ARTICLE |
Correspondence to: Linda J. Sandell, Washington U. School of Medicine, Dept. of Orthopaedic Surgery, Mail Stop 90-34-674, 216 South Kingshighway, St Louis, MO 63110. E-mail: sandelll@msnotes.wustl.edu
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Summary |
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The control of extracellular matrix (ECM) production is important for the development, maintenance, and repair of cartilage tissues. Matrix molecule synthesis is generally regulated by the rate of gene transcription determined by DNA transcription factors. We have shown that transcription factors Sox9, AP-2, and [delta]EF1 are able to alter the rate of CD-RAP transcription in vitro: Sox9 upregulates, AP-2 exhibits biphasic effects, and [delta]EF1 represses expression of the CD-RAP gene. To correlate these in vitro activities in vivo, transcription factors were co-immunolocalized with ECM proteins in three different cartilage tissues in which the rates of biosynthesis are quite different: articular, meniscal, and growth plate. Immunoreactivities of type II collagen and CD-RAP were higher in growth plate than in either the articular or meniscal cartilages and correlated positively with Sox9 protein. Sox9 staining decreased with hypertrophy and was low in articular and meniscal cartilages. In contrast, AP-2 and [delta]EF1 were low in proliferating chondrocytes but high in lower growth plate, articular, and meniscal cartilages. This increase was also accompanied by intense nuclear staining. These immunohistochemical results are the first to localize both [delta]EF1 and AP-2 to adult articular, meniscal, and growth plate cartilages and provide in vivo correlation of previous molecular biological studies.
(J Histochem Cytochem 50:10591065, 2002)
Key Words: type II collagen, CD-RAP, AP-2, Sox9, [delta]EF1, cartilage
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Introduction |
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THE SKELETON is made up of highly differentiated cells and matrices such as bone, cartilage, muscle, and adipose tissue. The differentiation of these tissues is based on the gene regulation system in each cell type. Through enormous efforts to understand the molecular mechanism of skeletal development, several transcription factors have been identified as regulators for cell-specific genes. The four myogenic basic helix-loop-helix (bHLH) transcription factors MyoD, myogenin, Myf-5, and MRF4 were found capable of activating the program for muscle differentiation (for review see 2 (PPAR
2) and adipocyte determination and differentiation-dependent factor 1 (ADD1) play an important role in the development of the adipose lineage (
The mechanism of transcriptional regulation of chondrogenesis is not understood in detail. Significantly, however, transcription factor Sox9, a close relative of the sex-determining region Y protein (SRY), was shown to have an activating role in chondrogenesis by enhancing type II collagen gene expression (
Our studies have focused on three transcription factors, Sox9, AP-2 and [delta]EF1 (also called ZEB1), which are important for regulating the chondrocyte genes, type II collagen, and cartilage-derived-retinoic acid-sensitive protein (CD-RAP). We have shown that, like type II collagen, Sox9 enhances CD-RAP expression via binding to a corresponding consensus region in its promoter (
Previous reports on transgenic and knockout mice for each of these transacting factors clearly indicate that they are important for regulation of cartilage differentiation during development because mutations in each demonstrate pronounced skeletal defects (
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Materials and Methods |
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Tissue Preparation
Five 8-week C57BL/6J male mice were used in this study (Washington University School of Medicine animal protocol number 20000244). The mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Knee joint and proximal tibiae were dissected from each mouse and fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.4) for 16 hr. The samples were decalcified with Formical-2000 (Decal Chemical; Congers, NY) for 24 hr. After dehydration the tissues were embedded in paraffin, sectioned at 5 µm, and mounted on Superfrost/Plus Microscope slides (Fisher Scientific; Pittsburgh, PA).
Antibodies
The human AP-2 and rat [delta]EF1 (ZEB1 R-17) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody recognizing bovine type II collagen was kindly provided by Dr. Michael Cremer (
Immunohistological Analysis
Immunochemical stainings for CD-RAP/MIA, type II collagen, type X collagen, Sox9, and AP-2 were carried out using a DAKO LSAB Kit (DAKO; Carpinteria, CA). The sections were rehydrated in a serial series of ethanol and immersed in 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity. The tissue sections were digested with 1% hyaluronidase for 45 min at 37C. The primary antibody was applied and incubated for 45 min at room temperature (RT). Antibodies against human CD-RAP/MIA, bovine type II collagen, human type X collagen, human Sox9, and human AP-2
were used at dilutions of 1:500, 1:100, 1:500, 1:30, and 1:100, respectively. After linking with the second antibody for 30 min at RT, the slides were incubated in streptoavidin solution for 30 min at RT. The antigen site was visualized by 3-amino-9-ethylcarbazole, a chromogen resulting in a reddish-brown precipitate. Immunostaining for CD-RAP included a 20-min incubation in 2% ß-mercaptoethanol in PBS for unmasking the epitope before primary antibody application. Immunostaining for [delta]EF1 was performed using the Elite PK-6101 Rabbit IgG ABC Kit (Vector; Burlingame, CA). Tissue sections were processed as above except that the hyaluronidase digestion was replaced by a 15-min incubation in Triton X-100 (1%) before overnight incubation with primary antibody (1:100). Sections were then counterstained with hematoxylin before mounting. The proliferating cell nuclear antigen (PCNA) was detected using a ZYMED PCNA Staining Kit (ZYMED Laboratories; San Francisco, CA) to identify proliferating chondrocytes (
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Results |
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Growth Plate
Hematoxylineosin staining of the proximal tibiae from an 8-week postnatal mouse revealed a typical growth plate containing cartilage cells characteristic of various stages of development: the small chondrocytes of the less mature proliferation zone (p), active mature chondrocytes (m), and enlarged chondrocytes of the upper (uh) and lower (lh) hypertrophic zones (Fig 1A). PCNA, a marker of cell division, was detected in both the proliferation and maturation zones, verifying the high division rate of the cells in these areas (Fig 1B). PCNA was not detected in uh or lh chondrocytes. Type II collagen was detected uniformly throughout the growth plate from the zones of proliferation to hypertrophy (Fig 1D). The distribution of type II collagen paralleled that of toluidine blue, a general indicator of negatively charged proteoglycans. The distribution of CD-RAP throughout the growth plate was similar to that of type II collagen. However, it was particularly intense in the pericellular and interterritorial matrices of chondrocytes in the proliferating and resting zones (Fig 1E). The immunostaining for type X collagen was strictly limited to the enlarged chondrocytes typical of hypertrophic cartilage (
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Biochemical studies have shown a positive linear relationship between the presence of Sox9 and CD-RAP promoter activity (
Articular and Meniscal Cartilage
The signals for all ECM proteins found in the articular and meniscal cartilages were significantly lower than those found in the growth plate of the same sections (Fig 2A2D). This likely reflects the age of the matrix and its relative inaccessibility to antibodies, in contrast to newly synthesized matrix that has less crosslinking and is more easily detected. The articular (a) and meniscal (m) cartilages of the joint of the proximal tibiae in an 8-week mouse stained with toluidine blue is shown in Fig 2E. Even though the levels of type II collagen appeared to be less than that in the growth plate (Fig 2B and Fig 2G), the distribution was coincident with toluidine blue (Fig 2A and Fig 2E). We have previously shown that CD-RAP protein can be detected intracellularly in the articular surface and meniscus of an 8-week mouse (
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A faint signal for Sox9 was identified in the nuclei of chondrocytes of the articular and meniscal cartilages (Fig 2F). Like type II collagen and CD-RAP, the levels of Sox9 in these chondrocytes were significantly lower than the growth plate. In contrast, high levels of the transacting factors AP-2 and [delta]EF1 were detected in the nuclei of chondrocytes in both these same areas (Fig 2H and Fig 2J), similar to the intensity observed in hypertrophic chondrocytes (Fig 1H and Fig 1I).
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Discussion |
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The localization of cartilage-specific proteins (type II and X collagens and CD-RAP), DNA transcription factors (Sox9, AP-2, and [delta]EF1) and a cell proliferation marker (PCNA) were examined in single sections containing adult murine growth plate, articular, and meniscal cartilages. Consistent with previous mRNA data, the detection of type II collagen in the growth plate was higher than either the articular or meniscal cartilages and correlated well with levels of Sox9 protein (
An inverse correlation was observed between the localization of the transcription factors AP-2 and [delta]EF1 in nuclei, and CD-RAP and type II collagen in the matrix. Our in vitro studies on CD-RAP gene regulation indicate that both AP-2 (
The fact that CD-RAP was discovered as a gene downregulated by retinoic acid (
In this study we also examined the distribution of type X collagen in the knee joint. The presence of type X collagen correlated positively with AP-2 and [delta]EF1 in the hypertrophic chondrocytes of the growth plate. However, both of these factors were also intensely expressed throughout the articular surface and meniscus, whereas type X collagen was virtually undetectable in these same areas. This discrepancy between the presence of AP-2 and [delta]EF1 and type X collagen suggests that the expression of type X collagen is not likely to be directly regulated by either factor and is regulated differentially from type II collagen and CD-RAP. This is consistent with previous studies in chick chondrocytes that show differential regulation between types II and X collagens in response to parathyroid hormone-related protein (PTHrP) (
In summary, the presence of the transacting factors Sox9, AP-2, and [delta]EF1 in the nuclei of chondrocytes is directly correlated with the transcriptional effects observed in vitro (
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Acknowledgments |
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Supported by NIH grants R01AR36994 and R01AR45550.
Received for publication January 11, 2002; accepted March 20, 2002.
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