F.J. Lovicu

Transforming Growth Factor-β-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 (TGFβ) superfamily have also been implicated to play a role in lens fiber differentiation, inappropriate TGFβ 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 TGFβ-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, α-smooth muscle actin) and various integrins (α2, α5, α7B), as well as the loss of epithelial genes [Pax6, Cx43, CP49, α-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 TGFβ 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 TGFβ, and integrin signaling, possibly via ILK, appears to mediate some of the morphological features of EMT initiated by TGFβ. 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.

The role of fibroblast growth factor in eye lens development.

In this review we have presented evidence that FGF plays an important role in inducing events in lens morphogenesis and growth. Our studies show that FGF stimulates lens epithelial cells in explants to proliferate, migrate, and differentiate into fibers at low, medium, and high concentrations, respectively. This has some important implications for understanding the behavior of lens cells in the eye. The fact that aFGF is detected in the equatorial region of the lens where cells are actively proliferating, possibly migrating, and differentiating into fibers suggests that these events may be under autocrine control in vivo, at least to some extent. Because FGF is also present in the ciliary and iridial region of retina and in the vitreous, paracrine control may also be involved. Cell proliferation, fiber differentiation, and (possibly) cell migration occur in characteristic spatial patterns that are related to distinct compartments of the lens. We suggest that cells in the germinative zone receive only a low level of FGF stimulation arising from the cells themselves and possibly also from the ciliary and iridial regions of the retina but, whatever the source, this is only sufficient to stimulate proliferation. Lens epithelial cells that migrate or are displaced into the transitional zone below the lens equator receive some FGF from these sources but in addition receive a strong stimulus from the high level of FGF in the vitreous; thus, fiber differentiation is induced. Cells at the junction between these two zones may receive an intermediate level of FGF stimulation, sufficient to induce cell migration. In essence, we are proposing that, in the eye, FGF acts as a lens morphogen in the sense that different levels of FGF stimulation elicit different lens cell responses. Hence its characteristic distribution in the eye establishes lens polarity and growth patterns. Since FGF has an inductive effect on lens cells from mature age animals, we also propose that this specific distribution of FGF in the eye is also important for maintenance of a normal lens throughout life. Finally the synergistic effects of insulin/IGF on the FGF-induced responses highlight the importance of considering the distribution of members of the insulin/IGF family of molecules in vivo. Mechanisms that control levels of both the FGF and insulin/IGF families of factors in the eye are probably of crucial importance in the formation and maintenance of a normal lens.

SPATIAL AND TEMPORAL EXPRESSION OF P57KIP2 DURING MURINE LENS DEVELOPMENT

The expression patterns of p57(KIP2), an important cyclin-dependent kinase inhibitor in the lens, is investigated. This study shows that the expression of p57 mRNA throughout lens morphogenesis and growth correlates with lens cell withdrawal from the cell cycle (shown by changing patterns of BrdU incorporation) and the onset of lens fibre differentiation (shown by beta-crystallin expression). p57 expression at the early stages of fibre differentiation make it a useful marker for the initiation of this process.

FGF receptor-1 (flg) expression is correlated with fibre differentiation during rat lens morphogenesis and growth.

Our previous studies indicate an important role for fibroblast growth factor (FGF) in lens development. Here we study the expression of the flg variant of FGF receptor 1 (FGFR1) during lens development by immunohistochemistry and in situ hybridisation. FGFR1 was expressed throughout lens development. Prominent FGFR1 immunoreactivity was associated with cell nuclei, particularly in differentiating lens fibres, suggesting internalisation and nuclear translocation of the receptor. FGFR1 immunoreactivity was also associated with basolateral membranes of cells in the equatorial region and at lens sutures. FGFR1 mRNA was only weakly expressed during early lens morphogenesis but expression increased with the onset of lens fibre differentiation. Once the lens acquired its distinct polarity, an anteroposterior gradient in both protein reactivity and mRNA signal was evident. Anteriorly, central epithelial cells showed weak expression for FGFR1, whereas more posteriorly, in the germinative and transitional zones of the lens where cells maximally proliferate and undergo early stages of fibre differentiation, respectively, expression was significantly stronger. The anteroposterior gradient of increased expression of FGFR1 in the lens coincides with the previously documented anteroposterior gradient of FGF stimulation. In lens epithelial explants, FGF stimulation was found to upregulate FGFR1 expression. Such upregulation may be an important mechanism for generating a high level of FGF stimulation and ensuring a fibre differentiation response. In postnatal rat lenses, there was a significant age‐related decline in FGFR1 expression; this correlates with the reduced rate of lens fibre differentiation with age. Overall, these studies support the hypothesis that FGF and FGFR1 are important for regulation of lens fibre differentiation throughout lens development.

TGFβ 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.

Peter Bishop Lecture: growth factors in lens development and cataract: key roles for fibroblast growth factor and TGF-beta.

Growth factors play key roles in influencing cell behaviour and cell fates during development. Studies in our laboratory indicate that members of the fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β) growth factor families have central roles in lens developmental biology. The first section of this brief review presents evidence that indicates a role for FGF in normal lens development. The second section describes the varied effects of TGF-β on lens cells. This includes the induction of changes characteristic of certain types of cataract as well as evidence for a role in lens fibre cell maturation and/or survival.

Expression of Crim1 during murine ocular development

Crim1 (cysteine-rich motor neuron 1), a novel gene encoding a putative transmembrane protein, has recently been isolated and characterized (Kolle, G., Georgas, K., Holmes, G.P., Little, M.H., Yamada, T., 2000. CRIM1, a novel gene encoding a cysteine-rich repeat protein, is developmentally regulated and implicated in vertebrate CNS development and organogenesis. Mech. Dev. 90, 181-193). Crim1 contains an IGF-binding protein motif and multiple cysteine-rich repeats, analogous to those of chordin and short gastrulation (sog) proteins that associate with TGFbeta superfamily members, namely Bone Morphogenic Protein (BMP). High levels of Crim1 have been detected in the brain, spinal chord and lens. As members of the IGF and TGFbeta growth factor families have been shown to influence the behaviour of lens cells (Chamberlain, C.G., McAvoy, J. W., 1997. Fibre differentiation and polarity in the mammalian lens: a key role for FGF. Prog. Ret. Eye Res. 16, 443-478; de Iongh R.U., Lovicu, F.J., Overbeek, P.A., Schneider, M.D., McAvoy J.W., 1999. TGF-beta signalling is essential for terminal differentiation of lens fibre cells. Invest. Ophthalmol. Vis. Sci. 40, S561), to further understand the role of Crim1 in the lens, its expression during ocular morphogenesis and growth is investigated. Using in situ hybridisation, the expression patterns of Crim1 are determined in murine eyes from embryonic day 9.5 through to postnatal day 21. Low levels of transcripts for Crim1 are first detected in the lens placode. By the lens pit stage, Crim1 is markedly upregulated with high levels persisting throughout embryonic and foetal development. Crim1 is expressed in both lens epithelial and fibre cells. As lens fibres mature in the nucleus, Crim1 is downregulated but strong expression is maintained in the lens epithelium and in the young fibre cells of the lens cortex. Crim1 is also detected in other developing ocular tissues including corneal and conjunctival epithelia, corneal endothelium, retinal pigmented epithelium, ciliary and iridial retinae and ganglion cells. During postnatal development Crim1 expression is restricted to the lens, with strongest expression in the epithelium and in the early differentiating secondary fibres. Thus, strong expression of Crim1 is a distinctive feature of the lens during morphogenesis and postnatal growth.

Fibrosis in the lens. Sprouty regulation of TGFβ-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 β (TGFβ) 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 TGFβ-Smad signaling responsible for EMT and anterior subcapsular cataract, this indicates a role for the ERK/MAPK pathway in TGFβ-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.

List of Speakers and Titles

Session 2: EMT in Development Chair Donald F. Newgreen (Melbourne) Regulation of Epithelial-Mesenchymal Cell Transformation in the Embryonic Chick Heart Runyan RB, Shoemaker S, Person A, Berkompas J, Scholz M, Mercado-Pimental M, Doyle SE, Stanislaw S (Tucson, Ariz.) Neural Epithelial-Mesenchymal Transitions: Many Ways to the Same End Newgreen D, Lewis S, Minichiello J, Farlie P (Melbourne) Intravital Imaging of Neural Crest Cells Kulesa PM (Kansas City, Mo.) Regulation of Neural Crest Formation by Sox2, Slug and BMP4 Wakamatsu Y, Endo Y, Sakai D, Osumi N (Sendai) Transforming Growth Factor3 (TGF 3) Upregulates Lymphoid Enhancing Factor-1 (LEF-1) Gene to Induce Epithelial-Mesenchymal Transformation (EMT) during Mouse Palate Development Nawshad A, Hay ED (Boston, Mass.)

042. The role of TGF-β in normal and pathological lens development

How the lens develops its highly ordered architecture and growth patterns is a major question in developmental biology. During embryogenesis, cells in the anterior and posterior segments of the lens vesicle, differentiate into the epithelial and fibre cells, respectively. Our research has aimed to identify the molecules and mechanisms that regulate the divergent fates of lens cells. We have studied the roles of various growth factors in regulating lens cell fates using rat lens epithelial explant cultures and transgenic and mutant mouse models. Our research has shown that members of the FGF growth factor family are key initiators of lens fibre differentiation in mammals and there is now compelling evidence that a gradient of FGF in the eye controls lens polarity and growth patterns. Recent evidence also supports a role for TGF-β signalling in this process and indicates that a cascade of growth factor signalling may be required for normal fibre differentiation. Less is known about the anterior segment; however, our recent studies point to an important role for the Wnt growth factor family in epithelial differentiation. Growth factor signalling can also cause pathological changes; e.g. TGF-β can destabilise the normal epithelial phenotype and induce aberrant growth and differentiation that mimics the epithelial-mesenchymal transition characteristic of some forms of cataract. These studies highlight the importance of growth factor signalling in regulating the ordered growth and differentiation of the lens. It is also clear that the bioavailabity of some growth factors needs to be tightly regulated so that they act in the appropriate cellular compartment. Some cataracts may be a consequence of disturbed growth factor, particularly TGF-β, regulatory mechanisms.