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Keratoconus in vitro and the key players of the TGF-β pathway.

Priyadarsini S, McKay TB, Sarker-Nag A, Karamichos D - Mol. Vis. (2015)

Bottom Line: We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05).Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7.Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK.

ABSTRACT

Purpose: Keratoconus (KC) is a corneal thinning disease of unknown etiology whose pathophysiology is correlated with the presence of a thin corneal stroma and altered extracellular matrix (ECM). Transforming growth factor-β (TGF-β) signaling is a key regulator of ECM secretion and assembly in multiple tissues, including the anterior segment of the eye, and it has been linked to KC. We have previously shown that human keratoconus cells (HKCs) have a myofibroblast phenotype and altered ECM assembly compared to normal human corneal fibroblasts (HCFs). Moreover, TGF-β3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-β signaling that contribute to the observed fibrotic phenotype in HKCs.

Methods: HCFs and HKCs were stimulated with TGF-β1, TGF-β2, or TGF-β3 isoforms (0.1 ng/mL) in the presence of a stable vitamin C derivative (0.5 mM) for 4 weeks. All samples were examined using RT-PCR and western blotting to quantify changes in the expressions of key TGF-β signaling molecules between HCFs and HKCs.

Results: We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05). Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7. HCFs also showed an upregulation in TGF-βRI, TGF-βRII, and TGF-βRIII following TGF-β3 treatment, whereas HKCs showed a significant two-fold downregulation.

Conclusions: Overall, our data shows the decreased expressions of the regulatory SMADs SMAD6 and SMAD7 by HKCs contribute to the pathological ECM structure observed in KC, and TGF-β3 may attenuate this mechanism by downregulating the expression of the key profibrotic receptor, TGF-βRII. Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.

No MeSH data available.


Related in: MedlinePlus

Quantification of TGF-βR1, TGF-βR2, and TGF-βR3 expression in HCFs and HKCs following stimulation with all three TGF-β isoforms. RT-PCR analysis shows gene expression for (A) TGF-βR1, (B) TGF-βR2, and (C) TGF-βR3. Western blot analysis shows protein expression for (D) TGF-βR1, (E) TGF-βR2, and (F) TGF-βR3. G: Representative Western blots from three independent experiments. All samples were repeated at least three times. p<0.05 was considered to be statistically significant (****p<0.0001, ***p<0.001, *p<0.05).
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f2: Quantification of TGF-βR1, TGF-βR2, and TGF-βR3 expression in HCFs and HKCs following stimulation with all three TGF-β isoforms. RT-PCR analysis shows gene expression for (A) TGF-βR1, (B) TGF-βR2, and (C) TGF-βR3. Western blot analysis shows protein expression for (D) TGF-βR1, (E) TGF-βR2, and (F) TGF-βR3. G: Representative Western blots from three independent experiments. All samples were repeated at least three times. p<0.05 was considered to be statistically significant (****p<0.0001, ***p<0.001, *p<0.05).

Mentions: The TGFβ receptors, TGF-βRI, TGF-βRII, and TGF-βRIII, showed substantial variations in expressions in the diseased HKCs versus the normal HCFs. We used RT–PCR to measure changes in receptor expressions in both cell types (Figure 2A-C). Our results show the TGF-β2 stimulation significantly upregulates the expression of TGF-βR1 in HKCs (2.5-fold, p≤0.05) compared to control HKC levels (Figure 2A). In contrast, HCFs did not significantly upregulate the expressions of the TGF-β receptors with TGF-β1 and TGF-β2 treatment, suggesting HKCs are more responsive to TGF-β3 isoform stimulation, as indicated by the increased expressions of the profibrotic receptors. The reduction in TGF-βRI in HKCs following TGF-β3 treatment correlates with the reduction in the profibrotic TGF-β1 ligand under the same conditions, suggesting TGF-β3 favors an antifibrotic mechanism by downregulating the expressions of both the profibrotic receptor and ligand in HKCs. In addition, the TGF-βR2 and TGF-βR3 expressions are also significantly downregulated (p≤0.05) in HKCs following TGF-β3 treatment (Figure 2B-C), suggesting TGF-β3 regulates the expressions of TGF-β receptors in KC.


Keratoconus in vitro and the key players of the TGF-β pathway.

Priyadarsini S, McKay TB, Sarker-Nag A, Karamichos D - Mol. Vis. (2015)

Quantification of TGF-βR1, TGF-βR2, and TGF-βR3 expression in HCFs and HKCs following stimulation with all three TGF-β isoforms. RT-PCR analysis shows gene expression for (A) TGF-βR1, (B) TGF-βR2, and (C) TGF-βR3. Western blot analysis shows protein expression for (D) TGF-βR1, (E) TGF-βR2, and (F) TGF-βR3. G: Representative Western blots from three independent experiments. All samples were repeated at least three times. p<0.05 was considered to be statistically significant (****p<0.0001, ***p<0.001, *p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4443584&req=5

f2: Quantification of TGF-βR1, TGF-βR2, and TGF-βR3 expression in HCFs and HKCs following stimulation with all three TGF-β isoforms. RT-PCR analysis shows gene expression for (A) TGF-βR1, (B) TGF-βR2, and (C) TGF-βR3. Western blot analysis shows protein expression for (D) TGF-βR1, (E) TGF-βR2, and (F) TGF-βR3. G: Representative Western blots from three independent experiments. All samples were repeated at least three times. p<0.05 was considered to be statistically significant (****p<0.0001, ***p<0.001, *p<0.05).
Mentions: The TGFβ receptors, TGF-βRI, TGF-βRII, and TGF-βRIII, showed substantial variations in expressions in the diseased HKCs versus the normal HCFs. We used RT–PCR to measure changes in receptor expressions in both cell types (Figure 2A-C). Our results show the TGF-β2 stimulation significantly upregulates the expression of TGF-βR1 in HKCs (2.5-fold, p≤0.05) compared to control HKC levels (Figure 2A). In contrast, HCFs did not significantly upregulate the expressions of the TGF-β receptors with TGF-β1 and TGF-β2 treatment, suggesting HKCs are more responsive to TGF-β3 isoform stimulation, as indicated by the increased expressions of the profibrotic receptors. The reduction in TGF-βRI in HKCs following TGF-β3 treatment correlates with the reduction in the profibrotic TGF-β1 ligand under the same conditions, suggesting TGF-β3 favors an antifibrotic mechanism by downregulating the expressions of both the profibrotic receptor and ligand in HKCs. In addition, the TGF-βR2 and TGF-βR3 expressions are also significantly downregulated (p≤0.05) in HKCs following TGF-β3 treatment (Figure 2B-C), suggesting TGF-β3 regulates the expressions of TGF-β receptors in KC.

Bottom Line: We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05).Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7.Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK.

ABSTRACT

Purpose: Keratoconus (KC) is a corneal thinning disease of unknown etiology whose pathophysiology is correlated with the presence of a thin corneal stroma and altered extracellular matrix (ECM). Transforming growth factor-β (TGF-β) signaling is a key regulator of ECM secretion and assembly in multiple tissues, including the anterior segment of the eye, and it has been linked to KC. We have previously shown that human keratoconus cells (HKCs) have a myofibroblast phenotype and altered ECM assembly compared to normal human corneal fibroblasts (HCFs). Moreover, TGF-β3 treatment promotes assembly of a more normal stromal ECM and modulates the fibrotic phenotype in HKCs. Herein, we identify alterations in TGF-β signaling that contribute to the observed fibrotic phenotype in HKCs.

Methods: HCFs and HKCs were stimulated with TGF-β1, TGF-β2, or TGF-β3 isoforms (0.1 ng/mL) in the presence of a stable vitamin C derivative (0.5 mM) for 4 weeks. All samples were examined using RT-PCR and western blotting to quantify changes in the expressions of key TGF-β signaling molecules between HCFs and HKCs.

Results: We found a significant downregulation in the SMAD6 and SMAD7 expressions by HKCs when compared to HCFs (p≤0.05). Moreover, stimulation of HKCs with any of the three TGF-β isoforms did not significantly alter the expressions of SMAD6 or SMAD7. HCFs also showed an upregulation in TGF-βRI, TGF-βRII, and TGF-βRIII following TGF-β3 treatment, whereas HKCs showed a significant two-fold downregulation.

Conclusions: Overall, our data shows the decreased expressions of the regulatory SMADs SMAD6 and SMAD7 by HKCs contribute to the pathological ECM structure observed in KC, and TGF-β3 may attenuate this mechanism by downregulating the expression of the key profibrotic receptor, TGF-βRII. Our study suggests a significant role of altered regulation of TGF-β signaling in KC progression and that it may enable novel therapeutic developments targeting TGF-β receptor regulation.

No MeSH data available.


Related in: MedlinePlus