Dataset Information

Calpain expression and activity during lens fiber cell differentiation.

ABSTRACT: In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was alphaII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.

SUBMITTER: De Maria A

PROVIDER: S-EPMC2679455 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Calpain expression and activity during lens fiber cell differentiation.

De Maria Alicia A Shi Yanrong Y Kumar Nalin M NM Bassnett Steven S

The Journal of biological chemistry 20090306 20

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained ...[more]

PMID: 19269960

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Project description:To identify transcripts expressed late in lens fiber cell maturation that might regulate fiber cell fusion, organelle degradation, or other events associated with the maturation of lens fiber cells.cDNA libraries were prepared from microdissected regions of chicken embryo lenses using a PCR-based method. Subtractive hybridization was used to identify transcripts expressed exclusively in fiber cells that had detached from the lens capsule. Database searches and PCR amplification with degenerate primers were used to identify human, mouse, rat, rabbit, and bovine orthologs of one such sequence and to confirm its expression in the lenses of these animals. The ability of in vitro-transcribed and translated protein to bind DNA was assessed by mobility shift assays. The locus encoding this transcript and an area about 6 kb upstream of the translation start site were sequenced. The microscopic morphology of lenses from mice in which the locus encoding this protein had been disrupted by the insertion of a nuclear-targeted bacterial lacZ sequence were analyzed. Gene expression was analyzed by PCR, in situ hybridization, and by staining for beta-galactosidase activity in lenses expressing lacZ in place of the coding sequence. Knockout lenses expressing green fluorescent protein in a mosaic pattern were sectioned in the equatorial plane and viewed with a confocal microscope to assess the presence of cell-cell fusions during fiber cell maturation.Subtractive hybridization identified transcripts encoding Hop, a short, atypical homeodomain-containing protein that had previously been shown to be an important regulator of gene expression in the heart and lung. Chicken Hop did not bind to known homeodomain-binding sequences in DNA. In chicken embryos, Hop transcripts were first detected at E6. At all stages analyzed, Hop mRNA was only detected in cells that had detached from the lens capsule. Mice in which the Hop coding sequence was replaced with nuclear-targeted beta-galactosidase showed that Hop was expressed in the mouse lens in a similar pattern to the chicken lens. Characterization of lenses from mice lacking Hop revealed no morphological phenotype and no apparent defects in the degradation of nuclei or fiber cell fusion during fiber cell maturation.The expression pattern of Hop provides the first evidence that new transcription is initiated in lens fiber cells after they detach from the capsule. Hop may be the first of a class of genes with this pattern of expression. Although lens abnormalities have yet to be identified in mice lacking Hop, the genomic sequences that regulate Hop expression in the lens may be useful for expressing exogenous transcripts selectively in fiber cells just before they fuse with their neighbors and degrade their organelles.

Project description:Background: FGF signaling controls numerous processes during cell lineage specification, organogenesis and terminal differentiation. In lens, FGF signaling was implicated as the key pathway that controls lens fiber cell differentiation, but little is known about its full range and spectrum of regulated genes. Results: Herein, we employed rat lens epithelial explant system and performed RNA and microRNA expression profiling in cells induced to differentiate by FGF2. The primary data were collected at explants grown overnight in the presence of 5 ng/ml of FGF2, followed by a treatment with 100 ng/ml of FGF2 and collection of samples at 2, 4, 12 and 24 hours. Global analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on microRNAs (miRNAs). We identified a total number of 131 FGF2-regulated miRNAs. Forty-four of these microRNAs had at least two predicted and inversely regulated target RNA molecules. The genes regulated by the highest number of miRs include Nfib, Nfat5, c-Maf, Ets1 and N-Myc, all encoding DNA-binding transcription factors. Analysis of RNA data revealed that activated FGF signaling influenced other major signaling pathways known to regulate lens differentiation including BMP/TGF-M-NM-2, Notch, and Wnt signaling. In the M-bM-^@M-^\earlyM-bM-^@M-^] response phase (2-4 hours), miRNAs targeted expression of batteries of genes that control transcription, cell death, cell proliferation, cell junction, and protein serine/threonine kinase activity. In M-bM-^@M-^\lateM-bM-^@M-^] stages (12-24 hours), the main miRNA targets included regulators of cell cycle arrest and cellular differentiation. Specific miRNA:mRNA interaction networks were identified for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Dicer1, Fbx33 and Wdr47 (RNA processing); Ash1l, Med1/PBP and Kdm5b (chromatin remodeling); and c-Maf, Ets1 and Stc1 (FGF signaling). MicroRNAs including miR-9, -143, -155, -455 and -543 downregulated expression of c-Maf in the 3M-bM-^@M-^Y-UTR luciferase reporter asssays. The functional requirement for miRNAs in lens was further demonstrated via disrupted lens fiber cell differentiation in lenses with inactivated Dicer1. Conclusions: These studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and identified novel gene regulatory networks (GRNs) connected by multiple miRNAs. Differential gene expression of FGF2-treated lens cell cultures was analyzed at 0, 2, 4, 12 and 24 hours following the treatment. Two biological replicate experiments were performed.

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Calpain expression and activity during lens fiber cell differentiation.

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Calpain expression and activity during lens fiber cell differentiation.

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