Search
Gene Regulation
Decoding the Formula for Cartilage Growth
Target Conditions: Several conditions cause by bone and cartilege defects
Traditional Approach: Medication, surgery
New Approach: Identifying cellular elements that regulate cell function
Progress: Substances that govern the development of cartilege have been identified
Investigator Dr. Chuan-ju Liu
A clearer understanding of regulation of cartilage growth and differentiation has potent clinical applications in designing biologically based treatment for several orthopaedic and rheumatologic conditions, including fracture nonunion, large segmental bone or cartilage defects, and osteoarthritis.
Research in this area seeks to identify the regulatory agents—transcription factors—that are responsible for signaling undifferentiated cells to create cartilage. In the human embryo, certain of these mesenchymal stem cells are pluripotential: they have the ability to develop at different locations into connective or supporting tissues, smooth muscle, vascular endothelium, or blood cells. Until recently, the molecular events governing the differentiation of stem cells into chondrocytes (cartilage cells) and the expression of cartilage marker genes have been poorly understood.
The differentiation of stem cells into chondrocytes is a fundamental molecular event essential for growth, repair, and regeneration of cartilage and bone. After commitment to the chondrocyte lineage, these cells undergo condensation, cease expression of type I collagen, and differentiate into a chondrocytic phenotype characterized by expression of collagens type II, IX, XI, the noncollagenous protein cartilage oligomeric matrix protein (COMP), and the proteoglycan aggrecan.
A key component in this process is COMP, a noncollagenous extracellular matrix protein with a relatively cartilage-specific spatial and temporal expression pattern. We have identified the COMP promoter—that portion of the cartilage gene that regulates the expression of COMP. Work is now under way to identify the transcription factors that, by binding to the COMP promoter, direct cartilage cells’ function and phenotype.
Recent studies examining transcriptional regulation of the genes for subunits of types II and XI collagen have identified members of the Sox family of transcription factors (L-Sox-5, Sox-6, Sox-9) as direct regulators of these genes. Mutations in the human Sox-9 gene are associated with the development of camptomelic dysplasia, a human dwarfism condition that affects all cartilage-derived structures. These findings suggest that Sox-9, and members of the Sox gene family, may participate in the activation of a genetic program designed to coordinately regulate the expression of genes responsible for the chondrocytic phenotype. Similar analysis of the transcriptional regulation of other cartilage-specific genes, encoding both collagenous and noncollagenous proteins, provides a useful strategy for identifying additional transcription factors that control chondrocyte specification and differentiation.
The gene for cartilage oligomeric matrix protein encodes a noncollagenous pentameric matrix protein expressed predominantly in articular cartilage. Mutations in the human COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia autosomal dominant forms of short-limb dwarfism characterized by short stature and early-onset osteoarthritis. During embryonic and adult stages in several different species, including mouse, rat and human, COMP expression appears to be spatially and temporally restricted primarily to chondrocytes. This relatively cartilage-specific expression pattern, taken together with the observation that COMP mRNA levels are elevated by the chondrogenic factor TGFb-1 suggest that COMP gene expression may be regulated by the chondrocyte differentiation state.
To begin to understand the mechanisms governing cartilage-specific expression of COMP, we cloned the mouse COMP promoter and identified genetic elements necessary for cartilage-specific expression in the chondrocytic cell line. This cell line, established from long-term culture of the transplantable Swarm rat chondrosarcoma, displays a stable chondrocytic phenotype. We have identified two regions in the promoter sequence, an element situated proximally (–125 to –75) and a region located distally (–1925 to –592), that are necessary for COMP expression. Sequences within the proximal region are conserved between the mouse and human promoters. Analysis of nucleotides within this conserved region suggests possible regulation by the high-mobility group class of transcription factors. Identification of cartilage-specific enhancers in the COMP promoter provides genetic probes for cloning transcription factors that regulate chondrocyte-specific gene expression.
COMP mRNA expression in RCS and mouse cartilage cells Northern blot analysis was performed using total RNA and a digoxigenin-labeled mouse COMP riboprobe. RNA was obtained from RCS Cells (10 mg) (lane 1), NIH/3T3 cells (10 mg) (lane 2) and mouse cartilage (5 mg) (lane 3).
Our laboratory is in the process of localizing the active element(s) in the distal region. The next step will be to determine the nature of possible DNA-binding proteins by gel mobility shift assays. The functional importance of these elements for transcriptional activity must then be determined by site-directed mutagenesis of these regions within the context of the wild-type promoter. Identification of nucleotides within the element(s) that are required for both protein binding and promoter activity should provide molecular probes for cloning cartilage-specific transcriptional regulators. These experiments promise to further our understanding of how stem cells differentiate into cartilage.
Decoding the Formula for Cartilage Growth
Target Conditions: Several conditions cause by bone and cartilege defects
Traditional Approach: Medication, surgery
New Approach: Identifying cellular elements that regulate cell function
Progress: Substances that govern the development of cartilege have been identified
Investigator Dr. Chuan-ju Liu
A clearer understanding of regulation of cartilage growth and differentiation has potent clinical applications in designing biologically based treatment for several orthopaedic and rheumatologic conditions, including fracture nonunion, large segmental bone or cartilage defects, and osteoarthritis.
Research in this area seeks to identify the regulatory agents—transcription factors—that are responsible for signaling undifferentiated cells to create cartilage. In the human embryo, certain of these mesenchymal stem cells are pluripotential: they have the ability to develop at different locations into connective or supporting tissues, smooth muscle, vascular endothelium, or blood cells. Until recently, the molecular events governing the differentiation of stem cells into chondrocytes (cartilage cells) and the expression of cartilage marker genes have been poorly understood.
The differentiation of stem cells into chondrocytes is a fundamental molecular event essential for growth, repair, and regeneration of cartilage and bone. After commitment to the chondrocyte lineage, these cells undergo condensation, cease expression of type I collagen, and differentiate into a chondrocytic phenotype characterized by expression of collagens type II, IX, XI, the noncollagenous protein cartilage oligomeric matrix protein (COMP), and the proteoglycan aggrecan.
A key component in this process is COMP, a noncollagenous extracellular matrix protein with a relatively cartilage-specific spatial and temporal expression pattern. We have identified the COMP promoter—that portion of the cartilage gene that regulates the expression of COMP. Work is now under way to identify the transcription factors that, by binding to the COMP promoter, direct cartilage cells’ function and phenotype.
Recent studies examining transcriptional regulation of the genes for subunits of types II and XI collagen have identified members of the Sox family of transcription factors (L-Sox-5, Sox-6, Sox-9) as direct regulators of these genes. Mutations in the human Sox-9 gene are associated with the development of camptomelic dysplasia, a human dwarfism condition that affects all cartilage-derived structures. These findings suggest that Sox-9, and members of the Sox gene family, may participate in the activation of a genetic program designed to coordinately regulate the expression of genes responsible for the chondrocytic phenotype. Similar analysis of the transcriptional regulation of other cartilage-specific genes, encoding both collagenous and noncollagenous proteins, provides a useful strategy for identifying additional transcription factors that control chondrocyte specification and differentiation.
The gene for cartilage oligomeric matrix protein encodes a noncollagenous pentameric matrix protein expressed predominantly in articular cartilage. Mutations in the human COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia autosomal dominant forms of short-limb dwarfism characterized by short stature and early-onset osteoarthritis. During embryonic and adult stages in several different species, including mouse, rat and human, COMP expression appears to be spatially and temporally restricted primarily to chondrocytes. This relatively cartilage-specific expression pattern, taken together with the observation that COMP mRNA levels are elevated by the chondrogenic factor TGFb-1 suggest that COMP gene expression may be regulated by the chondrocyte differentiation state.
To begin to understand the mechanisms governing cartilage-specific expression of COMP, we cloned the mouse COMP promoter and identified genetic elements necessary for cartilage-specific expression in the chondrocytic cell line. This cell line, established from long-term culture of the transplantable Swarm rat chondrosarcoma, displays a stable chondrocytic phenotype. We have identified two regions in the promoter sequence, an element situated proximally (–125 to –75) and a region located distally (–1925 to –592), that are necessary for COMP expression. Sequences within the proximal region are conserved between the mouse and human promoters. Analysis of nucleotides within this conserved region suggests possible regulation by the high-mobility group class of transcription factors. Identification of cartilage-specific enhancers in the COMP promoter provides genetic probes for cloning transcription factors that regulate chondrocyte-specific gene expression.
COMP mRNA expression in RCS and mouse cartilage cells Northern blot analysis was performed using total RNA and a digoxigenin-labeled mouse COMP riboprobe. RNA was obtained from RCS Cells (10 mg) (lane 1), NIH/3T3 cells (10 mg) (lane 2) and mouse cartilage (5 mg) (lane 3).
Our laboratory is in the process of localizing the active element(s) in the distal region. The next step will be to determine the nature of possible DNA-binding proteins by gel mobility shift assays. The functional importance of these elements for transcriptional activity must then be determined by site-directed mutagenesis of these regions within the context of the wild-type promoter. Identification of nucleotides within the element(s) that are required for both protein binding and promoter activity should provide molecular probes for cloning cartilage-specific transcriptional regulators. These experiments promise to further our understanding of how stem cells differentiate into cartilage.