TI - Discussion . AB - We have demonstrated that AND-34-/- mice develop lens rupture approximately one month after birth while AND-34+/- mice have histologically normal lenses . Despite widespread expression of AND-34 in the brain , liver , lungs , spleen and thymus , the phenotype we have observed following the loss of AND-34 expression is thus far restricted to the ocular lens . The obvious questions that this phenotype raises are why the lens rupture occurs and what accounts for the specificity of the tissue affected . The characteristics of the lens rupture we observe in AND-34-/- mice are different from those of most previously reported forms of molecularly characterized lens defects . Among the four major forms of mouse cataracts , lens extrusion cataracts are the least common [29] . While several spontaneous murine lens extrusion cataract mutations have been described , the majority have not been characterized as to the specific gene affected . Further , among those in which the genetic locus has been identified , the degree of lens rupture has not typically been as severe as that observed in AND-34-/- mice [30-33] . A strain of mice with a spontaneously occurring recessive "lens rupture" ( lr ) mutation were reported in 1950 and has some characteristics similar to AND-34-/- mice [30] . This albino strain of mice had posterior rupture of the entire nucleus of the lens with 100% phenotypic penetrance at three months . In striking similarity to AND-34-/- mice , either the displaced nucleus or the remaining soft degenerate lens passed into the anterior chamber in 18% of cases . In a second strain of mice reported in 1963 , a radiation-induced "ectopic" ( ec ) lens rupture mutation resulted [31] . Unfortunately , the genetic basis for the lr and ec phenotypes was never established , and both strains of mice are now extinct . Secreted protein rich in cysteine ( SPARC ) is a matricellular protein whose elimination results in lens opacity at one month of age and cataract formation and lens capsule rupture at seven to eight months of age . However , overall , the severity of the SPARC-/- phenotype appears considerably less than that observed in AND-34-/- mice [34] . Loss of Abi2 , a member of the Abl-interactor family of adaptor proteins , results in abnormal lens fiber orientation during development , failure of anterior and posterior lens suture formation and posterior lens rupture at postnatal day 1 [35] . We have not observed major abnormalities in lens fiber orientation or suture formation in AND-34-/- mice , suggesting that the rupture may not occur as a result of improper alignment of developing lens fibers . Since the first lens abnormality observed is cortical lens fiber vacuolization followed by lens fiber liquefaction , we hypothesize that disruption of normal focal adhesion complex dynamics in AND-34-/- lens epithelial cells and their differentiated progeny result in defective adhesion between lens fibers . Cortical lens fiber vacuolization and liquefaction is eventually followed by lens capsule rupture and extrusion of cortical lens material into the posterior chamber of the eye . As is the case with most prior reports , lens rupture in AND-34-/- mice occurs posteriorly where the capsule is weakest . The lens capsule is one of the thickest basement membranes in the body and is composed of collagen , laminin , fibronectin , entactin-1 , and sulfated proteoglycans [36] . The capsular matrix is produced and remodeled throughout life by lens epithelial cells anteriorly and by the basal ends of elongating fibers posteriorly [37] . As overexpression of AND-34 augments fibronectin deposition in breast cancer epithelial cell lines , we also hypothesize that loss of AND-34 in lens epithelial cells alters the composition and strength of the lens capsule [9] . Of note , since other animal models of cataract characterized by fiber breakdown do not necessarily undergo capsular rupture , it would appear that the altered synthesis of the lens capsule is likely to play a pivotal role in the lens rupture observed in AND-34-/- mice . As lens fibers elongate , they maintain an apical cell cell interaction with lens epithelial cells and a posterior cell-extracellular matrix ( ECM ) interaction with the lens capsule . Given that we observe an extrusion of ruptured cortical material posteriorly , loss of AND-34 may dysregulate lens fiber interaction with and maintenance of the posterior lens capsule . Our observation that loss of AND-34 expression leads to the rupture of the adult lens is unexpected and suggests that interaction of AND-34 with other components of the focal adhesion complex plays a non-redundant role in maintaining the integrity of the lens . Focal adhesion kinase ( FAK ) transmits signals from integrins to the p130Cas complex by binding to the p130Cas SH3 domain . FAK expression in the adult lens is restricted to those areas where lens epithelial cells exit the cell cycle and initiate differentiation ( posterior germinative zone and transitional zone , respectively ) [38] . FAK is also present in the basal membrane complex of lens fiber cells where they attach to the lens capsule [39] . FAK in turn regulates Src -mediated regulation of p130Cas signaling . Src family kinases bind by their SH2 domains to the FAK Y397 autophosphorylaTION site , facilitating Src kinase activation and subsequent Src -mediated phosphorylaTION of multiple tyrosine motifs in the SUBstrate domain of p130Cas [40] . Similar to the studies noted above with FAK , activated Src ( PHOSphorylated Tyr527 ) is selectively observed in equatorial lens epithelia [41] . Our studies suggest that loss of AND-34 leads to lens rupture as a result of altered p130Cas complex signaling in the same equatorial lens epithelial population identified as the site of lens-associated FAK and Src signaling . Western blot analysis confirms AND-34 expression in lens epithelial cells and to a lesser extent in lens fiber cells when normalized for total protein loaded . In situ hybridization demonstrates that within the lens epithelium , the predominant site of AND-34 expression is at the lens equator . Altered PAGE mobility of p130Cas in preparations derived from AND-34-/- mice is consistent with reduced p130Cas PHOSphorylation . However , while most studies of p130Cas have focused on Src -mediated tyrosine phosphorylaTION of the p130Cas subSTRate domain , our own recent studies suggest that AND-34 regulates p130Cas serine PHOSphorylation ( unpublished observation ) . Given limitations in the amount of protein we could obtain from primary AND-34-/- lens epithelia , studies with cultured lens epithelial cells from wild type and AND-34-/- mice will be required to confirm whether loss of AND-34 does in fact reduce lens epithelial p130Cas serine phosphorylaTION as well as the functional implications of such reduced PHOSphorylation . As the absence of AND-34 transcript expression in lens fibers is an expected consequence of the general loss of transcripts from such cells and as we detect AND-34 protein expression in lens fibers by western blot analysis , it remains possible that loss of AND-34 expression has an important impact on lens fiber function as well as lens epithelial cell function . AND-34 induced Rac activation in breast cancer epithelial cell lines is dependent upon PI3K activation , most likely as a result of activation of pleckstrin homology domain -containing Rac GEFs . The same AND-34 mediated PI3K activation is associated with Akt signaling as judged by Akt Ser 473 PHOSphorylation [19] . Both PI3K itself and PI3K dependent Rac activation are known to play important roles in lens epithelial differentiation and survival [26] . Rac activation antagonizes Rho , resulting in the disassembly of actin stress fibers , a critical event in lens epithelial cell differentiation [42] . Among PI3K effector proteins , Akt is required for IGF-1 ( insulin-like growth factor 1 ) mediated protection of lens epithelial cells from apoptotic stimuli [43] . PI3K signaling and Akt has also been implicated in differentiation of lens epithelial cells into secondary fiber cells in response to vitreal factors [44,45] . To examine whether an AND-34 dependent PI3K signaling pathway might be active in murine lens epithelia , we examined levels of Akt Ser 473 PHOSphorylation in lens epithelial lysates from wild type and AND-34-/- mice . Akt Ser473 PHOSphorylation was markedly diminished in lysates derived from AND-34 null mice despite conservation of total levels of Akt . These results support the hypothesis that loss of AND-34 disrupts a PI3K and Rac -mediated signaling pathway required for appropriate differentiation of lens epithelial cells . Further studies of AND-34 dependent PI3K and Rac signaling in cultured lens cells should help to elucidate the relationship of such signaling to lens rupture in AND-34-/- mice .