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Skeletal Growth and Cell Differentiation
Defining the Role of COMP in Skeletal Development
Target Condition: Abnormal skeletal diagnosis
Traditional Approach: Medication, surgery
New Approach: Early identification and intervention employing genetic engineering technology
Progress: The function and structure of the key protein have been characterized
Investigator Dr. Chuan-ju Liu
In the 1990s researchers found that disruption of the cartilage oligomeric matrix protein (COMP) gene is responsible for two types of dwarfism, multiple epiphyseal dysplasia and pseudoachondroplasia. Patients with these disorders are characterized by short stature and the early onset of osteoarthritis.
Discovery of the link between COMP and these conditions has suggested new lines of research involving this key protein. Dr. Liu is conducting studies to determine the underlying mechanism of this phenomenon in the hope of gaining new insights into skeletal development and growth in general. The research now under way has two aims: (1) to characterize the function of COMP in both its normal and mutated manifestations; and (2) to develop a transgenic mouse that exhibits the characteristics of COMP-altered dwarfism and can thus be used to learn how COMP mutations disrupt growth.
Our recent work in this area indicates that COMP is an important biochemical component of extracellular matrix (the “scaffolding” in musculoskeletal tissues), particularly in its interactions with another, important extracellular matrix protein, fibronectin. Laboratory studies undertaken so far have revealed that these protein interactions are of high affinity and occur at specific sites on each protein.
Interaction of COMP with fibronectin Electron microscopy reveals that specific globular domains of cartilage oligomeric matrix protein bind to fibronectin (arrows), thus providing an important clue to understanding COMP’s role in skeletal development.
We discovered that the interaction is concentration-dependent and saturable and appears to occur under physiologically relevant conditions. Rotary shadowing and molecular electron microscopy and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nanometers (a nanometer is a millionth of a millimeter) from the N-terminal domain of fibronectin. The fact that these molecular species bind in vivo was demonstrated by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry.
COMP molecule — schematic The molecular domains of cartilage oligomeric matrix protein (COMP)..
It has also been demonstrated that COMP can mediate cell-matrix interactions as well as matrix-matrix interactions. In our laboratory experiments, articular chondrocytes exhibited both a time- and dose-dependent attachment to COMP, fibronectin, and vitronectin. Antibodies to the vitronectin receptor were able to significantly inhibit chondrocyte attachment to both COMP and vitronectin, although they had no inhibitory effect on cell attachment to fibronectin. Iodinated chondrocyte cell membrane receptors were bound to an affinity column of COMP. Once bound material was eluted, autoradiography revealed two prominent bands that were confirmed to be the vitronectin receptor. We were thus able to demonstrate for the first time that COMP is a adhesion molecule for chondrocytes and that this interaction is mediated by the integrin receptor.
Cartilage surface of a long-bone growth plateImmunolocalization of COMP and FN using a dual labeling: (A) staining for COMP; (B) staining for FN; (C) staining for both COMP and FN.
In preparation for creating a transgenic mouse model, we studied the normal sequence and distribution of COMP in the developing mouse. To begin, we cloned and sequenced mouse COMP cDNA. The encoded mouse product of 755 amino acids shares a high degree of identity to and possesses all the characteristic molecular features of both rat and human COMP. The significance of this finding derives from the concept of conservation of sequence during evolution, implying as it does biological, nonredundant protein function.
Co-localization of COMP with fibronectin Confocal laser microscopy of chondrocytes in a three-dimensional culture. Red staining indicates COMP; green staining indicates fibronectin; dual staining (as in upper right panel) demonstrates co-localization of the molecules in specific areas on the cell surface.
In adult mouse tissues, COMP was found to be highly expressed in cartilage and tendon, moderately expressed in trachea, bone, skeletal muscle, eye, heart, and placenta, and minimally expressed in testis. Immunohistology revealed that COMP expression began as early as 10 days postcoitus in predifferentiated mouse embryo mesenchyme. COMP was detected in all cartilaginous tissues and the skeletal muscles in the embryo at day 13. As development progressed, expression of COMP was marked in the growth plate. At 19 days postcoitus, COMP was prominently expressed in the hypertrophic zone of the growth plate, perichondrium, and periosteum as well as in the superficial layer of articular cartilage surface, but it was absent in the more central areas of the epiphyseal cartilage. The restricted tissue distribution and expression of COMP in developing as well as adult mouse tissues suggest the regulation of this protein at the transcriptional level. These findings regarding COMP expression during normal mouse skeletal development are critical to determining how its mutation perturbs growth.
Experiments are under way in vitro to determine normal and mutant protein function. Knowledge of the mouse genomic sequence has enabled us to produce a vector to introduce the mutated COMP and develop a transgenic mouse that will express the phenotype seen in human dwarfisms. These animals will be instrumental in a detailed developmental analysis.
Defining the Role of COMP in Skeletal Development
Target Condition: Abnormal skeletal diagnosis
Traditional Approach: Medication, surgery
New Approach: Early identification and intervention employing genetic engineering technology
Progress: The function and structure of the key protein have been characterized
Investigator Dr. Chuan-ju Liu
In the 1990s researchers found that disruption of the cartilage oligomeric matrix protein (COMP) gene is responsible for two types of dwarfism, multiple epiphyseal dysplasia and pseudoachondroplasia. Patients with these disorders are characterized by short stature and the early onset of osteoarthritis.
Discovery of the link between COMP and these conditions has suggested new lines of research involving this key protein. Dr. Liu is conducting studies to determine the underlying mechanism of this phenomenon in the hope of gaining new insights into skeletal development and growth in general. The research now under way has two aims: (1) to characterize the function of COMP in both its normal and mutated manifestations; and (2) to develop a transgenic mouse that exhibits the characteristics of COMP-altered dwarfism and can thus be used to learn how COMP mutations disrupt growth.
Our recent work in this area indicates that COMP is an important biochemical component of extracellular matrix (the “scaffolding” in musculoskeletal tissues), particularly in its interactions with another, important extracellular matrix protein, fibronectin. Laboratory studies undertaken so far have revealed that these protein interactions are of high affinity and occur at specific sites on each protein.
Interaction of COMP with fibronectin Electron microscopy reveals that specific globular domains of cartilage oligomeric matrix protein bind to fibronectin (arrows), thus providing an important clue to understanding COMP’s role in skeletal development.
We discovered that the interaction is concentration-dependent and saturable and appears to occur under physiologically relevant conditions. Rotary shadowing and molecular electron microscopy and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nanometers (a nanometer is a millionth of a millimeter) from the N-terminal domain of fibronectin. The fact that these molecular species bind in vivo was demonstrated by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry.
COMP molecule — schematic The molecular domains of cartilage oligomeric matrix protein (COMP)..
It has also been demonstrated that COMP can mediate cell-matrix interactions as well as matrix-matrix interactions. In our laboratory experiments, articular chondrocytes exhibited both a time- and dose-dependent attachment to COMP, fibronectin, and vitronectin. Antibodies to the vitronectin receptor were able to significantly inhibit chondrocyte attachment to both COMP and vitronectin, although they had no inhibitory effect on cell attachment to fibronectin. Iodinated chondrocyte cell membrane receptors were bound to an affinity column of COMP. Once bound material was eluted, autoradiography revealed two prominent bands that were confirmed to be the vitronectin receptor. We were thus able to demonstrate for the first time that COMP is a adhesion molecule for chondrocytes and that this interaction is mediated by the integrin receptor.
Cartilage surface of a long-bone growth plateImmunolocalization of COMP and FN using a dual labeling: (A) staining for COMP; (B) staining for FN; (C) staining for both COMP and FN.
In preparation for creating a transgenic mouse model, we studied the normal sequence and distribution of COMP in the developing mouse. To begin, we cloned and sequenced mouse COMP cDNA. The encoded mouse product of 755 amino acids shares a high degree of identity to and possesses all the characteristic molecular features of both rat and human COMP. The significance of this finding derives from the concept of conservation of sequence during evolution, implying as it does biological, nonredundant protein function.
Co-localization of COMP with fibronectin Confocal laser microscopy of chondrocytes in a three-dimensional culture. Red staining indicates COMP; green staining indicates fibronectin; dual staining (as in upper right panel) demonstrates co-localization of the molecules in specific areas on the cell surface.
In adult mouse tissues, COMP was found to be highly expressed in cartilage and tendon, moderately expressed in trachea, bone, skeletal muscle, eye, heart, and placenta, and minimally expressed in testis. Immunohistology revealed that COMP expression began as early as 10 days postcoitus in predifferentiated mouse embryo mesenchyme. COMP was detected in all cartilaginous tissues and the skeletal muscles in the embryo at day 13. As development progressed, expression of COMP was marked in the growth plate. At 19 days postcoitus, COMP was prominently expressed in the hypertrophic zone of the growth plate, perichondrium, and periosteum as well as in the superficial layer of articular cartilage surface, but it was absent in the more central areas of the epiphyseal cartilage. The restricted tissue distribution and expression of COMP in developing as well as adult mouse tissues suggest the regulation of this protein at the transcriptional level. These findings regarding COMP expression during normal mouse skeletal development are critical to determining how its mutation perturbs growth.
Experiments are under way in vitro to determine normal and mutant protein function. Knowledge of the mouse genomic sequence has enabled us to produce a vector to introduce the mutated COMP and develop a transgenic mouse that will express the phenotype seen in human dwarfisms. These animals will be instrumental in a detailed developmental analysis.