Abstract
Purpose.: Transforming growth factor (TGF)-β can mediate fibrotic responses via Smad4-dependent and -independent signaling pathways. TGFβ-induced transdifferentiation of lens epithelial cells to myofibroblasts and matrix contraction contribute to posterior capsule opacification (PCO) after cataract surgery. The primary objective of the study was to determine the importance of Smad4 in TGFβ2-induced transdifferentiation, matrix contraction, and Smad signaling by human lens epithelial cells.
Methods.: The human lens cell line FHL 124 was used. Smad4 knockdown was achieved by using siRNA technology and efficiency tested at the message and protein level by real-time PCR and Western blots, respectively. Smad4 and Smad2/3 nuclear distribution after TGFβ2 stimulation (10 ng/mL) was determined by immunocytochemistry. Gene expression of transdifferentiation markers, α-smooth muscle actin and fibronectin and the inhibitory Smad, Smad7, in the presence and absence of TGFβ2 (10ng/mL) was determined with real-time PCR. TGFβ2-induced contraction was assessed with a patch contraction assay. Suspended bead array technology was used to determine pERK, pP38, and pJNK levels in response to TGFβ2 over a 2-hour time course.
Results.: SiRNA targeted against Smad4, successfully reduced expression of Smad4 message and protein after both the initial transfection period and removal of transfection conditions. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 after siSmad4 treatment relative to control siRNA in both the presence and absence of 10 ng/mL TGFβ2. Smad4 knockdown cells exhibited TGFβ2-induced Smad2/3 nuclear translocation which did not differ significantly from Smad4-expressing cells. Real-time PCR analysis showed significant induction of αSMA and fibronectin transcript in the Smad4-expressing cell line (control siRNA group). These inductions were suppressed in Smad4-knockdown cells. TGFβ-induced mSmad7 expression in FHL 124 cells was unaffected by Smad4 knockdown. Smad4-expressing cells did not exhibit a significant contractile response after 24 hours of culture in the presence or absence of 10 ng/mL TGFβ2. However, Smad4-knockdown cells demonstrated a significant reduction in patch area (i.e., contraction), after TGFβ2 treatment. Bead array analysis showed that TGFβ2 significantly increased pERK and pP38 levels relative to unstimulated control. No significant change was observed with pJNK.
Conclusions.: Smad4 is critical for the expression of transdifferentiation markers, αSMA, and fibronectin; in contrast, Smad4 signaling is not essential for TGFβ-induced Smad7 expression. Smad4 does not appear to be necessary for TGFβ-induced matrix contraction. TGFβ does activate Smad-independent pathways in human lens epithelial cells. Therefore, TGFβ2 regulation of transdifferentiation and matrix contraction appears to be mediated by distinct TGFβ signaling pathways.
Methods.: The human lens cell line FHL 124 was used. Smad4 knockdown was achieved by using siRNA technology and efficiency tested at the message and protein level by real-time PCR and Western blots, respectively. Smad4 and Smad2/3 nuclear distribution after TGFβ2 stimulation (10 ng/mL) was determined by immunocytochemistry. Gene expression of transdifferentiation markers, α-smooth muscle actin and fibronectin and the inhibitory Smad, Smad7, in the presence and absence of TGFβ2 (10ng/mL) was determined with real-time PCR. TGFβ2-induced contraction was assessed with a patch contraction assay. Suspended bead array technology was used to determine pERK, pP38, and pJNK levels in response to TGFβ2 over a 2-hour time course.
Results.: SiRNA targeted against Smad4, successfully reduced expression of Smad4 message and protein after both the initial transfection period and removal of transfection conditions. These findings were confirmed by immunocytochemistry for Smad4, which showed a significant reduction in nuclear Smad4 after siSmad4 treatment relative to control siRNA in both the presence and absence of 10 ng/mL TGFβ2. Smad4 knockdown cells exhibited TGFβ2-induced Smad2/3 nuclear translocation which did not differ significantly from Smad4-expressing cells. Real-time PCR analysis showed significant induction of αSMA and fibronectin transcript in the Smad4-expressing cell line (control siRNA group). These inductions were suppressed in Smad4-knockdown cells. TGFβ-induced mSmad7 expression in FHL 124 cells was unaffected by Smad4 knockdown. Smad4-expressing cells did not exhibit a significant contractile response after 24 hours of culture in the presence or absence of 10 ng/mL TGFβ2. However, Smad4-knockdown cells demonstrated a significant reduction in patch area (i.e., contraction), after TGFβ2 treatment. Bead array analysis showed that TGFβ2 significantly increased pERK and pP38 levels relative to unstimulated control. No significant change was observed with pJNK.
Conclusions.: Smad4 is critical for the expression of transdifferentiation markers, αSMA, and fibronectin; in contrast, Smad4 signaling is not essential for TGFβ-induced Smad7 expression. Smad4 does not appear to be necessary for TGFβ-induced matrix contraction. TGFβ does activate Smad-independent pathways in human lens epithelial cells. Therefore, TGFβ2 regulation of transdifferentiation and matrix contraction appears to be mediated by distinct TGFβ signaling pathways.
| Original language | English |
|---|---|
| Pages (from-to) | 5318-5327 |
| Number of pages | 10 |
| Journal | Investigative Ophthalmology & Visual Science |
| Volume | 50 |
| Issue number | 11 |
| DOIs | |
| Publication status | Published - 2009 |