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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.

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Normal apoptosis and reduced expression of proliferation markers in mutant (fl/fl, Cre) mice. (A and B) Immunostaining of caspase-cleaved cytokeratin 18. A comparable signal can be observed in control (A) and mutant fl/fl,Cre (B) mice. (C and D) Indirect immunofluorescence staining with anti–cyclin D1 antibody of growth plate from neonatal mouse femur. (C) Control mouse (fl/+, Cre). (D) Mutant littermate (fl/fl, Cre). E and F show the DAPI signal for the fields shown in C and D, respectively. (G and H) Percentage of PCNA- (G) or BrdU- (H) positive cells in the growth plate of neonatal control and mutant mouse femurs. Mean ± SEM of three sections from two control and two mutant mice are shown. *P < 0.05; ***P < 0.001. (I) Growth curves of control (fl/+,Cre; ▪) and mutant (fl/fl,Cre; •) primary chondrocytes. Cells were seeded at low density and viable cells (trypan blue exclusion) were counted daily with a hemocytometer. Results are shown as the percentage of increase relative to number of cells seeded.
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fig6: Normal apoptosis and reduced expression of proliferation markers in mutant (fl/fl, Cre) mice. (A and B) Immunostaining of caspase-cleaved cytokeratin 18. A comparable signal can be observed in control (A) and mutant fl/fl,Cre (B) mice. (C and D) Indirect immunofluorescence staining with anti–cyclin D1 antibody of growth plate from neonatal mouse femur. (C) Control mouse (fl/+, Cre). (D) Mutant littermate (fl/fl, Cre). E and F show the DAPI signal for the fields shown in C and D, respectively. (G and H) Percentage of PCNA- (G) or BrdU- (H) positive cells in the growth plate of neonatal control and mutant mouse femurs. Mean ± SEM of three sections from two control and two mutant mice are shown. *P < 0.05; ***P < 0.001. (I) Growth curves of control (fl/+,Cre; ▪) and mutant (fl/fl,Cre; •) primary chondrocytes. Cells were seeded at low density and viable cells (trypan blue exclusion) were counted daily with a hemocytometer. Results are shown as the percentage of increase relative to number of cells seeded.

Mentions: The normal differentiation pattern of chondrocytes from ILKfl/fl,Cre embryos suggested that the observed dwarfism phenotype could be due to decreased chondrocyte proliferation and/or increased apoptotic rates. We examined the effect of ILK inactivation on expression levels of caspase-cleaved cytokeratin, a marker of apoptosis. In the growth plate, apoptosis is restricted to regions of terminal differentiation, the prehypertrophic and hypertrophic zones. There was no difference in the staining for caspase-cleaved cytokeratin in the prehypertrophic and hypertrophic zones between mutant and control animals (Fig. 6, A and B)Figure 6.


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)

Normal apoptosis and reduced expression of proliferation markers in mutant (fl/fl, Cre) mice. (A and B) Immunostaining of caspase-cleaved cytokeratin 18. A comparable signal can be observed in control (A) and mutant fl/fl,Cre (B) mice. (C and D) Indirect immunofluorescence staining with anti–cyclin D1 antibody of growth plate from neonatal mouse femur. (C) Control mouse (fl/+, Cre). (D) Mutant littermate (fl/fl, Cre). E and F show the DAPI signal for the fields shown in C and D, respectively. (G and H) Percentage of PCNA- (G) or BrdU- (H) positive cells in the growth plate of neonatal control and mutant mouse femurs. Mean ± SEM of three sections from two control and two mutant mice are shown. *P < 0.05; ***P < 0.001. (I) Growth curves of control (fl/+,Cre; ▪) and mutant (fl/fl,Cre; •) primary chondrocytes. Cells were seeded at low density and viable cells (trypan blue exclusion) were counted daily with a hemocytometer. Results are shown as the percentage of increase relative to number of cells seeded.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Normal apoptosis and reduced expression of proliferation markers in mutant (fl/fl, Cre) mice. (A and B) Immunostaining of caspase-cleaved cytokeratin 18. A comparable signal can be observed in control (A) and mutant fl/fl,Cre (B) mice. (C and D) Indirect immunofluorescence staining with anti–cyclin D1 antibody of growth plate from neonatal mouse femur. (C) Control mouse (fl/+, Cre). (D) Mutant littermate (fl/fl, Cre). E and F show the DAPI signal for the fields shown in C and D, respectively. (G and H) Percentage of PCNA- (G) or BrdU- (H) positive cells in the growth plate of neonatal control and mutant mouse femurs. Mean ± SEM of three sections from two control and two mutant mice are shown. *P < 0.05; ***P < 0.001. (I) Growth curves of control (fl/+,Cre; ▪) and mutant (fl/fl,Cre; •) primary chondrocytes. Cells were seeded at low density and viable cells (trypan blue exclusion) were counted daily with a hemocytometer. Results are shown as the percentage of increase relative to number of cells seeded.
Mentions: The normal differentiation pattern of chondrocytes from ILKfl/fl,Cre embryos suggested that the observed dwarfism phenotype could be due to decreased chondrocyte proliferation and/or increased apoptotic rates. We examined the effect of ILK inactivation on expression levels of caspase-cleaved cytokeratin, a marker of apoptosis. In the growth plate, apoptosis is restricted to regions of terminal differentiation, the prehypertrophic and hypertrophic zones. There was no difference in the staining for caspase-cleaved cytokeratin in the prehypertrophic and hypertrophic zones between mutant and control animals (Fig. 6, A and B)Figure 6.

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