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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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Targeted inactivation of ILK in chondrocytes. (A) Structure of the floxed ILK allele. Excision by the Cre recombinase deletes the kinase domain. (black triangles), loxP sites; E, exon; TAG, translational stop codon. (B and C) Cre expression in wild-type (B, ILKfl/+) and transgenic (C, ILKfl/fl,Cre) littermates. Indirect immunofluorescence staining of growth plate from neonatal mouse femur with anti-Cre antibody. (D) Cre-mediated excision of the floxed ILK allele in chondrocytes. DNA was prepared from tail clippings and analyzed by PCR with appropriate primers. The wild-type (w.t.,1.9 kb), floxed (fl, 2.1 kb) and Cre excised (230 bp) bands are indicated. Genotypes of a representative litter are indicated above lanes 1–4. M, DNA size markers. (E and F) ILK protein expression. Indirect immunofluorescence staining of growth plate from neonatal mouse femur. Note strong ILK signal in proliferating wild-type chondrocytes (E) that express type II collagen and reduced ILK expression in ILKfl/fl,Cre chondrocytes (F). Bar, 100 μm.
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fig3: Targeted inactivation of ILK in chondrocytes. (A) Structure of the floxed ILK allele. Excision by the Cre recombinase deletes the kinase domain. (black triangles), loxP sites; E, exon; TAG, translational stop codon. (B and C) Cre expression in wild-type (B, ILKfl/+) and transgenic (C, ILKfl/fl,Cre) littermates. Indirect immunofluorescence staining of growth plate from neonatal mouse femur with anti-Cre antibody. (D) Cre-mediated excision of the floxed ILK allele in chondrocytes. DNA was prepared from tail clippings and analyzed by PCR with appropriate primers. The wild-type (w.t.,1.9 kb), floxed (fl, 2.1 kb) and Cre excised (230 bp) bands are indicated. Genotypes of a representative litter are indicated above lanes 1–4. M, DNA size markers. (E and F) ILK protein expression. Indirect immunofluorescence staining of growth plate from neonatal mouse femur. Note strong ILK signal in proliferating wild-type chondrocytes (E) that express type II collagen and reduced ILK expression in ILKfl/fl,Cre chondrocytes (F). Bar, 100 μm.

Mentions: Chondrocyte-specific Cre-mediated recombination at the ROSA26 reporter locus. The Col2-Cre transgenic strain (Col2-Cre Tg) was crossed to the ROSA26 reporter (Rosa +/−) strain. Cre-mediated recombination allowed expression of the β-Gal gene, which was monitored using LacZ staining in newborns (left) and tissue sections (right). Bar, 500 μm.


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)

Targeted inactivation of ILK in chondrocytes. (A) Structure of the floxed ILK allele. Excision by the Cre recombinase deletes the kinase domain. (black triangles), loxP sites; E, exon; TAG, translational stop codon. (B and C) Cre expression in wild-type (B, ILKfl/+) and transgenic (C, ILKfl/fl,Cre) littermates. Indirect immunofluorescence staining of growth plate from neonatal mouse femur with anti-Cre antibody. (D) Cre-mediated excision of the floxed ILK allele in chondrocytes. DNA was prepared from tail clippings and analyzed by PCR with appropriate primers. The wild-type (w.t.,1.9 kb), floxed (fl, 2.1 kb) and Cre excised (230 bp) bands are indicated. Genotypes of a representative litter are indicated above lanes 1–4. M, DNA size markers. (E and F) ILK protein expression. Indirect immunofluorescence staining of growth plate from neonatal mouse femur. Note strong ILK signal in proliferating wild-type chondrocytes (E) that express type II collagen and reduced ILK expression in ILKfl/fl,Cre chondrocytes (F). Bar, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Targeted inactivation of ILK in chondrocytes. (A) Structure of the floxed ILK allele. Excision by the Cre recombinase deletes the kinase domain. (black triangles), loxP sites; E, exon; TAG, translational stop codon. (B and C) Cre expression in wild-type (B, ILKfl/+) and transgenic (C, ILKfl/fl,Cre) littermates. Indirect immunofluorescence staining of growth plate from neonatal mouse femur with anti-Cre antibody. (D) Cre-mediated excision of the floxed ILK allele in chondrocytes. DNA was prepared from tail clippings and analyzed by PCR with appropriate primers. The wild-type (w.t.,1.9 kb), floxed (fl, 2.1 kb) and Cre excised (230 bp) bands are indicated. Genotypes of a representative litter are indicated above lanes 1–4. M, DNA size markers. (E and F) ILK protein expression. Indirect immunofluorescence staining of growth plate from neonatal mouse femur. Note strong ILK signal in proliferating wild-type chondrocytes (E) that express type II collagen and reduced ILK expression in ILKfl/fl,Cre chondrocytes (F). Bar, 100 μm.
Mentions: Chondrocyte-specific Cre-mediated recombination at the ROSA26 reporter locus. The Col2-Cre transgenic strain (Col2-Cre Tg) was crossed to the ROSA26 reporter (Rosa +/−) strain. Cre-mediated recombination allowed expression of the β-Gal gene, which was monitored using LacZ staining in newborns (left) and tissue sections (right). Bar, 500 μm.

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