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Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens.

Near RI, Smith RS, Toselli PA, Freddo TF, Bloom AB, Vanden Borre P, Seldin DC, Lerner A - Mol. Vis. (2009)

Bottom Line: We sought to establish the effects of the loss of AND-34 expression in a mammalian organism.A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber.Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston Medical Center, Boston, MA 2118, USA.

ABSTRACT

Purpose: AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism.

Methods: AND-34(-/-) mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues.

Results: Western analyses confirmed total loss of expression in AND-34(-/-) splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34(-/-) mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34(-/-) mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34(+/+) lens epithelial cells, it was markedly reduced in the AND-34(-/-) lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium.

Conclusions: These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.

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Related in: MedlinePlus

AND-34 targeting. A: Exon structure of AND-34 cDNA is shown. B: Production of the recombinant ES clone is demonstrated. A genomic phage clone containing exons 4 and 5 was mapped as shown. The area between the indicated SmaI sites (3.5 kb) was deleted, and the two surrounding “arms”, 5.3 kb of the 5′ sequence and 1.8 kb of the 3′ sequence, were cloned into the pPNT vector. Location of wild type and knockout PCR primers are shown as well as the hybridization probe used for Southern blotting. C: Screening for homologous recombination. DNA prepared from ES clones was purified, aliquots from four clones per lane were pooled, and PCR was performed to detect the recombined KO allele. The first lane shows a pool that contains an ES clone that has the KO allele (arrows indicate lanes of PCRs with clones that contain no AND-34−/− allele). The control PCR used primers specific for Neo. Homologous recombination was confirmed by Southern blot analysis as shown. DNA from individual clones was cleaved with BamHI and analyzed with the indicated probe. Clones 165 and 169 display the recombinant allele. Wild type 129 mouse DNA serves as a control. M=marker lane.
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f1: AND-34 targeting. A: Exon structure of AND-34 cDNA is shown. B: Production of the recombinant ES clone is demonstrated. A genomic phage clone containing exons 4 and 5 was mapped as shown. The area between the indicated SmaI sites (3.5 kb) was deleted, and the two surrounding “arms”, 5.3 kb of the 5′ sequence and 1.8 kb of the 3′ sequence, were cloned into the pPNT vector. Location of wild type and knockout PCR primers are shown as well as the hybridization probe used for Southern blotting. C: Screening for homologous recombination. DNA prepared from ES clones was purified, aliquots from four clones per lane were pooled, and PCR was performed to detect the recombined KO allele. The first lane shows a pool that contains an ES clone that has the KO allele (arrows indicate lanes of PCRs with clones that contain no AND-34−/− allele). The control PCR used primers specific for Neo. Homologous recombination was confirmed by Southern blot analysis as shown. DNA from individual clones was cleaved with BamHI and analyzed with the indicated probe. Clones 165 and 169 display the recombinant allele. Wild type 129 mouse DNA serves as a control. M=marker lane.

Mentions: Targeting vectors were created within plasmid pPNT [21] that contains Neo (positive selection) and Hsv-Tk (negative selection). An initial targeting vector (not shown) eliminated exons 1 and 2 (Figure 1A) with the strategy of deleting the start codon. Given that this initial approach resulted in a strain of mice that expressed a truncated form of AND-34, a subsequent alternate strategy was undertaken to eliminate exons 4 and 5 that contain the SH2 region of AND-34 (Figure 1B). The 5.3 kb SmaI fragment 5′ of exon 4 was cloned into the BamHI site of pPNT using BamHI linkers. The 1.8 kb SmaI-NotI fragment containing a small portion (122 nt) of exon 5 (446 nt) was cloned into the XhoI-NotI portion of pPNT using a XhoI linker. These two “arms” encompassed a 3.1 kb segment bound by SmaI sites, which was omitted from the vector, thereby, eliminating the entire SH2 region.


Loss of AND-34/BCAR3 expression in mice results in rupture of the adult lens.

Near RI, Smith RS, Toselli PA, Freddo TF, Bloom AB, Vanden Borre P, Seldin DC, Lerner A - Mol. Vis. (2009)

AND-34 targeting. A: Exon structure of AND-34 cDNA is shown. B: Production of the recombinant ES clone is demonstrated. A genomic phage clone containing exons 4 and 5 was mapped as shown. The area between the indicated SmaI sites (3.5 kb) was deleted, and the two surrounding “arms”, 5.3 kb of the 5′ sequence and 1.8 kb of the 3′ sequence, were cloned into the pPNT vector. Location of wild type and knockout PCR primers are shown as well as the hybridization probe used for Southern blotting. C: Screening for homologous recombination. DNA prepared from ES clones was purified, aliquots from four clones per lane were pooled, and PCR was performed to detect the recombined KO allele. The first lane shows a pool that contains an ES clone that has the KO allele (arrows indicate lanes of PCRs with clones that contain no AND-34−/− allele). The control PCR used primers specific for Neo. Homologous recombination was confirmed by Southern blot analysis as shown. DNA from individual clones was cleaved with BamHI and analyzed with the indicated probe. Clones 165 and 169 display the recombinant allele. Wild type 129 mouse DNA serves as a control. M=marker lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666772&req=5

f1: AND-34 targeting. A: Exon structure of AND-34 cDNA is shown. B: Production of the recombinant ES clone is demonstrated. A genomic phage clone containing exons 4 and 5 was mapped as shown. The area between the indicated SmaI sites (3.5 kb) was deleted, and the two surrounding “arms”, 5.3 kb of the 5′ sequence and 1.8 kb of the 3′ sequence, were cloned into the pPNT vector. Location of wild type and knockout PCR primers are shown as well as the hybridization probe used for Southern blotting. C: Screening for homologous recombination. DNA prepared from ES clones was purified, aliquots from four clones per lane were pooled, and PCR was performed to detect the recombined KO allele. The first lane shows a pool that contains an ES clone that has the KO allele (arrows indicate lanes of PCRs with clones that contain no AND-34−/− allele). The control PCR used primers specific for Neo. Homologous recombination was confirmed by Southern blot analysis as shown. DNA from individual clones was cleaved with BamHI and analyzed with the indicated probe. Clones 165 and 169 display the recombinant allele. Wild type 129 mouse DNA serves as a control. M=marker lane.
Mentions: Targeting vectors were created within plasmid pPNT [21] that contains Neo (positive selection) and Hsv-Tk (negative selection). An initial targeting vector (not shown) eliminated exons 1 and 2 (Figure 1A) with the strategy of deleting the start codon. Given that this initial approach resulted in a strain of mice that expressed a truncated form of AND-34, a subsequent alternate strategy was undertaken to eliminate exons 4 and 5 that contain the SH2 region of AND-34 (Figure 1B). The 5.3 kb SmaI fragment 5′ of exon 4 was cloned into the BamHI site of pPNT using BamHI linkers. The 1.8 kb SmaI-NotI fragment containing a small portion (122 nt) of exon 5 (446 nt) was cloned into the XhoI-NotI portion of pPNT using a XhoI linker. These two “arms” encompassed a 3.1 kb segment bound by SmaI sites, which was omitted from the vector, thereby, eliminating the entire SH2 region.

Bottom Line: We sought to establish the effects of the loss of AND-34 expression in a mammalian organism.A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber.Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Boston Medical Center, Boston, MA 2118, USA.

ABSTRACT

Purpose: AND-34/BCAR3 (Breast Cancer Anti-Estrogen Resistance 3) associates with the focal adhesion adaptor protein, p130CAS/BCAR1. Expression of AND-34 regulates epithelial cell growth pattern, motility, and growth factor dependence. We sought to establish the effects of the loss of AND-34 expression in a mammalian organism.

Methods: AND-34(-/-) mice were generated by homologous recombination. Histopathology, in situ hybridization, and western blotting were performed on murine tissues.

Results: Western analyses confirmed total loss of expression in AND-34(-/-) splenic lymphocytes. Mice lacking AND-34 are fertile and have normal longevity. While AND-34 is widely expressed in wild type mice, histologic analysis of multiple organs in AND-34(-/-) mice is unremarkable and analyses of lymphocyte development show no overt changes. A small percentage of AND-34(-/-) mice show distinctive small white eye lesions resulting from the migration of ruptured cortical lens tissue into the anterior chamber. Following initial vacuolization and liquefaction of the lens cortex first observed at postnatal day three, posterior lens rupture occurs in all AND-34(-/-) mice, beginning as early as three weeks and seen in all mice at three months. Western blot analysis and in situ hybridization confirmed the presence of AND-34 RNA and protein in lens epithelial cells, particularly at the lens equator. Prior data link AND-34 expression to the activation of Akt signaling. While Akt Ser 473 phosphorylation was readily detectable in AND-34(+/+) lens epithelial cells, it was markedly reduced in the AND-34(-/-) lens epithelium. Basal levels of p130Cas phosphorylation were higher in AND-34(+/+) than in AND-34(-/-) lens epithelium.

Conclusions: These results demonstrate the loss of AND-34 dysregulates focal adhesion complex signaling in lens epithelial cells and suggest that AND-34-mediated signaling is required for maintenance of the structural integrity of the adult ocular lens.

Show MeSH
Related in: MedlinePlus