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Roles of TGF? and FGF signals during growth and differentiation of mouse lens epithelial cell in vitro.

ABSTRACT: Transforming growth factor ? (TGF?) and fibroblast growth factor (FGF) signaling pathways play important roles in the proliferation and differentiation of lens epithelial cells (LECs) during development. Low dosage bFGF promotes cell proliferation while high dosage induces differentiation. TGF? signaling regulates LEC proliferation and differentiation as well, but also promotes epithelial-mesenchymal transitions that lead to cataracts. Thus far, it has been difficult to recapitulate the features of germinative LECs in vitro. Here, we have established a LEC culture protocol that uses SB431542 (SB) compound to inhibit TGF?/Smad activation, and found that SB treatment promoted mouse LEC proliferation, maintained LECs' morphology and distinct markers including N-cadherin, c-Maf, Prox1, and ?A-, ?B-, and ?-crystallins. In contrast, low-dosage bFGF was unable to sustain those markers and, combined with SB, altered LECs' morphology and ?-crystallin expression. We further found that Matrigel substrate coatings greatly increased cell proliferation and uniquely affected ?-crystallin expression. Cultured LECs retained the ability to differentiate into ?-crystallin-positive lentoids by high-dosage bFGF treatment. Thus, a suppression of TGF?/Smad signaling in vitro is critical to maintaining characteristic features of mouse LECs, especially expression of the key transcription factors c-Maf and Prox1.

SUBMITTER: Wang D

PROVIDER: S-EPMC5544739 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Roles of TGFβ and FGF signals during growth and differentiation of mouse lens epithelial cell in vitro.

Wang Dong D Wang Eddie E Liu Kelsey K Xia Chun-Hong CH Li Song S Gong Xiaohua X

Scientific reports 20170804 1

Transforming growth factor β (TGFβ) and fibroblast growth factor (FGF) signaling pathways play important roles in the proliferation and differentiation of lens epithelial cells (LECs) during development. Low dosage bFGF promotes cell proliferation while high dosage induces differentiation. TGFβ signaling regulates LEC proliferation and differentiation as well, but also promotes epithelial-mesenchymal transitions that lead to cataracts. Thus far, it has been difficult to recapitulate the features ...[more]

PMID: 28779082

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Project description:PurposeInsulin 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.MethodsExpression 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.ResultsNormal 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.ConclusionsElevated 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.

Project description:PURPOSE: Epidemiological and experimental studies have revealed that exposure to ultraviolet B (UVB) light can induce cataractogenesis. The objective of this study was to determine gene expression changes in human lens epithelial cells in response to UVB exposure and identify factors that can be involved in UVB-induced cataractogenesis. METHODS: SV40 T-antigen-transformed human lens epithelial cells (SRA01/04) were irradiated at various UVB-energy levels (10-80 mJ/cm²) and checked for viability. An irradiation condition of 30 mJ/cm² was adopted for transcriptome analysis. Total RNAs isolated from UVB-exposed and unexposed cells at 12 h and 24 h after UVB exposure were examined for global gene expression changes using Affymetrix Human Gene 1.0 ST array. mRNA levels of specific genes were examined by RT-PCR and real-time PCR, and protein levels in the conditioned media were assayed by ELISA. To examine mRNA expression in human lens, primary cultured human lens epithelial (HLE) cells were prepared from surgically removed lens epithelium, and used for UVB-irradiation and expression analysis. Effects of certain gene products on SRA01/04 cell metabolism were examined using commercially available recombinant proteins. RESULTS: Expression of most the genes analyzed was essentially unchanged (between 0.5 and 2.0 fold) in UVB-irradiated cells compared to non-irradiated cells at both 12 and 24 h after UVB exposure. Sixty one and 44 genes were upregulated more than twofold by UVB exposure at 12 h and 24 h, respectively. Emphasis was placed on genes encoding extracellular proteins, especially growth factors and cytokines. A total of 18 secreted protein genes were upregulated more than twofold at either or both time points. Amphiregulin (AREG) and growth differentiation factor 15 (GDF15) were chosen because of their higher upregulation and novelty, and their upregulation was confirmed in SRA01/04 cells using RT-PCR and real-time PCR analysis. AREG and GDF15 protein levels in conditioned media significantly increased at all UVB-energy points at 24 h, while they were scarcely detectable at 12 h. AREG and GDF15 mRNA levels were also significantly upregulated in UVB-irradiated primary cultured HLE cells compared with the corresponding control culture. AREG significantly stimulated ³H-thymidine and ³H-leucine uptake in SRA01/04 cells as did a positive control epidermal growth factor (EGF). Recombinant GDF15 did not stimulate ³H-thymidine incorporation at any concentration tested, but significantly stimulated ³H-leucine uptake. RT-PCR analysis demonstrated that primary cultured HLE and SRA01/04 cells expressed not only epidermal growth factor receptor (EGFR) mRNA but also transforming growth factor ? receptors (TGFBR1 and TGFBR2) mRNAs. CONCLUSIONS: These results indicate that AREG and GDF15 produced in response to UVB exposure can affect the growth and protein synthesis of lens epithelial cells, suggesting that they have autocrine and paracrine roles related to pathological changes of lens tissue during long-term UVB exposure.

Project description:Purpose:Ocular lens fiber cell elongation, differentiation, and compaction are associated with extensive reorganization of cell adhesive interactions and cytoskeleton; however, our knowledge of proteins critical to these events is still evolving. This study characterizes the distribution pattern of neuronal-specific ?-catenin (?N-catenin) and its interaction with the N-cadherin-associated adherens junctions (AJs) and their stability in the mouse lens fibers. Methods:Expression and distribution of ?N-catenin in developing mouse and adult human lenses was determined by RT-PCR, immunoblot, and immunofluorescence analyses. Characterization of ?N-catenin and N-cadherin interacting proteins and colocalization analyses were performed using immunoprecipitation, mass spectrometry, and confocal imaging. Effects of periaxin deficiency on the stability of lens fiber cell AJs were evaluated using perixin-null mice. Results:?N-catenin exhibits discrete distribution to lens fibers in both mouse and human lenses, undergoing a robust up-regulation during fiber cell differentiation and maturation. Epithelial-specific ?-catenin (?E-catenin), in contrast, distributes primarily to the lens epithelium. ?N-catenin and N-cadherin reciprocally coimmunoprecipitate and colocalize along with ?-catenin, actin, spectrin, vinculin, Armadillo repeat protein deleted in velo-cardio-facial syndrome homolog, periaxin, and ankyrin-B in lens fibers. Fiber cells from periaxin-null mouse lenses revealed disrupted N-cadherin/?N-catenin-based AJs. Conclusions:These results suggest that the discrete shift in ?-catenin expression from ?E-catenin to ?N-catenin subtype that occurs during lens epithelial cell differentiation may play a key role in fiber cell cytoarchitecture by regulating the assembly and stability of N-cadherin-based AJs. This study also provides evidence for the importance of the fiber cell-specific cytoskeletal interacting periaxin, in the stability of N-cadherin/?N-catenin-based AJs in lens fibers.

Dataset Information

Roles of TGF? and FGF signals during growth and differentiation of mouse lens epithelial cell in vitro.

Publications

Roles of TGFβ and FGF signals during growth and differentiation of mouse lens epithelial cell in vitro.

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