Role of 5′TG3′-interacting factors (TGIFs) in Vorinostat (HDAC inhibitor)-mediated Corneal Fibrosis Inhibition

Human corneal fibroblast and myofibroblast cultures

Primary corneal fibroblast cultures were generated from donor human corneas procured from an eye bank (Heartland Eye Bank, Kansas City, MO). Based on the slit lamp, specular microscopy, physical examination, and donor medical record data provided by the eye bank, it was ensured that only healthy and disease-free corneas were used in the present study. Corneal tissues were washed with a sterile cell culture medium, and the epithelium and endothelium were removed by gentle scraping with a #15 scalpel blade. The corneal stroma was cut into small sections, placed on a culture dish, and incubated in a humidified 5% CO₂ incubator at 37 °C in minimum essential medium (MEM) supplemented with 10% fetal bovine serum for two to three weeks. The primary human corneal fibroblasts harvested from these corneal buttons were seeded in six-well plates in MEM supplemented with 10% fetal bovine serum and allowed to reach 60%–70% confluence. To generate myofibroblast cultures, the fibroblasts were seeded using MEM containing 10% fetal bovine serum, switched to serum-free medium containing TGFβ1 (5 ng/ml) after 12 h, and incubated for 5 days. The cultures were fed with fresh serum-free TGFβ1-containing medium every 24 h.

Vorinostat, siRNA and RNAi treatment

A 10 mM stock solution of vorinostat (Cayman Chemical Company, Ann Arbor, MI) was prepared in dimethylsulfoxide (DMSO) and diluted with MEM to achieve a final concentration of 2.5 µM in the culture medium. The equivalent amount of DMSO (0.5 µl in 2 ml culture medium) served as controls. For the knockdown of TGIF1 and TGIF2 gene expression, prevalidated siRNA (Ambion Life technologies, Grand Island, NY) were used at a 30 nM concentration (Table 1). For the knockdown of Smad2/3/4, the prevalidated RNA interference (RNAi) oiligos (Table 1) were cloned into a pcDNA 6.2 miR RNAi expression vector under the control of Pol II promoter (Life Technologies, Grand Island, NY). The siRNA and RNAi plasmid transfections were performed using the commercially available transfection reagent lipofectamine3000 (Life technologies corporation, Grand Island, NY) as per the manufacturer’s instructions. Briefly, a transfection solution was prepared by adding 10 µl lipofectamine3000 in 200 µl of optiMEM medium to the appropriate amounts of siRNA/RNAi plasmid in 200 µl of opitiMEM. The mixture was incubated for 5 min at room temperature. The above transfection mixture was added to the human corneal fibroblasts in 1 ml of a serum-free culture medium.

RNA extraction and cDNA synthesis

Total RNA from cells was extracted with an RNeasy kit (Qiagen, Valencia, CA) and reverse transcribed to cDNA following the vendor’s instructions (Promega, Madison, WI). PCR was performed for the detection of TGIF1 and TGIF2. A 50 µl reaction mixture containing 2 µl cDNA, 2 µl forward (200 nM) and 2 µl reverse (200 nM) primer, 0.2 mM of each deoxynucleotide triphosphates (dNTP), and 1.25 U of Taq polymerase was run one cycle at 95 °C for 3 min, then 40 cycles of 95 °C for 30 s, followed by 55 °C for 30 s and 72 °C for 60 s, using a thermocycler (Bio-Rad Laboratories, Hercules, CA) and primers listed in Table 2. The cDNA samples were prepared from fibroblasts originating from two different human donor corneas. Each PCR was repeated at least three times.

Gene expression quantification by qPCR

The relative quantification of TGIFs mRNA was performed using the StepOne Plus real-time PCR system (Applied Biosystems, Carlsbad, CA). A 20 µl reaction mixture containing 2 µl of cDNA, 2 µl of forward primer (200 nM), 2 µl of reverse primer (200 nM), and 10 µl of 2X SYBR green super mix (Bio-Rad Laboratories, Hercules, CA) was run at a universal cycle (95 °C for 10 min, 40 cycles at 95 °C for 15 s, and 60 °C for 60 s) in accordance with the manufacturer’s instructions, using primers listed in Table 2.

Immunoblotting

Both the treated and untreated HCF cultures were lysed in a radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) followed by centrifugation at 10,000 g for 10 min. Samples were suspended in a NuPAGE LDS buffer containing a reducing agent (Life Technologies Corporation, Grand Island, NY) and heated at 70 °C for 10 min. Protein samples were resolved by NuPAGE Novex Bis-Tris mini gels (Invitrogen) and transferred onto the polyvinylidene difluoride membranes using wet transfer at 25 V. The transferred proteins were detected by incubating the membrane with primary antibodies: TGIF1, TGIF2 (Santa Cruz Biotechnology, Santa Cruz, CA), acetylhistone H3, acetyl histone H4 (EMD Millipore, Billerica, MA), alpha smooth muscle actin (αSMA Dako, Carpinteria, CA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology), followed by alkaline phosphatase conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibody. After washing three times in 0.05% Tween-20 in Tris-buffered saline of pH 8.0 for 5 min each, the blot was developed using the nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate method. Three separate western blots were performed for each experiment. The digital quantification of western blots was performed using NIH Image J software.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde, and immunofluorescence staining for α–smooth muscle actin (1:200 dilution) and acetyl histone H3 (1:100 dilution EMD Millipore, Billerica, MA) was performed. Samples were incubated with 5% BSA for 30 min at room temperature, followed by primary antibody incubation for 90 min. The secondary antibodies Alexa 488 donkey anti-rabbit and Alexa 594 donkey anti-mouse IgG secondary antibody (1:500 dilution) were incubated for 1 h. The cells were washed three times in PBS buffer, mounted in Vectashield containing 4'-6-diamidino-2-phenylindole (DAPI; Vector Laboratories), and viewed and photographed with a Leica fluorescent microscope (Leica DM 4000B) equipped with a digital camera (SpotCam RT KE).

Statistical analyses

Results were expressed as a mean ± standard error. Statistical analysis was performed using one-way ANOVA and Tukey’s test. A value of p<0.05 was considered significant.

Detection of TGIF1 and TGIF2 in human corneal fibroblasts

To investigate whether human corneal fibroblasts express TGIFs, PCR was performed to detect the presence from mRNA transcripts for TGIF1 and TGIF2. Figure 1 shows the agarose gel electrophoresis of the PCR demonstrating the presence of amplified bands for TGIF1 and TGIF2 in the cDNA obtained from human corneal fibroblasts. An appropriately sized amplification product of 608 bp was detected for TGIF1 in two cDNA samples of human corneal fibroblasts originating from two separate donor human corneas. Amplification products for TGIF2 corresponding to the product size of 646 bp were also present in the human corneal fibroblast cDNA samples. No PCR product was detected in the negative controls that contained primers for TGIFs but no cDNA (Figure 1).

Effect of vorinostat on TGIF1 and TGIF2 expression and histone acetylation

After confirming that human corneal fibroblasts express TGIF1 and TGIF2, we next tested if vorinostat treatment of human corneal fibroblasts increased TGIF expression. The mRNA and protein lysates were obtained from the human corneal fibroblasts +/− vorinostat. As is evident from Figure 2A, treatment of human corneal fibroblasts with vorinostat (2.5 µM) caused a 2.8±0.81-fold increase (p<0.05) in TGIF1 mRNA and 3.3±0.25-fold increase (p<0.01) in TGIF2 mRNA levels as compared to DMSO-treated vehicle control groups (–vorinostat). Figure 2B shows the western blot detection of TGIF protein levels in DMSO-treated vehicle control and vorinostat (2.5 µM)-exposed human corneal fibroblasts. The immunoblotting data demonstrated that vorinostat caused a 1.4-fold increase (p<0.05) in TGIF1 and 1.8-fold increase (p<0.05) in TGIF2 protein in comparison to vehicle-treated control human corneal fibroblasts. Further, we also tested the effect of vorinostat on TGIF1 and TGIF2 expression in corneal keratocytes. The corneal keratocytes were isolated using dispase and collagenase enzymatic digestions and grown in serum-free medium in the presence or absence of vorinostat. Our results demonstrate that vorinostat increased the TGIF1 and TGIF2 mRNA in human corneal keratocytes as observed in human corneal fibroblasts (data not shown).

We further postulated that the observed increase in TGIFs was likely mediated by enhanced transcription due to histone acetylation and DNA unfolding caused by vorinostat. To test whether the 2.5 µM dose of vorinostat used in the present study was sufficient to cause histone acetylation, we performed immunoblotting for acetylated histone H3 and acetylated histone H4. As can be seen in Figure 3A, vorinostat treatment of human corneal fibroblasts caused an 8-fold increase (p<0.001) in acetylhistone H3 and a 50-fold increase (p<0.001) in acetylhistone H4. Figure 3B demonstrated that only 36±4% of nuclei stained +ve for acetylhistone H3 in DMSO-treated vehicle control corneal fibroblasts (–vorinostat). On the other hand, vorinostat treatment of human corneal fibroblasts significantly increased the acetylhistone H3 to 75±3.4% (p<0.001) nuclei showing +ve staining. A similar trend demonstrating the increase in acetylhistone H4-stained nuclei was also observed with vorinostat (data not shown). No statistically significant difference in the total number of DAPI-stained nuclei (blue) was observed in the control and vorinostat-exposed human corneal fibroblasts (Figure 3B).

Effect of Smad knockdown on TGFβ1-induced myofibroblasts formation

TGIFs are negative regulators of TGFβ1-mediated Smad signaling. Therefore, we next tested the hypothesis that TGFβ1-mediated transdifferentiation of human corneal fibroblasts to myofibroblasts is largely mediated through Smad signaling. To test this hypothesis, we transfected the human corneal fibroblasts with plasmids expressing Smad2/Smad3 or Smad4 RNAi. Figure 4 shows the effect of RNAi-mediated knockdown of Smad2/Smad3 or Smad4 on TGFβ1-evoked SMA formation. As is evident from Figure 4, the knockdown of Smad2/Smad3 or Smad4 markedly attenuated TGFβ1-mediated increase in αSMA in cultured human corneal fibroblasts tested using immunofluorescence (Figure 4A-E) and immunoblotting (Figure 4F). This suggests that TGFβ1-mediated transdifferentiation of human corneal fibroblasts to myofibroblasts is largely mediated through Smad signaling.

Effect of vorinostat on myofibroblast formation

Next, we tested the hypothesis that vorinostat-induced increases in TGIF1 and TGIF2 were accompanied by a concurrent decrease in corneal fibrosis. An in vitro model of corneal fibrosis in which the application of TGFβ1 induces myofibroblasts formation was used to test this hypothesis. The transformation of corneal fibroblasts to myofibroblasts is a key pathological feature in corneal fibrosis. Myofibroblasts are contractile, metabolically active, and opaque cells containing intracellular microfilament bundles of αSMA, which serves as a maker for fibrosis. As expected, TGFβ1treatment of human corneal fibroblasts caused a 3.8±0.2-fold increase (p<0.001) in αSMA mRNA (Figure 5A) and a 4.3-fold increase (p<0.01) in SMA protein (Figure 5B). Vorinostat treatment significantly attenuated TGFβ 1-induced increases in αSMA mRNA by 83±7.7% (p<0.001, Figure 5A) and protein levels by 97±5% (p<0.001, Figure 5B).

The antifibrotic effects of vorinostat were further investigated by αSMA immuno-cytochemistry (Figure 6). As anticipated, human corneal fibroblasts cultured in the absence of TGFβ1 (–TGFβ) under serum-free conditions showed minimal αSMA expression (Figure 6). TGFβ1 (5 ng/ml) treatment caused a robust myofibroblast formation, as indicated by an intense αSMA immunostaining in 82%–94% of cells (p<0.001). Following vorinostat treatment, only 6%–8% of cells (p<0.001) showed αSMA immunostaining, confirming that vorinostat remarkably attenuates TGFβ1-evoked myofibroblast formation (Figure 6).

Effect of TGIFs knockdown on the antifibrotic effect of vorinostat

Finally, we tested whether the increase in TGIF expression is an essential step for the antifibrotic effect of vorinostat. If so, then the TGIF knockdown should impede the antifibrotic effect of vorinostat. To test this hypothesis, we designed a reverse experiment wherein we tested the antifibrotic effect of vorinostat along with siRNA-mediated knockdown of TGIFs. Figures 5A and B show that TGIF1 and TGIF2 knockdown prevented vorinostat-mediated decreases in αSMA mRNA by 31%–45% (p<0.05) and protein levels by 12%–23% (p<0.05), respectively. Similarly, immunostaining data for αSMA shows that TGIF1 or TGIF2 siRNA knockdown significantly prevented vorinostat-induced decreases in myofibroblast formation. Only 6%–8% of cells (p<0.05) showed αSMA immunostaining when exposed to vorinostat alone, whereas 12%–18% of cells (p<0.05) stained for αSMA when cells were exposed to vorinostat after either TGIF1 or TGIF2 knockdown (Figure 6). Overall, our data suggest that the knockdown of either TGIF1 or TGIF2 partially blocks the antifibrotic effect of vorinostat.

To test whether the simultaneous knockdown of both TGIF1 and TGIF2 had any additive or synergistic effect on blocking the antifibrotic effect of vorinostat, we treated the cells with siRNA of both TGIF1 and TGIF2. However, the simultaneous knockdown of TGIF1 and TGIF2 did not cause any additive neutralization of vorinostat on αSMA mRNA (Figure 5A) or protein (Figure 5B and Figure 6) as compared to the knockdown of either the TGIF1 or TGIF2 alone.