Expression and activity of the CDK inhibitor p57Kip2 in chondrocytes undergoing hypertrophic differentiation.
Growth plates of p57-null mice exhibit several abnormalities, including loss of collagen type X (CollX) expression. The phenotypic consequences of p57 expression were assessed in an in vitro model of hypertrophic differentiation. Adenoviral p57 expression was not sufficient for CollX expression but did augment induction of CollX by BMP-2.


BACKGROUND
During hypertrophic differentiation, chondrocytes pass from an actively proliferative state to a postmitotic, hypertrophic phenotype. The induction of growth arrest is a central feature of this phenotypic transition. Mice lacking the cyclin dependent-kinase inhibitor p57Kip2 exhibit several developmental abnormalities including chondrodysplasia. Although growth plate chondrocytes in p57-null mice undergo growth arrest, they do not express collagen type X, a specific marker of the hypertrophic phenotype. This study was carried out to investigate the link between p57 expression and the induction of collagen type X in chondrocytes and to determine whether p57 overexpression is sufficient for the induction of hypertrophic differentiation.


METHODS
Neonatal rat epiphyseal or growth plate chondrocytes were maintained in an aggregate culture model, in defined, serum-free medium. Protein and mRNA levels were monitored by Western and Northern blot analyses, respectively. Proliferative activity was assessed by fluorescent measurement of total DNA and by 3H-thymidine incorporation rates. An adenoviral vector was used to assess the phenotypic consequences of p57 expression.


CONCLUSIONS
During in vitro hypertrophic differentiation, levels of p57 mRNA and protein were constant despite changes in chondrocyte proliferative activity and the induction of hypertrophic-specific genes in response to bone morphogenetic protein (BMP)-2. Adenoviral p57 overexpression induced growth arrest in prehypertrophic epiphyseal chondrocytes in a dose-dependent manner but was not sufficient for the induction of collagen type X, either alone or when coexpressed with the related CDKI p21Cip1. Similar results were obtained with more mature tibial growth plate chondrocytes. p57 overexpression did augment collagen type X induction by BMP-2. These data indicate that p57-mediated growth arrest is not sufficient for expression of the hypertrophic phenotype, but rather it occurs in parallel with other aspects of the differentiation pathway. Our findings also suggest a contributing role for p57 in the regulation of collagen type X expression in differentiating chondrocytes.
Stewart
Matthew C
MC
Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA. matt1@uiuc.edu
Kadlcek
Renata M
RM
Robbins
Paul D
PD
MacLeod
James N
JN
Ballock
R Tracy
RT
D000256:Adenoviridae / Q000235:genetics; D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D024043:Collagen Type II / Q000235:genetics; D024121:Collagen Type X / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D050761:Cyclin-Dependent Kinase Inhibitor p57; D016213:Cyclins / Q000032:analysis / Q000235:genetics; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006132:Growth Plate / Q000166:cytology; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D014162:Transfection; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases
C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; C493958:Cdkn1a protein, rat; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; D024043:Collagen Type II; D024121:Collagen Type X; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050761:Cyclin-Dependent Kinase Inhibitor p57; D016213:Cyclins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004247:DNA; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase