Expression of superficial zone protein in mandibular condyle cartilage

Abstract

Objective: Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle.

Methods: The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage.

Results: SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-beta in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1beta. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-beta.

Conclusions: These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies.

Figure 1

Distribution of SZP and hyaluronan in mandibular condyle cartilage. Frozen sections of articular cartilages from bovine mandibular condyle were incubated with SZP antibody (A) or control IgG (D). SZP distribution was visualized by FITC, and nuclei were stained with DAPI (blue fluorescence) (B). Panels C and E show the original tissue structure of section of A, B and D, respectively. (SL, superficial layer; FZ, fibrous zone; PZ, proliferative zone; TZ, transitional zone; bars 100 μm)

Figure 2

Effects of IL-1β and TGF-β on the SZP level in mandibular condyle cartilage. Condyle cartilages from bovine mandible were incubated in serum free medium without (A, D) or with IL-1β (1 ng/ml) (B, E) or TGF-β (5 ng/ml) (C, F) for 4 days. Tissues were treated with monensin for the last 4 hr of the incubation (D, E, and F). Frozen sections of the explants were incubated with SZP antibody. SZP distribution was visualized by FITC conjugated Streptavidin. (Arrowhead, superficial layer; FZ, fibrous zone; bars 100 μm)

Figure 3

Effects of IL-1β and TGF-β on the expression of SZP mRNA in the cultured MSF. Dose response (24 hr; A, C) and time course response (1 ng/ml for IL-1β; B, 5 ng/ml for TGF-β; D) for SZP mRNA induction were assessed by quantitative real time RT-PCR. Reverse transcription products (80 ng) of total RNA from chondrocytes +/− IL-1β (A, B) or TGF-β (C, D) treatment were amplified by using a specific primer set for SZP. The fold increase in treated (black bars) relative to controls (hatched bars) were calculated from CP values using equation 1 described above. The ratio of the copy numbers between experimental samples to time-zero controls are shown as closed circles. Data represent the mean ± SD of three experiments with different batches of cells. Statistical significance was shown between controls (open bars) and experimental samples. (*; p < 0.05, **; p < 0.01)

Figure 4

Effects of IL-1β and TGF-β on the SZP protein level in the conditioned medium of MSF cultures. SZP protein levels were assessed in the conditioned medium of MSF cultures treated without (○) or with IL-1β (▲) at 1.0 ng/ml or TGF-β (■) at 5 ng/ml, using ELISA. The effect of dose (A) and time course (B) of IL-1β and TGF-β effects on SZP protein are shown. SZP values were normalized to total DNA content. Data represent the mean ± the SD of three experiments with different batches of cells. Statistical significance was shown between control (dose 0) and experimental samples at each time point. (*; p < 0.05, **; p < 0.01)