Critical Role of Klf5 in Regulating Gene Expression during Post-Eyelid Opening Maturation of Mouse Corneas

Abstract

BackgroundKlf5 plays an important role in maturation and maintenance of the mouse ocular surface. Here, we quantify WT and Klf5-conditional null (Klf5CN) corneal gene expression, identify Klf5-target genes and compare them with the previously identified Klf4-target genes to understand the molecular basis for non-redundant functions of Klf4 and Klf5 in the cornea.Methodology/Principal FindingsPostnatal day-11 (PN11) and PN56 WT and Klf5CN corneal transcriptomes were quantified by microarrays to compare gene expression in maturing WT corneas, identify Klf5-target genes, and compare corneal Klf4- and Klf5-target genes. Whole-mount corneal immunofluorescent staining was employed to examine CD45+ cell influx and neovascularization. Effect of Klf5 on expression of desmosomal components was studied by immunofluorescent staining and transient co-transfection assays. Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases and 72 concordant decreases. PN56 Klf5CN corneas shared 241 increases and 98 decreases with those previously described in Klf4CN corneas. Xenobiotic metabolism related pathways were enriched among genes decreased in Klf5CN corneas. Expression of angiogenesis and immune response-related genes was elevated, consistent with neovascularization and CD45+ cell influx in Klf5CN corneas. Expression of 1574 genes was increased and 1915 genes decreased in WT PN56 compared with PN11 corneas. Expression of ECM-associated genes decreased, while that of solute carrier family members increased in WT PN56 compared with PN11 corneas. Dsg1a, Dsg1b and Dsp were down-regulated in Klf5CN corneas and their corresponding promoter activities were stimulated by Klf5 in transient co-transfection assays.Conclusions/SignificanceDifferences between PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in functions of Klf5 during corneal maturation. Klf5 contributes to corneal epithelial homeostasis by regulating the expression of desmosomal components. Klf4- and Klf5-target genes are largely distinct, consistent with their non-redundant roles in the mouse cornea.

Ocular Surface Development and Gene Expression

“…By comparing the WT corneal transcriptomes at PN11 and PN56, we identified 1574 and 1915 genes whose expression decreased and increased, respectively, by more than 2-fold between PN11 and PN56 [32]. Apart from validating the previously identified changes in gene expression during corneal maturation [30, 31], this study identified additional genes whose expression was modulated in the cornea following eyelid opening.…”

The Ets Transcription Factor EHF as a Regulator of Cornea Epithelial Cell Identity

“…The inclusion of expression data from separated epithelium, separated stroma, and other epithelial tissues further refined the data. Validating this approach, we observed that known target genes of the transcription factors KLF4 and KLF5 (58,59,72) were located within the same supercluster as Klf4 and Klf5. Our clustering approach allowed us to gain insights into the previously published gene expression data for mice deleted for Klf4 and Klf5 in the cornea.…”

“…5). For example, cluster A contains the well characterized epithelial differentiation genes Krt12, Aldh3a1, and Upk1b (55-57) and known transcriptional regulators for epithelial differentiation, including Klf4 and Klf5 (58,59). In fact, the KLF5 target genes Dsp and Dsg1b (59) are both found in this cluster.…”

150th Anniversary Series: Desmosomes and the Hallmarks of Cancer

“…Recent years identified a number of transcriptional factors that modulate 4 O. HUBER & I. PETERSEN desmosomal cadherin expression including Stat6, Klf5, Smad4, grainy head-like 1, Foxn1, Cdx1/Cdx2, c-Rel, lymphocyte enhancer factor/transcription factor (LEF/ TCF), and p53/p63 (Ferone et al, 2013;Funakoshi et al, 2008;Johnson et al, 2014;Kenchegowda et al, 2012;Mlacki et al, 2014;Omori-Miyake et al, 2014;Oshiro et al, 2003;Owens et al, 2008;Tokonzaba et al, 2013;Wilanowski et al, 2008). Interestingly, plakoglobin in a complex with LEF-1 is able to bind to the proximal promoter regions of both Dsc2 and Dsc3, however with opposite effects.…”