Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy

Colin Farquharson, Douglas Lester, E Seawright, David Jefferies, Brian Houston

    Research output: Contribution to journalArticle

    43 Citations (Scopus)

    Abstract

    Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100% identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

    Original languageEnglish
    Pages (from-to)405-12
    Number of pages8
    JournalBone
    Volume25
    Issue number4
    DOIs
    Publication statusPublished - Oct 1999

    Fingerprint

    Growth Plate
    Chondrocytes
    Microtubules
    Hypertrophy
    Tubulin
    Colchicine
    Cell Size
    Collagen Type X
    Staining and Labeling
    Density Gradient Centrifugation
    3' Untranslated Regions
    Population Density
    Glycosaminoglycans
    Cytoskeleton
    Cell Communication
    Sepharose
    Thymidine
    Reverse Transcription
    Alkaline Phosphatase
    Organism Cloning

    Cite this

    Farquharson, C., Lester, D., Seawright, E., Jefferies, D., & Houston, B. (1999). Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy. Bone, 25(4), 405-12. https://doi.org/10.1016/S8756-3282(99)00187-8
    Farquharson, Colin ; Lester, Douglas ; Seawright, E ; Jefferies, David ; Houston, Brian. / Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy. In: Bone. 1999 ; Vol. 25, No. 4. pp. 405-12.
    @article{bf8dab71725140b7bcb135dd1248aa7f,
    title = "Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy",
    abstract = "Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100{\%} identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0{\%}; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1{\%}; p < 0.001), collagen (49.1{\%}; p < 0.01), and glycosaminoglycan (43.3{\%}; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.",
    author = "Colin Farquharson and Douglas Lester and E Seawright and David Jefferies and Brian Houston",
    year = "1999",
    month = "10",
    doi = "10.1016/S8756-3282(99)00187-8",
    language = "English",
    volume = "25",
    pages = "405--12",
    journal = "Bone",
    issn = "8756-3282",
    publisher = "Elsevier Inc.",
    number = "4",

    }

    Farquharson, C, Lester, D, Seawright, E, Jefferies, D & Houston, B 1999, 'Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy', Bone, vol. 25, no. 4, pp. 405-12. https://doi.org/10.1016/S8756-3282(99)00187-8

    Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy. / Farquharson, Colin; Lester, Douglas; Seawright, E; Jefferies, David; Houston, Brian.

    In: Bone, Vol. 25, No. 4, 10.1999, p. 405-12.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy

    AU - Farquharson, Colin

    AU - Lester, Douglas

    AU - Seawright, E

    AU - Jefferies, David

    AU - Houston, Brian

    PY - 1999/10

    Y1 - 1999/10

    N2 - Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100% identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

    AB - Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100% identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

    U2 - 10.1016/S8756-3282(99)00187-8

    DO - 10.1016/S8756-3282(99)00187-8

    M3 - Article

    VL - 25

    SP - 405

    EP - 412

    JO - Bone

    JF - Bone

    SN - 8756-3282

    IS - 4

    ER -