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Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells.

ABSTRACT: The Krüppel-like transcription factor KLF4 is among the most highly expressed transcription factors in the mouse cornea (B. Norman, J. Davis, and J. Piatigorsky, Investig. Ophthalmol. Vis. Sci. 45:429-440, 2004). Here, we deleted the Klf4 gene selectively in the surface ectoderm-derived structures of the eye (cornea, conjunctiva, eyelids, and lens) by mating Klf4-LoxP mice (J. P. Katz, N. Perreault, B. G. Goldstein, C. S. Lee, P. A. Labosky, V. W. Yang, and K. H. Kaestner, Development 129:2619-2628, 2002) with Le-Cre mice (R. Ashery-Padan, T. Marquardt, X. Zhou, and P. Gruss, Genes Dev. 14:2701-2711, 2000). Klf4 conditional null (Klf4CN) embryos developed normally, and the adult mice were viable and fertile. Unlike the wild type, the Klf4CN cornea consisted of three to four epithelial cell layers; swollen, vacuolated basal epithelial and endothelial cells; and edematous stroma. The conjunctiva lacked goblet cells, and the anterior cortical lens was vacuolated in Klf4CN mice. Excessive cell sloughing resulted in fewer epithelial cell layers in spite of increased cell proliferation at the Klf4CN ocular surface. Expression of the keratin-12 and aquaporin-5 genes was downregulated, consistent with the Klf4CN corneal epithelial fragility and stromal edema, respectively. These observations provide new insights into the role of KLF4 in postnatal maturation and maintenance of the ocular surface and suggest that the Klf4CN mouse is a useful model for investigating ocular surface pathologies such as dry eye, Meesmann's dystrophy, and Steven's-Johnson syndrome.

SUBMITTER: Swamynathan SK

PROVIDER: S-EPMC1800665 | biostudies-literature | 2007 Jan

REPOSITORIES: biostudies-literature

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Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells.

Swamynathan Shivalingappa K SK Katz Jonathan P JP Kaestner Klaus H KH Ashery-Padan Ruth R Crawford Mary A MA Piatigorsky Joram J

Molecular and cellular biology 20061023 1

The Krüppel-like transcription factor KLF4 is among the most highly expressed transcription factors in the mouse cornea (B. Norman, J. Davis, and J. Piatigorsky, Investig. Ophthalmol. Vis. Sci. 45:429-440, 2004). Here, we deleted the Klf4 gene selectively in the surface ectoderm-derived structures of the eye (cornea, conjunctiva, eyelids, and lens) by mating Klf4-LoxP mice (J. P. Katz, N. Perreault, B. G. Goldstein, C. S. Lee, P. A. Labosky, V. W. Yang, and K. H. Kaestner, Development 129:2619-2 ...[more]

PMID: 17060454

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Project description:To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression.cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest.The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb.Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that are potentially active in the function of corneal and conjunctival epithelia.

Project description:Introduction:Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-? signalling pathway, which is involved in corneal epithelial wound healing. Methods:HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-? secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-?2 treatment. Results:KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-? canonical signalling pathway, TGF-?2 secretion by HCECs increased up to 5 folds, and several TGF-?-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-? signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-?2 treatment. Conclusions:These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-?2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-? signalling.

Dataset Information

Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells.

Publications

Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells.

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