Regulation of corneal epithelial barrier function by Kruppel-like transcription factor 4.
OBJECTIVE
Previously, the authors showed that Klf4-conditional null (Klf4CN) corneas display epithelial fragility. Here, they investigated the mechanism by which Klf4 regulates corneal epithelial barrier function.


METHODS
Klf4CN mice were generated by breeding Le-Cre with Klf4-LoxP mice. Fluorescein staining was used to test the corneal barrier function. RT-PCR, immunoblots, and immunofluorescence were used to detect the expression of cell junctional proteins. The effect of Klf4 on promoter activities was measured by transient cotransfection assays. Trans-epithelial electrical resistance (TEER) was used to measure the barrier-forming ability of control or anti-KLF4 siRNA-treated cells.


RESULTS
Increased fluorescein staining and decreased tight junction protein Tjp1 expression demonstrated that the Klf4CN corneal epithelial barrier function is defective. Expression of desmosomal components Dsp, Dsg-1a, and Dsg-1b was downregulated in the Klf4CN corneas, and their corresponding promoter activities were upregulated by Klf4 in transient cotransfection assays. Hemidesmosomal α3- and β4-integrin levels were not affected even though there were fewer hemidesmosomes in the Klf4CN corneas. The basement membrane components laminin-α5, -α3, -β3, and -β1-1 were downregulated, suggesting that the disrupted basement membrane is responsible for fewer hemidesmosomes in the Klf4CN cornea. Tight junction proteins OCLN1 and TJP1were downregulated in anti-KLF4 siRNA-treated cells, which failed to develop epithelial barrier function as measured by TEER.


CONCLUSIONS
Klf4 contributes to corneal epithelial barrier function by upregulating the expression of functionally related subsets of cell junctional proteins and basement membrane components.
Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Sudha
S
Swamynathan
Doreswamy
D
Kenchegowda
Joram
J
Piatigorsky
Shivalingappa
S
Swamynathan
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010539:Permeability; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation / Q000502:physiology; D016335:Zinc Fingers / Q000502:physiology
C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D019793:Fluorescein