PURPOSE: 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.
PURPOSE: 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.
Authors: Shivalingappa K Swamynathan; Jonathan P Katz; Klaus H Kaestner; Ruth Ashery-Padan; Mary A Crawford; Joram Piatigorsky Journal: Mol Cell Biol Date: 2006-10-23 Impact factor: 4.272
Authors: C Yan Cheng; Pearl Py Lie; Ka-Wai Mok; Yan-Ho Cheng; Elissa Wp Wong; Jayakanthan Mannu; Premendu P Mathur; Helen H N Yan; Dolores D Mruk Journal: Spermatogenesis Date: 2011-07-01
Authors: Divya Gupta; Stephen A K Harvey; Doreswamy Kenchegowda; Sudha Swamynathan; Shivalingappa K Swamynathan Journal: Exp Eye Res Date: 2013-09-25 Impact factor: 3.467