Molecular Vision 2007; 13:2248-2262 <http://www.molvis.org/molvis/v13/a255/> Received 23 May 2007 | Accepted 27 November 2007 | Published 29 November 2007

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The role of Hsp70 and Hsp90 in TGF-β-induced epithelial-to-mesenchymal transition in rat lens epithelial explants

Alice Banh,¹ Paula A. Deschamps,² Mathilakath M. Vijayan,³ Jacob G. Sivak,¹ Judith A. West-Mays²
 
 

¹School of Optometry, University of Waterloo, Waterloo, ON, Canada; ²Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada; ³Biology, University of Waterloo, Waterloo, ON, Canada

Correspondence to: Alice Banh, School of Optometry, University of Waterloo, Waterloo, ON, N2L 3G1 Canada; Phone: (650) 725-7805; FAX: (650) 723-7382; email: abanh@scimail.uwaterloo.ca

Abstract

Purpose: This study investigates the effects of heat shock treatment and the role of Hsp70 and Hsp90 on tranforming growth factor beta 2 (TGF-β2)-induced epithelial-to-mesenchymal transition (EMT) in rat lens epithelial explants.

Methods: Rat lens epithelial explants from 7-10 day-old Wistar rats were dissected and incubated for 24 h before treatment. The explants were divided into eight treatment groups: control (culture medium), fibroblast growth factor-2 (FGF-2), TGF-β2, and TGF-β2+FGF-2 under normal culture conditions and heat shocked conditions. The explants were heat shocked at 45 °C before treatment with the respective media. H&E staining was performed on whole-mount epithelial explants from each group. Immunofluorescence staining for α-smooth muscle actin (α-SMA), F-actin, and E-cadherin was also used to determine EMT and fibrotic plaque formation in the lens epithelial explants. Apoptotic cell death was determined using the TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) assay. Confocal microscopy was used to visualize immunoreactivity in the whole-mount epithelial explants. Western blot analysis of α-SMA, E-cadherin, Hsp70, and Hsp90 were also performed.

Results: TGF-β2-induced EMT and plaque formation in the lens epithelial explants. The simultaneous treatment of epithelial explants with TGF-β2+FGF-2-induced the most significant morphological changes and EMT. Heat shock treatment of lens epithelial explants before TGF-β2 treatment did not inhibit plaque formation, but there was significant reduction of α-SMA expression and greater E-cadherin expression when compared to the non-heat shocked TGF-β2-treated explants. Interestingly, TGF-β-induced apoptotic cell death was significantly lower in the heat shocked explants compared to the non-heat shock lens explants. Heat-induced accumulation of Hsp70 and Hsp90 expression was reduced in the heat shocked groups at day 4 of treatment.

Conclusions: TGF-β2-induced EMT was significantly reduced in the heat shocked TGF-β2 lens epithelial explants. After four days of culture, there is a reduction in expression of Hsp70 and Hsp90 in the heat-shocked groups, indicating that the lens epithelial cells are under a less stressful condition than the non-heat shocked groups. In conclusion, molecular chaperones can play a protective role against TGF-β2-induced EMT and enhance cell survival.

Introduction

The ocular lens is a transparent structure that provides part of the refractive power needed to focus images on the human retina. A cataract results in reduced transparency of the lens, which leads to vision loss. Recently, there has been great interest in human anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO), both of which are secondary cataracts formed from residual lens epithelial cells (LECs) after cataract surgery [1,2]. Understanding the mechanisms involved in these types of cataract development can lead to prevention and treatment.

Transforming growth factor beta (TGF-β) is a secreted polypeptide that is involved in various cellular processes including cell proliferation, differentiation, apoptosis, migration, and extracellular matrix (ECM) formation [3-7]. Under physiologic conditions, TGF-β in the lens and ocular media mainly exists in its latent form whereas an increase of biologically active TGF-β has been detected in the ocular media from patients suffering with ASCs [8,9]. Previous in vivo and in vitro studies have shown that TGF-β-induced epithelial-to-mesenchymal transition (EMT) in transgenic mice and cultured rat LECs results in the formation of multilayer plaques and in the transdifferentiation of LECs to myofibroblasts/fibroblastic or spindle-like cells. The EMT is also accompanied by capsular wrinkling, apoptosis, α-smooth muscle actin (α-SMA) expression, and an aberrant deposition of ECM such as collagen type I and III, fibronectin, and tenacin [2,3,10-13]. It has been established that these TGF-β-induced cataractous changes are associated with ASC and PCO development [1-3,13-15]. However, the exact mechanisms involved in TGF-β-induced EMT in LECs are still under investigation.

Heat shock proteins (Hsps) are molecular chaperones that were initially identified as proteins expressed after exposure of cells to environmental stress. However, it has been proven that Hsps also play a crucial role in proper protein assembly, folding, transport, and degradation under normal conditions [16,17]. The heat shock proteins are divided into families that are classified according to their molecular weight [17-19], and each family of Hsps recognizes and interacts with various non-native polypeptides through different modes of binding [20]. Previous studies have shown that molecular chaperoning activities of Hsp70 and Hsp90 within the lens are critical in maintaining the lens transparency [21-23]. However, the role of heat shock proteins in LECs during EMT is unclear.

Hsp70 is another heat shock protein present in the lens epithelium and superficial cortical fibers of the adult human lens [22]. Hsp70 is required for correct folding, assembly, intracellular targeting, and degradation of polypeptides and oligomeric proteins [24]. The constitutive form, Hsc70, and the inducible forms of Hsp70 are present in the lens under normal unstressed conditions. It is suggested that the normal microenvironment of the lens is stressful, therefore, requiring continuous expression of inducible Hsp70 [22,25,26]. Studies using bovine lenses, mouse LECs, and rat lenses show that Hsp70 expression is upregulated under heat, oxidative, osmotic, and mechanical stresses [23,27].

Hsp90 is one of the most abundant proteins in unstressed eukaryotic cells (1%-2% of all cellular protein). In mammalian cells, there are two functionally similar Hsp90 isoforms, Hsp90α and Hsp90β [28]. Hsp90 is involved in regulating the activity of intracellular proteins such as steroid hormone receptors and protein kinase [28,29]. Interestingly, TGF-β has been shown to regulate the expression levels of both Hsp70 and Hsp90 in cultured chicken embryo cells (CEC) [30]. Hence, heat shock proteins may play a protective role in the LECs during TGF-β-induced EMT.

The present study investigates the effects of heat shock treatment and the role of molecular chaperones in TGF-β2-induced EMT in rat lens epithelial explants. Fibroblast growth factor-2 (FGF-2) was also used to exacerbate the effects of TGF-β as described in previous studies [31-33]. The morphological and molecular changes of the rat lens epithelial explants were examined, and the expressions of α-SMA, F-actin, and E-cadherin were used as indicators of EMT. The protein expression levels of Hsp70, Hsp90, and apoptotic cell loss were also analyzed. The findings of this study show that heat shock treatment reduces the effects of TGF-β-induced EMT in explanted rat LECs. Heat shocked TGF-β epithelial explants demonstrated lower α-SMA expression and greater retention of E-cadherin expression and organization than non-heat shocked TGF-β explants. TGF-β-induced apoptosis was also reduced in the heat shock rat explants. Interestingly, this study also shows that Hsp70 and Hsp90 protein expressions in the heat shock treated FGF-2, TGF-β, and TGF-β2/FGF-2 rat LECs are significantly lower than their respective non-heat shocked groups at four days after initial treatment.

Methods

In vitro rat lens epithelial explants

All animal studies were performed according to the Canadian Council on Animal Care Guidelines and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. Lenses were dissected from Wistar rats (7-10 days old) after euthanization by cervical dislocation. The lens was placed with its anterior side down onto a 35 mm laminin-coated culture dish (BD Biosciences, Mississauga, ON, Canada) containing specialized serum-free medium M199 with antibiotics [34]. Lens epithelial explants were prepared by peeling the posterior pole of the lens capsule and pinning the anterior capsule to the culture dish with the epithelial cells facing upwards. The lens fiber mass was removed and discarded [12,32]. This provided a primary rat LEC culture on an intact anterior lens capsule and served as the substratum for the cells. The lens epithelial explants were incubated at 37 °C with 4.0% CO₂ for 24 h before any treatment to ensure that they were not damaged during the dissection. Only explants with a confluent monolayer of LECs were used. The concentration of FGF-2 and TGF-β2 used for treatment was 10 ng/ml (FGF-2; PeproTech Inc., Rocky Hill, NJ) and 8 ng/ml (TGF-β2; Cedarlane Laboratories Ltd., Burlington, ON, Canada). The explants were divided into eight treatment groups: under normal culture conditions, control (culture medium), FGF-2, TGF-β2, and TGF-β2/FGF-2 (simultaneous treatment with both TGF-β2 and FGF-2) and under heat shocked conditions, control (culture medium), FGF-2, TGF-β2, and TGF-β2/FGF-2. The culture medium was replaced 24 h after dissection with 3.5 ml of the appropriate treatment medium. The normal conditioned groups (control, FGF-2, TGF-β2, and TGF-β2/FGF-2) were placed back into the incubator. The heat shocked explants were heat shocked at 45 °C for 1 h and stabilized at 37 °C with 4.0% CO₂ for 3 h before treatment with the respective media as mentioned above. The explants were cultured for four days in the treatment media and then prepared for histological, immunohistochemical, western blot, and TUNEL analysis.

Histology and immunohistochemistry staining

A total of 128 rat epithelial explants were used for histological and immunohistochemical analysis. Four explants from each of the eight treatment groups were prepared for each of the four different staining procedures. The whole-mount of lens epithelial explants was used for either hematoxylin and eosin (H&E) or immunohistochemistry staining (F-actin, α-SMA, and E-cadherin). The lens epithelial explants were fixed in 4% paraformaldahyde for 30 min, then permeablized with 0.1% TritonX-100 in 1X phosphate buffered saline (PBS) solution for 15 min at room temperature. H&E staining procedures were performed on the epithelial explants. The epithelial explants were examined with a Leica microscope, and images were captured using a high-resolution camera and associated software (OpenLab; Quorum Technologies Inc., Guelph, ON, Canada). Images were reproduced for publication using Adobe Photoshop 9.0.1 (Adobe Systems Inc., San Jose, CA).

For α-SMA and E-cadherin immunofluorescence localization, the whole-mount lens epithelial explants were incubated with 5% normal goat serum for 30 min at room temperature. The explants were then incubated overnight at 4 °C either with mouse anti-α-SMA monoclonal antibody (1:400; Sigma-Aldrich Ltd., Oakville, ON, Canada) or mouse anti-E-cadherin antibody (1:100; BD Biosciences). An Alexa Fluor 488 conjugated goat anti-mouse secondary antibody (1:100; Invitrogen, Burlington, ON, Canada) was used for detection of the bound primary antibodies. All whole-mount explants were mounted in Vectashield mounting medium with 4', 6-Diaminodino-2-Phenylindol (DAPI; Vector Laboratories Inc., Burlingame, CA) to visualize the nuclei. A Zeiss scanning laser confocal microscope (LSM 510 META; Carl Zeiss Canada Ltd., Toronto, ON, Canada) equipped with an Argon-Krypton laser was used to examine immunoreactivity of the epithelial explants. Cross-sections of the explants were visualized with confocal z-stack function with an optical slice thickness of 0.3 μm. The images were captured using the Zeiss LSM software. Adobe Photoshop 9.0.1 was also used to reproduce images for publication.

For F-actin localization, the whole-mount lens epithelial explants were incubated with 5% normal goat serum for 30 min at room temperature. The explants were then incubated with Alexa 546 phalloidin (1:50; Invitrogen, Inc.) for 30 min at room temperature. The epithelial explants were mounted in Vectashield mounting medium with DAPI to visualize the nuclei. Laser scanning confocal microscopy was performed as described above.

TUNEL assay

TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) labeling was used to examine cell death in three lens epithelial explants of each treatment group. A total of 24 explants were used. The whole-mount epithelial explants were fixed and permeabilized as described above. The ApopTag plus flourescein in situ apoptosis detection kit (Millipore, Billerica, MA) was used to detect apoptotic nuclei, and the TUNEL procedure was performed in accordance with the manufacturer's instructions. A positive control was prepared by treating a sample with DNaseI before TUNEL staining. All sections were mounted in Vectashield mounting medium with DAPI and photographed using the laser scanning confocal microscope. For quantitative analysis, the percentage of TUNEL-positive cells among 150 lens epithelial cells in three fields per section was determined at 250 fold magnification.

Western blot analysis

Seventy-two epithelial explants from each of the eight treatment groups were collected and pooled (in groups of four explants) for western blot analysis of α-SMA and E-cadherin. In addition, both Hsp70 and Hsp90 protein expression were analyzed 24 h and four days after initial treatment. The lens epithelial explants were homogenized in Triton-X100 lysis buffer containing protease inhibitor cocktail (Roche Applied Science, Laval, QC, Canada). Total protein concentration was determined with the Bradford protein assay [35]. Equal amounts of total protein from each group of explants were electrophoresed on 10% SDS polyacrylamide gels. In addition, a heat shocked HeLa cell lysate (Assay Designs, Ann Arbor, MI) was loaded and used as a positive control for heat shocked proteins. The proteins were electro-transferred onto a nitrocellulose membrane (Pall Corporation, East Hills, NY). Membranes were blocked with 5% skimmed milk powder in Tris-buffered saline (50 mM Tris base, NaCl pH 8.5) + 0.1% Tween-20 and then incubated overnight at 4 °C with either a mouse anti-α-SMA monoclonal antibody (1:1000), a mouse anti-E-cadherin antibody (1:2500), rabbit anti-Hsp70 polyclonal antibody (1:2000), or rabbit anti-Hsp90 polyclonal antibody (1:2000). Following this incubation, membranes were probed with the appropriate HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:5000; Amersham Biosciences, Piscataway, NJ) and enhanced chemiluminescent (ECL) detection reagents (Amersham Biosciences). The western blots were visualized by X-ray film exposure. The Hsp70 and Hsp90 antibodies were purchased from Assay Designs. The membranes were stripped and reprobed with a mouse anti-β-actin antibody (1:1000; Cedarlane Laboratories Ltd.) as a loading control. The secondary detection for β-actin followed the same procedures as mentioned above. The bands were quantitated by densitometry and normalized with β-actin using ImageJ software (software developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). Protein expression is expressed as a percentage of normal control ±standard error of the mean (%±SEM).

Statistical analysis

The analysis of variance (ANOVA; SPSS^TM 11.0 statistical software) was used to assess the treatment effects on protein expression and apoptotic cell death of rat lens epithelial explants. The Tukey's post-hoc test was use to determine the significance between treatment groups. A p-value that was less than or equal to 0.05 was considered to be significant.

Results

Histological and immunohistochemical analysis of the rat lens epithelial explants

Histological examination of cultured lens epithelial explants from 7-10 day-old rats treated with TGF-β2 and TGF-β2/FGF-2 under both normal and heat shocked conditions revealed distinct evidence of EMT including extensive multi-layering of cells forming plaques on the anterior lens capsule (Figure 1 and Figure 2E-H). In contrast to the plaques formed on the TGF-β2/FGF-2 treated explants (Figure 2G,H), which are typically large and diffuse, the plaques in the TGF-β2 explants (Figure 2E,F) were substantially smaller. Capsular wrinkling as well as fibroblastic and spindle shaped cell formation also occurred in the TGF-β-treated explants (not shown). The videos associated with Figure 1 and Figure 2 also demonstrate the different morphology of a monolayer control explant and multilayering effect of TGF-β2/FGF-2 treated explant on a y-axis confocal projection. The control and FGF-2 epithelial explants demonstrated a confluent monolayer of LECs with a cobblestone pattern and which did not form multilayer plaques (Figure 2A-D). However the FGF-2-treated explants exhibited a more rounded cellular appearance and greater cell density per area than the controls. The cellular morphology of heat shocked groups (Figure 2B,D,F,H) did not differ significantly from the normal condition groups (Figure 2A,C,E,G). Heat shock treatment did not inhibit TGF-β-induced plaque formation. But the plaques formed on the heat shocked TGF-β2/FGF-2-treated explants were also less severe and spanned a smaller area of the explants when compared to the non-heat shocked TGF-β2/FGF-2 plaques.

The expression of F-actin and α-SMA were examined next since these are commonly used as markers for TGF-β-induced EMT (Figure 3 and Figure 4) [36]. The control explants (Figure 3A,B) demonstrate normal polygonal arrays of F-actin stress fibers underlying the apical location of the LEC membranes [37]. Interestingly, treatment with FGF-2 significantly reduced the F-actin immunoreactivity in the LEC cytoplasm, and the F-actin expression was mainly confined to the cell borders. The TGF-β2-treated explants (Figure 3E,F)demonstrated a slight increase in F-actin immunoreactivity and stress fiber extension when compared to the controls (Figure 3A,B). By comparison, the simultaneous treatment of epithelial explants with TGF-β2 and FGF-2 (Figure 3G,H) induced the formation of substantially longer peripheral extension of F-actin filaments in the plaques. There was no significant difference in F-actin immunoreactivity between the normal cultured explants (Figure 3A,C,E,G) and heat shocked explants (Figure 3B,D,F,H). The increase in stress fiber formation and reorganization in the lens epithelial explants demonstrates the occurrence of TGF-β-induced EMT in the LECs.

Expression of α-SMA was also observed in the multilayer plaques of the TGF-β2 and TGF-β2/FGF-2 lens epithelial explants whereas no α-SMA expression was detected in the FGF-2 treated explants (Figure 4). The control explants showed diffuse staining of α-SMA in the cytoplasm of some LECs (Figure 4A,B). The TGF-β2-treated explants (Figure 4E,F) demonstrated greater α-SMA immunoreactivity and more filamentous expression of α-SMA in the apical portion of the cells. The simultaneous treatment of epithelial explants with TGF-β2 and FGF-2 (Figure 4G,H) induced the greatest α-SMA immunoreactivity with the formation of substantially longer and extended α-SMA filaments in the plaques. Interestingly, the heat shocked epithelial explants (Figure 4B,F,H) showed significantly lower α-SMA immunoreactivity than the same treatment groups under normal culture conditions (Figure 4A,E,G, respectively). Fewer cells from the heat shocked control explants show α-SMA staining when compared to the non-heat shocked control explants. The heat shocked TGF-β2 and TGF-β2/FGF-2 explants show less filamentous α-SMA staining than the non-heat shocked TGF-β2 and TGF-β2/FGF-2 explants. The results from western blot analysis of α-SMA (42 kDa) expression in lens epithelial explants confirmed the immunostaining results (Figure 5). Statistical analysis (ANOVA: p is less than or equal to 0.05) shows that there is a significant treatment effect. The heat shocked control epithelial explants (lane 2, 55.7%±5.6%) show significantly lower levels of α-SMA protein expression compared to the normal control epithelial explants (lane 1, 100%) while the FGF-2-treated explants (normal; lane 3, 6.7%±3.5%) and heat shocked-treated explants (lane 4, 1.4%±0.7%) show negligible amounts of α-SMA. The treatment with TGF-β2 (normal, lane 5, 255.2%±3.3%; heat shock, lane 6, 170.0%±8.4%) and with TGF-β2/FGF-2 (normal, lane 7, 363.0%±4.8%; heat shock, lane 8, 293.4%±2.3%) caused a significant increase in α-SMA protein expression with the greatest increase of α-SMA expression in the TGF-β2/FGF-2 explants. In addition, the α-SMA expression of heat shocked-treated TGF-β2 and TGF-β2/FGF-2 explants is significantly lower than that of the same treatment groups under normal cultured conditions. Therefore, in contrast to F-actin expression, α-SMA expression is reduced in the heat shocked TGF-β groups.

E-cadherin is an epithelial marker, which is localized at the adherence junctions and the disassembly of E-cadherin is associated with TGF-β-induced EMT [38]. Both the control (Figure 6A,B) and FGF-2 (Figure 6C,D) treated explants show normal E-cadherin expression at the cell-to-cell junction locations. The TGF-β2-treated explants (Figure 6E,F) demonstrated lower E-cadherin immunoreactivity and a loss of E-cadherin in some LECs when compared to the controls. The simultaneous treatment of epithelial explants with TGF-β2 and FGF-2 (Figure 6G,H) induced considerable loss of E-cadherin organization and diffuse staining. Heat shock treatment did not affect the E-cadherin expression levels in both the control and FGF-2 epithelial explants. However, heat shock TGF-β2 and TGF- β2/FGF-2 (Figure 6F,H) demonstrated higher immunoreactivity and organization of E-cadherin than normal TGF-β2 and TGF-β2/FGF-2 (Figure 6E,G) explants, respectively. Results from western blot analysis of E-cadherin (120 kDa) expression in lens epithelial explants (Figure 7) confirmed the immunostaining results. Statistical analysis (ANOVA: p is less than or equal to 0.05) showed that there is a significant treatment effect. Both the normal control explants (lane 1, 100%) and heat shocked control explants (lane 2, 100.3%±1.8%) express similar levels of E-cadherin protein. FGF-2 treatment (normal, lane 3, 119.5%±8.5%; heat shocked, lane 4, 126.6%±5.9%) induced significant increase of E-cadherin expression when compared to the normal control epithelial explants. Both treatment with TGF-β2 (normal, lane 5, 70.0%±1.8%; heat shocked, lane 6, 86.7%±3.1%) and TGF-β2/FGF-2 (normal, lane 7, 45.6%±2.6%; heat shocked, lane 8, 72.6%±3.4%) demonstrated a significant decrease in E-cadherin protein expression with the greatest loss of E-cadherin expression in the TGF-β2/FGF-2 explants. However, the E-cadherin expression of heat shocked TGF-β2 and TGF-β2/FGF-2 explants were significantly higher than the same treatment groups under normal culture conditions. The decrease in E-cadherin expression coincided with increased expression of F-actin and α-SMA in TGF-β-treated explants. Therefore, these results show that TGF-β induced EMT in the rat LECs. Although heat shock treatment did not inhibit TGF-β-induced multilayer plaque formation, it reduced the severity of EMT as shown in the α-SMA and E-cadherin results.

Heat shock treatment did not induce an observable effect on cytoskeletal rearrangements at 24 h (data not shown). This is largely due to the age of the animals used for this experiment. The younger animals used in this study are less responsive than the older animals, which show greater response to TGF-β at 24 h (data not shown). Thus, it could be hypothesized that explants from older animals would show a greater heat shock effect on cytoskeletal reorganization at 24 h.

Apoptotic cell death in rat lens epithelial explants

Since TGF-β-induced multilayer plaque formation in lens epithelial explants and apoptosis has been reported to occur in TGF-β-induced anterior subcapsular cataracts (ASC) [39,40], we used TUNEL labeling to examine the level of apoptosis in the different experimental groups (Figure 8). The photographs in Figure 8 represent TUNEL staining in a normal control explant (Figure 8A) and a TGF-β2/FGF-2 explant (Figure 8B). Both TGF-β2/FGF-2 and TGF-β2 (not shown) showed an increase of TUNEL-positive nuclei in the plaques when compared to the control whereas the FGF-2 lens epithelial explants showed negligible amounts of TUNEL-positive nuclei (photograph not shown). Statistical analysis (ANOVA: p is less than or equal to 0.05) showed that there was a significant treatment effect. The heat shocked control epithelial explants (2.3%±0.6%) demonstrated less apoptotic cell death than the control explants (4.3%±0.5%) under normal culture conditions, but the decrease was not significant. FGF-2 treatment (normal, 1.9%±0.6%; heat shocked, 0.8%±0.2%) induced significant decreases in apoptotic cell death relative to the normal control epithelial explants. The normal TGF-β2 explants (7.5%±0.8%) and TGF-β2/FGF-2 (normal, 9.6%±0.6%; heat shocked, 6.8%±0.5%) demonstrated a significant increase in cell death. The increase of cell death in the heat shocked TGF-β2 (4.6%±0.7%) explants was not significantly different from normal control explants. In addition, the heat shocked TGF-β2 and TGF-β2/FGF-2 explants demonstrated significantly lower apoptotic cell death than the same treatment groups under normal culture conditions. Thus, TGF-β-induced apoptosis was reduced by heat shock treatment in rat lens epithelial explants.

Effect of the TGF-β2 on heat shock protein expressions in rat lens epithelial explants

Western blot analysis of Hsp70 expression at 24 h (70 kDa, Figure 9) and four days after initial treatment (Figure 10) showed that there was a significant treatment effect (ANOVA: p is less than or equal to 0.05) between the different groups. A heat shocked HeLa cell lysate was used as a positive control. β-Actin was used as a loading control and for normalization of band intensity. At 24 h after heat shock and initial treatment, all heat shocked-treated explants demonstrated a significant increase in Hsp70 expression when compared to the normal control. In addition, the heat shocked-treated explants: control (120.6%±4.8%), FGF-2 (121.7%±3.7%), TGF-β2 (118.9%±3.7%), and TGF-β2/FGF-2 (120.6%±6.4%) also demonstrate a significant increase of Hsp70 expression when compared to their respective normal conditioned explants: control (100%), FGF-2 (108.0%±3.7%), TGF-β2 (100.1%±4.1%), and TGF-β2/FGF-2 (97.5%±5.0%).

In contrast, Hsp70 expression levels after four days of treatment (Figure 10) showed opposing trends in Hsp70 accumulation at 24 h. Heat shock treatment did not affect Hsp70 levels in the control epithelial explants (normal, lane 1, 100%; heat shocked, lane 2, 99.6%±2.7%). However, Hsp70 expression significantly decreased in normal FGF-2 (lane 3, 73.5%±4.7%) and in heat shocked FGF-2 (lane 4, 54.6%±3.7%). The heat shocked TGF-β2 (lane 6, 83.5%±1.6%) treated explants also showed significant decrease in Hsp70 expression when compared to the control explants. In contrast, the normal TGF-β2 (lane 5, 118.0%±3.3%) and TGF-β2/FGF-2 (normal, lane 7, 211.6%±4.5%; heat shocked, lane 8, 124.7%±4.0%) explants showed significant increase of Hsp70 protein expression with the greatest increase in the TGF-β2/FGF-2 explants. In addition, the Hsp70 expression of heat shocked FGF-2, TGF-β2, and TGF-β2/FGF-2 explants are significantly lower than the same treatment groups under normal culture conditions.

Similar to Hsp70 protein expression, heat shock treatment induced a significant increase (ANOVA: p is less than or equal to 0.05) of Hsp90 expression in all treatment groups after 24 h (90 kDa, Figure 11). As mentioned above, the HeLa cell lysate was used as a positive control. β-Actin was used as a loading control and for normalization of band intensity. The FGF-2 (normal, 130.4%±11.4%), heat shocked (141.4%±6.7%), heat shocked control (120.2%±0.2%), heat shocked TGF-β2 (139.6%±0.8%), and heat shocked TGF-β2/FGF-2 (139.5%±5.0%) explants showed a significant increase of Hsp90 protein expression when compared to the normal control explants (100%). In contrast, Hsp90 expression of the normal conditioned TGF-β2 (112.0%±5.6%) and TGF-β2/FGF-2 (111.5%±5.4%) explants showed no significant difference when compared to the normal control. In addition, the Hsp90 expression of heat shocked control TGF-β2 and heat shocked control TGF-β2/FGF-2 explants was significantly higher than the same treatment groups under normal culture conditions while there was no significant difference between the normal and heat shocked FGF-2 treated explants.

The results from the Hsp90 protein analysis at four days after initial treatment (Figure 12) also showed significant treatment effect (ANOVA: p is less than or equal to 0.05). Heat shock treatment significantly decreased the Hsp90 expression levels in the control epithelial explants (lane 2, 55.6%±1.1%) and TGF-β2 epithelial explants (lane 6, 73.6%±6.0%) when compared to normal control epithelial explants (lane 1, 100%). Although there was also a decrease of Hsp90 expression in the normal TGF-β2 explant (lane 5, 93.9%±2.5%), it was not significant. The FGF-2 (normal, lane 3, 172.6%±2.5%; heat shocked, lane 4, 129.0%±3.3%) and TGF-β2/FGF-2 explants (normal, lane 7, 187.0%±5.3%; heat shocked, lane 8, 157.0%±5.3%) showed significant increase of Hsp90 protein expression with the greatest increase in the TGF-β2/FGF-2 explants. Heat shock treatment also significantly decreased the Hsp90 expression levels of FGF-2, TGF-β2, and TGF-β2/FGF-2 explants when compared to the same treatment groups under normal cultured conditions. Interestingly, the heat shocked TGF-β explants (TGF-β2 and TGF-β2/FGF-2) demonstrated lower levels of Hsp70 and Hsp90 than the TGF-β explants under normal culture conditions. This may be an indication that heat shock treatment reduced TGF-β-induced stress conditions in the epithelial explants. Thus, there was less upregulation of molecular chaperone activities in the heat shocked TGF-β explants when compared to the TGF-β explants under normal conditions.

Discussion

Overall, the results from the current study demonstrated some abatement of TGF-β-induced EMT in cells pre-conditioned by a heat shock treatment as also indicated by a reduction in the relative expression levels of Hsp70 and Hsp90 at four days after initial treatment which suggests lower stress levels.

The expression of α-SMA served as the main EMT marker in this study (Figure 4 and Figure 5). The control cultured epithelial explants demonstrated a few positive α-SMA stained LECs. Previous studies have also described α-SMA expression in primary cultured LECs including those derived from bovine, rabbit, and human [41]. The presence of α-SMA expression in control LECs has been attributed to elevated stress levels caused by culture conditions. Yet, non-cataractous human lenses have also shown low-level expression of α-SMA mRNA and protein expression in the LECs in vivo [42]. Interestingly, FGF-2 treated explants demonstrated a negligible amount of α-SMA immunoreactivity. These results are in accordance with another study using 21-day-old rat lens epithelial explants, which show no α-SMA expression in the FGF-2-treated explants and minimal α-SMA expression in the control explants [14]. Treatment with TGF-β2 substantially increased α-SMA immunoreactivity and filamentous actin morphology with the greatest effect induced in the TGF-β2/FGF-2 explants. Previously, it was shown that TGF-β and FGF have opposing effects on α-SMA expression. While TGF-β2 increased α-SMA expression, FGF-2 decreased α-SMA expression in cultured bovine LECs [43]. Furthermore, the results demonstrated a substantial reduction of α-SMA immunoreactivity and protein expression in the heat shocked control, TGF-β2, and TGF-β2/FGF-2 explants when compared to their respective non-heat shocked treatment groups. Thus, along with the morphological findings, the results of this study show that TGF-β-induced EMT is also reduced by heat shock treatment.

The loss of E-cadherin and F-actin stress fiber rearrangement are phenotypic changes associated with TGF-β-mediated EMT [36,38,44-46]. Thus, it was shown in the current study that TGF-β treatment induced the loss of E-cadherin expression and delocalization of E-cadherin from the cell junctions of the cultured rat LECs with the greatest loss found in the TGF-β2/FGF-2 treated explants (Figure 6 and Figure 7) [38,44,45]. However, the heat shocked TGF-β (TGF-β and TGF-β2/FGF-2) rat explants demonstrated significantly greater levels of E-cadherin expression and less severe E-cadherin delocalization than the non-heat shocked TGF-β groups (Figure 7). Our results are consistent with another study that demonstrated an upregulation of E-cadherin expression in human pancreatic adenocarcinoma cell lines treated with FGF [47]. In addition to E-cadherin delocalization, substantial F-actin stress fiber formation and reorganization was also seen in TGF-β2/FGF-2 treated lens epithelial explants (Figure 3). Although heat shock treatment did not prevent TGF-β-induced stress fiber reorganization, it did diminish the loss of E-cadherin expression and organization in the TGF-β-treated rat LECs.

Hsp70 and Hsp90 exhibit chaperoning activities in the lens [21]. A basal level of inducible Hsp70 is expressed in the normal control rat epithelial explants [22,25]. Treatment of lens explants with FGF-2 demonstrated reduced Hsp70 protein expression when compared to the control explants. By contrast, TGF-β induced a significant increase in Hsp70 protein expression with the greatest accumulation of Hsp70 found in the TGF-β2/FGF-2 explants. The increase in expression of Hsp70 in the TGF-β explants may be related to the increase in chaperoning functions needed to prevent aggregation and denaturation of proteins as well as to increase cell survival during EMT. This is supported by a previous study, which demonstrated that an increase of Hsp70 expression is associated with lens cell differentiation in embryonic chicken lens cells. Furthermore, Hsp70 induction is also observed in apoptotic cells [26].

Hsp90 is another major chaperone present in normal cultured rat LECs. FGF-2 stimulated an upregulation of Hsp90 expression in the rat lens epithelial explants with the greatest accumulation of Hsp90 found in the TGF-β2/FGF-2 treated explants. Treatment with TGF-β alone showed similar levels of Hsp90 expression as the control explants. Hsp90 is required for the translocation of FGF-2 from the endosomes into the cytosol and cell nucleus where nuclear FGF-2 can increase cell proliferation and survival of carcinoma cells [48]. Thus, the upregulation of Hsp90 is related to increased cell proliferation and survival in FGF-2 treated rat epithelial explants. It has been reported that inhibition of Hsp90 prevented FGF-2 induced mitogenic activity in human breast cancer epithelial cells. Hence, it is suggested that Hsp90 chaperone activity is necessary for FGF-2 stimulated cell proliferation [49]. Hsp90 expression is lowered in the heat shock-treated explants of all treatment groups in comparison to the respective non-heat shocked groups. This also serves as an indication that heat shocked LECs are under less stress than the non-heat shocked LECs.

Interestingly, both Hsp90 and Hsp70 can interact with cytoskeletal elements including microtubules, microfilaments, actin, and tublin [28,30,50], and it has been suggested that Hsp90 and Hsp70 proteins are required during extensive cytoskeletal rearrangement and disassembly as was shown to occur in the TGF-β2/FGF-2 treated rat lens epithelial explants [30]. Therefore TGF-β-induced α-SMA and F-actin disorganization as well as E-cadherin disassembly are concurrent with the upregulation of Hsp70 and Hsp90 protein expression.

Similar to previous findings, treatment with TGF-β induced a significant increase in apoptotic cell death in the rat lens epithelial explants (Figure 8) [39,40]. The TGF-β2/FGF-2 treated explants showed the greatest amount of apoptotic cell death. In contrast, the FGF-2 treated explants demonstrated the least amount of TUNEL positive nuclei in comparison to the control explants. All cultured rat lens epithelial explants that were preconditioned to heat stress showed a reduction of apoptotic cell death with respect to the non-heat shock explants. Although the reduction between the normal and heat shocked control is not significant, the heat shock treatment appeared to significantly enhance the survival of rat LECs following TGF-β treatment.

Hsps are also known to be involved in regulating apoptosis. Hsp70 is reported to protect cells from apoptosis [51] while Hsp90 can either promote or prevent apoptosis, depending on the apoptotic stimuli, tissue type, stages of development, and the client proteins being chaperoned. For example, the inhibition of Hsp90 blocked lens apoptosis in the blind cavefish [24]. Hsp90 together with a client protein Akt (protein kinase B) can protect human vascular endothelial cells from stress-induced apoptosis by inhibiting apoptosis signal-regulating kinase 1 (ASK1) activity [52].

Hsp70 can also inhibit apoptosis by inhibiting caspase-3 and stress activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) activation [53-55]. Hence, the upregulation of Hsp70 expression observed may be due to an increased need for anti-apoptotic functions of Hsp70 in the TGF-β-treated explants. Hsp70 protein expression was diminished in the heat shocked TGF-β and FGF-2 explants when compared to the non-heat shocked explants. This corresponds with the reduction of apoptotic cell death found in the heat shocked TGF-β2 and TGF-β2/FGF-2 treated explants. Thus, less Hsp70 chaperoning activity is needed in the heat shocked TGF-β lens explants with respect to the non-heat shocked TGF-β lens explants. It has been reported that heat shock-induced Hsp70 mRNA accumulation in rat LECs progressively declines after three hours of recovery [27]. Similarly, the increased accumulation of Hsp70 expression at 24 h in the heat shocked explants also declined at four days after initial treatment. The results of our study demonstrated that heat shock treatment enhanced the tolerance of LECs to TGF-β-induced effects even four days after initial exposure of the explant culture.

The proteasome is a multicatalytic cytoplasmic and nuclear complex that is responsible for intracellular protein degradation and modulations of cell signaling proteins, including those associated with TGF-β [56]. TGF-β-induced apoptosis and α-SMA mRNA expression was blocked by proteasomal inhibition in human LECs. The proteasome inhibitor induced the accumulation of SnoN (Smad inhibitor protein) and repressed TGF-β signal progression in LECs [56]. Another study showed that Hsp90 plays a protective role in human lens proteasome activities against oxidative insults [57]. Therefore, increased Hsp90 expression in the TGF-β2/FGF-2 treated explants may serve to protect proteasomal activities and enhanced TGF-β-induced response.

In conclusion, the findings of this study show that preconditioning of cultured primary rat LECs to heat stress reduced the TGF-β-induced EMT effect on the LECs. Further investigation is required to determine the specific roles that heat shock proteins play during TGF-β-induced EMT in LECs.

Acknowledgements

This research was supported by NIH Grant EY015006 (J.W.M.) and Natural Sciences and Engineering Research Council of Canada (J.G.S.).

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