Limits...
Downregulation of β-actin and its regulatory gene HuR affect cell migration of human corneal fibroblasts.

Joseph R, Srivastava OP, Pfister RR - Mol. Vis. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL.

ABSTRACT

Purpose: 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.

Show MeSH

Related in: MedlinePlus

Localization of human antigen R after gene silencing. The normal corneal fibroblasts were treated with GAPDH siRNA, β-actin siRNA, or human antigen R (HuR) siRNA. The immunoreactivity of HuR was analyzed at 72 h post-transfection. A: Fibroblasts stained with Hoechst nuclear stain after a treatment with GAPDH siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with GAPDH siRNA. C: Overlay of (A) and (B). D: Fibroblasts stained with Hoechst nuclear stain after a treatment with β-actin siRNA. E: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with β-actin siRNA. F: Overlay of (D) and (E). G: Fibroblasts stained with Hoechst nuclear stain after treatment with HuR siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after treatment with HuR siRNA. C: Overlay of (G) and (H). Note that the GAPDH siRNA and β-actin siRNA treatments had no effect on HuR expression and its nuclear localization. HuR gene silencing significantly reduced the expression of HuR, as shown by the absence of green fluorescence in nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4016806&req=5

f2: Localization of human antigen R after gene silencing. The normal corneal fibroblasts were treated with GAPDH siRNA, β-actin siRNA, or human antigen R (HuR) siRNA. The immunoreactivity of HuR was analyzed at 72 h post-transfection. A: Fibroblasts stained with Hoechst nuclear stain after a treatment with GAPDH siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with GAPDH siRNA. C: Overlay of (A) and (B). D: Fibroblasts stained with Hoechst nuclear stain after a treatment with β-actin siRNA. E: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with β-actin siRNA. F: Overlay of (D) and (E). G: Fibroblasts stained with Hoechst nuclear stain after treatment with HuR siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after treatment with HuR siRNA. C: Overlay of (G) and (H). Note that the GAPDH siRNA and β-actin siRNA treatments had no effect on HuR expression and its nuclear localization. HuR gene silencing significantly reduced the expression of HuR, as shown by the absence of green fluorescence in nuclei.

Mentions: To further verify the western blot results, we used confocal imaging to localize the individual proteins after siRNA treatment. We analyzed whether β-actin gene silencing affected HuR expression in corneal fibroblasts (Figure 2). Figure 2 shows the immunoreactivity to the anti-HuR antibody of the corneal fibroblasts transfected with GAPDH siRNA, HuR siRNA, or β-actin siRNA. The effect on HuR gene expression was analyzed after the GAPDH gene was silenced. Figure 2A,D,G show Hoechst nuclear staining (blue fluorescent staining), and Figure 2B,E,H shows the immunoreactivity to the anti-HuR antibody. Green fluorescent staining in the nuclear region (punctate in appearance) was observed on immunoreactivity with the anti-HuR antibody in the cells treated with siGAPDH (Figure 2B), and with β-actin siRNA (Figure 2E). Figure 2C,F,I is an overlay of Figure 2A,B,D,E,G,H. GAPDH gene silencing had no effect on HuR gene expression. β-actin gene silencing had no effect on the expression of HuR as represented by the presence of green fluorescent staining in the nuclear region (Figure 2E). The effect of β-actin gene silencing on HuR expression was in concordance with our previously described RT–PCR and western blot results. In contrast, HuR gene silencing resulted in the loss of HuR expression, which representing a loss of green punctate fluorescent staining (Figure 2H). Together, the results show that HuR expression was downregulated in corneal fibroblasts after transfection with HuR siRNA (Figure 2H), whereas β-actin and GAPDH gene silencing had no effect on HuR expression (Figure 2B,E).


Downregulation of β-actin and its regulatory gene HuR affect cell migration of human corneal fibroblasts.

Joseph R, Srivastava OP, Pfister RR - Mol. Vis. (2014)

Localization of human antigen R after gene silencing. The normal corneal fibroblasts were treated with GAPDH siRNA, β-actin siRNA, or human antigen R (HuR) siRNA. The immunoreactivity of HuR was analyzed at 72 h post-transfection. A: Fibroblasts stained with Hoechst nuclear stain after a treatment with GAPDH siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with GAPDH siRNA. C: Overlay of (A) and (B). D: Fibroblasts stained with Hoechst nuclear stain after a treatment with β-actin siRNA. E: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with β-actin siRNA. F: Overlay of (D) and (E). G: Fibroblasts stained with Hoechst nuclear stain after treatment with HuR siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after treatment with HuR siRNA. C: Overlay of (G) and (H). Note that the GAPDH siRNA and β-actin siRNA treatments had no effect on HuR expression and its nuclear localization. HuR gene silencing significantly reduced the expression of HuR, as shown by the absence of green fluorescence in nuclei.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016806&req=5

f2: Localization of human antigen R after gene silencing. The normal corneal fibroblasts were treated with GAPDH siRNA, β-actin siRNA, or human antigen R (HuR) siRNA. The immunoreactivity of HuR was analyzed at 72 h post-transfection. A: Fibroblasts stained with Hoechst nuclear stain after a treatment with GAPDH siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with GAPDH siRNA. C: Overlay of (A) and (B). D: Fibroblasts stained with Hoechst nuclear stain after a treatment with β-actin siRNA. E: Immunoreactivity of fibroblasts with anti-HuR antibody after a treatment with β-actin siRNA. F: Overlay of (D) and (E). G: Fibroblasts stained with Hoechst nuclear stain after treatment with HuR siRNA. B: Immunoreactivity of fibroblasts with anti-HuR antibody after treatment with HuR siRNA. C: Overlay of (G) and (H). Note that the GAPDH siRNA and β-actin siRNA treatments had no effect on HuR expression and its nuclear localization. HuR gene silencing significantly reduced the expression of HuR, as shown by the absence of green fluorescence in nuclei.
Mentions: To further verify the western blot results, we used confocal imaging to localize the individual proteins after siRNA treatment. We analyzed whether β-actin gene silencing affected HuR expression in corneal fibroblasts (Figure 2). Figure 2 shows the immunoreactivity to the anti-HuR antibody of the corneal fibroblasts transfected with GAPDH siRNA, HuR siRNA, or β-actin siRNA. The effect on HuR gene expression was analyzed after the GAPDH gene was silenced. Figure 2A,D,G show Hoechst nuclear staining (blue fluorescent staining), and Figure 2B,E,H shows the immunoreactivity to the anti-HuR antibody. Green fluorescent staining in the nuclear region (punctate in appearance) was observed on immunoreactivity with the anti-HuR antibody in the cells treated with siGAPDH (Figure 2B), and with β-actin siRNA (Figure 2E). Figure 2C,F,I is an overlay of Figure 2A,B,D,E,G,H. GAPDH gene silencing had no effect on HuR gene expression. β-actin gene silencing had no effect on the expression of HuR as represented by the presence of green fluorescent staining in the nuclear region (Figure 2E). The effect of β-actin gene silencing on HuR expression was in concordance with our previously described RT–PCR and western blot results. In contrast, HuR gene silencing resulted in the loss of HuR expression, which representing a loss of green punctate fluorescent staining (Figure 2H). Together, the results show that HuR expression was downregulated in corneal fibroblasts after transfection with HuR siRNA (Figure 2H), whereas β-actin and GAPDH gene silencing had no effect on HuR expression (Figure 2B,E).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL.

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus