Dataset Information

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

ABSTRACT:

Purpose

The Fat family of atypical cadherins, originally identified in Drosophila, play diverse roles during embryogenesis and adult tissue maintenance. Among four mammalian members, Fat1 is essential for kidney and muscle organization, and is also essential for eye development; Fat1 knockout causes partial penetrant microphthalmia or anophthalmia. To account for the partial penetrance of the Fat1 phenotype, involvement of Fat4 in eye development was assessed. Lens phenotypes in Fat1 and 4 knockouts were also examined.

Methods

Fat1 and Fat4 mRNA expression was examined by in situ hybridization. Knockout phenotypes of Fat1 and Fat4 were analyzed by hematoxylin and eosin (H&E) and immunofluorescent staining.

Results

We found Fat4 knockout did not affect eye induction or enhance severity of Fat1 eye defects. Although Fat1 and Fat4 mRNAs are similarly expressed in the lens epithelial cells, only Fat1 knockout caused a fully penetrant lens epithelial cell defect, which was apparent at embryonic day 14.5 (E14.5). The columnar structure of the lens epithelial cells was disrupted and in some regions cell aggregates were formed. In these multilayered regions, apical cell junctions were fragmented and the apical-basal polarity was lost. EdU incorporation assay also showed enhanced proliferation in the lens epithelial cells. Interestingly, these defects were found mainly in the central zone of the epithelial layer. The lens epithelial cells of the germinative zone maintained their normal morphology and fiber differentiation occurred normally at the equator.

Conclusions

These observations indicate that Fat1 is essential for lens epithelial cell polarity and proliferation but not for terminal differentiation.

SUBMITTER: Sugiyama Y

PROVIDER: S-EPMC4484397 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Publications

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

Sugiyama Yuki Y Shelley Elizabeth J EJ Badouel Caroline C McNeill Helen H McAvoy John W JW

Investigative ophthalmology & visual science 20150601 6

<h4>Purpose</h4>The Fat family of atypical cadherins, originally identified in Drosophila, play diverse roles during embryogenesis and adult tissue maintenance. Among four mammalian members, Fat1 is essential for kidney and muscle organization, and is also essential for eye development; Fat1 knockout causes partial penetrant microphthalmia or anophthalmia. To account for the partial penetrance of the Fat1 phenotype, involvement of Fat4 in eye development was assessed. Lens phenotypes in Fat1 and ...[more]

PMID: 26114487

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Project description:BACKGROUND:Overexpression of FAT1 gene and its oncogenic effects have been reported in several cancers. Previously, we have documented upregulation of FAT1 gene in glioblastoma (GBM) tumors which was found to increase the expression of proinflammatory markers, HIF-1α, stemness genes and EMT markers in glioma cells. Here, we reveal NFкB (RelA)/RelA/p65 as the transcriptional regulator of FAT1 gene in GBM cells. METHODS:In-silico analysis of FAT1 gene promoter was performed using online bioinformatics tool Promo alggen (Transfac 8.3) to identify putative transcription factor(s) binding motifs. A 4.0 kb FAT1 promoter (- 3220 bp to + 848 bp w.r.t. TSS + 1) was cloned into promoter less pGL3Basic reporter vector. Characterization of FAT1 promoter for transcriptional regulation was performed by in-vitro functional assays using promoter deletion constructs, site directed mutagenesis and ChIP in GBM cells. RESULTS:Expression levels of NFкB (RelA) and FAT1 were found to be increased and positively correlated in GBM tumors (n = 16), REMBRANDT GBM-database (n = 214) and TCGA GBM-database (n = 153). In addition to glioma, positive correlation between NFкB (RelA) and FAT1 expression was also observed in other tumors like pancreatic, hepatocellular, lung and stomach cancers (data extracted from TCGA tumor data). A 4.0 kb FAT1-promoter-construct [- 3220 bp/+ 848 bp, transcription start site (TSS) + 1, having 17 NFкB (RelA) motifs] showed high FAT1 promoter luciferase-activity in GBM cells (U87MG/A172/U373MG). FAT1 promoter deletion-construct pGL3F1 [- 200 bp/+ 848 bp, with 3-NFкB (RelA)-motifs] showed the highest promoter activity. Exposure of GBM cells to known NFкB (RelA)-activators [severe-hypoxia/TNF-α/ectopic-NFкB (RelA) + IKBK vectors] led to increased pGL3F1-promoter activity and increased endogenous-FAT1 expression. Conversely, siRNA-mediated NFкB (RelA) knockdown led to decreased pGL3F1-promoter activity and decreased endogenous-FAT1 expression. Deletion of NFкB (RelA)-motif at - 90 bp/- 80 bp [pGL3F1δ1-construct] showed significant decrease in promoter activity. Site directed mutagenesis at -90 bp/- 80 bp and ChIP assay for endogenous-NFкB (RelA) confirmed the importance of this motif in FAT1 expression regulation. Significant reduction in the migration, invasion as well as colony forming capacity of the U87MG glioma cells was observed on siRNA-mediated knockdown of NFкB (RelA). CONCLUSION:Since FAT1 and NFкB (RelA) are independently known to promote pro-tumorigenic inflammation and upregulate the expression of HIF-1α/EMT/stemness in tumors, targeting the NFкB (RelA)-FAT1 axis may attenuate an important tumor-promoting pathway in GBM. This may also be applicable to other tumors.

Project description:Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair, as well as homeostatic mechanisms that counteract ageing and neurodegeneration, cardiovascular disease, and cancer. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular 'brake' on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1KO) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1KO cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease, this Fat1 function may have importance in other settings of altered cell growth and metabolism.

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:Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2?) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs.To examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific ?A-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5) wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase II?, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation, for expression of DNase II?, for lens fiber cell denucleation and indirectly for retinal development.These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase II? as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens.

Dataset Information

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

Purpose

Methods

Results

Conclusions

Publications

Atypical Cadherin Fat1 Is Required for Lens Epithelial Cell Polarity and Proliferation but Not for Fiber Differentiation.

Similar Datasets

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