Down-regulation of 14-3-3 Zeta Inhibits TGF-β1–Induced Actomyosin Contraction in Human Trabecular Meshwork Cells Through RhoA Signaling Pathway

Abstract

Purpose: The aim of this study was to describe the expression and distribution of 14-3-3 zeta in trabecular meshwork (TM) cells and its regulatory role in the actomyosin system.

Methods: The expression of 14-3-3 zeta was detected using Western blot analysis, RT-PCR, and immunofluorescence staining. TGF-β1 was used to induce cell contraction. Changes in the levels of 14-3-3 zeta, total RhoA, and the phosphorylation of myosin light chain (MLC) and cofilin were determined using Western blot analysis. The effects of 14-3-3 zeta knockdown on the actin cytoskeleton and focal adhesion were determined using immunofluorescence. The mRNA levels of fibronectin and collagen I and III were examined using quantitative RT-PCR. The contraction of TM cells was detected using collagen gel contraction (CGC) assays. The activation of the RhoA pathway was analyzed using a specific kit.

Results: The 14-3-3 zeta protein was highly expressed in TM cells. Down-regulation of 14-3-3 zeta resulted in the following: a decrease in the phosphorylation of both MLC and cofilin, a decrease in the formation of stress fibers and focal adhesion, alteration of the mRNA composition of the extracellular matrix (ECM), and the inhibition of TGF-β1-induced cell contraction. In addition, silencing of 14-3-3 zeta directly decreased total RhoA levels in TM cells.

Conclusions: Collectively, our data suggest that 14-3-3 zeta plays a crucial role in regulating cytoskeletal structures, ECM homeostasis, and TGF-β1-induced contraction in TM cells by acting through the RhoA signaling pathway.

Glaucoma is a major cause of blindness worldwide, and this disease is predicted to affect approximately 79 million people by 2020.¹ Primary open-angle glaucoma (POAG), which is characterized by elevated intraocular pressure (IOP) and the associated progressive loss of retinal ganglion cells, is the most common form of glaucoma.² Among the various factors known to contribute to the onset of POAG, elevated IOP is the primary risk factor, and lowering IOP remains the only proven clinical treatment for this condition.^3,4 In human eyes, the balance between the production and the outflow of the aqueous humor (AH) regulates IOP. Approximately 80% of AH outflows through the trabecular meshwork (TM), which therefore plays a key role in maintaining normal IOP.⁵ The TM is made up of layers of beams containing the extracellular matrix and endothelial cell-like TM cells. Contraction of TM cells reduces the size of the intercellular space, which decreases TM permeability and AH outflows.⁶ Recent studies have demonstrated that inhibitors of the actomyosin system effectively relax the contraction of TM cells, which increases outflow and lowers IOP.^7,8 However, the specific mechanisms that regulate the contraction of TM cells are not fully understood. 

One of the important ways in which posttranslational modifications alter the structure and function of many proteins is the phosphorylation of Ser, Thr, and/or Tyr residues by different protein kinases.⁹ 14-3-3 family are adapter proteins, and they include seven isoforms named zeta (ζ), eta (η), gamma (γ), sigma (σ), epsilon (ε), theta (θ), and beta (β).¹⁰ The 14-3-3 proteins recognize and specifically bind to certain sites on phosphorylated proteins, and more than 300 protein targets have been shown to interact with 14-3-3 proteins.¹¹ The members of the 14-3-3 family of proteins are ubiquitous, and they participate in numerous molecular processes.¹² 

Recent reports have provided evidence showing that 14-3-3 proteins play an important role in the regulation of cytoskeleton.^13–15 These proteins interact with phosphorylated cofilin and upstream regulators (e.g., LIM kinase and TES kinase), through which they are conserved in phosphorylated and stabilized actin filaments.¹⁶ The 14-3-3 proteins also participate in the phosphorylation of myosin regulatory light chains, which regulate the contractile activity of smooth muscle and nonmuscle cells. Phosphorylated Ser472 in the largest subunit of the myosin light chain phosphatase interacts with 14-3-3 proteins, leading to the inhibition of its activity and provoking the contraction of smooth muscles.¹⁷ When mechanical stretching was induced, the expression of 14-3-3 zeta was decreased in TM cells.¹⁸ However, very little is known about the regulatory roles of the 14-3-3 proteins in the cytoskeleton of TM cells. 

Our previous study showed that the zeta isoform of the 14-3-3 family was highly expressed in the TM, and the 14-3-3 zeta was down-regulated in the TM in a rodent model of dexamethasone-induced ocular hypertension and in a dexamethasone-treated TM cell line (data not published). However, we have not determined the underlying cellular mechanisms involved in these processes. In the present study, we investigated the function of 14-3-3 zeta in modulating the cytoskeleton of TM cells. We demonstrated that 14-3-3 zeta regulated cytoskeletal rearrangement and inhibited TGF-β 1-induced contraction by decreasing the total RhoA level. 

Materials and Methods

Cell Culture and Treatment

The primary human TM cell line was obtained from ScienCell Research Labs (Catalog#6590; Carlsbad, CA, USA). Primary human TM cells were grown in Fibroblast Medium (Catalog No. 2301; ScienCell Research Labs) and were used at the third to sixth passage. For maintenance, the cells were incubated at 37°C in a 5% CO₂ environment. To identify the primary TM cells, the expression of known markers, Matrix Gla Protein, Chitinase-3-Like-1, dexamethasone-induced cross-linked actin networks (CLANs), and up-regulation of myocilin were analyzed using immunofluorescence staining; the mRNA levels of Matrix Gla Protein, Chitinase-3-Like-1, and dexamethasone-induced up-regulation of myocilin were also determined using RT-PCR. iHTM was a gift from Vincent Raymond (Laboratory of Ocular Genetics and Genomics, Quebec City, Canada), and GTM-3 was obtained from Yuhao Peng (Glaucoma Research; Alcon Laboratory, Fort Worth, TX, USA). These cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) F-12 containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 ng/mL streptomycin) at 37°C in 5% CO₂. All experiments were performed using primary TM cells, excepted for those shown in Figure 1D. Y-27632 was purchased from ENZO (Enzo Life Sciences, Farmingdale, NY, USA). TGF-β1 was obtained from Cell Signaling Technology (Beverly, MA, USA). TM cells were serum-starved for 24 hours, and TGF-β1 (5 ng/mL) was then added into the serum-free medium and the cells were incubated for 24 hours or 48 hours. 

Figure 1

 

14-3-3 zeta is expressed in human TM tissue and cultured TM cells. (Ai) The expression of 14-3-3 zeta was observed in TM cells on the beam and in the juxtacanalicular region. Some stainings were observed in the ECM and in the wall of Schlemm's canal. (Aii) No staining was observed in the sections on the negative control slides. (B) Gene expression of 14-3-3 isoforms in TM cells. The ζ, β, θ, and ε isoforms were highly expressed, while the γ and η isoforms were expressed at low levels. The σ isoform was not detected. (C) 14-3-3 zeta was highly expressed and was distributed throughout the cytoplasm in TM cells. (D) The expression of 14-3-3 zeta in primary TM cells, iHTM cells, and GTM-3 cells. Scale bars: 50 μm.

Reverse Transcription–Polymerase Chain Reaction

Total RNA was extracted using Trizol (Invitrogen Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. RNA was reverse transcribed to cDNA using a Super First-Strand Synthesis System (Takara, Tokyo, Japan). PCR amplification of cDNAs was performed using TaKaRa Ex Taq DNA Polymerase (Takara). PCR products were electrophoresed on 2% agarose gels and visualized using Goldview staining. The primer sequences and expected product sizes are shown in Table 1. 

Table 1

 

The Primer Sequences and Expected Product Sizes for RT-PCR

 

Knockdown of 14-3-3 Zeta Using Small Interfering RNA (siRNA)

The siRNA-14-3-3 zeta and nonsilencing siRNAs were designed and synthesized by Ribo Biotech (Guangzhou, China). The siRNA sequences are shown in Table 2. 

Table 2

 

The Sequences of siRNA

 

One hundred micromolars of nonsilencing siRNA and siRNA-14-3-3 zeta were transfected into TM cells using Lipofectamine RNAimax (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocols. At 24 hours before transfection, the cell medium was removed and replaced with antibiotic-free medium. When the cells were at 20% to 30% confluence, siRNA transfection was performed. Briefly, in six-well plates, 5 μL of Lipofectamine RNAimax and 10 μL of stored siRNA were each dissolved in 250 μL of Opti-MEM (Invitrogen; Gibco) for 5 minutes. Next, these two solutions were mixed together and incubated for 15 minutes, and then 1.5 mL of growth DMEM containing 0.5 mL of transfection mixture were added to each well. After transfection for 48 hours, quantitative RT-PCR and Western blot analysis were used to determine the silencing efficiency. 

Measurement of Cytoskeleton Organization

Analysis of the cytoskeleton and the staining for vinculin (focal adhesion) were performed using an actin cytoskeleton and focal adhesion staining kit (FAK100; Millipore, Billerica, MA, USA). Cells were seeded onto glass slides. After siRNA transfection for 48 hours, cells were fixed immediately for 15 minutes in 4% PFA at room temperature and then permeabilized using 0.25% Triton X-100 in PBS for 10 minutes. After blocking the cells in 1% BSA for 30 minutes, cells were incubated with anti-vinculin (1:200) at 4°C overnight. Then, the slides were washed in PBS for 15 minutes and incubated with FITC goat anti-mouse IgG secondary antibody and TRITC-conjugated phalloidin at room temperature for 1 hour. The slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 minutes and then mounted with antifade fluorescence mounting medium. Micrographs were recorded using a Zeiss laser confocal fluorescent microscope (LSM 510 META, Carl Zeiss Jena GmbH, Jena, Germany). 

Immunofluorescence Staining

Healthy human eyes were obtained from Zhongshan Ophthalmic Center Eye Bank (Guangzhou, China) within 24 hours postmortem. The anterior segment of the eye was cut into an approximately 10 × 10 mm tissue block including the cornea, limbus, TM, and sclera. The tissues were fixed in 4% paraformaldehyde at 4°C overnight, cryo-preserved in 30% sucrose, and embedded in optimal cutting temperature compound (OCT) to prepare the tissue for frozen sectioning. Then, immunofluorescent staining was performed. Briefly, the slides were washed with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 minutes, and then blocked in normal goat serum for 20 minutes. Next, the slides were incubated with 14-3-3 zeta antibody (1:100; Abcam, Cambridge, MA, USA) at 4°C overnight. After washing the tissue in PBS for 15 minutes, the slides were incubated with fluorescence-conjugated specific secondary antibodies at room temperature for 90 minutes, washed again with PBS for 15 minutes, and stained with DAPI for 5 minutes. Finally, the slides were mounted using anti-fade mounting medium and imaged using a Zeiss laser confocal fluorescent microscope (LSM 510 META, Carl Zeiss Jena GmbH). 

Western Blot Analysis

Following application of the appropriate treatment, TM cells were rinsed and lysed in lysis buffer (Cell Signaling Technology) that included a protease inhibitor cocktail (Roche Applied Science, Roche, Basilea, Switzerland) and PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science). A total of 20 μg of each sample was separated in a 12% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking in 5% BSA for 2 hours, anti-14-3-3 zeta antibody (1:1000; Abcam), anti-p-cofilin antibody (1:1000; Abcam), anti-p-myosin light chain (MLC) antibody (1:1000; Abcam), anti-total MLC (1:1000; Cell Signaling Technology), or anti-beta-actin antibody (1:2000; Abcam) was applied and the membranes were incubated at 4°C overnight. Next, the membranes were incubated with anti-rabbit secondary antibody (1:1000; Cell Signaling Technology) for 1 hour at room temperature. Signals were detected using chemiluminescence substrates (Perkin Elmer, Inc., Covina, CA, USA). Blots were scanned and analyzed using a Kodak Image Station 4000 MM (Kodak, USA). The experiment was repeated at least in three independent tests. 

RNA Isolation and Quantitative RT-PCR (qRT-PCR)

Total RNA was extracted using Trizol according to the manufacturer's instructions. The primers for target genes were obtained from the NCBI GenBank database. QRT-PCR was performed using the FastStart Universal SYBR Green Master reagent (Roche, Basel, Switzerland) and a Roche 480 real-time PCR system. Target gene expression was calculated using the 2^{−(ΔΔC[t])} method using β-actin as the housekeeping gene. The results were presented as a relative value compared to the control group. 

The primer sequences of the target genes are shown in Table 3. 

Table 3

 

The Primer Sequences for RT-PCR

 

Collagen Gel Contraction (CGC) Assays

Collagen gel contraction assays were performed using a Cell Contraction Assay Kit (CBA-201, Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions, with minor modifications. After transfection for 48 hours, the wells of 24-well plates were incubated with 1% BSA at 37°C for 1 hour. Next, cells were trypsinized and resuspended in serum-free culture medium at 1 × 10⁶ cells/mL. Collagen type I (final concentration, 1.9 mg/mL), 5× DEME, reconstitution buffer, and suspensions of TM cells (final cell density, 2 × 10⁵ cells/mL) were mixed in an ice bath. Then, the BSA was removed, 0.5 mL of the cell mixture was transferred to each well, and the gel was incubated for 90 minutes at 37°C. After collagen polymerization, 0.5 mL of culture medium with or without Y-27632 (10 μM) was added on top of each collagen gel lattice. After 1 hour, 0.5 mL of culture medium containing TGF-β1 (5 ng/mL) was added, and the gels were separated from the bottom of the wells using a 10-μL tip. The area of the gels was examined at 24 hours and 48 hours. The area was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). For normalization, the area of the collagen gel containing untreated TM cells was set at 100%, and the fold changes in the areas for the different treatment groups are shown as a bar graph. 

RhoA Activation Assay

RhoA activation assays were performed using a specific kit (STA-403A; Cell Biolabs). Briefly, following the appropriate treatment, cells were rinsed and lysed in lysis buffer (included in the kit) for 15 minutes in an ice bath. The lysate was centrifuged for 10 minutes at 12,000 rpm at 4°C. The supernatant was then incubated with 40 μL of rho-binding domain (RBD)-beads for 1 hour in an ice bath. Next, the beads were centrifuged for 20 seconds at 12,000 rpm at 4°C, the supernatant was abandoned, and 0.5 mL of lysis buffer was added to the beads. This step was repeated three times, and 40 μL of ×SDS sample buffer was then added to the immunoprecipitate, which was then boiled at 95°C for 5 minutes. After centrifugation at 12,000 rpm at 4°C for 30 seconds, 20 μL of the sample was used for Western blot analysis. The amount of activated RhoA was determined using an anti-RhoA antibody (1:200, included in the kit). 

Statistical Analysis

Results were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Multiple groups were compared using one-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant. Graphs were constructed using GraphPad Prism6 (GraphPad Prism, Inc., La Jolla, CA, USA). 

Results

The Expression of 14-3-3 Zeta in Human TM Tissue and Cultured TM Cells

RT-PCR, Western blot analysis, and immunofluorescence were performed to evaluate the expression of 14-3-3 zeta in TM tissue. In immunofluorescence staining, 14-3-3 zeta was highly expressed in TM cells on the beam and in the juxtacanalicular region. Positive staining for 14-3-3 zeta was also observed in the ECM and the wall of Schlemm's canal (Fig. 1Ai). No staining was observed in negative control sections that were incubated with nonimmune goat IgG (Fig. 1Aii). RT-PCR was used to detect the zeta isoform and the other six isoforms in primary cultures of TM cells. As shown in Figure 1B, the mRNA expression of the zeta (ζ), beta (β), theta (θ), and epsilon (ε) isoforms were detected, while the bands for the gamma (γ) and eta (η) isoforms were weak. The band for the sigma (σ) isoform was barely detectable. Immunofluorescence staining demonstrated that 14-3-3 zeta was expressed and distributed throughout the cytoplasm in TM cells (Fig. 1C), and the Western blot analysis showed that it was strongly expressed in all three types of TM cells (primary TM cells, iHTM cells, and GTM-3 cells) (Fig. 1D). 

Effect of the Down-Regulation of 14-3-3 Zeta on Myosin Light Chain and Cofilin Phosphorylation

Myosin light chain and cofilin play pivotal roles in the regulation of dynamic rearrangements of the cytoskeleton. Their phosphorylation induces actin filament stabilization and the formation of stress fibers. As shown in Figure 2, at 48 hours and 72 hours after transfection, 14-3-3 zeta expression was decreased by approximately 50% compared to the level in the control and NC groups (P < 0.05). Down-regulation of the phosphorylation of MLC and cofilin were accompanied by a decrease in 14-3-3 zeta at 48 hours and 72 hours after siRNA transfection (P < 0.01). 

Figure 2

 

Down-regulation of 14-3-3 zeta decreases the phosphorylation of cofilin and myosin light chain in TM cells. After 48 hours and 72 hours of siRNA transfection (in normal serum culture), the expression levels of 14-3-3 zeta (A), phosphorylated cofilin (B), and MLC (C) were significantly decreased compared to the control group and NC group in TM cells. Fold changes are shown as the mean ± SD (n = 3, **P < 0.01).

Down-Regulation of 14-3-3 Zeta Induces Cytoskeletal Changes in TM Cells

After transfection for 24 hours, TM cells were serum-starved for another 24 hours or 48 hours, and immunofluorescence staining was then performed to examine changes in actin stress fibers. As shown in Figure 3, after transfection of the cells for 48 hours and 72 hours, compared to the untreated cells (control group), knockdown of 14-3-3 zeta markedly reduced the formation of actin stress fibers (F-actin). The TM cells transfected with siRNAs with a randomized sequence (NC group) showed no significant difference in the amount of phalloidin staining of F-actin compared to the untreated cells. 

Figure 3

 

Down-regulation of 14-3-3 zeta induces cytoskeletal changes in TM cells. After siRNA transfection for 24 hours and subsequent serum starvation for another 24 hours or 48 hours, immunofluorescence staining showed that down-regulation of 14-3-3 zeta markedly reduced the number of actin stress fibers (F-actin, green). The TM cells transfected with a randomized siRNA sequence (NC group) were not significantly different compared to the untreated cells. The images are representative of three independent experiments. Scale bars: 50 μm.

The Down-Regulation of 14-3-3 Zeta Affects the Expression of ECM mRNAs in TM Cells

After transfection for 48 hours, the mRNA levels of fibronectin (FN), collagen I (COL1A1), and collagen III (COL3A1) were examined using qRT-PCR. As shown in Figure 4, compared to the control and NC groups, the down-regulation of 14-3-3 zeta significantly increased the mRNA level of COL3A1 by 1.99 ± 0.14-fold (P < 0.01). In addition, the mRNA level of FN was decreased to approximately 0.64 ± 0.11-fold (P < 0.01). There was no obvious difference in the mRNA level of COL1A1 (P > 0.05) in comparison to the control and NC groups. 

Figure 4

 

Down-regulation of 14-3-3 zeta changes the mRNA level of COLA1 and FN. After transfection for 48 hours (in normal serum culture), the mRNA level of FN (A) was decreased to approximately 0.64 ± 0.11-fold (n = 3, *P < 0.01), and the mRNA level of COL3A1 (B) was increased by 1.99 ± 0.14-fold (n = 3, *P < 0.01). There was no significant difference in the mRNA level of COL1A1 (n = 3, P > 0.05) (C) compared to the control group and NC group. Fold changes are shown as the mean ± SD.

The Effect of the Down-Regulation of 14-3-3 Zeta on TGF-β1-Induced Actomyosin Contraction

After transfection with siRNA-14-3-3 zeta for 24 hours and incubation in serum-free culture for an additional 24 hours, TM cells were embedded in collagen gels, and the gel areas were measured at 24 hours and 48 hours after detachment from the bottom of the wells. Cell contraction was assessed in serum-free medium and in serum-free medium containing TGF-β1 with or without Y-27632 (10 μM). TGF-β1 (5 ng/mL) was chosen, because at this concentration it was known to induce contraction of the TM cells. Y-27632, a classic Rho kinase inhibitor, was used as a positive control, because it was shown in previous studies that it could effectively block the influence of TGF-β1. Figure 5 shows that TGF-β1 induced marked contraction in the gels containing untreated and randomized siRNA sequence-transfected cells. The effect of TGF-β1 was blocked by Y-27632 and down-regulation of 14-3-3 zeta (P < 0.01). The relaxation produced by transfection with siRNA-14-3-3 zeta was comparable to that produced by Y-27632 in medium containing TGF-β1 (P < 0.01); However, compared to the effect of Y-27632 alone, the siRNA-mediated down-regulation of 14-3-3 zeta did not affect the contraction of TM cells in serum-free culture. 

Figure 5

 

The effect of 14-3-3 zeta down-regulation on CGC. After transfection with siRNA-14-3-3 zeta for 24 hours followed by incubation in serum-free culture for 24 hours, TM cells were embedded in collagen gels and treated with different medium for 24 hours and 48 hours. (A) At 24 hours, CGC was significantly induced by TGF-β1 (5 ng/mL) in the control group and NC group (n = 3, P < 0.01). TGF-β1 did not induce significant contractions in the siRNA-14-3-3 zeta-transfected group and Y-27632-treated group (n = 3, P > 0.05). After treatment with TGF-β1 for 48 hours, TGF-β1 induced a marked increase in the contraction of gels in the control group and NC group (n = 3, P < 0.01). The siRNA-14-3-3 zeta-transfected group and Y-27632-treated group showed a significant decrease in contraction compared to the control group and NC group (n = 3, P < 0.01), and the relaxation produced by siRNA-14-3-3 zeta transfection was comparable to that produced by Y-27632 in medium containing TGF-β1. Silencing of 14-3-3 zeta did not affect the contraction of TM cells in serum-free culture compared to the Y-27632 group (n = 3, P < 0.01). (B) The results of three independent experiments are expressed as the mean ± SD. The gel area was calculated using ImageJ software.

Down-Regulation of 14-3-3 Zeta Inhibits TGF-β1-Induced Remodeling of the Actin Cytoskeleton, Increased Focal Adhesion and Changes in Morphology

As shown in Figure 6, in the control and NC groups, TGF-β1 induced a marked accumulation of stress fibers that traversed the cell body, increases of phosphorylated MLC staining and punctuate staining for vinculin were also observed. This vinculin staining pattern is associated with focal adhesion complexes, which lie along stress fibers as well as diffuse cytoplasmic distribution. When cells were pretreated with Y-27632 (10 μM for 1 hour) or transfected with siRNA-14-3-3 zeta, a decrease of stress fibers, the intensity of immunostaining for vinculin, and phosphorylated MLC were observed. Consistent with these results, in the groups pretreated with Y-273632 or transfection with siRNA-14-3-3 zeta, significant shrinkage was observed, and cells assumed a more stellate appearance than the cells in the control groups. 

Figure 6

 

The down-regulation of 14-3-3 zeta blocks the TGF-β1-induced increase in stress fibers, focal adhesions, and changes in morphology. After transfection for 24 hours followed by serum starvation for another 24 hours, treatment with TGF-β1 (5 ng/mL) resulted in the accumulation of stress fibers (red), intense punctate vinculin staining (green), and P-MLC staining (green). In the siRNA-14-3-3 zeta-transfected group, these changes were blocked, and the results were similar to the Y-27632 (10 μM) group. SiRNA-14-3-3 zeta transfection and treatment with Y-27632 also led to shrinkage and retraction in the cells, which took on a stellate appearance. Images are representative of three independent experiments. Scale bars: 50 μm.

The Down-Regulation of 14-3-3 Zeta Inhibits the TGF-β1-Induced Phosphorylation of MLC and Cofilin

To further explore the up-stream regulation of actomyosin contraction and cytoskeletal rearrangements, phosphorylation of MLC and cofilin were detected by Western blot analysis. The data (Fig. 7) showed that treatment with TGF-β1 led to a significant increase in the phosphorylation of cofilin and MLC (P < 0.01) compared to the serum-free culture groups. Silencing of 14-3-3 zeta effectively inhibited TGF-β1-induced phosphorylation (P < 0.01). 

Figure 7

 

The down-regulation of 14-3-3 zeta inhibits the TGF-β1-induced phosphorylation of MLC and cofilin. Western blot analysis showed that treatment with TGF-β1 (5 ng/mL) led to a significant increase in the phosphorylation of cofilin and MLC compared to the serum-free culture groups. Silencing of 14-3-3 zeta effectively inhibited the phosphorylation of both cofilin (A) and MLC (C) that were induced by TGF-β1. (B, D) Quantitative analysis of the amount of phosphorylated cofilin and MLC. Fold changes are shown as the mean ± SD (n = 3, **P < 0.01).

The Effect of Down-Regulating 14-3-3 Zeta on RhoA Activation

As shown in Figure 8, treatment with TGF-β1 resulted in a marked increase in activated RhoA. Down-regulation of 14-3-3 zeta and treatment with Y-27632 led to a decrease in the TGF-β1-induced activation of RhoA. Interestingly, the down-regulation of 14-3-3 zeta resulted in a decrease in the total RhoA level (P < 0.05) (Figs. 8A–C). To avoid the nonspecific knockdown of total RhoA by siRNA transfection, two other siRNA sequences were investigated. The results showed that all three siRNA-14-3-3 zeta sequences effectively decreased the expression of total RhoA (P < 0.05) and 14-3-3 zeta (P < 0.01) (Figs. 8D–F). 

Figure 8

 

The effect of down-regulation of 14-3-3 zeta on RhoA activation. (A) Treatment with TGF-β1 (5 ng/mL) induced a marked increase in activated RhoA in the NC group but not in the siRNA-14-3-3 zeta or the Y-27632 group. Silencing of 14-3-3 zeta decreased the expression of total RhoA. (B–C) Quantitative analysis of activated RhoA (B) (n = 3, **P < 0.01) and total RhoA (C) (n = 5, *P < 0.05). (D) Western blot analysis showing that all three of the siRNA sequences significantly decreased the expression of 14-3-3 zeta and total RhoA in a normal serum culture environment. (E–F) Quantitative data were obtained from Western blot analysis of three independent experiments. Fold changes are shown as the mean ± SD (*P < 0.05, **P < 0.01).

Discussion

The dynamic structure of the actin cytoskeleton in TM cells plays an important role in the regulation of AH outflow. However, the underlying molecular mechanism involved in this process is not completely understood. In the present study, we demonstrated the expression of 14-3-3 zeta in TM cells and the structures of the AH outflow pathway. Our data also revealed that it played a crucial role in regulating cytoskeletal structures and TGF-β1-induced contraction in primary TM cells. 

The 14-3-3 protein family comprises seven isoforms that are ubiquitously expressed in mammalian cells.¹⁰ Although the expression profile and functions of the members of the 14-3-3 family in some ocular tissues have been reported in previous studies,^19–21 little is known about the distribution and function of 14-3-3 family in human TM and TM cells. Here, our results showed that 14-3-3 zeta was highly expressed in TM cells and AH outflow pathway tissues. With the exception of the sigma isoform, the other six isoforms were expressed in TM cells. Shankardas et al.²¹ have described the presence and distribution of 14-3-3 in human ocular surface tissues and primary culture cells. Although the TM and corneal endothelium (CE) are derived from the neural crest, the expression profiles of 14-3-3 proteins in TM cells were different from their profiles in the CE, suggesting that the TM cells used in our study might not be contaminated by other cell types. In addition, 14-3-3 isoforms in iHTM and GTM-3 cell lines showed identical profiles to those in primary TM cells (data not shown), which were also consistent with the profiles in smooth muscle cells.²² As we know, ample evidences support that TM possesses smooth muscle-like properties.²³ Taken together, our data indicate that 14-3-3 family members exhibit a tissue specific expression profiles in TM cells. 

The small GTPase Rho is a key factor in the regulation of cytoskeleton rearrangement and focal adhesion. Activated Rho interacts with Rho-associated protein kinase (ROCK) to activate its kinase activity, it then phosphorylates and inactivates MLC phosphatase, and finally phosphorylates MLC directly.^24,25 The phosphorylation of MLC leads to the formation of stress fibers and actomyosin contraction. Meanwhile, ROCK stimulates LIM kinase, which in turn phosphorylates cofilin at Ser3. The phosphorylation of cofilin results in the stabilization of F-actin.^7,26 In this study, our results revealed that silencing of 14-3-3 zeta not only caused the activation of cofilin, but also decreased the phosphorylation of MLC. Transfection with siRNA-14-3-3 zeta markedly reduced the accumulation of actin stress fibers in TM cells. Consistently, Christine E. Holt¹⁹ reported that R18 (an antagonist of 14-3-3/14-3-3 zeta) treatment significantly down-regulated the phosphorylation of cofilin in retinal ganglion cells. Thus we speculated that 14-3-3 zeta might participate in actin cytoskeletal remodeling. Moreover, silencing of 14-3-3 zeta modulated the mRNA levels of FN and COL3A1, which were synthesized by cultured TM cells, demonstrating that 14-3-3 zeta might be involved in the control of ECM synthesis in TM cells. Interestingly, knockdown of 14-3-3 zeta up-regulated the mRNA level of COL3A1. Another isoform, 14-3-3 sigma, has been reported to stimulate collagenase, which breaks down collagen type I and III in fibroblasts.²⁷ However, the TM cells did not express 14-3-3 sigma, the reasons for this activity are difficult to identify, and further research is required. Several studies have shown that sustained activation of Rho GTP signaling is associated with an increase in phosphorylated MLC, accumulation of stress fibers, and increases of the expression of ECM factors.²⁸ We therefore wondered whether 14-3-3 zeta could directly influence the activation of the RhoA pathway in TM cells. 

The concentrations of bioactive molecules such as TGF-β and endothelin-1, which activate the RhoA pathway, have been reported to be elevated in the AH of glaucomatous eyes.^29–31 However, TGF-β 2 is increased in the AH of POAG patients, its main biological function in TM cell is the stimulation of ECM secretion.^32,33 TGF-β1 has been shown to induce the contraction of collagen gels containing various types of cells, including bovine TM cells.^34–36 Given that the present study was focused on the relationship between actomyosin contraction and 14-3-3 zeta, we chose TGF-β1 for our experiments. 

In the CGC assay, silencing of 14-3-3 zeta significantly blocked TGF-β1-induced contraction, suggesting that the relaxation in the gels that was produced by silencing of 14-3-3 zeta might be the result of inhibited actomyosin contraction. To explore the mechanisms underlying these effects, we examined factors known to regulate actomyosin contraction. Consistent with previous studies, treatment with TGF-β1 led to a significant increase in the phosphorylation of MLC and cofilin, as well as increases in stress fiber formation and focal adhesion. As expected, down-regulation of 14-3-3 zeta attenuated those changes. TGF-β1 is known to signal through noncanonical pathway included Rho GTPase signaling pathways,^37,38 which is a key regulator of actomyosin contraction. Thus above results might strongly support our previous hypothesis that 14-3-3 zeta is involved in the RhoA signaling pathway. 

In the RhoA activation assay, the ROCK inhibitor Y-27632 markedly attenuated the activation of RhoA without changing the level of total RhoA.³⁹ Surprisingly, knockdown of 14-3-3 zeta decreased not only the level of activated RhoA but also the total RhoA level. These phenomena have not been previously reported in any cell type. Therefore, two other 14-3-3 zeta siRNA sequences were used to eliminate the possibility of nonspecific siRNA silencing, but there was no significant difference in the effects of those three sequences, which indicated that the down-regulation of 14-3-3 zeta specifically altered the total RhoA level. Taken together, we concluded that the relaxation of actomyosin contraction induced by knockdown of 14-3-3 zeta was partly attributable to decreases in the level of total RhoA in TM cells. 

Similar to other GTPases, RhoA contains both GDP-bound and active GTP-bound states, but the mechanisms by which RhoA is activated are incompletely understood. RhoA is activated primarily by guanine nucleotide exchange factors (GEFs) via phosphorylation, and it is then sequestered by Rho GDIs that remove the protein from the membrane while preventing its further interaction with other downstream effectors.⁴⁰ Dario Diviani et al⁴¹ showed that the Rho-GEF complex is inhibited when both PKA and 14-3-3 are anchored, because Rho then fails to interact with 14-3-3 or PKA to induce Rho-GEF activity. Ngok et al⁴² also showed that 14-3-3 isoforms interact with Syx, another Rho-GEF, in a phosphorylation-dependent manner in which GEF activity is inhibited by Syx. The difference between the above results and the results of our study could be the methods chosen to alter the function of 14-3-3. The mechanisms involved in these processes are difficult to characterize, and more in-depth studies will be conducted in the future. 

In conclusion, the data presented here suggest that the down-regulation of 14-3-3 zeta leads to the inhibition of TGF-β1-induced contraction by decreasing the expression of total RhoA in TM cells. Our study provides novel insight into the relationship between 14-3-3 zeta and cytoskeleton. These results lay a foundation for exploring the potential role of 14-3-3 zeta in the regulation of AH outflow and pathogenesis of glaucoma. 

Acknowledgments

Supported by grants from the National Natural Science Foundation of China (81170848, 81200685) and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology (NO.2014QN06). 

Disclosure: Y. Ye, None; Y. Yang, None; X. Cai, None; L. Liu, None; K. Wu, None; M. Yu, None