* Department of Cell Biology and the Vanderbilt Cancer Center, and Department of Pathology, Vanderbilt University,
Nashville, Tennessee 37232; § Department of Growth and Development and Anatomy, Programs in Cell Biology and
Developmental Biology, University of California, San Francisco, California 94143;
Division of Reproductive and
Developmental Toxicology Laboratory, National Center for Toxicological Research, Jefferson, Arkansas 72709
Members of the TGF- superfamily are important regulators of skeletal development. TGF-
s
signal through heteromeric type I and type II receptor
serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation,
thereby acting as a dominant-negative mutant (DNIIR).
To determine the role of TGF-
s in the development
and maintenance of the skeleton, we have generated
transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression
was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR
mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid
process and sternum. They also developed progressive
skeletal degeneration, resulting by 4 to 8 mo of age in
kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or
hypertrophic cartilage as judged by the expression of
type X collagen, a marker of hypertrophic cartilage
normally absent from articular cartilage. The synovium
was hyperplastic, and cartilaginous metaplasia was observed in the joint space.
We then tested the hypothesis that TGF- is required
for normal differentiation of cartilage in vivo. By 4 and
8 wk of age, the level of type X collagen was increased
in growth plate cartilage of transgenic mice relative to
wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix
of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog
(IHH) expression. IHH is a secreted protein that is
expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a
feedback loop that signals through the periosteum/
perichondrium to inhibit cartilage differentiation. The
data suggest that TGF-
may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-
promotes chondrocyte terminal differentiation and results in development of degenerative
joint disease resembling osteoarthritis in humans.
ENDOCHONDRAL bone develops through a complex
process whereby a cartilage model is replaced with
bone (for reviews see 9, 20, 25). The cartilage template is formed from undifferentiated mesenchymal cells
which condense and differentiate into chondrocytes. These cells then progress through a program of cell proliferation,
maturation, and hypertrophy. Hypertrophic chondrocytes
represent the terminally differentiated phenotype and are
replaced by bone cells in the final stages of endochondral
bone development. While most of the cartilage model is
eventually replaced with bone, cartilage is maintained on
the articular surface (articular cartilage) and, in mouse, a
small amount of the cartilage model persists in the growth
plate. Longitudinal growth proceeds from the epiphyseal growth plate and must be intimately coordinated with appositional growth to maintain the shapes of individual
bones. Thus, chondrocyte differentiation has to be strictly
regulated so that the proper length and shape of the bone
is maintained. The rate and extent of endochondral bone
growth is regulated by Indian hedgehog (IHH)1 and parathyroid hormone-related peptide (PTHrP), two secreted peptides (9, 35, for review see 74). IHH is expressed by
cells that are committed to becoming hypertrophic and
acts to increase PTHrP expression in the perichondrium
and periarticular region. PTHrP inhibits further chondrocyte differentiation in cells that express the PTH receptor
and are not yet committed to becoming hypertrophic.
Control of skeletal development and maintenance is complex and likely involves several additional mediators.
Members of the TGF- Persistence of cartilage on the articular surface is necessary for proper joint function. Osteoarthritis is a noninflammatory disorder of synovial joints, often referred to as
osteoarthrosis. Degeneration of the articular cartilage results in osteoarthritis, which is characterized by decreased
proteoglycan and increased type X collagen in the cartilage matrix (for review see 23). That TGF- Members of the TGF- To study the role of TGF- Generation and Identification of Transgenic Mice
The MT-DNIIR expression plasmid was used to generate transgenic mice
(see Fig. 1). The EcoRI/XbaI fragment of p102 containing the truncated
human TGF-
Whole Mount Skeletal Preparation
Whole mount skeletal preparations of adult mice were prepared according to Selby (57). Briefly, mice that had been skinned and eviscerated
were soaked in a series of KOH solutions over several days. Skeletal tissue was stained with 0.004% alizarin red in 1.9% KOH. The specimens
were cleared in glycerin, benzyl alcohol, and ethanol and then stored in
glycerin. Skeletons from 17.5-d post-coital fetal mice were double stained
for cartilage and bone with alcian blue and alizarin red (33, 38). All carcasses were skinned and fixed in 95% ethanol for 72 h. Carcasses were then placed in 95% ethanol-alcian blue solution (20 h) for cartilage staining, followed by a 95% ethanol wash (8 h) and maceration in 0.35% KOH
overnight. Bone was stained with alizarin red S in 0.2% KOH (4 to 6 h)
followed by clearing in 95% ethanol:glycerin (1:1).
RNA Analysis
Mouse hindlimbs were removed and skin and muscle were trimmed away.
The remaining skeletal tissue was frozen in liquid nitrogen and crushed
with a mortar and pestal into a fine powder. RNA was extracted from the
bone powder by the procedure of Chomerymski and Sacchi (13). RNA
was treated with RQ1 RNase-free DNAse (Promega Biotech, Madison,
WI) for 30 min at 37°C to remove contaminating genomic DNA. RNA
was then extracted by phenol:chloroform extraction and ethanol precipitation. RNA concentration was determined spectrophotometrically. For
RT PCR analysis, cDNA was synthesized from 1 µg of total RNA pooled
from the hind limbs of two to four mice using oligo dT primers as described in the GeneAmp RNA PCR kit (Perkin Elmer, Norwalk, CT).
PCR amplification was performed using 5 µl of the cDNA mix. The same
conditions and primers described above for the amplification of DNIIR
from genomic DNA were used, except primers to glyceraldehyde-3-phosphate dehydrogenase were added as an internal loading control. (3 In Situ Hybridization
In situ hybridization was performed as described (47) on sections from
mouse knee joints decalcified in EDTA. Briefly, hindlimbs were removed,
and skin and muscles were trimmed away. The long bone was trimmed
close to the joint. The joint was fixed overnight at 4°C in fresh 4%
paraformaldehyde. The specimens were rinsed in DEPC-treated water
and soaked in 0.1 M Tris, pH 7.5, 10% EDTA tetrasodium salt, 7.5% polyvinyl pyrolidione, and 1 µl/ml DEPC for 2 to 7 d at 4°C. The joints were
dehydrated through ethanol and xylene and then embedded in paraffin. If
the tissue remained too hard to section, the block was soaked in the
EDTA decalcification solution for an additional day. Sections were hybridized to 35S-labeled sense and antisense riboprobes. The MT-DNIIR
plasmid was linearized with EcoRI, and the antisense probe was made
with T7 polymerase. The sense probe was made from XbaI-linearized
plasmid and T3 polymerase. The IHH plasmid was the kind gift of Andy
McMahon (Harvard University, Cambridge, MA). The antisense probe
was made from XbaI-linearized plasmid using the T7 polymerase. The
PTH receptor probe was the kind gift of Henry Kronenberg (Havard
Medical School, Boston, MA). The antisense probe was made from the
EcoRI-linearized plasmid using T3 polymerase. Slides were exposed to
photographic emulsion at 4°C for 2 wk and then developed with D19 developer, fixed in 1% acetic acid, and cleared in 30% sodium thiosulfate. Sections were counterstained with 0.2% toluidine blue. Kodak Ektachrome film was used to take photographs under phase contrast, bright field, and
dark field illumination using a microscope (Zeiss, Thornwood, NY).
Histology and Immunohistochemistry
Mouse hindlimbs were fixed overnight at 4°C in 4% paraformaldehyde
and then decalcified overnight at 4°C in Surgipath decalcifying solution.
Specimens were dehydrated and embedded in paraffin. For routine histological analysis, sections were stained with hematoxylin and eosin using
standard procedures. To visualize proteoglycans, sections were stained
with safranine O. Briefly, deparafinized sections were rehydrated and
stained with hematoxylin for 3 min. The slides were washed in tap water,
and the hematoxylin was differentiated in acid alcohol and blued in lithium carbonate. Sections were then stained with a 1:5,000 dilution of aqueous fast green for 3 min, washed briefly in 1% acetic acid, and stained in
0.1% safranine O for 3 min. The sections were quickly dehydrated, cleared, and mounted.
Immunohistochemical staining of type X collagen was performed using
polyclonal antibodies to mouse type X collagen that were a generous gift
from Tim Pfordte and Bjorn Olsen (Harvard Medical School, Boston,
MA). Sections were dewaxed, rehydrated, and digested with 1 mg/ml hyaluronidase in PBS at 37°C for 45 min. Immunohistochemistry was performed using the Vectastain Elite immunoperoxidase staining kit (Vector
Laboratories, Hercules, CA) according to the manufacturer's instructions.
The color reaction was performed using the DAB substrate kit from Vector Laboratories. Sections were counterstained with hematoxylin.
Construction of MT-DNIIR Transgenic Mice
An expression plasmid (MT-DNIIR; Fig. 1) containing the
coding sequence for the truncated human TGF- Skeletal Defects in MT-DNIIR Transgenic Mice
Signs of skeletal abnormalities were observed in mice from
two of the MT-DNIIR transgenic lines, MT-DNIIR-4 and
MT-DNIIR-27, maintained on normal food and tap water.
Defects were observed in heterozygous mice. Skeletal defects were apparent by 3 mo of age and became progressively worse as the mice aged. MT-DNIIR-4 mice demonstrated kyphoscoliosis and stiffness in the hindlimb joints.
In some cases, hindlimbs were torqued laterally at varying angles. The xiphoid process of the sternum protruded out
from the chest and was visible under the skin. MT-DNIIR-27
mice demonstrated stiffness in the knees. No differences in
bone density were detected on X-ray films (data not shown).
To further characterize the nature of the skeletal defects,
alizarin red whole mount skeletal preparations of adult
mice (4 to 8 mo of age) were performed (Table I; Fig. 2).
A summary of skeletal defects observed in whole mount
skeletal preparation of adult wild-type and heterozygous
transgenic mice is shown in Table I. Representative preparations are shown from adult wild-type, MT-DNIIR-4, and
MT-DNIIR-27 mice (Fig. 2). The xiphoid process of adult
MT-DNIIR-4 transgenic animals was bifurcated. Knee
and shoulder joints of MT-DNIIR-4 and -27 mice were
disorganized and contained excess calcified tissue. MT-DNIIR-4 vertebrae were often misshapen and appeared
fused. Sternal defects and tumoral calcinosis were only
found in the MT-DNIIR-4 line; therefore, these observations must be considered tentative. However, the presence
of joint defects in two transgenic lines suggests the reasonable hypothesis that all the chondrocyte abnormalities in MT-DNIIR-4 mice are not simply insertional effects. Skeletal defects were not detected in wild-type mice.
Table I.
Summary of Skeletal Defects Identified by Alizarin
Red Whole Mount Skeletal Preparations
Expression and Localization of DNIIR mRNA
Since transgenic mice developed skeletal defects in the absence of exogenously added zinc, we sought to determine
if expression of the transgene in the MT-DNIIR-4 and -27 lines correlated with the skeletal phenotype and if the hybrid promoter allowed leaky expression in the absence of
zinc. We thus prepared adult skeletal mRNA from hind
limbs of mice maintained on normal food and tap water and
examined using RT-PCR analysis whether DNIIR mRNA
was expressed in transgenic (MTR4, -15, -27, -28, -30) and
wild-type mice (Fig. 3). Transgene-specific (DNIIR) primers were used to amplify mutant but not endogenous type
II receptor cDNA sequences. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to
normalize for the amount of cDNA used in each reaction. As shown in Fig. 3, DNIIR mRNA was expressed in skeletal tissue from MT-DNIIR-4 and -27 mice in the absence
of exogenously added zinc. DNIIR mRNA was not detected in MT-DNIIR-27 mice that did not exhibit skeletal
defects (data not shown). There was very little basal expression of the DNIIR transgene in skeletal tissue from
MT-DNIIR-15, -28, and -30 mice. We have also characterized DNIIR mRNA expression in other tissues in each of
the MT-DNIIR mouse lines (data not shown). Our analyses showed that DNIIR mRNA was expressed in different
sets of tissues depending on the individual MT-DNIIR
mouse line. This heterogeneity in expression pattern is
most likely due to the presence of only minimal gene regulatory elements in this version of the metallothionin promoter, which may make its transcriptional activity sensitive to DNA surrounding the transgene integration site.
However, the skeletal phenotype is most likely not due to
disruption of an unknown gene for several reasons. First,
the skeletal phenotype is observed in heterozygous mice.
Second, two separate lines of mice demonstrated a similar skeletal defect and, third, the sites of constituative expression of the DNIIR mRNA correlated with skeletal abnormalities. All of our experiments outlined below were performed with heterozygous MT-DNIIR-4 mice maintained
on normal food and tap water.
The expression of DNIIR mRNA in adult skeletal tissue
was localized using in situ hybridization (Fig. 4). Sections
of knee joints from wild-type and MT-DNIIR mice were
hybridized to an antisense 35S-labeled riboprobe corresponding to the extracellular domain of the human TGF-
Developmental Defects in MT-DNIIR Mice
To determine if skeletal defects were due to alterations in
embryonic skeletal development, alizarin red S/alcian blue
whole mount skeletal preparations from 17.5-d post-coital
wild-type and MT-DNIIR mice were performed. Fusion of
the sternum was incomplete in 64% (23/36) of MT-DNIIR
mice (Fig. 5, C and D). The degree of sternal bifurcation
varied. The most severe cases extended caudally from the
fifth rib, and the least severe cases involved only the xiphoid process. Defects in the size or shape of the long
bones or vertebrae were not detected in 17.5-d post-coital mice. Since sternal development and fusion occur between
12 and 15 d post-coitum, expression and localization of
DNIIR mRNA were examined by in situ hybridization in
12.5-d post-coital MT-DNIIR embryos (Fig. 5). Sections of
wild-type and transgenic embryos were hybridized to an
35S-labeled antisense DNIIR riboprobe. Expression was
localized to the mesenchyme of the thoracic body wall in
transgenic embryos (Fig. 5 B). There was no difference in
DNIIR mRNA expression in MT-DNIIR embryos from
mothers maintained on 25 mM ZnSO4 or tap water. No
hybridization was detected in sections from wild-type embryos (Fig. 5 A) or in sections hybridized to an 35S-labeled
sense riboprobe (data not shown). These data suggest expression of the DNIIR mRNA in the mesenchyme of the
thoracic body wall during sternal development results in
incomplete fusion of the sternum (Fig. 5 D). Since no
other skeletal defects were detected in 17.5-d post-coital
mice, and DNIIR mRNA in the embryo was limited to the
thoracic body wall, the other defects observed in adult
transgenic mice (Table I, Fig. 2) are probably not a secondary result of early developmental defects in patterning of the cartilage model.
Histology of Transgenic Skeletal Tissue
To determine the effects of DNIIR mRNA expression on
joint histology, sections from wild-type and MT-DNIIR
mouse knee joints at 4 wk (Fig. 6) and 6 mo (Fig. 7) of age
were stained with hematoxylin and eosin. Some degeneration of the articular surface was observed at 4 wk. Patches
of the articular surface were denuded of cartilage (Fig. 6
B), and hypertrophic cells were observed in the articular cartilage (Fig. 6 D). Furthermore, disorganized zones of
cartilage were often seen in the epiphyseal head of long
bones from mice at all ages (Fig. 6 B, black arrow). Alterations in the organization and histology of the growth
plate were apparent at 4 (Fig. 6, E and F), and 8 wk (Fig. 8,
C and D). In wild-type mice, cells in the growth plate exhibited normal columnar organization, and resting, proliferative, and hypertrophic zones were clearly demarcated (Fig. 6 E). In 4-wk-old transgenic mice, the hypertrophic
zone was thicker and the cells in this zone were not organized in columnar arrays (Fig. 6 F). Resting and proliferating zones were visible, but the cells in the proliferating
zone were sometimes grouped into clusters. A population
of small, round cells not readily detectable in wild-type
mice (Fig. 6 E) was located between the proliferating and
hypertrophic zones (Fig. 6 F, PHC). In 8-wk-old transgenic mice, very few proliferating cells were seen. Most of
the cells in the growth plate appeared hypertrophic and
abnormally round (see Fig. 8 D).
The histology of joints from older mice was very similar
to that observed in osteoarthritis (23; Fig. 7). In joints with
mild degeneration, articular cartilage appeared fibrillated
and disorganized (Fig. 7, B and D), and deeper chondrocytes were grouped into clusters (Fig. 7 D). As degeneration progressed, articular cartilage was replaced with hypertrophic cartilage and bone. Early osteophytes, which
represent areas of new endochondral ossification, were
present as outgrowths of chondroid tissue in the articular
margins (Fig. 7 F). Apparently detached fragments of
bone covered with cartilage were detected in the joint
space (Fig. 7, B and E). The synovium was hyperplastic
and thrown into folds (Fig. 7 E). Cartilaginous metaplasia
was observed in synovium filling the joint space (Fig. 4 E).
The growth plate was often disorganized or undetectable (Fig. 7 E) in bones from the older transgenic mice, while
age-matched wild-type mice maintained a small but organized growth plate. In the most severe cases (data not
shown), the femur was pressed into the tibia and the entire
surface of the tibia was destroyed. The data suggest that
altered responsiveness to TGF- DNIIR Expression Results in Altered Proteoglycan
and Type X Collagen Expression
Our analyses suggest that MT-DNIIR transgenic mice
demonstrate osteoarthritis, but we did not detect any
changes in the shape of long bones in newborn mice.
Based on this phenotype, we propose that altered responsiveness to TGF- Effects of Altered Responsiveness to TGF- IHH is a secreted protein expressed in chondrocytes committed to become hypertrophic and is thought to regulate
cartilage differentiation (73, 74). When misexpressed in
chick, IHH has been reported to induce PTHrP in perichondrial cells, which inhibits further differentiation of
chondrocytes that express the PTH receptor; therefore,
the negative-feedback effect of IHH on chondrocyte differentiation is indirect and is mediated by the perichondrium. Since the DNIIR is expressed in perichondrial cells
(Fig. 4, B and C) and to gain insight into the role of TGF-
Tumoral Calcinosis in MT-DNIIR-4 Mice
In humans, tumoral calcinosis is a heritable disease characterized by benign, calcified tumor-like periarticular masses
(19). Tumoral calcinosis-like lesions were found in MT-DNIIR-4 mice. The firm, rubbery masses were found most
often near the cervical vertebrae (Fig. 10, A and B), although masses were also seen all along the vertebral column, in the axilla and pelvis, and in the paws of transgenic
mice. Similar lesions were never seen in wild-type mice.
Alizarin red staining suggested that the lesions were calcified (Table I; Fig. 10 B). The encapsulated lesions consisted of a dense fibrous network with one or more spaces
filled with white, calcareous material. Histologically, these
spaces appeared necrotic (Fig. 10 C) and were surrounded
by poorly differentiated cells, calcified material (Fig. 10
D), and osteoclast-like multinucleated giant cells (Fig. 10
E). The tumoral calcinosis-like masses expressed DNIIR
mRNA as determined by RT-PCR analysis (Fig. 10 F).
RNA from Mv1Lu cells stably transfected with a DNIIR
expression plasmid, wild-type, and MT-DNIIR-4 skeletal
tissue was used as controls. The data suggest that alterations in responsiveness to TGF-
We have generated transgenic mice that express a truncated, kinase-defective TGF- Recently the dominant-negative strategy has been used
to characterize the role of TGF- Osteoarthritis
Osteoarthritis is a degenerative joint disease characterized
by destruction of the articular cartilage (for review see 23). Pathological features of osteoarthritis include fibrillation
of the articular cartilage, clustering and proliferation of articular chondrocytes, endochondral ossification of the articular surface (osteophytes), and cartilaginous metaplasia
in the synovium and joint space. Remarkably, joints of
MT-DNIIR mice display all of these pathological features.
Reduction in the proteoglycan content of articular cartilage is one of the first changes observed in osteoarthritis, a
feature also observed in our transgenic mice. It has been
shown that TGF- Most cells in normal articular cartilage are mature chondrocytes arrested at a stage before terminal hypertrophic
differentiation. Type X collagen is a marker for hypertrophic cartilage and has been detected in fibrillated cartilage from human osteoarthritis patients (72). This finding
suggests that focal premature chondrocyte differentiation
is present in osteoarthritic cartilage. TGF- The truncated TGF- Chondrocyte Differentiation
Changes in the shape of the epiphysis due to deregulated
chondrocyte differentiation would also contribute to the
formation of osteoarthritis. Indeed, loss of responsiveness
to TGF- Mice in which either the PTHrP or PTH receptor genes
have been inactivated demonstrate premature cartilage
hypertrophy resulting in a decrease in the amount of resting and proliferating cartilage and an overall increase in
the amount of endochondral bone formation (2, 35). Our
MT-DNIIR mice showed an increase in the amount of hypertrophic cartilage. Since PTHrP stimulates expression of bcl-2 and delays terminal differentiation and apoptosis in
chondrocytes (3), and TGF- Development of the Sternum
Besides an osteoarthritic phenotype in the joints, MT-DNIIR-4 mice also exhibit defects in sternal development.
The sternum normally develops from lateral mesoderm
that forms a pair of condensations between the clavicle at
the level of the first pair of ribs at 12 d post coitus (10).
Mesodermal condensations elongate and move ventro-medially over the next 3 d. As the condensations elongate caudally, cells differentiate into procartilage. The procartilage does not differentiate into cartilage until the two sternal rudiments have fused. Fusion and differentiation start
at the cranial end and move caudally, and this may account
for the range of sternal malformations observed in MT-DNIIR mice. Bifurcation of the sternum was most common at the caudal end of the sternum. Defects in fusion of
the xiphoid process were most common, but bifurcation
was detected up to the level of the fifth rib. While rare,
cases of congenital cleft sternum have been described in
humans (51, 63), but the molecular basis of this defect is
not known. Mice with mutations in the Bmp5 gene demonstrate bifurcation of the xiphoid process, and BMP-7 null
mice have holes in the xiphoid cartilage (27, 30). BMP-5 is
thought to regulate mesoderm condensation at the future
sites of cartilage rudiments, and defects in this process result in altered shape and size of specific bones (30, 60). Embryos homozygous for null mutations in both Bmp5
and Gdf5 result in disruption of sternebrae within the sternum and abnormal formation of fibrocartilage joints between the sternebrae and ribs (60). MT-DNIIR newborn
mice do not have detectable defects in the size or shape of
specific bones or defects in the formation of the fibrocartilagenous joints of the sternebrae. How defective TGF- Tumoral Calcinosis
In humans, tumoral calcinosis is a benign, soft tissue tumor
of uncertain origin. It is a heritable disease characterized by periarticular pseudotumors (19) primarily located near
the hip, shoulder, or elbow. Tumoral calcinosis involving
the vertebrae is rare in humans, but a few cases have been
reported (42). These benign masses are surrounded by a
well defined capsule surrounding a chalky fluid and consist
of fibrous tissue, inflammatory elements, and multi-nucleated, osteoclast-like giant cells. MT-DNIIR-4 mice developed pseudotumors of the paravertebral region, axilla,
pelvis, and paws that histologically resemble tumoral calcinosis. The tumors were primarily located around the cervical vertebrae but were also seen near thoracic vertebrae,
the pelvis, the axilla, and the paws. These lesions expressed
high levels of the truncated receptor. The dominant-negative inhibition of endogenous receptor signaling may be at
the basis of tumor development since inactivation of the
type II receptor has been detected in several types of tumors (37). Furthermore, restoration of the type II receptor by stable transfection suppressed the tumorigenicity of receptor negative cells (37, 62), suggesting that the type II
receptor may function as a tumor suppressor. It will be interesting to determine if mutations in members of the
TGF- In summary, we have generated transgenic mice that express a dominant loss of function mutation in the type II
receptor to address the role of endogenous TGF- superfamily are secreted growth
factors that regulate many aspects of development, including growth and differentiation (for reviews see 39, 44, 45).
Mice and humans with mutations in certain members of
the family (Bmp5 and Gdf5) display a wide range of skeletal defects including reduced size of specific bones, brachypodism, and chondrodysplasia (30, 60, 65). TGF-
s are expressed in developing and adult skeletal tissue. TGF-
1-3
mRNAs are expressed in condensing mesenchyme during
the early stages of chondrocyte differentiation in the mouse
and human (22, 41, 47, 52). TGF-
mRNAs were not detected in terminally differentiated chondrocytes, but TGF-
immunoreactivity was observed in the matrix surrounding
these cells (24, 48). TGF-
is thought to play an important
role in chondrogenic differentiation. Specifically, TGF-
promotes chondrogenesis in cultures of undifferentiated multipotent mesenchymal cells (14, 32, 36) but inhibits hypertrophic differentiation of chondrocyte cultures (4, 5, 29,
67) and in cultured mouse long bone rudiments (16).
might play a
role in the function and maintenance of articular cartilage
is suggested by its effects on chondrocyte differentiation and its expression in articular cartilage in vivo (18) and in organ culture (43). However, the effects of TGF-
on articular cartilage are not clear. TGF-
has been shown to
both inhibit or stimulate proteoglycan synthesis and growth
in articular chondrocytes in culture (for review see 66).
Since cells in the articular cartilage normally do not differentiate past the stage of resting chondrocytes, and TGF-
inhibits terminal differentiation, it is possible that TGF-
plays a role in the maintenance of functional articular cartilage. On the other hand, repeated intra-articular injections of TGF-
into mouse knee joints result in the formation of osteophytes on the articular surface, suggesting a
role for this peptide in the pathogenesis of osteoarthritis
(69, 70).
superfamily signal through a
family of serine/threonine kinase receptors (for reviews
see 15, 40, 64). Recently, a variety of type I and type II receptors for members of the TGF-
superfamily have been
identified and characterized. TGF-
and related factors
signal through a heteromeric cell surface receptor complex, which consists of two type II and two type I receptors. Overexpression of a cytoplasmically truncated type II
TGF-
receptor inhibits endogenous receptor function in
a dominant way, most likely by interfering with endogenous receptor complex formation and function (11). Truncated type II receptors have been used to block TGF-
signaling in cells in culture (7, 11, 12, 53, 55) and in transgenic mice (6, 75). In vivo, overexpression of a truncated
type II receptor is therefore thought to inactivate receptor
function in a tissue-specific manner, depending on the promoter used, and thus to inhibit the response to all three
TGF-
species while avoiding early embryonic lethality.
signaling in vivo, we have
generated transgenic mice that express a cytoplasmically
truncated, functionally inactive TGF-
type II receptor
under the control of a metallothionein-like promoter, MT-DNIIR. Two mouse lines (MT-DNIIR-4, -27) demonstrated
high basal levels of DNIIR expression in skeletal tissue.
Heterozygous mice from these lines maintained on normal
food and tap water developed joint abnormalities resembling osteoarthitis. We used this model system to test the
hypothesis that TGF-
signaling is required for normal
differentiation and maintenance of chondrocytes in vivo.
Our results suggest that loss of responsiveness to TGF-
overrides the IHH-feed back loop and promotes terminal
differentiation of chondrocytes resulting in osteoarthritis.
Materials and Methods
type II receptor (11) was inserted into the BamHI site of
MT-
(78) by blunt end ligation. The HindIII/BglI fragment containing
the transgene under the metallothionein promoter was microinjected into
the pronuclei of single cell embyos from crosses of C57BL/6 and DBA
mice (26). Mice were maintained on Purina mouse chow and tap water.
Transgenic mice were identified by Southern blot (59) and PCR analyses
of genomic DNA isolated from mouse tails by proteinase K digestion and
phenol/chloroform extraction. For Southern blots, genomic DNA was digested with PstI/EcoRI and the BamHI/EcoRI rabbit
-globin fragment
from MT-
was used as the probe. PCR was performed using primers to
the FLAG epitope sequence: ATC GTC ATC GTC TTT GTA GTC and
human TGF-
type II receptor: TCC CAC CGC ACG TTC AGA AG.
Genomic DNA was amplified for 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 45 s, and elongation for 2 min at 72°C in reaction buffer containing 2 mM MgCl, 1× PCR buffer (Perkin Elmer,
Blanchburg, NJ), 0.2 mM dNTPs (Pharmacia, Uppsala, Sweden), and 0.2 µM of each primer.
Fig. 1.
Map of the MT-DNIIR expression plasmid. The
EcoRI/XbaI fragment of the human TGF- type II receptor from
the plasmid p102 containing a FLAG epitope tag and the signal
sequence (SP), ligand binding, transmembrane (TM), and juxtamembrane (JM) domains of the receptor was inserted into the
BamHI site of the MT-
metallothionein expression vector by
blunt end ligation. The MT-
vector contains four metal responsive elements and the
globin TATA element, splice sites, and
polyadenylation signal (69). The HindIII/BglI fragment was injected into single cell embryos. Arrows mark the location of
primer sequences used for PCR and RT-PCR analysis.
[View Larger Version of this Image (17K GIF file)]
gapdh: CAT GTA GGC CAT GAG GTC CAC CAC; 5
gapdh: TGA
AGG TCG GTG TGA ACG GAT TTG GC; Clontech, Palo Alto, CA).
Samples from reactions performed in the absence of reverse transcriptase
were also amplified to demonstrate that there was no contaminating genomic DNA in the RNA samples.
Results
type II
receptor (11) and metal responsive regulatory elements
from the human metallothionein II promoter (78) was
constructed. The truncated receptor contained the extracellular, transmembrane, and juxtamembrane domains of the TGF-
type II receptor. Most of the cytoplasmic domain including the kinase domain was deleted. This mutant receptor is able to bind ligand and interact with type I
receptors and acts as a dominant-negative mutation in
cells in culture and in transgenic mice (11, 75). The promoter contained four metal responsive elements and a
-globin TATA element, splice sites, and polyadenylation
signal. This promoter has been shown to regulate gene expression with heavy metals in cells in culture (78). The
HindIII/BglI fragment of the MT-DNIIR expression plasmid was injected into single cell embryos, and five transgenic mouse lines (MT-DNIIR-4, -15, -27, -28, -30) were established.
Fig. 2.
Skeletal defects in MT-DNIIR transgenic mice. Photographs of alizarin red whole mount skeletal preparations from
adult wild-type (A, C, E, G, and I) MTR-DNIIR-4 (B, D, H, and
J), and MT-DNIIR-27 (F) mice. Arrows point to xiphoid process
(A and B), knee (C-F), shoulder (G and H) joints, and cervical
vertebrae (I and J).
[View Larger Version of this Image (82K GIF file)]
Fig. 3.
RT-PCR analysis of DNIIR mRNA expression in MT-DNIIR transgenic mouse lines. RNA isolated and pooled from
the hind limbs of two to four wild-type (WT) or transgenic (MT-DNIIR-4, -15, -27, -28, -30) mice maintained on normal food and
tap water was analyzed by RT-PCR. To specifically amplify the
truncated DNIIR cDNA, primers targeted to FLAG epitope sequences were used (Fig. 1). Amplifications of GAPDH was used
as an internal control. Two separate assays are shown separated
by the black bar.
[View Larger Version of this Image (20K GIF file)]
type II receptor. DNIIR mRNA expression was localized
to the articular cartilage, synovium, periosteum, and perichondrium of MT-DNIIR-4 mice at 8 wk of age (Fig. 4,
A-D). A low level of DNIIR mRNA was detected in the
lower hypertrophic zone growth plate of transgenic mice
(Fig. 4 D). At 6 mo of age, DNIIR mRNA was localized to
hyperplastic synovium filling the joint space, especially
near areas surrounding cartilage metaplasia (Fig. 4 E).
DNIIR was also localized to the periosteum (data not shown). No hybridization was detected in sections from
wild-type joints (Fig. 4 F). Hybridization was also not detected to an 35S-labeled sense probe in sections from wild-type or transgenic joints (data not shown).
Fig. 4.
Localization of DNIIR mRNA in skeletal tissue. Sections of knee joints from 8-wk MT-DNIIR transgenic mice were hybridized to an 35S-labeled antisense DNIIR riboprobe (A-D). Boxes shown in A delineate the approximate locations on the joints shown in
(B-D). DNIIR expression was detected in the articular cartilage (B, white arrow), synovium (B, black arrow), and periosteum/perichondrium (C and D, black arrowhead) of transgenic mice. Representative images from analysis of two different mice are shown. In 6-mo-old
transgenic mice, DNIIR expression was detected in hyperplastic synovium (E, small, black arrow) surrounding areas of cartilage metaplasia (E, large, black arrowhead). A representative image from two separate mice is shown. No hybridization was detected in wild-type
tissue (F) or in transgenic mice with an 35S-labeled sense riboprobe (data not shown). Toluidine blue-stained bright field (A-F) and
dark field (A-F
) images are shown. Bars: (A and F) 400 µm; (B-E) 100 µm.
[View Larger Version of this Image (101K GIF file)]
Fig. 5.
Expression of DNIIR mRNA in embryos. Sections of
12.5-d post-coital wild-type (A) and MTR-DNIIR transgenic (B)
embryos were hybridized to an 35S-labeled DNIIR riboprobe.
DNIIR nRNA expression was detected in the mesenchyme of the
thoracic body wall (A and B, arrow) in MT-DNIIR transgenic but
not wild-type embryos at the time the sternum begins to develop.
Toluidine blue-stained bright field (A and B) and dark field (A
and B
) images are shown. Li, liver, Ht, heart. Bifurcated sternum
in 17.5-d post-coital MT-DNIIR-4 mice. Alizarin red/alcian blue-stained skeletal preparations of wild-type (C) and MT-DNIIR
transgenic (D) embryos at 17.5 d post coitus. Bar, 200 µm.
[View Larger Version of this Image (202K GIF file)]
Fig. 6.
Knee joint histology in young MT-DNIIR mice. Images
of hematoxylin- and eosin-stained sections from wild-type (A, C,
and E) and MT-DNIIR (B, D, and F) knee joints at 4 wk of age.
Disorganized cartilage islands were often observed in the transgenic epiphysis (B, black arrowhead). Hypertrophic cells were located in the deep zones of the articular cartilage in transgenic (D)
but not wild-type (C) mice. Resting (RC), proliferating (PC), and
hypertrophic (HC) cells were easily detectable in the wild-type
growth plate (E). In transgenic mice, the histology of the growth
plate was altered. The hypertrophic zone was thicker, and two
distinct cell populations were observed, hypertrophic cells (HC)
and smaller, round prehypertrophic cells (PHC). Bars: (A and B)
400 µm; (C and D) 50 µm; (E and F) 77 µm.
[View Larger Version of this Image (203K GIF file)]
Fig. 7.
Knee joint histology in older MT-DNIIR mice. Joints
from 6-mo-old transgenic mice with joint damage (B and D-F)
and from wild-type mice (A and C) are shown. Cartilage was observed in the joint space (B and E, arrowheads) and the synovium
was hyperplastic (E, arrow) in transgenic mice. The articular surface of wild-type mice was smooth and organized (C). In transgenic mice, the articular surface was fibrillated and chondrocytes
were grouped into clusters (D, arrow). Early osteophytes were
also present on the articular surface (F). The growth plate was often undetectable or highly disorganized (E, white arrows) relative
to the wild-type growth plate (A). Bars: (A and B) 400 µm; (E)
270 µm; (C, D, and F) 50 µm.
[View Larger Version of this Image (208K GIF file)]
Fig. 8.
Localization of proteoglycans and type X collagen in
the knee joint. Sections from 8-wk-old wild-type (A and C) and
MT-DNIIR (B and D) knee joints stained with safranine O (A-D).
Images at 150× (A and B) show staining in the articular surface
(arrows). Images C and D focus on staining in the growth plate.
There was intense proteoglycan staining in the articular cartilage
of wild-type mice (A) while staining was less intense and patchy
on the articular surface of transgenic mice (B). Staining was also
less intense in the transgenic growth plate (D) relative to the
wild-type growth plate (C). Sections from 8 wk (E-G) and 6-mo-old
(H-J) wild-type (E and H) and MT-DNIIR (F, G, I, and J) were
used for immunohistochemical staining of type X collagen (E-J).
There was increased type X collagen staining in the transgenic
growth plate at 8 wk (F) relative to wild-type controls (E). Intracellular staining was detected in transgenic chondrocytes in the
upper zones of the growth plate (F, arrow). Type X collagen immunoreactivity was not readily detectable in articular cartilage from
wild-type mice at 6 mo of age (H) but was detected in fibrillated
cartilage (I, arrow) from older (6 mo) transgenic mice. Chondrocytes in osteophytes also stained for type X collagen (J). Arrows
represent the original joint lining (J). No staining was detected in
the absence of primary anitbody (G). Bars: (A and B) 66 µm; (C
and D) 25 µm; (E and F) 66 µm; (G) 200 µm; (H and I) 100 µm;
(J) 50 µm.
[View Larger Version of this Image (106K GIF file)]
results in cartilage disorganization leading to a progressive degeneration resembling osteoarthritis.
disrupts normal chondrocyte differentiation that later results in degeneration of the joint and the
osteoarthritis phenotype. To test this hypothesis we histologically characterized the expression of proteoglycan,
which is expressed by chondrocytes, and type X collagen
in joints of 8-wk-old wild-type and MT-DNIIR mice (Fig.
8, A-G). Safranine O stains proteoglycans in cartilage matrix. Articular cartilage in wild-type mice showed intense
staining of proteoglycan with safranine O (Fig. 8 A). In
contrast, staining was reduced and patchy in the articular cartilage of MT-DNIIR mice (Fig. 8 B). In wild-type mice,
safranine O stain extended throughout the growth plate
into the bone trabeculae (Fig. 8 C). However, the staining
intensity was decreased in MT-DNIIR mice from the resting and proliferative zones to the hypertrophic zone, and
staining was not detected in bone trabeculae of MT-DNIIR mice (Fig. 8 D). Type X collagen is a marker of
chondrocyte differentiation and is localized primarily to
the matrix of hypertrophic, terminally differentiated chondrocytes (54). Intracellular type X collagen is characteristic of nonproliferating, prehypertrophic chondrocytes. In
wild-type mice, type X collagen was localized to the matrix of the hypertrophic zone of the growth plate, which was
one or two cells thick (Fig. 8 E). Type X collagen was not
detected intracellularly in wild-type mice. In MT-DNIIR
mice, type X collagen staining was more intense and was
localized to a broad area of the growth plate matrix that
was four to six cells thick (Fig. 8 F). Type X collagen staining was also visible within cells throughout the growth
plate (Fig. 8 F). In humans, type X collagen is expressed in
osteoarthritic cartilage. It is localized to sites of newly formed osteophytic and repair cartilage, and marks areas
of endochondral bone formation (72). We therefore used
immunohistochemistry to determine the localization of
type X collagen in older (6 mo) transgenic mice with the
osteoarthritis phenotype (Fig. 8, I and J). Little type X collagen was detected in joints from wild-type mice (Fig. 8
H). By contrast, in the joints of older MT-DNIIR mice,
type X collagen was localized to fibrillated articular cartilage (Fig. 8 I), osteophytes (Fig. 8 J), and cartilage growing
in the joint space (data not shown). These data suggest
that expression of the DNIIR resulted in defects in chondrocyte differentiation so that there was less proteoglycan
but more type X collagen localized to the cartilage matrix.
This indicates that loss of responsiveness to TGF-
promotes terminal differentiation of chondrocytes.
on IHH Expression
in chondrocyte differentiation, we examined IHH and
PTH receptor expression in MT-DNIIR mice. Sections
from 8-wk-old wild-type and transgenic knee joints were
hybridized to 35S-labeled riboprobes. MT-DNIIR mice
demonstrated higher levels of IHH expression in the
growth plate relative to wild-type controls (Fig. 9, A and B).
Expression was higher in each cell and IHH localized to a
broader band of cells in the MT-DNIIR growth plate. PTH receptor was localized to prehypertrophic cells in
wild-type and MT-DNIIR mice (Fig. 9, C and D). There
was little difference in PTH receptor expression in the
growth plate; however, there appeared to be a higher level
of PTH receptor expression in osteoblasts in transgenic
mice (Fig. 9 C). The altered expression of IHH suggests loss of responsiveness to TGF-
s, overrides the IHH feedback loop, and promotes commitment to terminal differentiation.
Fig. 9.
IHH and PTH receptor expression. Sections
of knee joints from 8 wk
wild-type (A and C) and MT-DNIIR transgenic (B and D)
mice were hybridized to 35S-labeled antisense IHH (A and
B) and PTH receptor (C and
D) riboprobes. Bars, 100 µm.
[View Larger Version of this Image (78K GIF file)]
may contribute to the
formation of tumoral calcinosis.
Fig. 10.
Tumoral calcinosis-like lesions in MT-DNIIR transgenic mice. Photograph of a large tumoral calcinosis-like lesion
(arrow) from the cervical vertebrae of an MT-DNIIR transgenic
mouse (A). Whole mount skeletal preparation showing alizarin
red-stained tumoral carcinosis-like lesion (arrow) from the cervical vertebrae of an MT-DNIIR transgenic mouse (B). Images of
hematoxylin- and eosin-stained sections from tumoral calcinosis
lesion (C-E). The tumors were encapsulated and necrotic in the
center (C). The tumors consisted primarily of poorly differentiated mesencyme (B) and large multinucleated giant cells (E). Expression of DNIIR mRNA in tumoral calcinosis lesions (F).
RNA from Mv1Lu cells that expressed the truncated receptor
(CON), a tumoral calcinosis lesion (TC), and the hind limb of a
wild-type (WT) or MT-DNIIR-4 (MT4) transgenic mice was used
in RT-PCR analysis. Amplification of GAPDH cDNA was used
as an internal control. The tumoral calcinosis lesion expressed
high levels of the DNIIR mRNA. Bars: (C) 200 µm; (D) 100 µm;
(E) 50 µm.
[View Larger Version of this Image (189K GIF file)]
Discussion
type II receptor, which acts
as a dominant-negative inhibitor of TGF-
receptor signaling, in articular cartilage, periosteum/perichondrium,
synovium, and in the lower zones of the growth plate. Our
transgenic mice developed a progressive osteoarthritis-like disease. Besides defects in closure of the sternum, no
other developmental defects were detected, suggesting that the osteoarthritis was due to a defect in maintenance
of the skeletal system. Besides an anamalous, disorganized, and hypertrophic cartilage pattern, the young mice
had decreased proteoglycan and increased type X collagen
expression in the cartilage matrix. Our transgenic mice
also demonstrated increased IHH expression, suggesting
that loss of responsiveness to TGF-
results in a defect in
the coordination of chondrocyte differentiation such that
terminal differentiation is promoted.
s in homeostasis of skin
and pancreas (6, 75). In addition, dominant-negative FGF
receptor mutations have been used in transgenic mice to
characterize the role of these factors in skin and lung development (49, 77). The advantages of using the dominant-negative strategy include: (a) the function of the receptor can be inhibited in specific tissues at specific times
depending on the DNA regulatory elements employed.
This strategy reduces the problem of embryonic lethality
that is associated with targeted deletion of TGF-
ligands
(28, 31, 50, 58) or the TGF-
type II receptor (46) and allows for the characterization of TGF-
function in adult
mice. (b) Since signaling by all three TGF-
isoforms is
mediated by the TGF-
type II receptor, signaling by all
TGF-
isoforms is inhibited, avoiding problems with functional redundancy observed with targeted deletion of the
TGF-
ligands (28, 31, 50, 58). There are also several disadvantages to the dominant-negative strategy. First, the
dominant-negative effect requires high levels of expression of the mutant protein. Fortunately, the endogenous
TGF-
type II receptor is normally expressed at very low
levels. Second, the possibility exists that signaling by other
members of the TGF-
superfamily could be inhibited. A
dominant-negative activin receptor was shown to block
signaling by Vg-1, another TGF-
family member, in Xenopus embryos (56). However, the skeletal phenotype of
the MT-DNIIR-4 mice does not overlap with the skeletal
phenotype of mice with mutations in BMP-5, BMP-7, or
GDF-5 (27, 30, 60), suggesting that the TGF-
DNIIR
does not block signaling through these proteins.
1 stimulates proteoglycan synthesis in
normal and osteoarthrotic articular cartilage explants, suggesting that TGF-
plays a role in maintenence and repair
of articular cartilage (34). TGF-
1 suppresses arthritis in
some experimental animal models (8), but others have
proposed that TGF-
is pathogenic for osteoarthritis (17,
69, 70). Intra-articular injection of TGF-
1 into murine
knee joints stimulated proteoglycan synthesis but also resulted in disorganization of articular cartilage and formation of osteophytes (69, 70). Injection of TGF-
2 into rabbit joints resulted in decreased proteoglycan levels in the cartilage (17). Unfortunately, this model is complicated by the fact that TGF-
also induces inflammation in the joint
(1, 17, 21, 69), and inflammatory cytokines are known to
stimulate destruction of articular cartilage. In addition,
constant over-stimulation with TGF-
could result in negative feedback regulation of the TGF-
response. Shifts in
receptor expression and loss of responsiveness to various
growth factors have been detected in cartilage from inflamed knee joints (71). Thus the relevance of these findings to the pathogenesis of osteoarthritis is unclear. Our
data support the hypothesis that TGF-
plays a role in
maintaining articular cartilage. Loss of responsiveness to
TGF-
is likely the basis of the reduced proteoglycan expression and osteoarthritis observed in the MT-DNIIR mice.
has been
shown to inhibit terminal differentiation of chondrocytes in culture (4, 5, 29, 67). Accordingly, the articular surface
of joints in 4- and 8-wk-old MT-DNIIR mice had areas of articular cartilage that appeared hypertrophic and disorganized. Type X collagen was localized to fibrillated articular
cartilage, osteophytes, and cartilage in the joint space of
older MT-DNIIR mice. These observations suggest that
loss of responsiveness to TGF-
in the articular cartilage
results in inappropriate terminal differentiation of the
chondrocytes. These changes may be mechanistically involved in degeneration of the joint, since this may alter the biomechanical properties of the articular cartilage.
type II receptor was also expressed
in the synovium of the transgenic mice. As a result, the
synovium of the MT-DNIIR mice appeared hyperplastic,
and cartilage metaplasia was observed in the joint space.
The physiological effects of the transgene are most likely
direct and not due to inflammation, since no inflammation
was detected in the synovium or the joint space. Synovium
secretes proteases that are known to degrade the articular
matrix (25), and the expression of various proteases can be
downregulated by TGF-
. In addition to the consequences of reduced responsiveness to TGF-
in the articular cartilage, excess synovium could contribute to the formation of
osteoarthritis in MT-DNIIR mice by increasing the amount
of protease present in the joint space.
resulted in increased hypertrophic differentiation
as measured by an increase in type X collagen immunoreactivity and an increase in IHH expression in the MT-DNIIR
growth plate. These data are consistent with observations showing that TGF-
prevents hypertrophic differentiation
in chondrocytes grown in suspension and pellet cultures as
well as in long bone rudiment organ cultures (4, 5, 16, 29,
67). IHH is normally expressed in cells committed to becoming hypertrophic located in the region of transition between the proliferating and hypertrophic zones (73, 74).
Mis-expression of IHH in chick cartilage results in inhibition of chondrocyte hypertrophy and an increase in patched,
gli, and PTHrP expression in perichondrium and periarticular cartilage (73). Mice that overexpress PTHrP in cartilage exhibit delayed chondrocyte differentiation (3, 76).
PTHrP inhibits differentiation in cells that express the PTH receptor in the transition zone between proliferating
and IHH-expressing chondrocytes (3, 35, 73, 76). Thus the increased expression of IHH concominant with the increased hypertrophic differentiation in MT-DNIIR mice
suggests that loss of responsiveness to TGF-
overrides
this inhibitory feedback loop mediated by IHH and
PTHrP. In this context, TGF-
could act directly on components of the IHH feed back loop, including expression
of patched, gli, and PTHrP, that are located in the periosteum/perichondrium. TGF-
has been shown to induce
PTHrP expression in articular chondrocyte cultures (68).
Alternatively, loss of TGF-
responsiveness could promote differentiation through an independent pathway,
and IHH expression would be a consequence of increased
chondrocyte differentiation. In the latter case, the stimulatory actions of the DNIIR would be out of balance with
the normal inhibitory actions of IHH, resulting in a net increase of hypertrophic cartilage.
is known to induce apoptosis, it is likely that inhibition of TGF-
receptor signaling
may delay the normal program of cell death associated
with hypertrophic cartilage differentiation, and in this way
increase the amount of hypertrophic cartilage.
receptor signaling results in bifurcation of the xiphoid process is as yet unclear. However, TGF-
has been identified
as a factor in caudal sternum cells that inhibits terminal
differentiation (5). Therefore, loss of responsiveness to
TGF-
may stimulate terminal chondrocyte differentiation, which in turn may interfere with fusion of the sternal
rudiments in MT-DNIIR mice.
superfamily or their receptors exist in human tumoral calcinosis lesions.
s in
skeletal development and maintenance. Our data suggest
that endogenous TGF-
s maintain cartilage homeostasis
by preventing inappropriate chondrocyte differentiation.
Previous experiments using in vitro models of chondrocyte
differentiation have been difficult to interpret, with often
contradictory conclusions possibly due to variation in the
precise culture conditions used. Since interactions between cells, extracellular matrix, and growth factors are
complex, we believe experimentation in vivo may be more
informative, since these interactions are preserved. Our
transgenic mouse model can be used to further understand
the role of TGF-
s in chondrocyte differentiation and in
the pathogenesis of osteoarthritis.
Received for publication 7 May 1997 and in revised form 28 July 1997.
Address all correspondence to Rosa Serra, Department of Cell Biology, 649 MRBII Vanderbilt Cancer Center, Nashville, TN 37232-3868. Tel.: (615) 936-1507. Fax: (615) 936-1790.The authors are grateful to Dr. Bjorn Olsen for providing the mouse type X collagen antibody and to Drs. McMahon and Kronenberg for providing the IHH and PTHrP cDNA probes. We wish to thank Dr. Wayne J. Lennington for assistance with the bone and tumoral calcinosis pathology, George Holburn for radiological assistance, Philip Sohn for excellent technical assistance, Kim Newson for help with histology, Anna Chytil for help in RNA isolation, and Maureen McDonnell and Ray Dunn for their contributions during the early stages of this project. We would also like to thank Dr. Brigid Hogan for suggestions during the preparation of the manuscript.
This work was supported by grant numbers CA42572 and CA48799 from the National Cancer Institute and the Frances Williams Preston Laboratory funded by the T.J. Martell Foundation (H.L. Moses). R. Serra is also partially supported by grant NIH/NIAMS 5P30 AR4 1943 from the Vanderbilt Skin Diseases Research Center and grant IN-250366 from an American Cancer Society Institutional grant. R. Derynk is supported by National Institutes of Health grants AR41126 and DE10306. M. Johnson is supported by a Veterans Administration Merit award.
IHH, Indian hedgehog; PTHrP, parathyroid hormone-related peptide.
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