A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation.

Recent studies indicate an important role for the transcriptional co-activator Yes-associated protein (YAP), and its regulatory pathway Hippo, in controlling cell growth and fate during lens development; however, the exogenous factors that promote this pathway are yet to be identified. Given that fibroblast growth factor (FGF)-signaling is an established regulator of lens cell behavior, the current study investigates the relationship between this pathway and Hippo/YAP-signaling during lens cell proliferation and fibre differentiation. Rat lens epithelial explants were cultured with FGF2 to induce epithelial cell proliferation or fibre differentiation. Immunolabeling methods were used to detect the expression of Hippo-signaling components, Total and Phosphorylated YAP, as well as fibre cell markers, Prox-1 and ß-crystallin. FGF-induced lens cell proliferation was associated with a strong nuclear localisation of Total-YAP and low-level immuno-staining for phosphorylated-YAP. FGF-induced lens fibre differentiation was associated with a significant increase in cytoplasmic phosphorylated YAP (inactive state) and enhanced expression of core Hippo-signaling components. Inhibition of YAP with Verteporfin suppressed FGF-induced lens cell proliferation and ablated cell elongation during lens fibre differentiation. Inhibition of either FGFR- or MEK/ERK-signaling suppressed FGF-promoted YAP nuclear translocation. Here we propose that FGF promotes Hippo/YAP-signaling during lens cell proliferation and differentiation, with FGF-induced nuclear-YAP expression playing an essential role in promoting the proliferation of lens epithelial cells. An FGF-induced switch from proliferation to differentiation, hence regulation of lens growth, may play a key role in mediating Hippo suppression of YAP transcriptional activity.

Intrinsic and extrinsic regulatory mechanisms are required to form and maintain a lens of the correct size and shape.

Understanding how tissues and organs acquire and maintain an appropriate size and shape remains one of the most challenging areas in developmental biology. The eye lens represents an excellent system to provide insights into regulatory mechanisms because in addition to its relative simplicity in cellular composition (being made up of only two forms of cells, epithelial and fiber cells), these cells must become organized to generate the precise spheroidal arrangement that delivers normal lens function. Epithelial and fiber cells also represent spatially distinct proliferation and differentiation compartments, respectively, and an ongoing balance between these domains must be tightly regulated so that the lens achieves and maintains appropriate dimensions during growth and ageing. Recent research indicates that reciprocal inductive interactions mediated by Wnt-Frizzled and Notch-Jagged signaling pathways are important for maintaining and organizing these compartments. The Hippo-Yap pathway has also been implicated in maintaining the epithelial progenitor compartment and regulating growth processes. Thus, whilst some molecules and mechanisms have been identified, further work in this important area is needed to provide a clearer understanding of how lens size and shape is regulated.

Fibrosis in the lens. Sprouty regulation of TGFß-signaling prevents lens EMT leading to cataract.

Cataract is a common age-related condition that is caused by progressive clouding of the normally clear lens. Cataract can be effectively treated by surgery; however, like any surgery, there can be complications and the development of a secondary cataract, known as posterior capsule opacification (PCO), is the most common. PCO is caused by aberrant growth of lens epithelial cells that are left behind in the capsular bag after surgical removal of the fiber mass. An epithelial-to-mesenchymal transition (EMT) is central to fibrotic PCO and forms of fibrotic cataract, including anterior/posterior polar cataracts. Transforming growth factor ß (TGFß) has been shown to induce lens EMT and consequently research has focused on identifying ways of blocking its action. Intriguingly, recent studies in animal models have shown that EMT and cataract developed when a class of negative-feedback regulators, Sprouty (Spry)1 and Spry2, were conditionally deleted from the lens. Members of the Spry family act as general antagonists of the receptor tyrosine kinase (RTK)-mediated MAPK signaling pathway that is involved in many physiological and developmental processes. As the ERK/MAPK signaling pathway is a well established target of Spry proteins, and overexpression of Spry can block aberrant TGFß-Smad signaling responsible for EMT and anterior subcapsular cataract, this indicates a role for the ERK/MAPK pathway in TGFß-induced EMT. Given this and other supporting evidence, a case is made for focusing on RTK antagonists, such as Spry, for cataract prevention. In addition, and looking to the future, this review also looks at possibilities for supplanting EMT with normal fiber differentiation and thereby promoting lens regenerative processes after cataract surgery. Whilst it is now known that the epithelial to fiber differentiation process is driven by FGF, little is known about factors that coordinate the precise assembly of fibers into a functional lens. However, recent research provides key insights into an FGF-activated mechanism intrinsic to the lens that involves interactions between the Wnt-Frizzled and Jagged/Notch signaling pathways. This reciprocal epithelial-fiber cell interaction appears to be critical for the assembly and maintenance of the highly ordered three-dimensional architecture that is central to lens function. This information is fundamental to defining the specific conditions and stimuli needed to recapitulate developmental programs and promote regeneration of lens structure and function after cataract surgery.

Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism.

How tissues and organs develop and maintain their characteristic three-dimensional cellular architecture is often a poorly understood part of their developmental program; yet, as is clearly the case for the eye lens, precise regulation of these features can be critical for function. During lens morphogenesis cells become organized into a polarized, spheroidal structure with a monolayer of epithelial cells overlying the apical tips of elongated fiber cells. Epithelial cells proliferate and progeny that shift below the lens equator differentiate into new fibers that are progressively added to the fiber mass. It is now known that FGF induces epithelial to fiber differentiation; however, it is not fully understood how these two forms of cells assemble into their characteristic polarized arrangement. Here we show that in FGF-treated epithelial explants, elongating fibers become polarized/oriented towards islands of epithelial cells and mimic their polarized arrangement in vivo. Epithelial explants secrete Wnt5 into the culture medium and we show that Wnt5 can promote directed behavior of lens cells. We also show that these explants replicate aspects of the Notch/Jagged signaling activity that has been shown to regulate proliferation of epithelial cells in vivo. Thus, our in vitro study identifies a novel mechanism, intrinsic to the two forms of lens cells, that facilitates self-assembly into the polarized arrangement characteristic of the lens in vivo. In this way the lens, with its relatively simple cellular composition, serves as a useful model to highlight the importance of such intrinsic self-assembly mechanisms in tissue developmental and regenerative processes.

Understanding the role of growth factors in embryonic development: insights from the lens.

Growth factors play key roles in influencing cell fate and behaviour during development. The epithelial cells and fibre cells that arise from the lens vesicle during lens morphogenesis are bathed by aqueous and vitreous, respectively. Vitreous has been shown to generate a high level of fibroblast growth factor (FGF) signalling that is required for secondary lens fibre differentiation. However, studies also show that FGF signalling is not sufficient and roles have been identified for transforming growth factor-ß and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/ß-catenin and Notch signalling have been demonstrated in embryonic development, but it is not known if other factors are required for its formation and maintenance. This review provides an overview of current knowledge about growth factor regulation of differentiation and maintenance of lens cells. It also highlights areas that warrant future study.

TGFbeta promotes Wnt expression during cataract development.

TGFbeta induces lens epithelial cells to undergo epithelial mesenchymal transition (EMT) and many changes with characteristics of fibrosis including posterior capsular opacification (PCO). Consequently much effort is directed at trying to block the damaging effects of TGFbeta in the lens. To do this effectively it is important to know the key signaling pathways regulated by TGFbeta that lead to EMT and PCO. Given that Wnt signaling is involved in TGFbeta-induced EMT in other systems, this study set out to determine if Wnt signaling has a role in regulating this process in the lens. Using RT-PCR, in situ hybridization and immunolocalization this study clearly shows that Wnts 5a, 5b, 7b, 8a, 8b and their Frizzled receptors are upregulated in association with TGFbeta-induced EMT and cataract development. Both rat in vitro and mouse in vivo cataract models show similar profiles for the Wnt and Frizzled mRNAs and proteins that were assessed. Currently it is not clear if the canonical beta-catenin/TCF signaling pathway, or a non-canonical pathway, is activated in this context. Overall, the results from the current study indicate that Wnt signaling is involved in TGFbeta-induced EMT and development of fibrotic plaques in the lens.

Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition.

Posterior capsule opacification (PCO) is a common complication of cataract surgery caused by epithelial mesenchymal transition (EMT) and aberrant lens cell growth. One path to prevention depends on maintaining the quiescent lens epithelial phenotype. Here we report that lithium chloride (LiCl) is a potent stabilizer of the lens epithelial phenotype. In lens epithelial explants (controls), at low cell density, cells readily depolarized, spread out, and proliferated. By contrast, in the presence of LiCl, cells did not spread out or exhibit migratory behaviour. Using concentrations of 1-30 mM LiCl we also showed that cell proliferation is inhibited in a dose-dependent manner. Confocal microscopy and immunohistochemistry for ZO-1 and E-cadherin showed that LiCl treatment maintained tight junctions at the apical margins of cells. Taken together with measurements of cell heights, this showed that the cells in LiCl-treated explants maintained the apical baso-lateral polarity and cobblestone-like packing that is characteristic of lens epithelial cells in vivo. Significantly, the effects of LiCl also extended to blocking the potent EMT/cataract-promoting effects of transforming growth factor beta (TGFbeta) on lens epithelial cells. In TGFbeta-treated explants, cells progressively dissociated from one another, taking on various elongated spindle shapes and strongly expressing alpha-smooth muscle actin (alpha-SMA). These features are characteristic of PCO. In both rat and human capsulorhexis explants, LiCl treatment effectively blocked the accumulation of alpha-SMA and maintained the cells in a polarized, adherent, cobblestone-packed monolayer. These findings highlight the feasibility of applying molecular strategies to stabilize lens epithelial cells and prevent aberrant differentiation and growth that leads to cataract.

A role for Wnt/planar cell polarity signaling during lens fiber cell differentiation?

Wnt signaling through frizzled (Fz) receptors plays key roles in just about every developmental system that has been studied. Several Wnt-Fz signaling pathways have been identified including the Wnt/planar cell polarity (PCP) pathway. PCP signaling is crucial for many developmental processes that require major cytoskeletal rearrangements. Downstream of Fz, PCP signaling is thought to involve the GTPases, Rho, Rac and Cdc42 and regulation of the JNK cascade. Here we report on the localization of these GTPases and JNK in the lens and assess their involvement in the cytoskeletal reorganisation that is a key element of FGF-induced lens fiber cell differentiation.

Transforming growth factor-beta-induced epithelial-mesenchymal transition in the lens: a model for cataract formation.

The vertebrate lens has a distinct polarity and structure that are regulated by growth factors resident in the ocular media. Fibroblast growth factors, in concert with other growth factors, are key regulators of lens fiber cell differentiation. While members of the transforming growth factor (TGFbeta) superfamily have also been implicated to play a role in lens fiber differentiation, inappropriate TGFbeta signaling in the anterior lens epithelial cells results in an epithelial-mesenchymal transition (EMT) that bears morphological and molecular resemblance to forms of human cataract, including anterior subcapsular (ASC) and posterior capsule opacification (PCO; also known as secondary cataract or after-cataract), which occurs after cataract surgery. Numerous in vitro and in vivo studies indicate that this TGFbeta-induced EMT is part of a wound healing response in lens epithelial cells and is characterized by induced expression of numerous extracellular matrix proteins (laminin, collagens I, III, tenascin, fibronectin, proteoglycans), intermediate filaments (desmin, alpha-smooth muscle actin) and various integrins (alpha2, alpha5, alpha7B), as well as the loss of epithelial genes [Pax6, Cx43, CP49, alpha-crystallin, E-cadherin, zonula occludens-1 protein (ZO-1)]. The signaling pathways involved in initiating the EMT seem to primarily involve the Smad-dependent pathway, whereby TGFbeta binding to specific high affinity cell surface receptors activates the receptor-Smad/Smad4 complex. Recent studies implicate other factors [such as fibroblast growth factor (FGFs), hepatocyte growth factor, integrins], present in the lens and ocular environment, in the pathogenesis of ASC and PCO. For example, FGF signaling can augment many of the effects of TGFbeta, and integrin signaling, possibly via ILK, appears to mediate some of the morphological features of EMT initiated by TGFbeta. Increasing attention is now being directed at the network of signaling pathways that effect the EMT in lens epithelial cells, with the aim of identifying potential therapeutic targets to inhibit cataract, particularly PCO, which remains a significant clinical problem in ophthalmology.

Growth factor regulation of lens development.

Lens arises from ectoderm situated next to the optic vesicles. By thickening and invaginating, the ectoderm forms the lens vesicle. Growth factors are key regulators of cell fate and behavior. Current evidence indicates that FGFs and BMPs are required to induce lens differentiation from ectoderm. In the lens vesicle, posterior cells elongate to form the primary fibers whereas anterior cells differentiate into epithelial cells. The divergent fates of these embryonic cells give the lens its distinctive polarity. There is now compelling evidence that, at least in mammals, FGF is required to initiate fiber differentiation and that progression of this complex process depends on the synchronized and integrated action of a number of distinct growth factor-induced signaling pathways. It is also proposed that an antero-posterior gradient of FGF stimulation in the mammalian eye ensures that the lens attains and maintains its polarity and growth patterns. Less is known about differentiation of the lens epithelium; however, recent studies point to a role for Wnt signaling. Multiple Wnts and their receptors are expressed in the lens epithelium, and mice with impaired Wnt signaling have a deficient epithelium. Recent studies also indicate that other families of molecules, that can modulate growth factor signaling, have a role in regulating the ordered growth and differentiation of the lens.

Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development.

Recent studies indicate a role for Wnt signalling in regulating lens cell differentiation (Stump et al., 2003). To further our understanding of this, we investigated the expression patterns of Wnts and Wnt signalling regulators, the Dickkopfs (Dkks), during murine lens development. In situ hybridisation showed that Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt8a and Wnt8b genes are expressed throughout the early lens primordia. At embryonic day 14.5 (E14.5), Wnt5a, Wnt5b, Wnt7a, Wnt8a and Wnt8b are reduced in the primary fibres, whereas Wnt7b remains strongly expressed. This trend persists up to E15.5. At later embryonic stages, Wnt expression is predominantly localised to the epithelium and elongating cells at the lens equator. As fibre differentiation progresses, Wnt expression becomes undetectable in the cells of the lens cortex. The one exception is Wnt7b, which continues to be weakly expressed in cortical fibres. This pattern of expression continues through to early postnatal stages. However, by postnatal day 21 (P21), expression of all Wnts is distinctly weaker in the central lens epithelium compared with the equatorial region. This is most notable for Wnt5a, which is barely detectable in the central lens epithelium at P21. Dkk1, Dkk2 and Dkk3 have similar patterns of expression to each other and to the majority of the Wnts during lens development. This study shows that multiple Wnt and Dkk genes are expressed during lens development. Expression is predominantly in the epithelial compartment but is also associated, particularly in the case of Wnt7b, with early events in fibre differentiation.

Deregulation of lens epithelial cell proliferation and differentiation during the development of TGFbeta-induced anterior subcapsular cataract.

Normal lens development and growth is dependent on the tight spatial and temporal regulation of lens cell proliferation and fiber cell differentiation. The present study reports that these same cellular processes contribute to lens pathology as they become deregulated in the process of anterior subcapsular cataract development in a transgenic mouse model. During the formation and growth of transforming growth factor (TGF)beta-induced subcapsular plaques, lens epithelial cells lose key phenotypic markers including E-cadherin and connexin 43, they multilayer and subsequently differentiate into myofibroblastic and/or fiber-like cells. Growth of the subcapsular plaques in the transgenic mouse is sustained by an ordered process of cell proliferation, exit from the cell cycle and differentiation. As reiterating ordered growth and differentiation patterns is atypical of the direct effects of TGFbeta on lens cells in vitro, we propose that other growth factors in the eye, namely fibroblast growth factor, may also play a role in the establishment and regulation of the key cellular processes leading to lens pathology. Obtaining a better understanding of the molecular aspects and cellular dynamics of cataract formation and growth is central to devising strategies for slowing or preventing this disease.

TGFbeta-Smad signalling in postoperative human lens epithelial cells.

AIMS: To localise Smads3/4 proteins in lens epithelial cells (LECs) of fresh and postoperative human specimens. Smads3/4 are involved in signal transduction between transforming growth factor beta (TGFbeta) cell surface receptors and gene promoters. Nuclear localisation of Smads indicates achievement of endogenous TGFbeta signalling in cells. METHODS: Three circular sections of the anterior capsule, one lens, and 17 capsules undergoing postoperative healing were studied. Immunohistochemistry was performed for Smads3/4 in paraffin sections of the specimens. The effect of exogenous TGFbeta2 on Smad3 subcellular localisation was examined in explant cultures of extracted human anterior lens epithelium. RESULTS: The cytoplasm, but not the nuclei, of LECs of uninjured lenses was immunoreactive for Smads3/4. In contrast, nuclear immunoreactivity for Smads3/4 was detected in LECs during capsular healing. Nuclei positive for Smads3/4 were observed in monolayered LECs adjacent to the regenerated lens fibres of Sommerring's ring. Interestingly, the nuclei of LECs that were somewhat elongated, and appeared to be differentiating into fibre-like cells, were negative for Smads3/4. Fibroblast-like, spindle-shaped lens cells with nuclear immunoreactivity for nuclear Smads3/4 were occasionally observed in the extracellular matrix accumulated in capsular opacification. Exogenous TGFbeta induced nuclear translocation of Smad3 in LECs of anterior capsule specimens in explant culture. CONCLUSIONS: This is consistent with TGFbeta induced Smad signalling being involved in regulating the behaviour of LECs during wound healing after cataract surgery.

FGF-induced lens cell proliferation and differentiation is dependent on MAPK (ERK1/2) signalling.

Members of the fibroblast growth factor (FGF) family induce lens epithelial cells to undergo cell division and differentiate into fibres; a low dose of FGF can stimulate cell proliferation (but not fibre differentiation), whereas higher doses of FGF are required to induce fibre differentiation. To determine if these cellular events are regulated by the same signalling pathways, we examined the role of mitogen-activated protein kinase (MAPK) signalling in FGF-induced lens cell proliferation and differentiation. We show that FGF induced a dose-dependent activation of extracellular regulated kinase 1/2 (ERK1/2) as early as 15 minutes in culture, with a high (differentiating) dose of FGF stimulating a greater level of ERK phosphorylation than a lower (proliferating) dose. Subsequent blocking experiments using UO126 (a specific inhibitor of ERK activation) showed that activation of ERK is required for FGF-induced lens cell proliferation and fibre differentiation. Interestingly, inhibition of ERK signalling can block the morphological changes associated with FGF-induced lens fibre differentiation; however, it cannot block the synthesis of some of the molecular differentiation markers, namely, beta-crystallin. These findings are consistent with the in vivo distribution of the phosphorylated (active) forms of ERK1/2 in the lens. Taken together, our data indicate that different levels of ERK signalling may be important for the regulation of lens cell proliferation and early morphological events associated with fibre differentiation; however, multiple signalling pathways are likely to be required for the process of lens fibre differentiation and maturation.

Requirement for TGFbeta receptor signaling during terminal lens fiber differentiation.

Several families of growth factors have been identified as regulators of cell fate in the developing lens. Members of the fibroblast growth factor family are potent inducers of lens fiber differentiation. Members of the transforming growth factor beta (TGFbeta) family, particularly bone morphogenetic proteins, have also been implicated in various stages of lens and ocular development, including lens induction and lens placode formation. However, at later stages of lens development, TGFbeta family members have been shown to induce pathological changes in lens epithelial cells similar to those seen in forms of human subcapsular cataract. Previous studies have shown that type I and type II TGFbeta receptors, in addition to being expressed in the epithelium, are also expressed in patterns consistent with a role in lens fiber differentiation. In this study we have investigated the consequences of disrupting TGFbeta signaling during lens fiber differentiation by using the mouse alphaA-crystallin promoter to overexpress mutant (kinase deficient), dominant-negative forms of either type I or type II TGFbeta receptors in the lens fibers of transgenic mice. Mice expressing these transgenes had pronounced bilateral nuclear cataracts. The phenotype was characterized by attenuated lens fiber elongation in the cortex and disruption of fiber differentiation, culminating in fiber cell apoptosis and degeneration in the lens nucleus. Inhibition of TGFbeta signaling resulted in altered expression patterns of the fiber-specific proteins, alpha-crystallin, filensin, phakinin and MIP. In addition, in an in vitro assay of cell migration, explanted lens cells from transgenic mice showed impaired migration on laminin and a lack of actin filament assembly, compared with cells from wild-type mice. These results indicate that TGFbeta signaling is a key event during fiber differentiation and is required for completion of terminal differentiation.

Tgfbeta receptor expression in lens: implications for differentiation and cataractogenesis.

TGFbeta induces changes characteristic of some forms of cataract. However, the responsiveness of lens epithelial cells to TGFbeta is age-dependent; weanling and adult, but not neonatal, lens epithelial cells respond. This study investigated TGFbeta receptor (TbetaRI and TbetaRII) expression during rat lens development and the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. Immunofluorescence, immunoblotting, RT-PCR and in situ hybridization were used to examine the spatio-temporal expression patterns of TbetaR. Lens explants were used to investigate the effects of FGF-2 on TGFbeta responsiveness and TbetaR expression. In the lens epithelium, little or no immunoreactivity was detected at P3 but at P21 there was distinct reactivity for TbetaRI and TbetaRII. Reactivity for both receptors was also found in the differentiating fibers in the transitional zone and cortex at both ages. Western blotting of lens membrane extracts identified multiple molecular weight forms of TbetaRI (30, 50, 90 kDa) and TbetaRII (70-120 kDa). In situ hybridization with a rat probe for Alk5 (TbetaRI) showed that the lens expresses Alk5 mRNA in epithelium and fibers throughout development. A rat TbetaRII probe revealed distinct expression of a TbetaRII mRNA in lens fibers throughout development and in the lens epithelium at P21 but not at P3. In vitro studies showed that lens epithelial explants from P9 rats did not undergo cataractous changes in response to TGFbeta but P13 explants did. Addition of FGF-2 to P9 explants induced increased TbetaR immunoreactivity and enhanced the competency of lens epithelial cells to respond to TGFbeta. These data indicate that the overall increased expression of TGFbeta receptors in lens epithelium during postnatal development (P3-P21) underlies an age-related change in TGFbeta responsiveness. The results also suggest that lens cells may express multiple forms of TbetaR. Expression of TbetaR in lens fibers throughout lens development and the induction of enhanced TbetaR expression by FGF suggest a role for TGFbeta signaling during FGF-induced responses and fiber differentiation.

Smad translocation and growth suppression in lens epithelial cells by endogenous TGFbeta2 during wound repair.

To determine whether endogenous TGFbeta affects lens epithelial cells during repair after an anterior capsule injury in mice, we studied translocation of Smad proteins, which carry the TGFbeta signal from cell surface receptors to promoters in nuclei. We immunolocalized Smads in murine lenses at intervals up to 8 weeks following capsular injury. Effects of injecting TGFbeta neutralizing antibodies on Smad4 location and cell proliferation were examined at 24 hr after injury. Finally, we examined whether exogenous TGFbeta2 induced Smad nuclear translocation in murine lenses in organ culture. Cell proliferation was quantitated by 5-bromo-2'-deoxyuridine (BrdU) labelling. In uninjured lenses, Smads were located in the cytoplasm. In injured lenses, nuclear localization of Smads was observed in cells next to the capsular break from 8 to 24 hr after the injury, and was observed peripheral to the break at 48 hr. Nuclear Smads then continued to be observed occasionally in a minority of cells. Injection of antibodies neutralizing TGFbeta2, but not TGFbeta1 or TGFbeta3, inhibited Smad4 nuclear translocation and resulted in the appearance of BrdU-positive anterior epithelial cells. With the lenses in culture, transient nuclear localization of Smads occurred between 3 and 24 hr in response to continuous exposure to TGFbeta2. No nuclear translocation was seen at 48 hr. Endogenous TGFbeta2 affects lens cells during wound repair after anterior capsule injury, inhibiting lens cell proliferation during the early phase. Nuclear translocation of Smads in lens epithelial cells is transient even with continuous exposure to TGFbeta2.

Susceptibility to TGFbeta2-induced cataract increases with aging in the rat.

PURPOSE: Cataract is the most common cause of blindness in the world today, and yet there is no generally accepted treatment other than surgical intervention. Studies in rodent models designed to increase understanding of the molecular basis of cataract have shown that transforming growth factor (TGF)-beta induces morphologic and molecular changes similar to those associated with some forms of human cataract. Because aging is the most widely recognized risk factor for cataract, it is important that any animal model be examined in this context. This was a study of the effects of aging on susceptibility to TGFbeta-induced cataract. METHODS: Lenses from weanling, adult, and senile rats were cultured in defined serum-free medium with a range of concentrations of TGFbeta2. The lenses were cultured for up to 7 days, photographed daily, fixed, and prepared for histology and immunolocalization. Opacification was quantified by image analysis. RESULTS: Lenses from weanling, adult, and senile rats all underwent similar cataractous changes when exposed to TGFbeta. This included opacification, the formation of anterior subcapsular plaques, and accumulation of type I collagen and alpha-smooth muscle actin. Lenses from adult and senile animals, however, were generally more adversely affected by TGFbeta than lenses from weanlings. This study also showed that a low dose of TGFbeta administered over a prolonged period had an effect similar to that of a higher dose administered over a shorter period. CONCLUSIONS: An elevation of TGFbeta activity, either acute or chronic, and/or an age-related increase in lens cell susceptibility to TGFbeta may be triggering factors in the etiology of certain forms of cataract.

Inhibition of FGF-induced alphaA-crystallin promoter activity in lens epithelial explants by TGFbeta.

PURPOSE: Fibroblast growth factor (FGF) plays a key role in normal lens biology, and recent studies suggest that transforming growth factor (TGF)-beta is involved in the origin of certain forms of cataract. In the current study, the effects of FGF and TGFbeta on alphaA-crystallin promoter activity were investigated. METHODS: Rat lens epithelial explants were cultured with or without growth factors after transfecting with the firefly luciferase reporter gene driven by either the mouse alphaA-crystallin promoter region or a control simian virus (SV)40 promoter. RESULTS: FGF-2, at a concentration that induced lens fiber differentiation, strongly stimulated alphaA-crystallin promoter activity in explants at 3 to 4 days of culture, whereas SV40 promoter control specimens showed no comparable increase. At lower concentrations of FGF, sufficient to induce cell proliferation but not differentiation, there was only a slight increase in alphaA-crystallin promoter activity. Stimulation of alphaA-crystallin promoter activity induced by the fiber-differentiating concentration of FGF was virtually abolished by as little as 25 pg/ml TGFbeta2, but the onset of fiber-specific beta-crystallin accumulation was not prevented at this concentration. Phase-contrast microscopy revealed overt cataractous changes only at concentrations of TGFbeta more than 25 pg/ml. CONCLUSIONS: The stimulation of alphaA-crystallin promoter activity by FGF is consistent with its role in inducing accumulation of crystallins in explants. The blocking effect of TGFbeta on this process, even at a concentration too low to induce obvious pathologic changes, indicates the potential for TGFbeta to disturb alphaA-crystallin gene expression during early fiber differentiation.