Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene

Abstract

Tumor necrosis factor-related, activation-induced cytokine (TRANCE), a tumor necrosis factor family member, mediates survival of dendritic cells in the immune system and is required for osteoclast differentiation and activation in the skeleton. We report the skeletal phenotype of TRANCE-deficient mice and its rescue by the TRANCE transgene specifically expressed in lymphocytes. TRANCE-deficient mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation at several skeletal sites, including the limbs, skull, and vertebrae. These mice had marked chondrodysplasia, with thick, irregular growth plates and a relative increase in hypertrophic chondrocytes. Transgenic overexpression of TRANCE in lymphocytes of TRANCE-deficient mice rescued osteoclast development in two locations in growing long bones: excavation of marrow cavities permitting hematopoiesis in the marrow spaces, and remodeling of osteopetrotic woven bone in the shafts of long bones into histologically normal lamellar bone. However, osteoclasts in these mice failed to appear at the chondroosseous junction and the metaphyseal periosteum of long bones, nor were they present in tooth eruption pathways. These defects resulted in sclerotic metaphyses with persistence of club-shaped long bones and unerupted teeth, and the growth plate defects were largely unimproved by the TRANCE transgene. Thus, TRANCE-mediated regulation of the skeleton is complex, and impacts chondrocyte differentiation and osteoclast formation in a manner that likely requires local delivery of TRANCE.

Figure 1

Radiographs of 30-day-old wild-type mice (wt), TRANCE KO mice (ko), and TRANCE-null mice expressing TRANCE transgene (TRANCE TG/KO mice; tg/ko). Top row shows hind limb, pelvis, and vertebrae. Insets show higher magnification of the proximal tibia. (Lower) Skulls hemisected in the midline. Marrow cavities are absent in the osteopetrotic TRANCE KO mouse, but are restored in the TRANCE TG/KO mouse (arrows). The cartilage growth plate, seen as a radiolucent stripe across the proximal tibia in the Insets, is thicker in the TRANCE KO animal (arrow), whereas, in the TRANCE TG/KO mouse, there remains radio-opaque, sclerotic, mineralized tissue in the metaphyseal ends of the long bones (asterisk). Incisors, evident in wild type, fail to erupt in either the TRANCE KO or TG/KO mice (arrowheads), as do the molars.

Figure 2

Comparisons of tibial growth in 2-mo-old mice. Length of tibiae, measured on projected x-rays, is shown for wild-type (wt), TRANCE KO (ko), and TRANCE TG/KO (tg/ko) animals. The TRANCE KO mice exhibited significant growth retardation compared with wild-type mice (P < 0.0001). This defect was not significantly improved by the presence of the TRANCE transgene. Other ages and skeletal sites showed similar results (see text). Data are shown as mean + 1 SD, n ≥ 3.

Figure 3

Epiphyseal growth plate chondrodystrophy in proximal tibiae of TRANCE KO (ko) and TRANCE TG/KO (tg/ko) mice. Arrowheads denote the approximate lower limit of the proliferative chondrocyte zone and arrows the lower limit of the hypertrophic zone, the distance between thus being the thickness of the hypertrophic zone. Wild-type animals (wt) show a normal growth plate, with columns of chondrocytes undergoing proliferation and differentiation to hypertrophy in the lower region, followed by remodeling of cartilage into metaphyseal bone and the formation of a marrow cavity (m). TRANCE KO animals (ko) display abnormal chondrocyte organization and differentiation, with a reduced proliferating zone (arrowhead) and a thickened zone of hypertrophy, resulting in an increase in overall thickness of the growth plate (corresponding to the dark stripe in Fig. 1 ko Inset). The TRANCE transgene failed to correct the abnormal organization and differentiation of growth plate chondrocytes, but did reduce overall growth plate thickness. Extensive amounts of mineralized cartilage (asterisks) remained unresorbed. (Original magnification, ×800.) Toluidine-blue-stained, demineralized, Epon-embedded 1-μm sections.

Figure 4

Osteoclast distribution in proximal tibiae of wild-type (wt), TRANCE KO (ko), and TRANCE TG/KO (tg/ko) mice. (a and e) Toluidine blue stained, Epon-embedded sections from 1-mo-old wild-type and TRANCE TG/KO animals. (b–d) Osteoclasts (red) visualized by TRAP histochemistry on similarly oriented sections from wild-type (wt), TRANCE KO (ko), and TRANCE TG/KO (tg/ko) animals, respectively. Key areas of osteoclast activity in growing long bones are indicated by numbers 1–4 in a, and represent: 1, lower margin of epiphysis; 2, chondroosseous junction; 3, ends of metaphyseal trabeculae; and 4, metaphyseal periosteum. Osteoclasts are found in all four areas in wild type (b). No osteoclasts are present in TRANCE KO mice (c). In TRANCE TG/KO mice (d), osteoclasts are present in areas 1 and 3 only (arrows), with none in either the chondroosseous junction or the metaphyseal periosteum (asterisks). Marrow cavities (m) are seen in wild-type and TRANCE TG/KO, but not TRANCE KO mice. (f) A typical, multinucleated osteoclast resorbing mineralized cartilage (c) in area 3 of the TRANCE TG/KO mouse (s, venous sinus; m, marrow). (Original magnification: a–e, ×205; f, ×1,300.)

Figure 5

Cortical bone remodeling is rescued in TRANCE TG/KO mice. Comparison of remodeling of diaphyseal cortical bone in wild-type (wt), TRANCE KO (ko) and TRANCE TG/KO (tg/ko) mice. Periosteal surface is toward the top and endosteum toward the bottom in all panels. The TRANCE KO mice show typical osteopetrotic woven bone that has never been remodeled, with mineralized cartilage cores (c, purple) covered by bone (blue). Normal remodeled, lamellar bone (L) lacking cartilage cores is seen in the wild-type and TRANCE TG/KO diaphyses, with healthy, cuboidal osteoblasts (*) lining the endosteal bone surface, osteocytes in lacunae (arrows), and active marrow (m) in both specimens. Toluidine-blue-stained, 1-μm Epon sections, tibial diaphyses of 30-day-old animals. (Original magnification, ×1,300.)