systematically evaluated the presence and distribution of four SIBLING family members in rat femoral head cartilage: dentin matrix protein 1 (DMP1), bone sialoprotein (BSP), osteopontin (OPN), and dentin sialophosphoprotein (DSPP). First, non-collagenous proteins were extracted and then separated by ion-exchange chromatography. Next, the protein extracts eluted by chromatography were analyzed by Stains-all staining and Western immunoblotting. IHC was used to assess the distribution of these four SIBLING family members in the femoral head cartilage. Both approaches showed that all the four SIBLING family members are expressed in the femoral head cartilage. IHC showed that SIBLING members are distributed in various locations throughout the articular cartilage. The NH₂-terminal fragments of DMP1, BSP, and OPN are present in the cells and in the extracellular matrix, whereas the COOH-terminal fragment of DMP1 and the NH₂-terminal fragment of DSPP are primarily intracellularly localized in the chondrocytes. The presence of the SIBLING family members in the rat femoral head cartilage suggests that they may play important roles in chondrogenesis.

Figure 1

Identification of the small integrin-binding ligand, N-linked glycoprotein (SIBLING) family members in the femoral head cartilage by Stains-all staining and Western immunoblotting. Numbers at the top of each image represent fraction numbers of the Q-Sepharose chromatography. (A) Stains-all staining for every third fraction in the chromatographic region from fractions 16–90 is presented here to illustrate the existence of the four SIBLING family members. The blue protein bands in fractions 43–52 migrating between the 53- and 78-kDa molecular mass markers represent osteopontin (OPN; boxed). The dentin matrix protein 1 (DMP1)-C fragment (∼57 kDa, boxed) co-eluted with OPN in fractions 46–52. Bone sialoprotein (BSP; ∼90 kDa, boxed) mainly eluted in fractions 46–64. The identities of these SIBLING family members were confirmed by Western immunoblotting (B–G). (B) Control (Ctrl): 1 μg of DMP1 (including the full-length DMP1 and its processed fragments) extracted from long bone of rat. Western immunoblotting using the anti-DMP1-N-9B6.3 antibody demonstrated the presence of DMP1-N (37 kDa, white arrowhead) and full-length DMP1 (∼110 kDa, black arrow) in fractions 34–40. (C) Ctrl: 1 μg of DMP1 isolated from long bone of rat. Note the presence of DMP1-proteoglycan (DMP1-PG) recognized by the anti-DMP1-N-9B6.3 antibody in fractions 52–70. (D) Ctrl: 1 μg of DMP1 isolated from long bone of rat. DMP1-C (arrowhead) was detected by the anti-DMP1-C-857 antibody in fractions 46–52. (E) Ctrl: 1 μg of BSP isolated from long bone of rat. BSP (arrowhead) was recognized by the anti-BSP-10D9.2 antibody in fractions 46–64. (F) Ctrl: 1 μg of OPN isolated from long bone of rat. OPN (arrowhead) was detected by the anti-OPN antibody in fractions 43–49. (G) Ctrl: 0.3 μg of dentin sialoprotein (DSP) isolated from incisor dentin of rat. DSP was detected by the anti-DSP-2G7.3 antibody in fractions 22–31. Note that DSP extracted from the cartilage migrated faster than that isolated from rat dentin (i.e., the positive Ctrl). The lane for the positive Ctrl sample in the original Western immunoblotting gel was two lanes away from the lane of fraction 22; we horizontally moved the Ctrl lane to the position next to fraction 22 to better illustrate the results.

Figure 2

Hematoxylin and eosin (HE) staining of the femoral head cartilage from 4-, 8-, 16-, and 24-week-old rats. (A) Femoral head cartilage from a 4-week-old rat, (B) femoral head cartilage from an 8-week-old rat, (C) femoral head cartilage from a 16-week-old rat. (D) Femoral head cartilage including the secondary ossification center from a 24-week-old rat. In 4-, 8-, and 16-week-old rats, the femoral head cartilage was divided into three distinct layers: the superficial layer (S), the middle layer (M), and the deep layer (D). The deep layer is continuous to the growth plate (G). The structure of articular cartilage changes with aging. Compared with the younger rats (4-week-old; A), the cartilage of the older rats (16-week-old; C) has more matrix in the middle layer and a greater number of hypertrophic cells in the deep layer. The secondary ossification center (white arrowhead; C) was formed in the deep layer at 16 weeks after birth. At 24 weeks after birth, the secondary ossification center completely separated the articular cartilage from the growth plate (D). Bar = 100 μm.

Figure 3

IHC staining of the femoral head cartilage from 4-, 8-, and 16-week-old rats. Column 1, 4-week-old rat; column 2, 8-week-old rat; column 3, 16-week-old rat. (A–C) IHC for DMP1-N/DMP1-PG. The signal for DMP1-N/DMP1-PG was mainly observed in the chondrocytes and in the extracellular matrix (ECM) of the superficial layer. The intensity of IHC staining in the superficial layer increased with aging. At 16 weeks (C), the signal for DMP1-N/DMP1-PG was very strong in the ECM near the fovea (arrowhead) of the femoral head and in the secondary ossification center (arrow). (D–F) IHC for DMP1-C. The signal for DMP1-C was primarily observed in the cell nuclei in all layers. The staining intensity of DMP1-C is not as strong as DMP1-N/DMP1-PG in the same area. Arrow indicates the secondary ossification center (F). (G–I) IHC for BSP. BSP was observed in the cells of all three layers and in the ECM of the superficial layer at 4 weeks after birth (G). At 8 weeks (H), BSP signal was also observed in the ECM of the middle layer. At 16 weeks (I), the signal for BSP became stronger in the ECM beneath the superficial layer and in the chondrocytes in all layers of the cartilage. The staining was also intense in the secondary ossification center (arrow, I). (J–L) IHC for OPN. At 4 weeks after birth, OPN was observed in the cells of all layers and in the ECM of the superficial layer (J). At 8 weeks (K), the signal for OPN was also observed in the ECM of the middle layer. At 16 weeks (L), the signal for OPN became stronger in the ECM of the superficial and middle layers and in the chondrocytes in all layers of the cartilage. The staining was also strong in the secondary ossification center (arrow, L). (M–O) IHC for DSP. A weak signal for DSP was observed in the cells of all layers and in the ECM of the superficial layer at 4, 8, and 16 weeks. The staining for DSP was observed in the secondary ossification center (arrow, O). (P–R) Negative controls. In these IHC experiments, normal mouse IgG was used to replace the corresponding primary antibodies. Arrow indicates secondary ossification center (R). Bar = 200 μm.

Figure 4

IHC—higher magnification of the deep layer in the femoral head cartilage from an 8-week-old rat. (A) HE staining: the microphotograph is a higher magnification of boxed area in inset; (B) IHC for DMP1-N/DMP1-PG; (C) IHC for DMP1-C; (D) IHC for BSP; (E) IHC for OPN; and (F) IHC for DSP. Note the presence of BSP (D) and OPN (E) in the matrix surrounding chondrocytes in the deep layer. The staining for DMP1-N was weak in the matrix (B). DMP1-C and DSP were observed only within the cells (C,F). Bar = 100 μm.

Figure 5

IHC—higher magnification of the femoral head cartilage and the secondary ossification center of the cartilage in the 16-week-old rats. (A) HE staining; the microphotograph is a higher magnification of the boxed area in inset. IHC staining (B–F) was performed on sections that were serial to A. (B) IHC for DMP1-N/DMP1-PG; (C) IHC for DMP1-C; (D) IHC for BSP; (E) IHC for OPN; (F) IHC for DSP. Note that the signals for DMP1-N/DMP1-PG, BSP, and OPN were strong in the chondrocytes and in the ECM (B,D,E), whereas the signals for DMP1-C and DSP were relatively weak in the chondrocytes but not in the ECM (C,F). A′–F′ is a higher magnification of the secondary ossification center in the femoral head cartilage. (A′) HE staining of the boxed area in inset. IHC staining (B′–F′) was performed on sections that were serial to A′. (B′) IHC for DMP1-N/DMP1-PG; (C′) IHC for DMP1-C; (D′) IHC for BSP; (E′) IHC for OPN; (F′) IHC for DSP. All these SIBLING family members were detected at a relatively high level in the newly formed bone matrix of the secondary ossification center. Bar = 200 μm.