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.     

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals.     

Equarin is involved as an FGF signaling modulator in chick lens differentiation

Lens growth involves the proliferation of epithelial cells, followed by their migration to the equator region and differentiation into secondary fiber cells. It is widely accepted that fibroblast growth factor (FGF) signaling is required for the differentiation of lens epithelial cells into crystallin-rich fibers, but this signaling is insufficient to induce full differentiation. To better understand lens development, investigatory and functional analyses of novel molecules are required. Here, we demonstrate that Equarin, which is a novel secreted molecule, was expressed exclusively in the lens equator region during chick lens development. Equarin upregulated the expression of fiber markers, as demonstrated using in ovo electroporation. In a primary lens cell culture, Equarin promoted the biochemical and morphological changes associated with the differentiation of lens epithelial cells to fibers. A loss-of-function analysis was performed using zinc-finger nucleases targeting the Equarin gene. Lens cell differentiation was markedly inhibited when endogenous Equarin was blocked, indicating that Equarin was essential for normal chick lens differentiation. Furthermore, biochemical analysis showed that Equarin directly bound to FGFs and heparan sulfate proteoglycan and thereby upregulated the expression of phospho-ERK1/2 (ERK-P) proteins, the downstream of the FGF signaling pathway, in vivo and in vitro. Conversely, the absence of endogenous Equarin clearly diminished FGF-induced fiber differentiation. Taken together, our results suggest that Equarin is involved as an FGF modulator in chick lens differentiation.     

Duration of ERK1/2 phosphorylation induced by FGF or ocular media determines lens cell fate

The ocular environment is important for the establishment and maintenance of lens growth patterns and polarity. In the anterior chamber of the eye, the aqueous humour regulates lens epithelial cell proliferation whereas in the posterior, the vitreous humour regulates the differentiation of the lens cells into fiber cells. Members of the fibroblast growth factor (FGF) growth factor family have been shown to induce lens epithelial cells to undergo cell division and differentiate into fibers, with a low dose of FGF able to induce cell proliferation (but not fiber differentiation), and higher doses required to induce fiber differentiation. Both these cellular events have been shown to be regulated by the MAPK/ERK1/2 signalling pathway. In the present study, to better understand the contribution of ERK1/2 signalling in regulating lens cell proliferation and differentiation, we characterized the ERK1/2 signalling profiles induced by different doses of FGF, and compared these to those induced by the different ocular media. Here, we show that FGF induced a dose-dependent sustained activation of ERK1/2, with both a high (fiber differentiating) dose of FGF and vitreous, stimulating and maintaining a prolonged (up to 18 hr) ERK1/2 phosphorylation profile. In contrast, a lower (proliferating) dose of FGF, and aqueous, stimulated ERK1/2 phosphorylation for only up to 6 hr. If we selectively reduce the 18 hr ERK1/2 phosphorylation profile induced by vitreous to 6 hr, by specifically blocking FGF receptor signalling, the vitreous now fails to induce lens fiber differentiation but retains the ability to induce lens cell proliferation. These findings not only provide insights into the important role that FGF plays in the different ocular media that bathe the lens, but enlighten us on some of the putative molecular mechanisms by which one specific growth factor, in this case FGF, can elicit a different cellular response in the same cell type.     

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-β and Wnt/Frizzled families in regulating aspects of fibre differentiation. In the case of the epithelium, key roles for Wnt/β-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.     

bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line.

There is increasing evidence that basic fibroblast growth factor (bFGF) plays an important role in cell proliferation, differentiation, and survival in various systems. In the eye, although a truncated, dominant negative bFGF receptor in transgenic mice induced defective lens development and caused lens fiber cells to display characteristics of apoptosis, there is little direct evidence of the effect of bFGF on lens epithelial cell apoptosis. Our study examines the effects of bFGF on programmed cell death induced by serum deprivation using a human lens epithelial cell line. Cells supplemented with 20% fetal bovine serum were used as normal controls. Over a period of 7 days, the addition of 100 ng/ml bFGF effectively suppressed serum-deprived apoptosis. The expression of gamma-crystallin and major intrinsic protein, which are markers of lens cell differentiation, was not detected. Also there was no significant difference in cell proliferation between serum-deprived cells with or without bFGF. ICE (caspase-1) was expressed under both the conditions, but the level of expression between the two groups was not substantially different. bcl-2 and c-myc were upregulated only in bFGF-treated cells. Thus we speculate that the inhibitory effect of bFGF on apoptosis is through the upregulation of the inhibitor of apoptosis, instead of downregulation of the initiator. This effect appears to be independent of lens cell differentiation and proliferation.     

FGF signaling in chick lens development

The prevailing concept has been that an FGF induces epithelial-to-fiber differentiation in the mammalian lens, whereas chick lens cells are unresponsive to FGF and are instead induced to differentiate by IGF/insulin-type factors. We show here that when treated for periods in excess of those used in previous investigations (>5 h), purified recombinant FGFs stimulate proliferation of primary cultures of embryonic chick lens epithelial cells and (at higher concentrations) expression of the fiber differentiation markers delta-crystallin and CP49. Surprisingly, upregulation of proliferation and delta-crystallin synthesis by FGF does not require activation of ERK kinases. ERK function is, however, essential for stimulation of delta-crystallin expression in response to insulin or IGF-1. Vitreous humor, the presumptive source of differentiation-promoting activity in vivo, contains a factor capable of diffusing out of the vitreous body and inducing delta-crystallin and CP49 expression in chick lens cultures. This factor binds heparin with high affinity and increases delta-crystallin expression in an ERK-insensitive manner, properties consistent with an FGF but not insulin or IGF. Our findings indicate that differentiation in the chick lens is likely to be mediated by an FGF and provide the first insights into the role of the ERK pathway in growth factor-induced signal transduction in the lens.     

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.     

Basic FGF induces neuronal differentiation, cell division, and NGF dependence in chromaffin cells: a sequence of events in sympathetic development

To define further the molecules that control sympathoadrenal differentiation, we have investigated the effects of FGF, NGF, and glucocorticoid on cultured neonatal rat adrenal chromaffin cells. Basic FGF (bFGF), like NGF, induces cell division and neurite outgrowth from these cells. Dexamethasone inhibits neuronal differentiation but not proliferation induced by bFGF. Unlike NGF, bFGF will not support the survival of chromaffin cell-derived sympathetic neurons. However, bFGF induces a dependence on NGF. The overlapping but distinct responses to NGF and bFGF may underlie a sequence of events in sympathetic differentiation. bFGF (or another factor) may act locally in developing ganglia to stimulate mitotic expansion and initial axon outgrowth. Subsequent survival and maturation are then controlled by NGF, which is provided by peripheral targets of innervation. In the adrenal gland, glucocorticoids may permit bFGF to amplify the chromaffin population, while preventing neuronal differentiation.     

Bone morphogenetic protein signaling and the initiation of lens fiber cell differentiation

Previous studies showed that the retina produces factors that promote the differentiation of lens fiber cells, and identified members of the fibroblast growth factor (FGF) and insulin-like growth factor (IGF) families as potential fiber cell differentiation factors. A possible role for the bone morphogenetic proteins (BMPs) is suggested by the presence of BMP receptors in chicken embryo lenses. We have now observed that phosphorylated SMAD1, an indicator of signaling through BMP receptors, localizes to the nuclei of elongating lens fiber cells. Transduction of chicken embryo retinas and/or lenses with constructs expressing noggin, a secreted protein that binds BMPs and prevents their interactions with their receptors, delayed lens fiber cell elongation and increased cell death in the lens epithelium. In an in vitro explant system, in which chicken embryo or adult bovine vitreous humor stimulates chicken embryo lens epithelial cells to elongate into fiber-like cells, these effects were inhibited by noggin-containing conditioned medium, or by recombinant noggin. BMP2, 4, or 7 were able to reverse the inhibition caused by noggin. Lens cell elongation in epithelial explants was stimulated by treatment with FGF1 or FGF2, alone or in combination with BMP2, but not to the same extent as vitreous humor. These data indicate that BMPs participate in the differentiation of lens fiber cells, along with at least one additional, and still unknown factor.     

Downregulated expression of integrin alpha6 by transforming growth factor-beta(1) on lens epithelial cells in vitro

Integrins represent the main cell surface receptors that mediate cell-matrix and cell-cell interactions. They play critical roles in adhesion, migration, morphogenesis, and the differentiation of several cell types. Previous studies have demonstrated that members of the fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-beta(1), and insulin growth factor (IGF)-1 play important roles in lens biology. In particularly, TGF-beta(1) appears to play a key role in extracellular matrix production, cell proliferation, and cell differentiation of lens epithelial cells. In this study we investigated the effects of FGF-2, TGF-beta(1), and IGF-1 on the modulation of integrin receptors using lens epithelial cell lines (HLE B-3 and alphaTN-4) and lens explants. We found that the expression of integrin alpha6 is downregulated by TGF-beta(1), but is not responsive to FGF-2 or IGF-1. The promoter activity of the integrin alpha6 gene decreased upon TGF-beta(1) treatment in a transient transfection assay, and flow cytometric analysis demonstrated the reduced expression of integrin alpha6 by TGF-beta(1), whereas significant changes were not observed in the level of integrin alpha6 after the addition of FGF-2. These findings suggest that the reduced expression of integrin alpha6 caused by TGF-beta(1) might play a role in the activation of the cell cycle genes required during the fiber differentiation of the lens.     

A cell polarity protein aPKCλ is required for eye lens formation and growth

The organisation of individual cells into a functional three-dimensional tissue is still a major question in developmental biology. Modulation of epithelial cell shape is a critical driving force in forming tissues. This is well illustrated in the eye lens where epithelial cells elongate extensively during their differentiation into fibre cells. It is at the lens equator that epithelial cells elongate along their apical-basal axis. During this process the elongating epithelial cells and their earliest fibre cell derivatives remain anchored at their apical tips, forming a discrete region or modiolus, which we term the lens fulcrum. How this is achieved has received scant attention and is little understood. Here, we show that conditional depletion of aPKCλ, a central effector of the PAR polarity complex, disrupts the apical junctions in elongating epithelial cells so that the lens fulcrum fails to form. This results in disorganised fibre cell alignment that then causes cataract. Interestingly, aPKCλ depletion also promotes epithelial-mesenchymal transition of the lens epithelial cells, reducing their proliferation, leading ultimately to a small lens and microphthalmia. These observations indicate that aPKCλ, a regulator of polarity and apical junctions, is required for development of a lens that is the correct size and shape.     

Activated Ras alters lens and corneal development through induction of distinct downstream targets

Background  

Mammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF) signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas.     

Basic fibroblast growth factor regulates extracellular matrix and contractile protein expression independent of proliferation in vascular smooth muscle cells

Summary Basic fibroblast growth factor (bFGF) can influence proliferation and differentiation in vascular smooth muscle cells. Basic FGF promotes some features of the synthetic phenotype (proliferation) but is known to inhibit others (collagen synthesis). Whether bFGF availability influences smooth muscle cell phenotype independent of proliferation is not known. The purpose of this study was to determine if the effects of bFGF on extracellular matrix and contractile protein expression are dependent on changes in proliferation. Basic FGF availability was manipulated by adding bFGF to cultured cells or by inhibiting bFGF expression using antisense RNA, and adjusting culture conditions such that proliferation was held constant. Compared to cells cultured in serum alone, smooth muscle α-actin and myosin heavy chain expression was markedly reduced by added bFGF, but was not influenced by antisense inhibition of bFGF expression. Under the same conditions, collagen synthesis was inhibited by added bFGF, and was stimulated by reduced bFGF expression. These consequences of altering bFGF availability were not associated with changes in FGF receptor expression. These findings demonstrate that alterations in bFGF availability can regulate smooth muscle cell phenotype independent of proliferation, which may be related to the regulation of smooth muscle cell phenotype in vivo.     

Synthesis of basic fibroblast growth factor upon differentiation of rat mammary epithelial to myoepithelial-like cells in culture

Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial-like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial-like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial-like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial-like rat mammary 29 cells was able to displace [125I]-bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell-associated. The cell-associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial-like cells with Triton X-100 and 2 M NaCl showed that 50-65% of the cell-associated bFGF was in a detergent-resistant but 2 M NaCl-labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.     

FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial–mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up‐regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F‐actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up‐regulation of α‐smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock‐down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ‐treated LECs perturbed EMT by reactivating the mitogen‐activated protein kinase (MAPK)/ extracellular signal‐regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2‐mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs‐driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.