Molecular Vision 2015; 21:577-588
<http://www.molvis.org/molvis/v21/577>
Received 26 February 2015 | Accepted 20 May 2015 | Published 21 May 2015
Molecular Vision 2015; 21:577-588
<http://www.molvis.org/molvis/v21/577>
Received 26 February 2015 |
Accepted 20 May 2015 |
Published 21 May 2015
Shrestha Priyadarsini,1 Tina B. McKay,2 Akhee Sarker-Nag,1 Dimitrios Karamichos1,2
1Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK
Correspondence to: Dimitrios Karamichos, Department of Ophthalmology/Dean McGee Eye Institute, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; Phone: (405) 271 4019; FAX: (405) 271 8128; email: dimitrios-karamichos@ouhsc.edu
Keratoconus (KC) is a corneal thinning disease characterized by the formation of a cone-like cornea that affects 1:2,000 people around the world [1-5]. KC is characterized by the loss of corneal integrity and decreased visual acuity due to altered extracellular matrix (ECM) assembly and cornea structure, which can lead to increased scarring and fibrosis [6,7]. Transforming growth factor-β (TGF-β) is a known regulator of ECM formation and has been linked to the development of various diseases, including cancer [8,9], rheumatoid arthritis [10], and corneal dystrophies [11]. Previous reports have suggested a role of aberrant TGF-β signaling in KC disease in conventional 2D in vitro models [12-14]. The primary goal of our study is to dissect the role of key TGF-β molecules in the fibrotic phenotype characteristic of KC using our 3D in vitro model.
TGF-β signaling has been well studied and is known to occur with initial binding of the TGF-β ligand to TGF-βR1, which then dimerizes with TGF-βR2 and stimulates phosphorylation of SMAD2/3 followed by translocation to the nucleus and activation of the transcription of TGF-β-target genes [15-17]. TGF-β signaling is tightly regulated by SMAD6 and SMAD7, which serve to negatively regulate TGF-β signaling by 1) competing for the binding of receptor-regulated SMAD3 to co-mediators [18], 2) promoting recruitment of ubiquitin E3 ligases that result in degradation of the TGF-β receptor [17], and 3) functioning as nuclear transcription repressors by binding histone deacetylases and inhibiting the transcription of TGF-β-responsive genes [19].
The three TGF-β isoforms, TGF-β1, -β2, and -β3, whose secretion and binding initiate signaling, are known to be important modulators of ECM remodeling [16], the matrix metalloproteinase expression [20,21], and terminal differentiation to the myofibroblast [22,23]. Of the three TGF-β isoforms, TGF-β1 and TGF-β2 have been identified as stimulating a profibrotic response following injury [22,24], whereas TGF-β3 is known for its antifibrotic effects [25,26]. We have previously reported that TGF-β3 stimulates human keratoconus cells (HKCs) to secrete and assemble a normal stroma-like ECM, mimicking what is seen in vivo with high expressions of Collagen I and V, and low expressions of Collagen III and α-smooth muscle actin [25]. Our current study was designed to identify key players of the TGF-β signaling pathway involved in KC-derived cells.
In this study, we identified significant variations in the TGF-β pathway in HKCs, suggesting alterations in TGF-β signaling contribute to developments in the fibrotic phenotype observed in KC. We show that HKCs have downregulated the SMAD6 and SMAD7 expressions compared to HCFs. Furthermore, we show that TGF-β3 significantly downregulates the expression of the key receptor TGF-βRII, which may lead to the attenuation of the profibrotic signaling characteristic of HKCs. Our primary goal is to understand the underlying molecular mechanism of KC progression to develop therapeutic methods to promote normal, stromal ECM assembly by HKCs. Our current study suggests that understanding the role of the TGF-β pathway in KC ECM assembly may prove fundamental to revealing the basis of this corneal thinning disease.
All procedures used in these studies adhered to the tenets of the Declaration of Helsinki. Healthy human corneas were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA). Keratoconus donor corneas were obtained from Jesper Hjortdal (Aarhus University Hospital, Aarhus, Denmark).
Human corneas of healthy patients without any ocular disease were used for HCF isolation and age-matched HKCs were isolated from corneas of patients with Keratoconus defects, as previously described [27,28]. Briefly, the corneal epithelium and endothelium were removed from the stroma by scraping it with a razor blade. Stromal tissues were cut into small pieces of size 2×2 mm and placed into T25 culture flaks. The explants were allowed to adhere to the bottom of the flask at 37 °C for about 30 min and Eagle’s Minimum Essential Medium (EMEM: ATCC: Manassas, VA) containing 10% fetal bovine serum (FBS: Atlantic Biologic’s; Lawrenceville, CA) and 1% Antibiotic (Gibco® Antibiotic-Antimycotic, Life technologies) was added carefully without disturbing the ex-plants. Cells were further passaged into T75 culture flasks upon 100% confluence after 1–2 weeks of cultivation at 37 °C, 5% CO2.
In addition, 1×106 cells of HCF and HKC cells/well was seeded and cultured in an EMEM 10% FBS medium stimulated with 0.5 mM 2-O-α-D-Glucopyranosyl-L-Ascorbic Acid (Vit C, American Custom Chemicals Corporation, San Diego, CA) [27,29]. The adherent cells were further stimulated with one of the three TGF-β isoforms: TGF-β1, TGF-β2, or TGF-β3. According to the previous optimized concentration of TGF-β isoforms, a 0.1 ng/mL concentration was used [27,29,30]. The experimental culture was grown for 4 weeks before further processing. Cultures without any growth factors served as the controls (C), and fresh media were supplied every other day for the duration of the experiment.
RNA was extracted using the Ambion RNA mini extraction kit (Ambion TRIzol® Plus RNA Purification Kit: Life technologies, Carlsbad, CA) followed by cDNA synthesis using SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA) as per the manufacturer’s protocol. The TaqMan gene expression assays (Applied Biosystems, Foster City) GAPDH (Hs99999905_m1) and 18S (Hs99999901_s1) were used as the control and SMAD3 (Hs00969210_m1), SMAD4 (Hs00929647_m1), SMAD6 (Hs00178579_m1), SMAD7 (Hs00998193_m1), TGFBR1 (Hs00610320_m1), TGFBR2 (Hs00234253_m1), TGFBR3 (Hs01114253_m1), TGFB1 (Hs00998133_m1), TGFB2 (Hs00234244_m1), TGFB3 (Hs01086000_m1) as the study probes (Table 1). Furthermore, 10 ng of cDNA was used for initiating the PCR reaction for a 20-µl reaction mixture containing our desired probes and the Taqman FaSt Advanced Master Mix (Applied Biosystems, Life technologies, Foster city, CA). Amplification of samples was performed using the StepOnePlusTM real-time PCR system (Life Technologies) in accordance with the manufacture’s protocol. Results were confirmed in three separate analyses and Graph Pad Prism 6 and MS-Excel were used for data analysis.
Western blot analyses of HCF and HKC cells were performed with lysis of cells, as previously described [27,31,32]. Protein concentration and purity were assessed by Bradford assay (Thermo Scientific, IL). As well, 4%–20% Tris-Glycine gels (Novex, Life technologies, Carlsbad, CA) was used for gel electrophoresis, to which equal amounts of proteins were loaded and a protein transfer was done using Nitrocellulose membrane (Novex, Nitrocellulose membrane filter par sandwich, Life Technologies). After incubation in a 5% BSA blocking solution (Thermo Scientific), the membranes were incubated with primary rabbit antibodies (Table 2): anti-Smad3 (Invitrogen, Camarillo, CA), anti-SMAD7 (Sigma-Aldrich, Saint Louis, MO), anti-SMAD4 (Abcam, Cambridge, MA), anti-SMAD6 (Abcam), Anti-TGF-βRI (Abcam), anti-TGF-βRII (Abcam), anti-TGF-βRIII (Abcam), anti-TGF-β1 (Abcam), anti-TGF-β2, and anti-TGF β3 (Abcam) at 1:1,000 dilution overnight at 4 °C separately. This was followed by washing of the membranes and incubation with a secondary antibody (Alexa Flour® 568 Donkey anti-Rabbit, IgG [H+L], Abcam) at 1:2,000 dilutions for 1 h. The Kodak imaging system was used for detecting the antibody binding to the membrane. GAPDH (Abcam) was used as the loading control and results were analyzed by normalizing the value to that of the loading control expression and plotting the fold expression.
All experiments were independently performed at least in triplicate and the statistical significance was evaluated using a non-parametric t test analysis, where a p value of p<0.05 was considered to be statistically significant. The Graph Pad Prism 6 software was used for statistical analysis.
Our previous study looking into the effect of TGF-β isoforms on ECM assembly by HCFs and HKCs showed that TGF-β3 stimulated HKCs to generate a more normal stromal ECM, consistent with low Collagen III and high Collagen I and V assembly [25]. In our current study, we investigated the role of key TGF-β regulator molecules in HKCs compared to normal HCFs in an effort to further define the role of TGF-β signaling in KC. We have compared expression changes in the HKCs to the control HCFs in an effort to define differences that contribute to the disease phenotype and the role of the TGF-β isoforms in regulating ECM assembly. Figure 1A-C shows RT–PCR data of the TGFβ1, TGFβ2, and TGFβ3 transcripts following stimulation of HCFs and HKCs with the three TGF-β isoforms. The gene expression of TGFβ1 was similar for all conditions in normal HCFs. However, the TGF-β1 expression was approximately two-fold higher (p≤0.05) in HCFs compared to HKCs with TGF-β3 treatment (Figure 1A). The TGF-β2 gene expression showed a significant expression reduction (3.5-fold, 1.8-fold, and 2.2-fold, p≤0.05) in HKCs compared to HCFs with TGF-β1, TGF-β2, and TGF-β3 treatment, respectively (Figure 1B). Furthermore, the TGF-β3 expression was significantly downregulated (2.2-fold, eight-fold, and 5.6-fold, p≤0.05) in HKCs in the control, TGF-β1-, TGF-β2-, and TGF-β3-treated samples, respectively (Figure 1C). These data suggest TGF-β3 reduces the expression of the profibrotic ligand TGF-β1 in HKCs, and this reduction contributes to attenuating the ECM generated by HKCs.
We compared the gene expressions with protein expressions using a western blot analysis following TGF-β isoform treatments (Figure 1D-G). Our western blot results showed the TGF-β1 protein expression was comparable in both cell types at basal levels and following stimulation with TGF-β1 and TGF-β2 (Figure 1D). In addition, TGF-β3 treatment in HKCs increased the TGF-β1 protein expression significantly (1.5-fold, p≤0.05) compared to control HCFs (Figure 1D). The TGF-β2 protein expression was lower (Figure 1E, two-fold, p≤0.05) in HKCs with TGF-β isoform treatments compared to HCFs, supporting our gene analysis results that the TGF-β1 expression is the more abundant isoform expressed in HKCs following stimulation with the three isoforms. Furthermore, we found a reduction in the TGF-β3 protein expression (two-fold, p≤0.35) in HKCs following TGF-β3 treatment (Figure 1F).
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.
We also analyzed the protein expressions of the TGF-β receptors by western blot (Figure 2D-G), showing TGF-β3 stimulation significantly downregulated TGF-βRI (1.6-fold, p≤0.0005), TGF-βRII (three-fold, p≤0.001), and TGF-βRIII (2.6-fold, p≤0.001) in HKCs compared to HCFs (Figure 2D-F). The TGF-βRII expression (Figure 2E) was significantly reduced in HKCs in the control, TGF-β1-, TGF-β2-, and TGF-β3-treated cells (two-fold, three-fold, three-fold, and three-fold, respectively, p≤0.05). We also saw a significant reduction in TGF-βRIII in HKCs in the control and TGF-β1-stimulated samples (2.7-fold and 1.5-fold, respectively, p≤0.05) compared to HCFs (Figure 2F). Moreover, TGF-β3 stimulation ameliorates the fibrotic response evident in HKCs by significantly downregulating the expressions of TGF-βRI and TGF-βRII without altering the expression of TGF-βRIII, suggesting a differential response to TGF-β3 between diseased versus normal corneal fibroblasts, with TGF-βRI and TGF-βRII being the dominant receptors for TGF-β3.
We measured the gene expressions of the inhibitory SMADs (SMAD6 and SMAD7) to identify whether alterations in the basal levels following isoform treatment contribute to the fibrotic phenotype observed in HKCs (Figure 3A-B). Our RT–PCR data showed HCFs have a significantly upregulated expression of SMAD6 in the control, TGF-β1-, TGF-β2-, and TGF-β3-stimulated cells compared to HKCs (1.7-fold, 2.2-fold, 2.4-fold, and 3.3-fold, respectively, p≤0.05, Figure 3A). A similar pattern was seen in the SMAD7 expression, where we found a significant downregulation in the control, TGF-β1, TGF-β2, and TGF-β3 treatments (5.4-fold, 4.4-fold, and 12-fold, respectively, p≤0.05) in HKCs compared to HCFs (Figure 3B). This data shows a significant reduction in the regulatory SMADs—SMAD6 and SMAD7—in HKCs, providing the first evidence of the author’s knowledge, associating a malfunction in TGF-β signaling regulation with KC.
We analyzed the protein expressions of regulatory SMAD6 and SMAD7 by western blot (Figure 3C-E). The SMAD6 expression was reduced in HKCs with TGF-β1 treatment (1.1-fold, p≤0.05, Figure 3C). The SMAD7 expression was reduced in HKCs following TGF-β1, TGF-β2, and TGF-β3 stimulation (two-fold, three-fold and 2.5-fold, respectively, p≤0.03) compared to HCFs (Figure 3D). Our results show that HKCs fail to increase the expressions of the regulatory SMAD6 or SMAD7 with TGF-β treatment, suggesting a significant malfunction in the activation of the TGF-β responsive genes associated with regulating TGF-β signaling.
To determine whether the altered expressions of TGF-β isoforms, receptors, and regulatory SMAD6 and SMAD7 contribute to TGF-β signaling modulation, we measured the expressions of SMAD3 and SMAD4 using RT–PCR (Figure 4A-B). TGF-β3 stimulation caused a significant reduction in SMAD3 and SMAD4 in HKCs (1.7-fold and 1.6-fold, respectively, p≤0.05) compared to HCFs (Figure 4A-B). HKCs showed a one-fold increase in the pSMAD3 expression, as measured by western blot, compared to HCFs following TGF-β3 treatment (Figure 4F). This data correlates with the observed increase in the TGF-β1 isoform expression in HKCs with TGF-β3 treatment and suggests the elevated expression of TGF-β1 is directly linked to the significant increase in pSMAD3. The lack of TGF-βRII with TGF-β3 stimulation in HKCs suggests this receptor is required for the activation of profibrotic genes, and its absence fails to promote differentiation to the fibrotic phenotype.
We measured differential expressions of SMAD3 and SMAD4 by western blot in both cell types (Figure 4C-D). HKCs showed a 3.2-fold (p≤0.007) increase in SMAD3 at basal levels compared to HCFs (Figure 4C). TGF-β1 treatment also showed an elevated SMAD3 expression in HKCs (2.4-fold, p≤0.006) compared to HCFs (Figure 4C). Moreover, TGF-β3 stimulation caused an increase in the SMAD3 expression by both cell types, suggesting TGF-β3 may also activate gene expressions via a SMAD-dependent mechanism similar to the other isoforms. SMAD4, which is an adaptor protein that colocalizes with pSMAD3 following TGF-β receptor activation, also showed an increase (1.6-fold, p≤0.02) in expression in HKCs compared to HCFs (Figure 4D). The SMAD4 expression was upregulated (2.8-fold, p≤0.01) in HKCs compared to HCFs following TGF-β2 stimulation (Figure 4D). Collectively, our data show TGF-β3 significantly downregulates TGF-β1, TGF-βRI, SMAD3, and SMAD4 expressions, suggesting a plausible mechanism by which TGF-β3 favors antifibrotic ECM assembly.
KC is a corneal thinning disease with a pathophysiology directly linked to altered ECM assembly, myofibroblast differentiation, and distorted corneal structure [27]. KC is known to be associated with scarring and altered ECM assembly that leads to structural thinning of the cornea in vivo [33,34]. As TGF-β signaling is an important modulator of ECM assembly, we focused our current study on identifying the role of key TGF-β signaling molecules in a 3D in vitro model of KC.
HKCs are isolated from severe cases of KC, in which the patients have undergone corneal transplantation due to the progressive nature of the disease. It has been shown that myofibroblasts are not expressed in the early stages of KC [35], but are present only in severe cases associated with scarring or disruptions to the Bowman’s membrane [36]. In this study, we used fibroblasts isolated from KC patients and normal human controls isolated in the presence of serum. Our approach ensures we focus on the defects between the healthy and diseased corneal stromal cells. Isolation of keratocytes from KC patients is ideal; however, preliminary observations from our laboratory have found this to be difficult without additives included in the media. There are differences between keratocytes isolated from healthy corneal stroma and those that are KC derived. We did not utilize altered conditioned media, as our primary goal was to identify the native expressions of key TGF-β mediators without altering pathways that may skew the data.
The TGF-β pathway is a known regulator of ECM secretion and assembly. The three TGF-β isoforms, TGF-β1, TGF-β2, and TGF-β3, are the primary ligands that bind to TGFβRII, which has been found to recruit and phosphorylate TGFβR1, promoting phosphorylation of cytoplasmic SMAD2 and SMAD3 [37]. The TGF-β1 and TGF-β2 isoforms are ligands associated with promoting a profibrotic response characterized by the secretion of excess Collagen secretion and α-SMA expression [16,22]. The three TGF-β isoforms have differential roles in wound healing, with TGF-β3 having an antifibrotic function [26,29,38,39]. We have previously shown that TGF-β3 promotes the assembly of a normal ECM by HKCs in vitro by decreasing Collagen III secretion and the expressions of profibrotic markers characteristic of the diseased phenotype [25,29]. Several previous clinical studies have shown that TGFβ3 is a key modulator during the wound healing process [29,40,41]. Briefly, in the skin, TGFβ3 was found to be a key component in improving the quality of healing and it has made it to the clinical trials stage [42]. TGFβ3 has also been proposed as an antiscarring factor in buccal mucosa [43]. Furthermore, in an embryonic wound microenvironment, high levels of the TGFβ3 expression have been reported to contribute to a scar-less healing mechanism [42].
Due to the direct relationship associated with excessive TGF-β signaling and the development of fibrosis, inhibitors have been developed, targeting TGF-β signaling in vivo [44,45]. However, no treatments have been developed that directly promote ECM secretion and assembly to treat corneal diseases. Moreover, studies have associated KC with variations in TGF-β signaling [12,13,37].
Due to this direct correlation of KC with ECM assembly, we sought to identify the role of TGF-β signaling in contributing to the fibrotic phenotype observed in HKCs and to explore the potential of TGF-β3 in promoting normal ECM assembly by HKCs. In our experimental set-up, HCFs and HKCs were treated with TGF-β isoforms in a 4-week study, where gene and protein expressions of TGF-β signaling molecules were measured for both cell types. TGF-β1 was found to be elevated in HKCs with TGF-β3 treatment in comparison to HCFs. Both TGFβ2 and TGFβ3 were reduced in HKCs, suggesting that ECM assembly in KC is directly modulated primarily by TGF-β1, rather than by TGF-β2 and TGF-β3. This data supports previous studies suggesting the importance of TGF-β1 in KC disease [12,46,47]. Mechanistically, studies have shown that TGF-β pathway activation is dependent on receptor expression, receptor kinase activity, ligand concentration, and adaptor protein expression [18,22,48,49].
We measured changes in both gene expressions and protein expressions in key TGF-β signaling mediators in HKCs compared to HCFs to characterize fully the role of the TGF-β pathway in the pathobiology of KC. In addition, the expression at the mRNA level is known to deviate from that at the protein level due to the regulation of mRNA stability, transport, and translation [50-52]. We found differences in gene expressions compared to protein expressions in both cell types, which suggests the TGF-β pathway is highly regulated. The mechanism of KC pathogenesis is still unknown; however, our work suggests TGF-β signaling may play a significant role in the altered ECM assembled by HKCs.
Our results showed substantial variations in the expressions of the TGFβ receptors TGF-βRI, TGF-βRII, and TGF-βRIII in HKCs compared to HCFs, where TGF-β3 stimulation caused a significant downregulation of the TGF-βRI, TGF-βRII, and TGF-βRIII expressions in HKCs when compared to HCFs. We speculate TGF-β3 favors an antifibrotic mechanism in HKCs by downregulating the expression of the profibrotic receptor, TGF-βRII, thereby antagonizing profibrotic signaling in the abundance of the TGF-β1 ligand.
The SMAD proteins, which are considered to be key regulators in the propagation of the TGF-β pathway, also showed significant regulation in HKCs compared to HCFs. Here, we measured the expressions of the regulatory SMAD6 and SMAD7 at basal levels, as well as following stimulation with the three TGF-β isoforms. TGF-β signaling is known to regulate directly the regulatory SMAD expressions by activating SMAD6 and SMAD7 gene transcriptions upon ligand binding, which provides a negative feedback loop to curb aberrant TGF-β signaling [18]. In our study, we found that stimulation with the TGF-β isoforms caused a significant increase in the SMAD7 expression in HCFs compared to HKCs, whereas HKCs lacked any increase in the expressions of SMAD6 or SMAD7 following TGF-β isoform treatments. These results suggest a significant malfunction in the activation of TGF-β responsive genes associated with the negative regulation of TGF-β signaling in HKCs that may contribute to the altered ECM generated in KC.
To evaluate whether the observed altered expressions of TGFβ receptors and regulatory SMAD6 and SMAD7 proteins by HKCs resulted in increased TGFβ signaling, we measured the pSMAD3 expression. We show a significant increase in pSMAD3 with TGF-β3 in HKCs. The lack of the TGF-βRII expression with TGF-β3 stimulation in HKCs suggests this receptor is required for the activation of profibrotic genes, and its absence fails to promote differentiation to the fibrotic phenotype. Mechanistically, our data also suggest TGF-β3 activates the TGF-β pathway in a SMAD-dependent manner following the SMAD2/3/4 axis that leads to the transcription of genes associated with ECM deposition without terminal differentiation to the myofibroblast, which may prove useful in targeting the TGF-β signaling pathway in the treatment of KC.
We would like to propose a new model/mechanism for the relationship between the altered expressions of key TGF-β signaling molecules and the HKC fibrotic phenotype. HKCs have reduced SMAD6/7 expressions following stimulation with the three TGF-β isoforms compared to HCFs. Normal corneal fibroblasts increase the expressions of SMAD6/7 upon TGF-β isoform treatment, which serves to curb successfully aberrant activation of the TGF-β pathway by decreasing receptor availability and recruitment of SMAD2/3. Phosphorylation of SMAD2/3 promotes translocation to the nucleus and activation of the genes associated with myofibroblast differentiation. The lack of inhibition of TGF-β signaling by SMAD6/7 results in increased TGF-β signaling that promotes the formation of a fibrotic ECM characteristic of HKCs.
Our data directly link the altered expressions of the regulatory SMAD6/7 with the observed aberrant TGF-β signaling and increased fibrotic phenotype observed in HKCs. In addition, we show that TGF-β3 decreases the expression of TGF-βRII in HKCs. This result gives a mechanistic role to the antifibrotic phenotype stimulated by TGF-β3 by suggesting TGF-β3 regulates directly profibrotic signaling in HKCs by downregulating the expression of the key TGF-β receptor responsible for promoting activation of the pathway. In this study, we have identified a significant role for the regulatory SMADs, SMAD6 and SMAD7, in regulating TGF-β signaling, as well as highlight how alterations in expressions can contribute to the HKC phenotype. We also propose a plausible mechanistic role of TGF-β3 in downregulating the receptor expression, thereby stimulating an antifibrotic response. Clearly, the TGF-β pathway plays an important role in the development and progression of KC and may provide clues to the development of therapeutics to treat KC.
This work was supported by the National Institutes of Health Grants 5R01EY023568 (DK) and R01EY020886 (DK) and, in part, by an unrestricted grant (DMEI) from Research to Prevent Blindness (New York, NY USA) and a core grant (DMEI) from National Institutes of Health EY021725. Research reported in this publication was supported by the National Eye Institute of the NIH under award number T32EY023202.