Downregulation of β-actin and its regulatory gene HuR affect cell migration of human corneal fibroblasts.
OBJECTIVE
In an earlier study, we showed that human antigen R (HuR) and β-actin expression levels were downregulated in fibroblasts isolated from human keratoconus stroma compared to normal corneal stroma. To further extend the finding, we determined whether HuR expression affects β-actin gene expression and in turn affects corneal fibroblast migration and wound healing.


METHODS
Stromal keratocytes from normal human corneas were cultured in the presence of serum. Cells were transfected with siRNA specific for β-actin or HuR. SiRNAs specific for GAPDH or a scrambled sequence were used as positive and negative controls (siCTR) for transfection, respectively. The effects of gene silencing were analyzed at the transcriptional and translational levels. Specific proteins were immunohistochemically localized using confocal imaging. The effects of gene silencing on cell migration and cell proliferation were analyzed using a modified Boyden chamber and with a wound healing assay, respectively.


RESULTS
Reverse-transcription PCR (RT-PCR) and western blot analyses showed that when the HuR gene was silenced, β-actin expression was significantly downregulated. This was further confirmed at the translational level with immunohistochemical-confocal analysis. However, when the β-actin gene was silenced, its expression was significantly decreased but showed no effect on HuR gene expression. When the β-actin or HuR gene was individually silenced, the motility and proliferation of corneal fibroblasts were significantly reduced.


CONCLUSIONS
The results show that downregulation of the HuR gene results in decreased β-actin gene expression, which in turn results in decreased motility and proliferation of corneal fibroblasts. We conclude that decreased β-actin expression in normal corneal stroma clearly disrupts the cytoskeletal structure and functions, including keratocyte motility and wound healing.
Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL.
R
R
Joseph
Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL.
O P
OP
Srivastava
Eye Research Laboratory, Eye Research Foundation, Birmingham, AL.
R R
RR
Pfister
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics*; D003315:Cornea / Q000166:cytology*; D003319:Corneal Stroma / Q000166:cytology; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D051959:ELAV Proteins / Q000235:genetics* / Q000378:metabolism; D000067780:ELAV-Like Protein 1; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D014176:Protein Biosynthesis / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D014158:Transcription, Genetic / Q000187:drug effects; D014945:Wound Healing / Q000187:drug effects / Q000235:genetics
D000199:Actins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; C098770:ELAVL1 protein, human