Effects of c-Src kinase on lens diseases associated with EMT of human lens epithelial cells

Background c-Src kinase regulated the signaling pathway of epithelial to mesenchymal transition (EMT) in many cells. The purpose of this study was to investigate the effects of c-Src kinase on EMT of human lens epithelial cells in vivo stimulated by different factors. Methods Human lens epithelial cells, HLE-B3 were exposure to inammatory factors including IL-1α, IL-6, TNF-α, and IL-1β at 10 ng/mL, and high glucose (35.5 mM) respectively, for 30 mins. Activity of c-Src kinase was evaluated by expression of p-Src418 with western blot assay. To investigate activation of c-Src on EMT, HLE-B3 cells were transfected with pCDNA3.1-SrcY530F to up regulate c-Src, and pSlience4.1-ShSrc to knock down it. The expressions of c-Src kinase and molecular markers of EMT such as E-cadherin, ZO-1, α-SMA and vimentine were examined at 48 hours by RT-PCR and western blot. At 48 hours and 72 hours of transfection, cell proliferation was detected by MTT, cell mobility and migration were determined by scratch and transwell assay. Results Activity of c-Src kinase, expression of p-Src418 was upregulated by different inammatory factors and high glucose in HLE-B3 cells. When HLE-B3 cells were transfected with pCDNA3.1-SrcY530F, the expression of c-Src kinase was upregulated on both mRNA and protein level, and activity of c-Src, p-Src418 increased. The expressions of E-cadherin and ZO-1 suppressed, while the expressions of vimentin and αSMA elevated on both mRNA and protein level at the same time. Cell proliferation, mobility and migration increased along with activation of c-Src kinase. Conversely, when HLE-B3 cells were transfected with pSlience4.1-ShSrc, both c-Src kinase and p-Src418 were knocked down. The expressions of

c-Src, one of the Src family kinases (SFKs), is activated by many stimulators, such as epidermal growth factor receptor (EGFR) [17], P2RY2 (a purinergic GPCR receptor) and reactive oxygen species (ROS) [18], high glucose [19], heterotrimeric G protein-coupled receptors [20], PKA signaling [21] and the pathways of IL-1and EGFR/integrin signaling [22]. Activation of c-Src kinase is required for cell differentiation, migration and changes of intercellular junction, including cadherin-based intercellular adhesions and integrin-mediated cell-matrix adhesions of epithelial cells, particularly during EMT [23,24]. Inhibition of SFKs with their speci c inhibitors attenuates brosis in lung, pancreas, and skin, which suggest that activation of Src kinase is an attractive trigger point of organ brosis [25,26]. In lens epithelial cells, activation of Src kinase induced by serum increased cell migration, weakened cell-cell junctions, and made lens epithelial cell acquire the phenotype of mesenchymal cells [27].
c-Src is comprised of lipophilic N-terminus, followed by the regulatory SH3 and SH2 domains, catalytic protein tyrosine kinase (PTK) core and c-terminus regulatory tail [28][29][30]. The PTK domain contains the kinase domain and a conserved tyrosine residue involved in autophosphorylation. Phosphorylation of the Tyr 418 residue of the PTK domain is required for maximum kinase activity [31]. A negative regulatory domain is adjacent to the PTK domain. Phosphorylated Tyr 530 interacts and binds with the SH2 domain to keep the SFK in the inactive conformation. In the other words, c-Src kinase is activated by phosphorylation at Tyr 418 or dephosphorylation at Tyr 530 [32].
In the present study we hypothesized that activation of c-Src kinase stimulated by a variety of factors, such as in ammatory factors or high glucose could be a trigger for EMT of lens epithelial cells. By transfecting HLE-B3 cells with c-Src activated vector or Sh RNA vector, the effects of c-Src kinase on cell proliferation, mobility, migration, and EMT were observed.
Cell culture HLE-B3 cells were grown adherently in Dulbecco's modi ed Eagle's medium (DMEM, with 5.5 mM glucose) with 10% fetal bovine serum and 2mM L-glutamine, and incubated at 37℃ with 5% CO 2 . All cells used in the experiments were taken in logarithmic phase.

Groups and treatment
Groups of stimulation with in ammatory factors: in the treatment groups, HLE-B3 cells were treated with IL-1α, IL-6, TNF-α, and IL-1β at 10 ng/mL, respectively, for 30 mins. In the control group, HLE-B3 cells were cultured in DMEM (Dulbecco's modi ed Eagle's medium) with 0.5% fetal bovine serum for 30 mins. Transfection HLE-B3 cells (2×10 5 cells per well) were seeded in 6-well plates and grown overnight to 80% con uence prior to transfection. All transfections for plasmids were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Brie y, plasmid DNA-lipid complexes (4μg plasmids in 8μl Lipofectamine 2000 per well) were prepared, and then incubated for 20 minutes at room temperature. The DNA-lipid complexes were added to HLE-B3 cells in 6-well plates and were cultured for 4-6 hours. Lastly, DNA-lipid complexes were discarded and 2 ml complete medium were added. 400μg G418 (Invitrogen, Carlsbad, CA, USA) was applied to select neomycin-resistant cells.
Quantitative real-time RT-PCR Total RNA was extracted from cells using Trizol reagent (Takara, Dal ian, China), and 1 μg total RNA was used as the template for cDNA synthesis in a reverse transcription kit (Takara, Dal ian, China). RT-PCR was performed in the SYBR Green kit (Takara, Dal ian, China) using speci c primers for c-Src, E-cadherin, ZO-1, vimentin and αSMA ( Table 1). The relative expression levels of genes were nor malized to the endogenous housekeeping gene GAPDH.

Western blot
Cells were harvested and lysis in RIRA cell lysate (20 mM Tris-HCl, 1% NP-40, 0.5 mM PMSF), and extracted for total proteins. The proteins were quanti ed by BCA assay kit (Beyotime Biotechnology, Beijing, China) and added with 5×loading buffer (250mM Tris-HCl (pH6.8), 10% SDS, 0.5% BPB, 50% Glycerine, 5% 2-Mercaptoethanol). 35μg total proteins from each sample were uploaded and separated in 12% SDS-PAGE at 120V voltage, then transferred onto PVDF membranes at 40 mA for 2.5 hours at room temperature. According to the standard protein marker, the PVDF membrane was cut according to the molecular weight of the target protein. After blocking by 5% defatted milk powder for 2 hours at room temperature, PVDF membrane bands were incubated with primary antibodies overnight at 4°C, such as, c- was replaced with 100 μl MTT (5 mg/ml), and the plate was incubated at 37 °C for another 4 hours. After incubation, the culture medium was removed gently, and 100 μl DMSO was added. Finally, the absorbance was determined on a microreader (Bio-Rad) at 570 nm. All experiments were performed 3 times independently. The cell proliferation diagram was plotted using the absorbance at each time point.
Scratch assay HLE-B3 cells (2×10 5 cells/well) were seeded in 6-well plates. On the second day, cells were transfected with pCDNA3.1-c-Src Y530F , pSlience4.1-ShSrc and control vectors. After 24 hours, cells in each well were scratched with 200μl pipette tip. Once scratch was made, the plates were gently washed with PBS for 3 times, and then added 2 ml serum-free medium. Cell mobility was examined after 24 hours and 48 hours, respectively. The images just after scratching 0 hour (T0), 24 hours (T24) and 48 hours(T48) were taken with a digital camera (Olympus DP71, Japan) connected to an inverted microscope (Olympus IX71, Japan). Ten elds of each plate were picked randomly and marked. Measurements of the width of gap were repeated 3 times at the same eld. Gap closure (%) = [Gap in width (T0-T24/48)/Gap in width T0] × 100%.

Transwell assay
Cell migration was determined using transwell assay (Corning incorporated, NY, USA). HLE-B3 cells (2×10 5 cells/well) were seeded in 6-well plates. On the following day, cells were transfected with pCDNA3.1-c-Src Y530F , pSlience4.1-ShSrc vector and control vectors. After transfection for 24 hours, cells in each group were trypsinized and seeded in matrigel coated lters (2x10 4 cells/per well) and cultured with 100 μl serum-free medium. 600 μl completed medium was added into the lower compartment of chamber. After incubation for 24 hours and 48 hours, cells on the upper surface of the lter were wiped off with a swab, while cells invaded through the lter were xed with 95% ethanol, stained with crystal violet and counted under the microscope. Relative migration was based on the average number of cells on the underside of the membrane in ten random images generated at 4 × magni cation under the microscope.

Data analysis and statistics
Each experiment was repeated 3 times independently and all results were presented as mean ± standard deviation (SD). All data were analyzed using SPSS 19.0 software. Multiple-group comparison was performed by analysis of variance (ANOVA), followed by LSD test for between-group comparison. Values of P < 0.05 were considered as signi cant and indicated by asterisks in the gures.

Results
In ammatory factors and high glucose activated c-Src kinase Using Western blot assay, we found that after treatment with in ammatory factors IL-1α, IL-6, TNF-α and IL-1β for 30 mins, the activity of c-Src kinase (gray ratio of p-Src 418 /c-Src) in HLE-B3 cells was enhanced signi cantly, compared with the control group. The effect of TNF-α on activation of c-Src kinase was the strongest one (Fig. 1A). The expression of p-Src 418 in 35.5 mM glucose group was signi cantly higher than that in 5.5 mM glucose group and mannitol group, while in mannitol group it was almost the same as in 5.5 mM glucose group (Fig. 1B). These results suggested that both in ammatory factors and high glucose stimulated activity of c-Src kinase in HLE-B3 cells.
Alteration of c-Src kinase activity in HLE-B3 transfected with pCDNA3.1-c-Src Y530F vector or pSlience4.1-ShSrc vector First, pCDNA3.1-c-Src Y530F and pSlience4.1-ShSrc vectors were constructed and transfected into HLE-B3 cells for 48 hours. To evaluate the active and inhibitive effects on c-Src kinase, respectively, RT-PCR and Western blot assay were applied to examine the expressions of c-Src kinase on mRNA and protein levels.
In HLE-B3 cells transfected with pCDNA3.1-c-Src Y530F (group pCDNA3.1-c-Src Y530F ), the expressions of c-Src mRNA and protein were higher than two control groups (group pCDNA3.1 and group control) signi cantly. Furthermore, active c-Src kinase, the expression of p-Src 418 was much higher than controls (Fig. 2A&B), suggesting that c-Src kinase was activated by transfected with pCDNA3.1-Src Y530F vectors in the cells. In HLE-B3 cells transfected with pSlience4.1-ShSrc vector (group ShSrc), obvious suppressions of c-Src in mRNA and protein level were demonstrated, and p-Src 418 protein expression were also decreased ( Fig. 2C&D), implying the silencing of endogenous c-Src expression by ShRNA.

Activation of c-Src kinase promoted EMT of HLE-B3 cells
To explore the biological roles of activation of c-Src kinase in EMT of LECs, we further examined the expression of the epithelial cell proteins such as E-cadherin and ZO-1, and the mesenchymal cell proteins such as vimentin and αSMA in cells of group pCDNA3.1-c-Src Y530F , which c-Src kinase was activated. It was shown that expressions of E-cadherin and ZO-1 reduced signi cantly, and vimentin and αSMA increased dramatically compared with cells in control groups (group pCDNA3.1 and group control) by RT-PCR and Western blot, respectively (Fig. 3A&B). In pSlience4.1-ShSrc vector transfected cells, which c-Src was knocked down, the expressions of E-cadherin and ZO-1 increased, while vimentin and αSMA reduced signi cantly compared with control cells (group ShNC and group control), respectively (Fig. 3C&D). Altogether, activation of c-Src kinase could induce EMT process in HLE-B3 cells.
Activation of c-Src kinase stimulated cell proliferation MTT assay showed that proliferation of cells in group pCDNA3.1-c-Src Y530F increased by 9%, 9%, 25% and 39% compared with that in group pCDNA3.1 at 12 hours, 24 hours, 48 hours and 72 hours, respectively. And the proliferation of cells in group pCDNA3.1-c-Src Y530F increased by 4%, 9%, 21% and 37% compared with that in group control at 12 hours, 24 hours, 48 hours and 72 hours, respectively (Fig.  4A). While in cells of group ShSrc, the proliferation did not change at 12 hours compared with two control groups, but reduced by 2%, 7% and 13% compared to group ShNC and 3%, 8% and 14% compared to group control at 24 hours, 48 hours and 72 hours, respectively (Fig. 4B). It indicated that activation of c-Src kinase stimulated cell proliferation.
Activation of c-Src kinase increased cell mobility and migration By scratch assay, gap closure in cells of group pCDNA3.1-c-Src Y530F increased by 84%, 60% compared with group pCDNA3.1 and 137%, 65% compared with group control at 24 hours and 48 hours, respectively, which suggested the enhancing of migration ability after activing c-Src kinase in HLE-B3 cells (Fig. 5A). While in HLE-B3 cells transfected with pSlience4.1-ShSrc vector (group ShRNA), gap closure reduced by 63%, 62% compared with group ShNC and 65%, 65% compared with group control at 24 hours and 48 hours, respectively (Fig. 5B).
In transwell assay, the migrating cell number in group pCDNA3.1-c-Src Y530F increased by 71%, 152.8% compared with group pCDNA3.1 and 83.9%, 177.9% compared with group control at 24 hours and 48 hours, respectively (Fig. 6A)., and vice versa, in cells of group ShSrc, this number reduced by 31.5%, 41.6% compared to group ShNC and 29.6%, 40.3% compared with group control at 24 hours and 48 hours, respectively (Fig. 6B). It suggested that activation of c-Src could induce cell mobility and migration.

Discussion
EMT is a conserved and essential process shared by developmental morphogenesis and carcinogenesis as well as physiological response to injury, operation, and tissue brotic diseases. In lens brotic diseases, EMT is an important pathological process, for instance, in ASC and PCO. Our present study showed that in ammatory factors and high glucose stimulated the activity of c-Src in HLE-B3 cells, and activation of c-Src kinase promoted EMT process, cell migration and proliferation in HLE-B3 cells, and vice versa, when c-Src kinase was inhibited. All these data suggested that c-Src may be a key regulator in the lens diseases associated with EMT.
Activation of c-Src kinase could promote EMT process, cell migration and proliferation in HLE-B3 cells, which is consistent with the role of c-Src kinase in tumor. Activation of c-Src kinase affected EMT process and enhanced cell migration and proliferation in lots of cancer cells [37,38]. In various cancers, such as breast cancer, pancreatic cancer and castration-resistant prostate cancer, activation of c-Src increased cell invasiveness through altering activity of cadherins, adhesion proteins and integrins [39].
Src activation stimulates downstream kinase such as extracellular signal-regulated kinase (ERK) [40] and GSK3 [41,42], which involved in the regulation of cell survival, proliferation, and promotion of EMT. Activated Src interaction with p120-catenin may cause dissociation of cell-cell junctions facilitating cell mobility [43]. Similarly, LECs in EMT process underwent losing of cell adhesion from the epithelial cells and gaining ability of proliferation and migration, transformed towards the mesenchymal cells [44]. In broblasts, the binding of integrins to their ligands leaded to activation of focal adhesion plaques adhesion kinase (FAK), which, in turn, recruits and activates c-Src [45]. Furthermore, activation of c-Src is required to disrupt cadherin-dependent cell-cell contacts [46].
Our results showed that activation of c-Src reduced the expression of E-cadherin at the protein and mRNA levels in HLE-B3 cells. This may be one of the mechanisms in which activation of c-Src induced EMT of lens epithelial cells. E-cadherin is the major cadherin molecule expressed in epithelial cells and is down-regulated in mesenchymal cells [47]. Loss of E-cadherin is the characteristic associated with increasing potential to invade surrounding tissues and disseminate to distant sites and is hallmark of EMT [48][49][50]. E-cadherin is a single-span transmembrane glycoprotein that maintains intercellular contacts and cellular polarity in epithelial tissues. In tumor cells, loss of E-cadherin is associated with cell invasion and metastasis [51]. In pancreatic ductal adenocarcinoma (PDAC) cell lines, overexpression of activated c-Src induced down-regulation of E-cadherin [52]. c-Src binding to E-cadherin, disrupted cell-cell interaction, enabled cancer cells to detach from their original site [53]. These supported our ndings that activation of Src kinase increased cell motility and induced EMT.

Availability of data and materials
All data generated or analysed during this study are included in this published article. More details are available from the corresponding author on reasonable request. Tables   Table 1 Quantitative