Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling.

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei.

MAPK1 is required for establishing the pattern of cell proliferation and for cell survival during lens development.

The mitogen-activated protein kinases (MAPKs; also known as ERKs) are key intracellular signaling molecules that are ubiquitously expressed in tissues and were assumed to be functionally equivalent. Here, we use the mouse lens as a model system to investigate whether MAPK1 plays a specific role during development. MAPK3 is known to be dispensable for lens development. We demonstrate that, although MAPK1 is uniformly expressed in the lens epithelium, its deletion significantly reduces cell proliferation in the peripheral region, an area referred to as the lens germinative zone in which most active cell division occurs during normal lens development. By contrast, cell proliferation in the central region is minimally affected by MAPK1 deletion. Cell cycle regulators, including cyclin D1 and survivin, are downregulated in the germinative zone of the MAPK1-deficient lens. Interestingly, loss of MAPK1 subsequently induces upregulation of phosphorylated MAPK3 (pMAPK3) levels in the lens epithelium; however, this increase in pMAPK3 is not sufficient to restore cell proliferation in the germinative zone. Additionally, MAPK1 plays an essential role in epithelial cell survival but is dispensable for fiber cell differentiation during lens development. Our data indicate that MAPK1/3 control cell proliferation in the lens epithelium in a spatially defined manner; MAPK1 plays a unique role in establishing the highly mitotic zone in the peripheral region, whereas the two MAPKs share a redundant role in controlling cell proliferation in the central region of the lens epithelium.

Frs2α enhances fibroblast growth factor-mediated survival and differentiation in lens development.

Most growth factor receptor tyrosine kinases (RTKs) signal through similar intracellular pathways, but they often have divergent biological effects. Therefore, elucidating the mechanism of channeling the intracellular effect of RTK stimulation to facilitate specific biological responses represents a fundamental biological challenge. Lens epithelial cells express numerous RTKs with the ability to initiate the phosphorylation (activation) of Erk1/2 and PI3-K/Akt signaling. However, only Fgfr stimulation leads to lens fiber cell differentiation in the developing mammalian embryo. Additionally, within the lens, only Fgfrs activate the signal transduction molecule Frs2α. Loss of Frs2α in the lens significantly increases apoptosis and decreases phosphorylation of both Erk1/2 and Akt. Also, Frs2α deficiency decreases the expression of several proteins characteristic of lens fiber cell differentiation, including Prox1, p57(KIP2), aquaporin 0 and ß-crystallins. Although not normally expressed in the lens, the RTK TrkC phosphorylates Frs2α in response to binding the ligand NT3. Transgenic lens epithelial cells expressing both TrkC and NT3 exhibit several features characteristic of lens fiber cells. These include elongation, increased Erk1/2 and Akt phosphorylation, and the expression of ß-crystallins. All these characteristics of NT3-TrkC transgenic lens epithelial cells depend on Frs2α. Therefore, tyrosine phosphorylation of Frs2α mediates Fgfr-dependent lens cell survival and provides a mechanistic basis for the unique fiber-differentiating capacity of Fgfs on mammalian lens epithelial cells.

Meibomian gland stem/progenitor cells: The hunt for gland renewal.

Meibomian glands (MGs) secrete lipid (meibum) onto the ocular surface to form the outermost layer of the tear film. Proper meibum secretion is essential for stabilizing the tear film, reducing aqueous tear evaporation, and maintaining the homeostasis of the ocular surface. Atrophy of MG as occurs with aging, leads to reduction of meibum secretion, loss of ocular surface homeostasis and evaporative dry eye disease (EDED). Since MGs are holocrine glands, secretion of meibum requires continuous self-renewal of lipid-secreting acinar meibocytes by stem/progenitor cells, whose proliferative potential is dramatically reduced with age leading to MG atrophy and an age-related meibomian gland dysfunction (ARMGD). Understanding the cellular and molecular mechanisms regulating meibocyte stem/progenitor cell maintenance and renewal may provide novel approaches to regenerating MG and treating EDED. Towards that end, recent label retaining cell and lineage-tracing experiments as well as knock-out transgenic mouse studies have begun to identify the location and identities of meibocyte progenitor cells and potential growth and transcription factors that may regulate meibocyte renewal. In addition, recent reports have shown that ARMGD may be reversed by novel therapeutics in mice. Herein, we discuss our current understanding of meibocyte stem/progenitor cells and the hunt for gland renewal.

Spontaneous acinar and ductal regrowth after meibomian gland atrophy induced by deletion of FGFR2 in a mouse model.

PURPOSE: We have demonstrated that deletion of fibroblast growth factor receptor 2 gene (Fgfr2) leads to Meibomian gland (MG) atrophy in an inducible conditional knockout mouse model, referred as Fgfr2CKO. Herein, we investigated whether MG spontaneously recovers after atrophy in this model. METHODS: Two months old Fgfr2CKO mice were injected peritoneally once or twice of doxycycline (Dox) at 80 µg/gm of body weight to induce MG atrophy of various severities via Fgfr2 deletion. Recovery of acinar and ductal tissues was monitored by meibography, lipid staining and immunofluorescence against keratin-6a in MG whole-mount. Biomarkers for acinar and ductal differentiation and proliferation were also examined by immunostaining. RESULTS: Single Dox injection in Fgfr2CKO mice caused severe acinar and moderate ductal atrophy. Severe ductal shortening or loss occurred after second Dox injection, presumably related to the reported slower cycling of the ductal epithelia. Spontaneous acinar regrowth after atrophy was observed over a period of 60 days in both injection regimens. However, less robust acinar recovery was associated with more disrupted ductal structures in twice injected Fgfr2CKO mice. CONCLUSIONS: Our current findings further substantiate the role of FGFR2 in MG homeostasis, and suggest that FGFR2-signaling may provide a potential strategy for regenerating acini from age-related MG dysfunction in humans. Our data demonstrated that spontaneous MG recovery depends on the extent of ductal atrophy, suggesting that ductal epithelia may provide the progenitor cells for acinar regeneration. Nonetheless, the role of ductal tissue as the source of acinar progenitors awaits further investigation.

Sef is a negative regulator of fiber cell differentiation in the ocular lens.

Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.

Histopathology and selective biomarker expression in human meibomian glands.

BACKGROUND/AIMS: Meibomian gland dysfunction (MGD) is the most common form of evaporative dry eye disease, but its pathogenesis is poorly understood. This study examined the histopathological features of meibomian gland (MG) tissue from cadaver donors to identify potential pathogenic processes that underlie MGD in humans. METHODS: Histological analyses was performed on the MGs in the tarsal plates dissected from four cadaver donors, two young and two old adults, including a 36-year-old female (36F) and three males aged 30, 63 and 64 years (30M, 63M and 64M). RESULTS: The MGs of 36F displayed normal anatomy and structure, whereas the MGs of 30M showed severe ductal obstruction with mild distortion. The obstruction was caused by increased cytokeratin levels in association with hyperproliferation, but not hyperkeratinisation. In two older males, moderate to severe MG atrophy was noted. Cell proliferation was significantly reduced in the MG acini of the two older donors as measured by Ki67 labelling index (6.0%±3.4% and 7.9%±2.8% in 63M and 64M, respectively) when compared with that of the two younger donors (23.2%±5.5% and 16.9%±4.8% in 30M and 36F, respectively) (p<0.001). The expression patterns of meibocyte differentiation biomarkers were similar in the older and younger donors. CONCLUSION: Our histopathological study, based on a small sample size, suggests potentially distinct pathogenic mechanisms in MGD. In the young male adult, hyperproliferation and aberrant differentiation of the central ductal epithelia may lead to the obstruction by overproduced cytokeratins. In contrast, in older adults, decreased cell proliferation in acinar basal epithelia could be a contributing factor leading to MG glandular atrophy.

Rho GDP dissociation inhibitor-mediated disruption of Rho GTPase activity impairs lens fiber cell migration, elongation and survival.

To explore the role of the Rho GTPases in lens morphogenesis, we overexpressed bovine Rho GDP dissociation inhibitor (Rho GDI alpha), which serves as a negative regulator of Rho, Rac and Cdc42 GTPase activity, in a lens-specific manner in transgenic mice. This was achieved using a chimeric promoter of delta-crystallin enhancer and alpha A-crystallin, which is active at embryonic day 12. Several individual transgenic (Tg) lines were obtained, and exhibited ocular specific phenotype comprised of microphthalmic eyes with lens opacity. The overexpression of bovine Rho GDI alpha disrupted membrane translocation of Rho, Rac and Cdc42 GTPases in Tg lenses. Transgenic lenses also revealed abnormalities in the migration pattern, elongation and organization of lens fibers. These changes appeared to be associated with impaired organization of the actin cytoskeleton and cell-cell adhesions. At E14.5, the size of the Rho GDI alpha Tg lenses was larger compared to wild type (WT) and the central lens epithelium and differentiating fibers exhibited an abnormal increase of bromo-deoxy-uridine incorporation. Postnatal Tg eyes, however, were much smaller in size compared to WT eyes, revealing increased apoptosis in the disrupted lens fibers. Taken together, these data demonstrate a critical role for Rho GTPase-dependent signaling pathways in processes underlying morphogenesis, fiber cell migration, elongation and survival in the developing lens.

Indoleamine 2,3-dioxygenase overexpression causes kynurenine-modification of proteins, fiber cell apoptosis and cataract formation in the mouse lens.

Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the kynurenine pathway. The kynurenines formed in this pathway chemically modify proteins and cause apoptosis in cells. Evidence suggests that kynurenines and their protein modifications are involved in cataract formation, but this has yet to be directly demonstrated. We generated transgenic (Tg) mouse lines that overexpress human IDO in the lens. Homozygous Tg (homTg) lenses had higher IDO immunoreactivity, approximately 4.5 times greater IDO mRNA, and approximately 8 times higher IDO activity compared to lenses from hemizygous Tg (hemTg) animals. The kynurenine content was threefold higher in homTg than in hemTg but was not detected in wild-type (Wt) lenses. Kynurenine modifications were approximately 2.6 times greater in homTg than in hemTg or Wt. HomTg lenses had vacuoles in the epithelium and cortical fiber cells. Kynurenine modifications coincided with apoptosis in the secondary fiber cells of homTg lenses. Caspase-3 and caspase-9 activities were markedly higher in homTg than in hemTg and Wt. The glutathione content was approximately 36% lower in homTg compared to hemTg and Wt lenses. HomTg animals also developed bilateral cataracts within 3 months of birth. Together these data demonstrate that IDO-mediated production of kynurenines results in defects in fiber cell differentiation and their apoptosis and suggest that IDO activity is kept low in the lens to prevent deleterious effects by kynurenines.

Elevated insulin signaling disrupts the growth and differentiation pattern of the mouse lens.

PURPOSE: Insulin and insulin-like growth factors (IGFs) are putative regulators of cell proliferation and differentiation during lens development. Transgenic mice that overexpress IGF-1 in the lens have been previously described. To further understand the ocular functions of this growth factor family, the in vivo effects of insulin expression on lens development were investigated using transgenic mice. METHODS: Expression of insulin receptor (IR) and IGF-1 receptor (IGF-1R) in mouse lens was examined by reverse-transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Transgenic mice that overexpress insulin in the lens were generated using two different promoters: a fiber-cell specific alphaA-crystallin (alphaA) promoter and a modified alphaA-promoter linked to the chicken delta1-crystallin enhancer (called the deltaenalphaA promoter). The deltaenalphaA promoter is active in both lens epithelial and fiber cells. The lens phenotypes were analyzed by histology and immunohistochemistry. Protein expression was examined by western blotting. RESULTS: Normal mouse lenses express both the insulin receptor (IR) and the IGF-1 receptor (IGF-1R), and their expression is highest at the lens periphery where the germinative and transitional zones are located. In transgenic mice, insulin expression in the lens induced cataract formation. The severity of the cataracts reflected the level of transgene expression, independent of the type of promoter used. In severely affected families, the spherical shape of the lens was altered and the lenses were smaller than normal. Histological analysis showed no evidence of premature differentiation of the anterior epithelial cells. In contrast to the IGF-1 mice, insulin transgenic mice exhibited an anterior shift in the location of the germinative and transitional zones, leading to a reduction of the lens epithelial compartment. Additional alterations included expansion of the lens transitional zone, variable nuclear positioning in the lens bow region, and inhibition of fiber cell denucleation and terminal differentiation. CONCLUSIONS: Elevated intraocular insulin does not enhance proliferation nor induce differentiation of mouse lens epithelial cells. Since an increase in IGF-1 causes a posterior shift of the lens geminative and transitional zones, while an increase in insulin causes an anterior shift of these zones, our results suggest that these two growth factors may work together to control the location of this structural domain during normal lens development. Our data also suggest that increased insulin-signaling activity in the lens can antagonize the endogenous signals that are responsible for fiber cell maturation and terminal differentiation.

Fibroblast Growth Factor Receptor 2 (FGFR2) Is Required for Meibomian Gland Homeostasis in the Adult Mouse.

Purpose: Little is known about the signaling mechanisms controlling meibomian gland (MG) homeostasis and the pathogenic processes leading to MG atrophy and dysfunction in dry eye disease (DED). We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in the MG homeostasis of adult mice. Methods: A triple transgenic mouse strain (Krt14-rtTA; tetO-Cre; Fgfr2flox/flox), referred to as Fgfr2CKO mice, was generated in which the Fgfr2 gene is ablated by Cre recombinase in keratin 14 (Krt14)-expressing epithelial cells on doxycycline (Dox) induction. FGFR2 expression in normal human and mouse MGs was evaluated by immunohistochemistry. Pathologic MG changes in transgenic mice with conditional deletion of FGFR2 were examined by lipid staining, histology, and immunostaining. Results: FGFR2 was highly expressed in normal human MGs and adult mouse MGs. Two-month-old Fgfr2CKO mice fed Dox-containing chow for 2 weeks developed severe MG atrophy. MG acinar atrophy in the Fgfr2CKO mice was associated with reduced lipid (meibum) production and the development of clinical findings similar to those in humans with evaporative DED related to MG dysfunction (MGD). Immunohistochemical analyses showed that FGFR2 deletion severely affected proliferation and differentiation of MG acinar cells but affected MG ductal cells to a lesser extent. Conclusions: FGFR2 deletion results in significant MG acinar atrophy and clinical manifestations of MGD in Fgfr2CKO mice, suggesting that MG homeostasis is FGFR2 dependent. The Fgfr2CKO mice with inducible MG atrophy can serve as a valuable animal model for investigating the pathogenesis of MGD and developing novel therapeutic strategies for MGD-related DED.

Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation.

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.

Fibroblast growth factor receptor 2 (FGFR2) is required for corneal epithelial cell proliferation and differentiation during embryonic development.

Fibroblast growth factors (FGFs) play important roles in many aspects of embryonic development. During eye development, the lens and corneal epithelium are derived from the same surface ectodermal tissue. FGF receptor (FGFR)-signaling is essential for lens cell differentiation and survival, but its role in corneal development has not been fully investigated. In this study, we examined the corneal defects in Fgfr2 conditional knockout mice in which Cre expression is activated at lens induction stage by Pax6 P0 promoter. The cornea in LeCre, Fgfr2(loxP/loxP) mice (referred as Fgfr2(CKO)) was analyzed to assess changes in cell proliferation, differentiation and survival. We found that Fgfr2(CKO) cornea was much thinner in epithelial and stromal layer when compared to WT cornea. At embryonic day 12.5-13.5 (E12.5-13.5) shortly after the lens vesicle detaches from the overlying surface ectoderm, cell proliferation (judged by labeling indices of Ki-67, BrdU and phospho-histone H3) was significantly reduced in corneal epithelium in Fgfr2(CKO) mice. At later stage, cell differentiation markers for corneal epithelium and underlying stromal mesenchyme, keratin-12 and keratocan respectively, were not expressed in Fgfr2(CKO) cornea. Furthermore, Pax6, a transcription factor essential for eye development, was not present in the Fgfr2(CKO) mutant corneal epithelial at E16.5 but was expressed normally at E12.5, suggesting that FGFR2-signaling is required for maintaining Pax6 expression in this tissue. Interestingly, the role of FGFR2 in corneal epithelial development is independent of ERK1/2-signaling. In contrast to the lens, FGFR2 is not required for cell survival in cornea. This study demonstrates for the first time that FGFR2 plays an essential role in controlling cell proliferation and differentiation, and maintaining Pax6 levels in corneal epithelium via ERK-independent pathways during embryonic development.

Chick delta1-crystallin enhancer influences mouse alphaA-crystallin promoter activity in transgenic mice.

PURPOSE: Both the -366/+43 and the -282/+43 mouse alphaA-crystallin (or alphaA) promoters have been effective at driving transgene expression in lens fiber cells, but not in lens epithelium. Because the chick delta1-crystallin gene is expressed in lens epithelial cells, an enhancer was borrowed from this gene and linked to the alphaA promoter. This heterogenic enhancer/promoter construct was tested in transgenic mice to see whether it was active in both lens epithelium and fiber cells while retaining lens specificity. METHODS: The third intron of the chick delta1-crystallin gene, which contains a lens enhancer element, was added to the 5' end of the mouse alphaA promoter. We refer to this chimeric regulatory element as the deltaenalphaA promoter. To test its activity, we inserted coding sequences for five different genes. Transgenic mice were generated by pronuclear microinjection. Transgene expression patterns were analyzed by either X-gal staining, in situ hybridization or immunohistochemical staining. RESULTS: When deltaenalphaA-lacZ transgenic embryos were stained with X-gal at embryonic day (E)11.5, beta-galactosidase activity was detected only in the eye. Histologic sections of the stained embryos revealed that lacZ was expressed exclusively in the lens, in both epithelial and fiber cells. Transgenic mice were also generated using either the original alphaA- or the new deltaenalphaA promoter linked to an insulin cDNA. In situ hybridizations confirmed that the short alphaA promoter targeted prenatal insulin expression specifically to the lens fiber cells, whereas the deltaenalphaA promoter was active in both lens epithelial and fiber cells. Developmental studies of the deltaenalphaA-insulin mice showed that the deltaenalphaA promoter became active at the lens pit stage and remained active in all lens cells, even at postnatal ages. The deltaenalphaA promoter also successfully directed expression of SV40 T-antigen (TAg), human E2F2, and dominant negative Sprouty2 (dn-Spry2) genes to lens epithelial and fiber cells. The lens specificity of the deltaenalphaA promoter was maintained in minigenes with different types of introns and polyadenylation signals. CONCLUSIONS: A new lens-specific regulatory element was generated-the deltaenalphaA promoter, which can drive high levels of transgene expression in both lens epithelium and fiber cells throughout development. This modified promoter can be used for future transgenic studies of signal transduction and cell cycle regulation in lens epithelial cells.

Ectopic Pax6 expression disturbs lens fiber cell differentiation.

PURPOSE: Pax6 is a transcription factor necessary for the specification and subsequent formation of the ocular lens. It is expressed in all lens cells at early stages of development. After lens formation, Pax6 expression is maintained in the lens epithelium, whereas its level abruptly decreases in differentiated fiber cells. This study is to test the hypothesis that normal fiber cell differentiation would be perturbed by sustained Pax6 expression. METHODS: Transgenic mice expressing the canonical form of mouse Pax6 were created under the control of a modified mouse alphaA-crystallin promoter. The phenotypic changes in the transgenic lens were analyzed by light and electron microscopy. The effect of ectopic Pax6 expression on the lens fiber cells was investigated by in situ hybridization, immunohistochemical staining, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and two-dimensional (2-D) gel electrophoresis. RESULTS: Transgenic mice from seven different lines all had cataracts with severity that correlated with the transgene expression level in lens fiber cells. In severely affected lines, a lumen was present between the apical surfaces of the epithelial and fiber cells, suggesting that secondary fiber cell elongation is incomplete. Electron microscopy analysis showed that the ball-and-socket interdigitations between neighboring fiber cells were underdeveloped or attenuated in the transgenic lens. Most interesting, elevated levels of Pax6 in fiber cells reduced the protein levels of transcription factor cMaf, which is known to be essential in fiber cell differentiation. Furthermore, the total amount of lens proteins was 60% less than normal in the Pax6 transgenic lens. Among the crystallins examined, the relative ratio of intact betaB1-crystallin protein to total lens protein was significantly reduced. Real-time reverse transcriptase PCR showed that the ratio of betaB1-crystallin transcript levels to total mRNA levels were reduced by 87%. CONCLUSIONS: The data demonstrate that high levels of Pax6 expression disrupt normal fiber cell differentiation and maturation.

Histone deacetylase inhibitors trichostatin A and vorinostat inhibit TGFß2-induced lens epithelial-to-mesenchymal cell transition.

PURPOSE: Posterior capsule opacification (PCO) after cataract surgery is due in part to proliferation of the adhering lens epithelial cells and transdifferentiation into mesenchymal cells. The histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and vorinostat (suberoylanilidehydroxamic acid [SAHA]) are known to modulate cell proliferation and epithelial-mesenchymal transition (EMT). Studies have shown that TGFß2 can induce EMT similar to that seen during PCO. This study evaluated the effects of TSA and SAHA on TGFß2-induced EMT in lens epithelial explants. METHODS: Epithelial cells adherent to lens capsules were isolated from fresh pig lenses and human donor lenses and cultured for 12 hours. Explants were pretreated with TSA or SAHA for 1 hour and then treated with TGFß2 for up to 3 days. Scratch wound healing assay was used to determine epithelial cell proliferation and migration in the samples. The effects of TSA and SAHA on histone acetylation and HDAC 1 to 6 levels were analyzed by Western blotting. RESULTS: Western blotting and immunocytochemistry demonstrated high expression of α-SMA in lens epithelial cells treated with TGFß2. The HDAC inhibitors exerted dose-dependent inhibition of α-SMA expression, with complete inhibition occurring with 0.5 µM of TSA and 2.5 µM of SAHA. Transforming growth factor ß2-induced EMT was suppressed by TSA and SAHA. Histone deacetylase inhibition in pig lens epithelia led to increased acetylation of histone 3 and 4 at multiple sites. CONCLUSIONS: Histone deacetylase inhibitors, TSA, and SAHA prevent EMT in lens epithelial explants. The results also suggest that the epigenetic modifiers are the potential targets to control PCO after cataract surgery.

Activation of unfolded protein response in transgenic mouse lenses.

PURPOSE: Overloading of unfolded or misfolded proteins in the endoplasmic reticulum (ER) can cause ER stress and activate the unfolded protein response (UPR) in the cell. The authors tested whether transgene overexpression in the mouse lens would activate the UPR. METHODS: Transgenic mice expressing proteins that either enter the ER secretory pathway or are synthesized in cytosol were selected. Activation of the UPR was assessed by determining the expression levels of the ER chaperone protein BiP, the spliced form of X-box binding protein-1 (Xbp-1) mRNA, and the transcription factor CHOP. Changes in the ubiquitin-proteasome system in the mouse lens were detected by ubiquitin immunofluorescence. RESULTS: BiP expression was upregulated in the fiber cells of transgenic mouse lenses expressing platelet-derived growth factor-A (PDGF-A), dominant-negative fibroblast growth factor receptor (DN-FGFR), or DN-Sprouty2 (DN-Spy2). BiP upregulation occurred around embryonic day 16.5, primarily in the fiber cells adjacent to the organelle free zone. Fiber cell differentiation was disrupted in the PDGF-A and DN-Spry2 lenses, whereas the fiber cells were degenerating in the DN-FGFR lens. High levels of UPR activation and ubiquitin-labeled protein aggregates were found in the DN-FGFR lens, indicating inefficient disposal of unfolded/misfolded proteins in the fiber cells. CONCLUSIONS: This study implies that overexpression of some transgenes in the lens can induce ER or overall cell stress in fiber cells, resulting in the activation of UPR signaling pathways. Therefore, investigators should assess the levels of UPR activation when they analyze the downstream effects of transgene expression in the lens.

Induction of corneal myofibroblasts by lens-derived transforming growth factor beta1 (TGFbeta1): a transgenic mouse model.

PURPOSE: Transforming growth factor beta (TGFbeta) is an important cytokine in corneal development and wound healing. Transgenic mice that express an active form of human TGFbeta1 driven by a lens-specific promoter were used in the current study to determine the biological effects of lens-derived TGFbeta1 on postnatal corneal development and homeostasis. METHODS: The postnatal corneal changes in the TGFbeta1 transgenic mice were examined by fluorescein labeling and histology. Epithelial/endothelial-to-mesenchymal transition (E/EnMT) in the transgenic mouse cornea was demonstrated by immunostaining for alpha-smooth muscle actin (alpha-SMA) and cadherin-11. Expression of E- and N-cadherin in the corneal epithelial and endothelial cells, respectively, was analyzed by in situ hybridization. RESULTS: Among the established TGFbeta1 transgenic lines, mice from line OVE853 and OVE917 had normal-sized eyeballs but developed a corneal haze after eyelid opening. Histological examination showed that prenatal corneal development appeared to be normal. However, after postnatal day 7 (P7), the corneal endothelial cells in transgenic line OVE853 began to lose normal cell-cell contact and basement membrane structure. The endothelial layer was eventually absent in the inner surface of the transgenic mouse cornea. The morphological changes in the cornea correlated with abnormal expression of alpha-SMA, a molecular marker of EMT, and stress fiber formation in myofibroblast-like cells, which initially appeared in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. The E/EnMT in the transgenic mouse cornea was further demonstrated by loss of E- and N-cadherin expression in the corneal epithelial and endothelial cells, respectively, and meanwhile increasing expression of cadherin-11 in both corneal epithelium and stroma. CONCLUSIONS: Elevated levels of active TGFbeta1 in the anterior chamber can lead to myofibroblast formation in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. Our data suggest that the levels of biologically active TGFbeta in the aqueous humor must be under tight control to maintain corneal homeostasis. TGFbeta1 is the major cytokine during wound healing. Therefore, our findings also suggest a potential mechanism to explain the loss of corneal endothelial barrier and corneal opacification after intraocular surgery or trauma.

Ras signaling is essential for lens cell proliferation and lens growth during development.

The vertebrate ocular lens is a simple and continuously growing tissue. Growth factor-mediated receptor tyrosine kinases (RTKs) are believed to be required for lens cell proliferation, differentiation and survival. The signaling pathways downstream of the RTKs remain to be elucidated. Here, we demonstrate the important role of Ras in lens development by expressing a dominant-negative form of Ras (dn-Ras) in the lens of transgenic mice. We show that lens in the transgenic mice was smaller and lens growth was severely inhibited as compared to the wild-type lens. However, the lens shape, polarity and transparency appeared normal in the transgenic mice. Further analysis showed that cell proliferation is inhibited in the dn-Ras lens. For example, the percentage of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in epithelial layer was about 2- to 3-fold lower in the transgenic lens than in the wild-type lens, implying that Ras activity is required for normal cell proliferation during lens development. We also found a small number of apoptotic cells in both epithelial and fiber compartment of the transgenic lens, suggesting that Ras also plays a role in cell survival. Interestingly, although there was a delay in primary fiber cell differentiation, secondary fiber cell differentiation was not significantly affected in the transgenic mice. For example, the expression of beta- and gamma-crystallins, the marker proteins for fiber differentiation, was not changed in the transgenic mice. Biochemical analysis indicated that ERK activity, but not Akt activity, was significantly reduced in the dn-Ras transgenic lenses. Overall, our data imply that the RTK-Ras-ERK signaling pathway is essential for cell proliferation and, to a lesser extent, for cell survival, but not for crystallin gene expression during fiber differentiation. Thus, some of the fiber differentiation processes are likely mediated by RTK-dependent but Ras-independent pathways.