Skeletal abnormalities in mice lacking extracellular matrix proteins, thrombospondin-1, thrombospondin-3, thrombospondin-5, and type IX collagen

Abstract

Thrombospondin-5 (TSP5) is a large extracellular matrix glycoprotein found in musculoskeletal tissues. TSP5 mutations cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia; both show a characteristic growth plate phenotype with retention of TSP5, type IX collagen (Col9), and matrillin-3 in the rough endoplasmic reticulum. Whereas most studies focus on defining the disease process, few functional studies have been performed. TSP5 knockout mice have no obvious skeletal abnormalities, suggesting that TSP5 is not essential in the growth plate and/or that other TSPs may compensate. In contrast, Col9 knockout mice have diminished matrillin-3 levels in the extracellular matrix and early-onset osteoarthritis. To define the roles of TSP1, TSP3, TSP5, and Col9 in the growth plate, all knockout and combinatorial strains were analyzed using histomorphometric techniques. While significant alterations in growth plate organization were found in certain single knockout mouse strains, skeletal growth was only mildly disturbed. In contrast, dramatic changes in growth plate organization in TSP3/5/Col9 knockout mice resulted in a 20% reduction in limb length, corresponding to similar short stature in humans. These studies show that type IX collagen may regulate growth plate width; TSP3, TSP5, and Col9 appear to contribute to growth plate organization; and TSP1 may help define the timing of growth plate closure when other extracellular proteins are absent.

Figure 1

Growth plates from single and compound knockout mice. A: H&E-stained upper tibial growth plates from single and compound knockout mice at 1 and 2 months of age are shown. Control growth plates (A, D, F, H, K, and M) are on the left for each strain. The loss of type IX collagen (B and I), TSP3 (E and L), or TSP5 (G and N) causes disturbance of growth plate organization. The Col9 growth plate appears wider and the chondrocytes columns are not as well defined as in the control growth plates. Loss of TSP5 (G and N) causes minor disruption of columnar patterning, whereas loss of TSP1 (C and J) has little effect. B: Control growth plates (A, C, E, and G) are on the left for each strain. The double knockout of TSP5/Col9 genes (D and H) produces growth plate pathology that is similar to that of the single Col9 knockout growth plate [A (B and I)], but loss of TSP3/5 (B and F) causes more growth plate disorganization than TSP5 alone [A (G and N)]. C: Control growth plates (A and F) are on the left. Loss of TSP1/3/5 (B and G) is similar to the TSP3/5 knockout growth plate [B (B and F)] but loss of type IX collagen in combination with loss of TSP3/5 (D and I) and TSP1/3/5 (E and J) has a significant impact on the organization of the growth plate and the hypertrophic zone. Loss of type IX collagen in combination with loss of TSP1/5 is shown in C and H. Scale bar = 100 μmol/L [A (A)]. Upper tibial growth plates were used in all studies.

Figure 2

Comparison of growth plate chondrocyte organization in knockout mice and controls. A: The method used in the classification of chondrocytes is shown and described in Materials and Methods. B: Comparison of percent chondrocytes organized into columns in all knockout strains and respective controls at 1 month of age. C: Comparison of percent chondrocytes organized into columns in all knockout strains and respective controls at 2 months of age. Black and gray bars are measurements for controls and strains, respectively. All knockout strains showed disrupted chondrocyte columns, with the TSP1 knockout the least affected and the TSP3/5/Col9 and TSP1/3/5/Col9 knockout strains the most affected. *P < 0.05. Upper tibial growth plates were used in all studies.

Figure 3

Loss of type IX collagen affects the width of growth plate. A: Measurements of growth plates was performed as shown on the images and described in Materials and Methods. PZ, proliferative zone: GP, growth plate. B: Comparison of growth plate width at 1 month. Black and gray bars are measurements for controls and strains, respectively, and the asterisk indicates a significant difference from the control (P < 0.05). The overall growth plate of the Col9 and combinatorial knockout strains lacking Col9 tend to be wider compared with the controls. C: By 2 months, there is no difference in the width of the Col9 growth plate compared with the control; however, the TSP1, TSP5, and TSP3/5 growth plates are all more narrow than the control, whereas the growth plates of the other combinatorial mouse strains are wider than the control. *P < 0.05. Upper tibial growth plates were used in all studies.

Figure 4

Measurement of bone length. A: Measurement of the TSP1/3/5/Col9 limb compared with control limb is shown and described in Materials and Methods. B: Comparison of hind limb length from all knockout strains. Black and gray bars are measurements for controls and strains, respectively, and the asterisk indicates a significant difference (P > 0.05) from the control. The long bones of the hind limb are significantly shorter in Col9, TSP3/5, TSP5/Col9, TSP1/3/5, TSP1/5/Col9, TSP3/5/Col9, and TSP1/3/5/Col9 mice. The combinatorial loss of TSP3, TSP5, and type IX collagen has the greatest effect on limb length. *P < 0.05.

Figure 5

Distribution of MATN3 in knockout growth plates. Growth plates were simultaneously immunostained with an antibody to MATN3, as described in Materials and Methods. Control sections are to the left for all strains (A, D, F, H, J, and L). MATN3 was markedly diminished in the Col9 knockout growth plate and in all compound knockout growth plate strains that do not synthesize type IX collagen (B, K, N, O, and P). Interestingly, the Col9 knockout growth plate shows light immunostaining throughout the proliferative zone and darker staining in the hypertrophic zone (B). Loss of TSP1, TSP3, and TSP5 does not affect the quantity or distribution of MATN3 (C, E, and G). Loss of TSP3/5 (I) and TSP1/3/5 (M) are also shown. Scale bar = 100 μmol/L. Upper tibial growth plates were used in all studies.

Figure 6

Articular cartilage from hind limbs from knockout mice subjected to exercise regimen. C0l9, TSP5, TSP5/Colp knockout, and control mice were subjected to a running exercise regimen, as described in Materials and Methods. The H&E-stained articular cartilage sections are shown. All of the wild-type running mice (A, B, and C) had normal articular cartilage. The nonrunner TSP5 knockout mice (E) had relatively normal articular cartilage, whereas the articular cartilage of the runners (H) was mildly flattened. At the end of the exercise regimen, the Col9 knockout nonrunner (D) had mild flattening, whereas the runner (G) had severe flattening (asterisk) of the articular cartilage. Interestingly, the TSP5/Col9 knockout nonrunner (F) had flattened articular cartilage (asterisk), and exercise caused additional flattening (asterisk) and fraying (arrow) of the cartilage in the runner (I). A closer view of the articular cartilage cap is shown below each panel (a–i). Scale bar = 100 μmol/L.