The lens equator: A platform for molecular machinery that regulates the switch from cell proliferation to differentiation in the vertebrate lens

The vertebrate lens is a transparent, spheroidal tissue, located in the anterior region of the eye that focuses visual images on the retina. During development, surface ectoderm associated with the neural retina invaginates to form the lens vesicle. Cells in the posterior half of the lens vesicle differentiate into primary lens fiber cells, which form the lens fiber core, while cells in the anterior half maintain a proliferative state as a monolayer lens epithelium. After formation of the primary fiber core, lens epithelial cells start to differentiate into lens fiber cells at the interface between the lens epithelium and the primary lens fiber core, which is called the equator. Differentiating lens fiber cells elongate and cover the old lens fiber core, resulting in growth of the lens during development. Thus, lens fiber differentiation is spatially regulated and the equator functions as a platform that regulates the switch from cell proliferation to cell differentiation. Since the 1970s, the mechanism underlying lens fiber cell differentiation has been intensively studied, and several regulatory factors that regulate lens fiber cell differentiation have been identified. In this review, we focus on the lens equator, where these regulatory factors crosstalk and cooperate to regulate lens fiber differentiation. Normally, lens epithelial cells must pass through the equator to start lens fiber differentiation. However, there are reports that when the lens epithelium structure is collapsed, lens fiber cell differentiation occurs without passing the equator. We also discuss a possible mechanism that represses lens fiber cell differentiation in lens epithelium.     

Elevated tropomyosin expression is associated with epithelial–mesenchymal transition of lens epithelial cells

Injury to lens epithelial cells (LECs) leads to epithelial–mesenchymal transition (EMT) with resultant fibrosis. The tropomyosin (Tpm) family of cytoskeleton proteins is involved in regulating and stabilizing actin microfilaments. Aberrant expression of Tpms leads to abnormal morphological changes with disintegration of epithelial integrity. The EMT of LECs has been proposed as a major cause of posterior capsule opacification (PCO) after cataract surgery. Using in vivo rodent PCO and human cataractous LECs, we demonstrated that the aberrant expression of rat Tpm and human Tpm1α/2β suggested their association in remodelling of the actin cytoskeleton during EMT of LECs. Expression analysis from abnormally growing LECs after lens extraction revealed elevated expression of α‐smooth muscle actin (α‐SMA), a marker for EMT. Importantly, these cells displayed increased expression of Tpm1α/2β following EMT/PCO formation. Expression of Tpm1α/2β was up‐regulated in LECs isolated from cataractous lenses of Shumiya Cataract Rats (SCRs), compared with non‐cataractous lenses. Also, LECs from human patients with nuclear cataract and anterior subcapsular fibrosis (ASF) displayed significantly increased expression of Tpm2β mRNA, suggesting that similar signalling invokes the expression of these molecules in LECs of cataractous SCR and human lenses. EMT was observed in LECs overexpressed with Tpm1α/2β, as evidenced by increased expression of α‐SMA. These conditions were correlated with remodelling of actin filaments, possibly leading to EMT/PCO and ASF. The present findings may help clarify the condition of the actin cytoskeleton during morphogenetic EMT, and may contribute to development of Tpm‐based inhibitors for postponing PCO and cataractogenesis.     

Endogenous voltage gradients as mediators of cell-cell communication: strategies for investigating bioelectrical signals during pattern formation

Alongside the well-known chemical modes of cell-cell communication, we find an important and powerful system of bioelectrical signaling: changes in the resting voltage potential (V_mem) of the plasma membrane driven by ion channels, pumps and gap junctions. Slow V_mem changes in all cells serve as a highly conserved, information-bearing pathway that regulates cell proliferation, migration and differentiation. In embryonic and regenerative pattern formation and in the disorganization of neoplasia, bioelectrical cues serve as mediators of large-scale anatomical polarity, organ identity and positional information. Recent developments have resulted in tools that enable a high-resolution analysis of these biophysical signals and their linkage with upstream and downstream canonical genetic pathways. Here, we provide an overview for the study of bioelectric signaling, focusing on state-of-the-art approaches that use molecular physiology and developmental genetics to probe the roles of bioelectric events functionally. We highlight the logic, strategies and well-developed technologies that any group of researchers can employ to identify and dissect ionic signaling components in their own work and thus to help crack the bioelectric code. The dissection of bioelectric events as instructive signals enabling the orchestration of cell behaviors into large-scale coherent patterning programs will enrich on-going work in diverse areas of biology, as biophysical factors become incorporated into our systems-level understanding of cell interactions.     

Vaccination Expands Antigen-Specific CD4+ Memory T Cells and Mobilizes Bystander Central Memory T Cells

CD4⁺ T helper memory (Th_mem) cells influence both natural and vaccine-boosted immunity, but mechanisms for their maintenance remain unclear. Pro-survival signals from the common gamma-chain cytokines, in particular IL-7, appear important. Previously we showed in healthy volunteers that a booster vaccination with tetanus toxoid (TT) expanded peripheral blood TT-specific Th_mem cells as expected, but was accompanied by parallel increase of Th_mem cells specific for two unrelated and non cross-reactive common recall antigens. Here, in a new cohort of healthy human subjects, we compare blood vaccine-specific and bystander Th_mem cells in terms of differentiation stage, function, activation and proliferative status. Both responses peaked 1 week post-vaccination. Vaccine-specific cytokine-producing Th_mem cells were predominantly effector memory, whereas bystander cells were mainly of central memory phenotype. Importantly, TT-specific Th_mem cells were activated (CD38^High HLA-DR⁺), cycling or recently divided (Ki-67⁺), and apparently vulnerable to death (IL-7Rα^Low and Bcl-2 ^Low). In contrast, bystander Th_mem cells were resting (CD38^Low HLA-DR^- Ki-67^-) with high expression of IL-7Rα and Bcl-2. These findings allow a clear distinction between vaccine-specific and bystander Th_mem cells, suggesting the latter do not derive from recent proliferation but from cells mobilized from as yet undefined reservoirs. Furthermore, they reveal the interdependent dynamics of specific and bystander T-cell responses which will inform assessments of responses to vaccines.     

Nonlinear ionizing radiation-induced changes in eye lens cell proliferation,cyclin D1 expression and lens shape

Elevated cataract risk after radiation exposure was established soon after the discovery of X-rays in 1895. Today, increased cataract incidence among medical imaging practitioners and after nuclear incidents has highlighted how little is still understood about the biological responses of the lens to low-dose ionizing radiation (IR). Here, we show for the first time that in mice, lens epithelial cells (LECs) in the peripheral region repair DNA double strand breaks (DSB) after exposure to 20 and 100 mGy more slowly compared with circulating blood lymphocytes, as demonstrated by counts of γH2AX foci in cell nuclei. LECs in the central region repaired DSBs faster than either LECs in the lens periphery or lymphocytes. Although DSB markers (γH2AX, 53BP1 and RAD51) in both lens regions showed linear dose responses at the 1 h timepoint, nonlinear responses were observed in lenses for EdU (5-ethynyl-2′-deoxy-uridine) incorporation, cyclin D1 staining and cell density after 24 h at 100 and 250 mGy. After 10 months, the lens aspect ratio was also altered, an indicator of the consequences of the altered cell proliferation and cell density changes. A best-fit model demonstrated a dose-response peak at 500 mGy. These data identify specific nonlinear biological responses to low (less than 1000 mGy) dose IR-induced DNA damage in the lens epithelium.     

Heat shock factor 4 regulates lens epithelial cell homeostasis by working with lysosome and anti-apoptosis pathways

Activation of Heat shock factor 4-mediated heat shock response is closely associated with postnatal lens development. HSF4 controls the expression of small heat shock proteins (e.g. HSP25 and CRYAB) in lens epithelial cells. However, their roles in modulating lens epithelium homeostasis remain unclear. In this paper, we find that HSF4 is developmentally expressed in mouse lens epithelium and fiber tissue. HSF4 and alpha B-crystallin can selectively protect lens epithelial cells from cisplatin and H2O2 induced apoptosis by stabilizing mitochondrial membrane potential (ΔY_m) and reducing ROS production. In addition, to our surprise, HSF4 is involved in upregulating lysosome activity. We found mLEC/HA-Hsf4 cells to have increased DLAD expression, lysosome acidity, cathepsin B activity, and degradation of plasmid DNA and GFP-LC3 protein when compared to mLEC/Hsf4-/- cells. Knocking down Cryab from mLEC/HA-Hsf4 cells inhibits HSF4-mediated lysosome acidification, while overexpression of CRYAB can upregulate cathepsin B activity in mLEC/Hsf4-/- cells. CRAYAB can interact with ATP6V1/A the A subunit of the H⁺ pump vacuolar ATPase, and is colocalized to lamp1 and lamp2 in the lysosome. Collectively, these results suggest that in addition to modulating anti-apoptosis, HSF4 is able to regulate lysosome activity by at least controlling alpha B-crystallin expression, shedding light on a novel molecular mechanism of HSF4 in regulating lens epithelial cell homeostasis.     

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.     

MAINTENANCE OF IONS,PROTEINS AND WATER IN LENS FIBER CELLS BEFORE AND AFTER TREATMENT WITH NON-IONIC DETERGENTS

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks ’ balanced salt solution in either absence or presence of non-ionic detergents (0.2 % Triton X-100 or 0.2 % Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K⁺, Mg^{2 +}, Na⁺, Ca^{2 +}, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 °C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K⁺, Mg^{2 +}, water and cytomatrix proteins.     

The roles of αV integrins in lens EMT and posterior capsular opacification

Posterior capsular opacification (PCO) is the major complication arising after cataract treatment. PCO occurs when the lens epithelial cells remaining following surgery (LCs) undergo a wound healing response producing a mixture of α‐smooth muscle actin (α‐SMA)‐expressing myofibroblasts and lens fibre cells, which impair vision. Prior investigations have proposed that integrins play a central role in PCO and we found that, in a mouse fibre cell removal model of cataract surgery, expression of α_V integrin and its interacting β‐subunits β1, β5, β6, β8 are up‐regulated concomitant with α‐SMA in LCs following surgery. To test the hypothesis that α_V integrins are functionally important in PCO pathogenesis, we created mice lacking the α_V integrin subunit in all lens cells. Adult lenses lacking α_V integrins are transparent and show no apparent morphological abnormalities when compared with control lenses. However, following surgical fibre cell removal, the LCs in control eyes increased cell proliferation, and up‐regulated the expression of α‐SMA, β1‐integrin, fibronectin, tenascin‐C and transforming growth factor beta (TGF‐β)–induced protein within 48 hrs, while LCs lacking α_V integrins exhibited much less cell proliferation and little to no up‐regulation of any of the fibrotic markers tested. This effect appears to result from the known roles of α_V integrins in latent TGF‐β activation as α_V integrin null lenses do not exhibit detectable SMAD‐3 phosphorylation after surgery, while this occurs robustly in control lenses, consistent with the known roles for TGF‐β in fibrotic PCO. These data suggest that therapeutics antagonizing α_V integrin function could be used to prevent fibrotic PCO following cataract surgery.     

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.     

The Analysis of Intracellular and Intercellular Calcium Signaling in Human Anterior Lens Capsule Epithelial Cells with Regard to Different Types and Stages of the Cataract

In this work we investigated how modifications of the Ca²⁺ homeostasis in anterior lens epithelial cells (LECs) are associated with different types of cataract (cortical or nuclear) and how the progression of the cataract (mild or moderate) affects the Ca²⁺ signaling. We systematically analyzed different aspects of intra- and inter-cellular Ca²⁺ signaling in the human LECs, which are attached to surgically isolated lens capsule (LC), obtained during cataract surgery. We monitored the temporal and spatial changes in intracellular Ca²⁺ concentration after stimulation with acetylcholine by means of Fura-2 fluorescence captured with an inverted microscope. In our analysis we compared the features of Ca²⁺ signals in individual cells, synchronized activations, spatio-temporal grouping and the nature of intercellular communication between LECs. The latter was assessed by using the methodologies of the complex network theory. Our results point out that at the level of individual cells there are no significant differences when comparing the features of the signals with regard either to the type or the stage of the cataract. On the other hand, noticeable differences are observed at the multicellular level, despite inter-capsule variability. LCs associated with more developed cataracts were found to exhibit a slower collective response to stimulation, a less pronounced spatio-temporal clustering of LECs with similar signaling characteristics. The reconstructed intercellular networks were found to be sparser and more segregated than in LCs associated with mild cataracts. Moreover, we show that spontaneously active LECs often operate in localized groups with quite well aligned Ca²⁺ activity. The presence of spontaneous activity was also found to affect the stimulated Ca²⁺ responses of individual cells. Our findings indicate that the cataract progression entails the impairment of intercellular signaling thereby suggesting the functional importance of altered Ca²⁺ signaling of LECs in cataractogenesis.     

Effects of aging in vitro on intracellular proteolysis in cultured rabbit lens epithelial cells in the presence and absence of serum

Summary Alterations in proteolytic capabilities have been associated with abnormalities in the aged eye lens, but in vivo tests of this hypothesis have been difficult to pursue. To simulate aging, we cultured cells from an 8-yr-old rabbit to early (population-doubling level 20 to 30) and late (population-doubling level > 125) passage. Long-lived (t_1/2>10 h) and short-lived (t_1/2<10 h) intracellular proteins were labeled with [³H]leucine, and the ability of the cells to mount a proteolytic response to the stress of serum withdrawal was determined. For early passage cells, the average t_1/2 of long-lived proteins in the presence and absence of serum was 62 and 39 h, respectively. For late-passage cells, the average t_1/2 of long-lived proteins in the presence and absence of serum was 58 and 43 h, respectively. The net increase in intracellular proteolysis in the absence of serum was 59 and 35% for early and late-passage cells, respectively. Thus, in vitro-aged rabbit lens epithelial cells mount only 60% the proteolytic response to serum removal shown in “younger” cells. The enhanced ability of early passage cells to respond to serum removal seems to involve lower homeostatic levels of proteolysis in the presence of serum and greater enhancement of proteolysis in the absence of serum. Less than 2% of the protein is in the pool of short-lived proteins. Rates of proteolysis of short-lived proteins in the presence and absence of serum were indistinguishable. With respect to basal proteolytic rates in the presence of serum and ability to mount a proteolytic response upon serum withdrawal, these rabbit lens epithelial cells are similar to bovine lens epithelial cells and fibroblasts. This work was supported in part by contract 53-3K06-5-10 U.S. Department of Agriculture, Washington, DC, Massachusetts Lions Eye Research FUnd, Inc., the Daniel and Florence Guggenheim Foundation, and a grant EY00362 from the National Eye Institute, Bethesda, MD.     

Unfolded Protein Response (UPR) is activated during normal lens development

The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2α were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis.     

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.     

Interferon‐γ suppresses Na+–H+ exchanger in cultured human endolymphatic sac epithelial cells

Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na⁺–H⁺ exchanger (NHE) is a major determinant of intracellular pH (pH_i), and facilitates Na⁺ and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN‐γ on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, ‐2, and ‐3 isoforms was determined by real‐time RT‐PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH‐sensitive fluorescent dye, 2′,7′‐bis(carbonylethyl)‐5(6)‐carboxyfluorescein (BCECF), and a NHE‐inhibitor, 3‐methylsulfonyl‐4‐piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, ‐2, and ‐3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and ‐2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na⁺‐dependent pH_i recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN‐γ (50 nM for 24 h) suppressed mRNA expression of NHE1 and ‐2. IFN‐γ also suppressed functional activity of both NHE1 and ‐2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN‐γ suppresses the expression and functional activity of NHE1 and ‐2. J. Cell. Biochem. 107: 965–972, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of c-Jun and a negative dominant mutation of c-Jun on differentiation and gene expression in lens epithelial cells

We have used a retroviral vector (RCAS) to overexpress wild-type chicken c-Jun or a deletion mutant of chicken c-Jun (JunΔ7) lacking the DNA binding region to investigate the possible role of c-Jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c-Jun increased the rate of cell proliferation and greatly delayed the appearance of “lentoid bodies,” structures which contain differentiated cells expressing fiber cell markers. Excess c-Jun expression also significantly decreased the level of β_A3/A1-crystallin mRNA, without affecting αA-crystallin mRNA. In contrast, the mutated protein, JunΔ7, had no effect no proliferation or differentiation but markedly increased the level of αA-crystallin mRNA in proliferating cell cultures. These results suggest that c-Jun or Jun-related proteins may be negative regulators of αA- and β_A3/A1-crystallin genes in proliferating lens cells.