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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 expression regulates p130Cas and Akt signaling in lens epithelial cells. Lenses were removed from the eyes of AND-34−/− and AND-34+/+ mice (four months old), and the capsular epithelial layer (EPI) was separated from the lens fibers (FIB) by dissection. A: The expression of AND-34/NSP2 and CHAT/NSP3 were examined in the lens tissues of AND-34+/+ mice by immunoblot. Lysates from HEK293T cells that were transfected with AND-34 (pAND) or CHAT (pCHAT) expression constructs were used as controls. Expression of crystallin, a component of lens fibers, and tubulin were also examined. To detect low level expression of AND-34 in lens fibers, fivefold more of the lens fiber sample was loaded in the first lane (Fib-5X) than in the fifth lane (Fib-1X). B: The phosphorylation status of p130Cas and AKT Ser 473 was examined in AND-34−/− and AND-34+/+ lens epithelial and lens fiber samples by western blot analysis. Phosphorylated p130Cas is characterized by reduced PAGE migration. Total levels of AKT expression were also assessed as a control. Similar results were obtained in three experiments.
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f9: AND-34 expression regulates p130Cas and Akt signaling in lens epithelial cells. Lenses were removed from the eyes of AND-34−/− and AND-34+/+ mice (four months old), and the capsular epithelial layer (EPI) was separated from the lens fibers (FIB) by dissection. A: The expression of AND-34/NSP2 and CHAT/NSP3 were examined in the lens tissues of AND-34+/+ mice by immunoblot. Lysates from HEK293T cells that were transfected with AND-34 (pAND) or CHAT (pCHAT) expression constructs were used as controls. Expression of crystallin, a component of lens fibers, and tubulin were also examined. To detect low level expression of AND-34 in lens fibers, fivefold more of the lens fiber sample was loaded in the first lane (Fib-5X) than in the fifth lane (Fib-1X). B: The phosphorylation status of p130Cas and AKT Ser 473 was examined in AND-34−/− and AND-34+/+ lens epithelial and lens fiber samples by western blot analysis. Phosphorylated p130Cas is characterized by reduced PAGE migration. Total levels of AKT expression were also assessed as a control. Similar results were obtained in three experiments.

Mentions: AND-34 is a member of a gene family of which there are two members in mice, AND-34/NSP2 and CHAT/NSP3. To determine whether CHAT expression could serve a redundant biological role in the murine lens, we assessed AND-34 and CHAT protein expression in the lens epithelium and lens fiber cells from wild type mice. AND-34 was readily detected in western blots of lens epithelial cell tissue but was not detected when comparable amounts of lens fiber cell protein was examined (Figure 9A). As expected, β-crystallin was readily detected in lens fiber samples but not in the lens epithelial samples. Given that lens fiber cells but not lens epithelial cells contain an abundant amount of crystallin (see Figure 9A), it is not easy to compare the expression of other less abundant proteins between these two cell types by western blot analysis. Upon loading fivefold more lens fiber samples, AND-34 expression was detected (Figure 9A, lane 1). Neither lens epithelial nor fiber cells showed detectible CHAT expression.


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 expression regulates p130Cas and Akt signaling in lens epithelial cells. Lenses were removed from the eyes of AND-34−/− and AND-34+/+ mice (four months old), and the capsular epithelial layer (EPI) was separated from the lens fibers (FIB) by dissection. A: The expression of AND-34/NSP2 and CHAT/NSP3 were examined in the lens tissues of AND-34+/+ mice by immunoblot. Lysates from HEK293T cells that were transfected with AND-34 (pAND) or CHAT (pCHAT) expression constructs were used as controls. Expression of crystallin, a component of lens fibers, and tubulin were also examined. To detect low level expression of AND-34 in lens fibers, fivefold more of the lens fiber sample was loaded in the first lane (Fib-5X) than in the fifth lane (Fib-1X). B: The phosphorylation status of p130Cas and AKT Ser 473 was examined in AND-34−/− and AND-34+/+ lens epithelial and lens fiber samples by western blot analysis. Phosphorylated p130Cas is characterized by reduced PAGE migration. Total levels of AKT expression were also assessed as a control. Similar results were obtained in three experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9: AND-34 expression regulates p130Cas and Akt signaling in lens epithelial cells. Lenses were removed from the eyes of AND-34−/− and AND-34+/+ mice (four months old), and the capsular epithelial layer (EPI) was separated from the lens fibers (FIB) by dissection. A: The expression of AND-34/NSP2 and CHAT/NSP3 were examined in the lens tissues of AND-34+/+ mice by immunoblot. Lysates from HEK293T cells that were transfected with AND-34 (pAND) or CHAT (pCHAT) expression constructs were used as controls. Expression of crystallin, a component of lens fibers, and tubulin were also examined. To detect low level expression of AND-34 in lens fibers, fivefold more of the lens fiber sample was loaded in the first lane (Fib-5X) than in the fifth lane (Fib-1X). B: The phosphorylation status of p130Cas and AKT Ser 473 was examined in AND-34−/− and AND-34+/+ lens epithelial and lens fiber samples by western blot analysis. Phosphorylated p130Cas is characterized by reduced PAGE migration. Total levels of AKT expression were also assessed as a control. Similar results were obtained in three experiments.
Mentions: AND-34 is a member of a gene family of which there are two members in mice, AND-34/NSP2 and CHAT/NSP3. To determine whether CHAT expression could serve a redundant biological role in the murine lens, we assessed AND-34 and CHAT protein expression in the lens epithelium and lens fiber cells from wild type mice. AND-34 was readily detected in western blots of lens epithelial cell tissue but was not detected when comparable amounts of lens fiber cell protein was examined (Figure 9A). As expected, β-crystallin was readily detected in lens fiber samples but not in the lens epithelial samples. Given that lens fiber cells but not lens epithelial cells contain an abundant amount of crystallin (see Figure 9A), it is not easy to compare the expression of other less abundant proteins between these two cell types by western blot analysis. Upon loading fivefold more lens fiber samples, AND-34 expression was detected (Figure 9A, lane 1). Neither lens epithelial nor fiber cells showed detectible CHAT expression.

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