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HSV-1 infection suppresses TGF-beta1 and SMAD3 expression in human corneal epithelial cells.

Nie Y, Cui D, Pan Z, Deng J, Huang Q, Wu K - Mol. Vis. (2008)

Bottom Line: Protein expression of TGF-beta1, TGF-beta2, SMAD3, and phospho-SMAD3 were analyzed by indirect immunofluorescence at 0 h, 12 h, and 24 h post-infection (p.i.) in HCE cells.Compared with normal cells, the mRNA levels of TGF-beta1 in HSV-1 infected HCE cells decreased significantly at 8 h, 12 h, and 24 h p.i. (p<0.01), and the expression of SMAD3 was also dramatically decreased 12 h and 24 h p.i. (p<0.01).Protein expression of TGF-beta1, SMAD3, and phospho-SMAD3 decreased in infected cells at 12 h and 24 h p.i. compared with normal cells, but TGF-beta2 had no change.

View Article: PubMed Central - PubMed

Affiliation: Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, PR China.

ABSTRACT

Purpose: The present study was undertaken to investigate whether transforming growth factor-beta (TGF-beta) isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) and SMADs (SMAD2 and SMAD3) are involved in herpes simplex virus type 1 (HSV-1) corneal infection.

Methods: Human corneal epithelial cells (HCE) were infected with HSV-1 at a multiplicity of infection of 5. Cell morphological changes were observed under phase-contrast microscopy. Levels of mRNA for TGF-beta isoforms 1, 2, and 3 as well as for SMAD2 and SMAD3 were measured by reverse transcription polymerase chain reaction (RT-PCR) at 0 h, 4 h, 8 h, 12 h, and 24 h after infection. Protein expression of TGF-beta1, TGF-beta2, SMAD3, and phospho-SMAD3 were analyzed by indirect immunofluorescence at 0 h, 12 h, and 24 h post-infection (p.i.) in HCE cells. Protein expression of TGF-beta1 was also evaluated by ELISA.

Results: Following HSV-1 infection, a cytopathic effect in HCE cells was observed at 8 h p.i. and became significant at 24 h p.i. Compared with normal cells, the mRNA levels of TGF-beta1 in HSV-1 infected HCE cells decreased significantly at 8 h, 12 h, and 24 h p.i. (p<0.01), and the expression of SMAD3 was also dramatically decreased 12 h and 24 h p.i. (p<0.01). No noticeable changes were found as a result of infection with respect to levels of TGF-beta2, TGF-beta3, and SMAD2 in HCE cells. Protein expression of TGF-beta1, SMAD3, and phospho-SMAD3 decreased in infected cells at 12 h and 24 h p.i. compared with normal cells, but TGF-beta2 had no change.

Conclusions: TGF-beta1 and SMAD3 may be involved in the pathology of corneal diseases associated with HSV-1 infection.

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Related in: MedlinePlus

Reverse transcription polymerase chain reaction analysis of the TGF-β isoforms in human corneal epithelial cells infected with HSV-1. A: Products of RT-PCR that were run on 2% agarose gel electrophoresis. The intensities of TGF-β1 bands decreased significantly at 8 h, 12 h, and 24 h p.i., while that of TGF-β2 and TGF-β3 bands unchanged. GAPDH was used as an internal control. B: The level of mRNA was expressed as the ratio of integrated optical density (IOD) of specific PCR products over GAPDH IOD. Each data was the mean value of three independent experiments. Single asterisks indicate significant differences (p<0.05).
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f2: Reverse transcription polymerase chain reaction analysis of the TGF-β isoforms in human corneal epithelial cells infected with HSV-1. A: Products of RT-PCR that were run on 2% agarose gel electrophoresis. The intensities of TGF-β1 bands decreased significantly at 8 h, 12 h, and 24 h p.i., while that of TGF-β2 and TGF-β3 bands unchanged. GAPDH was used as an internal control. B: The level of mRNA was expressed as the ratio of integrated optical density (IOD) of specific PCR products over GAPDH IOD. Each data was the mean value of three independent experiments. Single asterisks indicate significant differences (p<0.05).

Mentions: First, the mRNA level of TGF-β isoforms (i.e., TGF-β1, TGF-β2, and TGF-β3) in HCE cells infected with HSV-1 was estimated using RT–PCR (Figure 2). The mRNA level of TGF-β1 decreased significantly by 40.3%, 57.3%, and 70.4% at 8 h, 12 h, and 24 h p.i., respectively, when compared with uninfected cells (p<0.01). However, mRNA profiles of TGF-β2 and TGF-β3 in infected cells at 8 h, 12 h, and 24 h p.i were similar to that of uninfected cells (p>0.05).


HSV-1 infection suppresses TGF-beta1 and SMAD3 expression in human corneal epithelial cells.

Nie Y, Cui D, Pan Z, Deng J, Huang Q, Wu K - Mol. Vis. (2008)

Reverse transcription polymerase chain reaction analysis of the TGF-β isoforms in human corneal epithelial cells infected with HSV-1. A: Products of RT-PCR that were run on 2% agarose gel electrophoresis. The intensities of TGF-β1 bands decreased significantly at 8 h, 12 h, and 24 h p.i., while that of TGF-β2 and TGF-β3 bands unchanged. GAPDH was used as an internal control. B: The level of mRNA was expressed as the ratio of integrated optical density (IOD) of specific PCR products over GAPDH IOD. Each data was the mean value of three independent experiments. Single asterisks indicate significant differences (p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529468&req=5

f2: Reverse transcription polymerase chain reaction analysis of the TGF-β isoforms in human corneal epithelial cells infected with HSV-1. A: Products of RT-PCR that were run on 2% agarose gel electrophoresis. The intensities of TGF-β1 bands decreased significantly at 8 h, 12 h, and 24 h p.i., while that of TGF-β2 and TGF-β3 bands unchanged. GAPDH was used as an internal control. B: The level of mRNA was expressed as the ratio of integrated optical density (IOD) of specific PCR products over GAPDH IOD. Each data was the mean value of three independent experiments. Single asterisks indicate significant differences (p<0.05).
Mentions: First, the mRNA level of TGF-β isoforms (i.e., TGF-β1, TGF-β2, and TGF-β3) in HCE cells infected with HSV-1 was estimated using RT–PCR (Figure 2). The mRNA level of TGF-β1 decreased significantly by 40.3%, 57.3%, and 70.4% at 8 h, 12 h, and 24 h p.i., respectively, when compared with uninfected cells (p<0.01). However, mRNA profiles of TGF-β2 and TGF-β3 in infected cells at 8 h, 12 h, and 24 h p.i were similar to that of uninfected cells (p>0.05).

Bottom Line: Protein expression of TGF-beta1, TGF-beta2, SMAD3, and phospho-SMAD3 were analyzed by indirect immunofluorescence at 0 h, 12 h, and 24 h post-infection (p.i.) in HCE cells.Compared with normal cells, the mRNA levels of TGF-beta1 in HSV-1 infected HCE cells decreased significantly at 8 h, 12 h, and 24 h p.i. (p<0.01), and the expression of SMAD3 was also dramatically decreased 12 h and 24 h p.i. (p<0.01).Protein expression of TGF-beta1, SMAD3, and phospho-SMAD3 decreased in infected cells at 12 h and 24 h p.i. compared with normal cells, but TGF-beta2 had no change.

View Article: PubMed Central - PubMed

Affiliation: Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou, PR China.

ABSTRACT

Purpose: The present study was undertaken to investigate whether transforming growth factor-beta (TGF-beta) isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) and SMADs (SMAD2 and SMAD3) are involved in herpes simplex virus type 1 (HSV-1) corneal infection.

Methods: Human corneal epithelial cells (HCE) were infected with HSV-1 at a multiplicity of infection of 5. Cell morphological changes were observed under phase-contrast microscopy. Levels of mRNA for TGF-beta isoforms 1, 2, and 3 as well as for SMAD2 and SMAD3 were measured by reverse transcription polymerase chain reaction (RT-PCR) at 0 h, 4 h, 8 h, 12 h, and 24 h after infection. Protein expression of TGF-beta1, TGF-beta2, SMAD3, and phospho-SMAD3 were analyzed by indirect immunofluorescence at 0 h, 12 h, and 24 h post-infection (p.i.) in HCE cells. Protein expression of TGF-beta1 was also evaluated by ELISA.

Results: Following HSV-1 infection, a cytopathic effect in HCE cells was observed at 8 h p.i. and became significant at 24 h p.i. Compared with normal cells, the mRNA levels of TGF-beta1 in HSV-1 infected HCE cells decreased significantly at 8 h, 12 h, and 24 h p.i. (p<0.01), and the expression of SMAD3 was also dramatically decreased 12 h and 24 h p.i. (p<0.01). No noticeable changes were found as a result of infection with respect to levels of TGF-beta2, TGF-beta3, and SMAD2 in HCE cells. Protein expression of TGF-beta1, SMAD3, and phospho-SMAD3 decreased in infected cells at 12 h and 24 h p.i. compared with normal cells, but TGF-beta2 had no change.

Conclusions: TGF-beta1 and SMAD3 may be involved in the pathology of corneal diseases associated with HSV-1 infection.

Show MeSH
Related in: MedlinePlus