Limits...
Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.

Terpstra L, Prud'homme J, Arabian A, Takeda S, Karsenty G, Dedhar S, St-Arnaud R - J. Cell Biol. (2003)

Bottom Line: This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK).In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates.ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

View Article: PubMed Central - PubMed

Affiliation: Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal, Québec, Canada H3G 1A6.

ABSTRACT
Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

Show MeSH

Related in: MedlinePlus

Reduced growth and abnormal growth plate structure after chondrocyte-specific inactivation of ILK. (A) Phenotypic appearance of ILKfl/+,Cre (wild-type, left) and ILKfl/fl,Cre (mutant, right) littermates at 18 d. (B and C) Reduced femur length after chondrocyte-specific inactivation of ILK (B). Femurs were smaller at all ages examined, but the divergence in size was more evident in older animals (C). (D–G) Toluidine blue staining of distal femoral growth plate from control 18-d-old mouse (D and F) and corresponding sample from mutant littermate (ILKfl/fl,Cre, E and G). Note the perturbed columnar organization of the chondrocytes in the mutant sample. The disorganized arrangement of the chondrocytes at the secondary ossification center is also evident on the low power micrographs. Bars, 100 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172728&req=5

fig4: Reduced growth and abnormal growth plate structure after chondrocyte-specific inactivation of ILK. (A) Phenotypic appearance of ILKfl/+,Cre (wild-type, left) and ILKfl/fl,Cre (mutant, right) littermates at 18 d. (B and C) Reduced femur length after chondrocyte-specific inactivation of ILK (B). Femurs were smaller at all ages examined, but the divergence in size was more evident in older animals (C). (D–G) Toluidine blue staining of distal femoral growth plate from control 18-d-old mouse (D and F) and corresponding sample from mutant littermate (ILKfl/fl,Cre, E and G). Note the perturbed columnar organization of the chondrocytes in the mutant sample. The disorganized arrangement of the chondrocytes at the secondary ossification center is also evident on the low power micrographs. Bars, 100 μm.

Mentions: LoxP sites were inserted downstream from exons 4 and 12 at the ILK locus through homologous recombination in embryonic stem cells (Fig. 3 A). This strategy should delete all the kinase domain of ILK upon Cre-mediated excision and create a allele. By mating floxed ILK mice with Col2-Cre transgenic mice, mice heterozygous for the recombined floxed ILK allele were generated (genotype: ILKfl/+,Cre). These mice had no phenotype (Fig. 4Figure 4.


Reduced chondrocyte proliferation and chondrodysplasia in mice lacking the integrin-linked kinase in chondrocytes.

Terpstra L, Prud'homme J, Arabian A, Takeda S, Karsenty G, Dedhar S, St-Arnaud R - J. Cell Biol. (2003)

Reduced growth and abnormal growth plate structure after chondrocyte-specific inactivation of ILK. (A) Phenotypic appearance of ILKfl/+,Cre (wild-type, left) and ILKfl/fl,Cre (mutant, right) littermates at 18 d. (B and C) Reduced femur length after chondrocyte-specific inactivation of ILK (B). Femurs were smaller at all ages examined, but the divergence in size was more evident in older animals (C). (D–G) Toluidine blue staining of distal femoral growth plate from control 18-d-old mouse (D and F) and corresponding sample from mutant littermate (ILKfl/fl,Cre, E and G). Note the perturbed columnar organization of the chondrocytes in the mutant sample. The disorganized arrangement of the chondrocytes at the secondary ossification center is also evident on the low power micrographs. Bars, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172728&req=5

fig4: Reduced growth and abnormal growth plate structure after chondrocyte-specific inactivation of ILK. (A) Phenotypic appearance of ILKfl/+,Cre (wild-type, left) and ILKfl/fl,Cre (mutant, right) littermates at 18 d. (B and C) Reduced femur length after chondrocyte-specific inactivation of ILK (B). Femurs were smaller at all ages examined, but the divergence in size was more evident in older animals (C). (D–G) Toluidine blue staining of distal femoral growth plate from control 18-d-old mouse (D and F) and corresponding sample from mutant littermate (ILKfl/fl,Cre, E and G). Note the perturbed columnar organization of the chondrocytes in the mutant sample. The disorganized arrangement of the chondrocytes at the secondary ossification center is also evident on the low power micrographs. Bars, 100 μm.
Mentions: LoxP sites were inserted downstream from exons 4 and 12 at the ILK locus through homologous recombination in embryonic stem cells (Fig. 3 A). This strategy should delete all the kinase domain of ILK upon Cre-mediated excision and create a allele. By mating floxed ILK mice with Col2-Cre transgenic mice, mice heterozygous for the recombined floxed ILK allele were generated (genotype: ILKfl/+,Cre). These mice had no phenotype (Fig. 4Figure 4.

Bottom Line: This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK).In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates.ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

View Article: PubMed Central - PubMed

Affiliation: Genetics Unit, Shriners Hospital for Children, 1529 Cedar Avenue, Montréal, Québec, Canada H3G 1A6.

ABSTRACT
Chondrocyte proliferation and differentiation requires their attachment to the collagen type II-rich matrix of developing bone. This interaction is mediated by integrins and their cytoplasmic effectors, such as the integrin-linked kinase (ILK). To elucidate the molecular mechanisms whereby integrins control these processes, we have specifically inactivated the ILK gene in growth plate chondrocytes using the Cre-lox methodology. Mice carrying an ILK allele flanked by loxP sites (ILK-fl) were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type II promoter. Inactivation of both copies of the ILK-fl allele lead to a chondrodysplasia characterized by a disorganized growth plate and to dwarfism. Expression of chondrocyte differentiation markers such as collagen type II, collagen type X, Indian hedgehog and the PTH-PTHrP receptor was normal in ILK-deficient growth plates. In contrast, chondrocyte proliferation, assessed by BrdU or proliferating cell nuclear antigen labeling, was markedly reduced in the mutant growth plates. Cell-based assays showed that integrin-mediated adhesion of primary cultures of chondrocytes from mutant animals to collagen type II was impaired. ILK inactivation in chondrocytes resulted in reduced cyclin D1 expression, and this most likely explains the defect in chondrocyte proliferation observed when ILK is inactivated in growth plate cells.

Show MeSH
Related in: MedlinePlus