The transcription factor CREB acts as an important regulator mediating oxidative stress-induced apoptosis by suppressing αB-crystallin expression

Results

Treatment of rat lens organ culture with oxidative stress induces apoptosis of lens epithelial cells, which is linked to down-regulation of αB-crystallin but up-regulation of CREB

It is well established that oxidative stress has an initiating role in cataractogenesis [4, 31–32]. In deducing the underlying cellular mechanism, we have previously demonstrated that oxidative stress first induces apoptosis of lens epithelial cells followed by subsequent cataractogenesis [31, 32]. To further explore how oxidative stress causes apoptosis of lens epithelial cells, we treated rat lenses with 40 mU glucose oxidase (GO) for 0 to 3 hours. Consistent with our previous studies [31–32], GO treatment generated hydrogen peroxide (Figure 1A) and caused significant drop of the free thiol level (Figure 1B). As a result, the epithelial cells of the treated rat lens were induced to undergo apoptosis (Figure 1C and 1D). More importantly, we observed that GO treatment caused significant downregulation of αB-crystallin expression in 30 minutes (Figure 2A, 2B). Paralleling to downregulation of αB-crystallin expression, GO induced transient upregulation of CREB expression in 30 minutes (Figure 2C, 2D). Subsequently, as CREB expression became attenuated, expression of αB-crystallin appeared slightly restored in 180 minutes, suggesting that CREB seemed to negatively regulate αB-crystallin to promote apoptosis (Figure 2B, 2D).

Figure 1. Treatment of rat lens with 40 mU GO caused apoptosis of lens epithelial cells. (A) Dynamic H₂O₂ concentration generated from 40mU glucose oxidase (GO) in the M199 medium in which rat lenses were cultured in 10-cm culture dish with 30 ml medium. (B) Dynamic changes of free thiol levels in rat lens epithelial cells under 40 mU GO treatment. (C) Live/dead assays to reveal time-dependent apoptosis of rat lens epithelial cells under treatment of 40 mU GO. Green fluorescence represents live cells as detected by Calcein-AM, and red fluorescence detected by EthD-1 refers to dead cells. (D) Apoptotic rate of rat lens epithelial cells under 40 mU GO treatment. All experiments were repeated three times. Error bar represents standard deviation, N=3.

Figure 2. GO-induced apoptosis of rat lens epithelial cells is derived from downregulated expression of αB-crystallin caused by upregulation of CREB. (A) Western blot analysis of αB-crystallin in rat lens epithelium with 40mU glucose oxidase treated from 0 to 180 minutes. (B) Semi-quantification of the western blot results in (A). (C) Western blot analysis of total CREB (T-CREB) in rat lens epithelium with 40mU glucose oxidase treated from 0 to 180 minutes. (D) Semi-quantification of the western blot results in (C). Note the reverse relationship between expression of αB-crystallin with that of total CREB expression. All experiments were repeated three times. Error bar represents standard deviation, N=3. * p<0.05; ** p<0.01; NS, statistically not significant.

Mouse lens epithelial cells expressing CREB are more sensitive to oxidative stress-induced apoptosis

To test if CREB could suppress αB-crystallin expression to promote oxidative stress-induced apoptosis, we first established stable lines of lens epithelial cells expressing the empty vector, pCI-αTN4-1, or wild type CREB, pCI-CREB-αTN4-1. Expression of exogenous wild type CREB was determined using western blot analysis and immunofluorescence. As show in Figure 3A and 3B, wild type CREB was clearly overexpressed. Both endogenous and exogenous CREB were localized in the nuclei (Supplementary Figure 1).

Figure 3. The expression of exogenous CREB sensitizes mouse lens epithelial cells to 40 mU GO-induced apoptosis (C, F). (A) Western blot analysis of the CREB levels in αTN4-1, pCI-αTN4-1, and pCI-CREB-αTN4-1 cells. (B) Semi-quantification of the western blot results in (A). (C and F) The αTN4-1, pCI-αTN4-1, and pCI-CREB-αTN4-1 cells were grown to 90% confluence. Then, 40 mU GO was added into the 3 types of cells, and the 3 types of cells were treated for indicated time. At the end of treatment, the cells were harvested for either live/dead assays (C), or for CellTiter-Glo® Luminescent Cell Viability Assay analysis [89] (F) to determine the rate of apoptosis. Note that pCI-CREB-αTN4-1 cells displayed the highest level of apoptosis (nearly 100%) in the 40mU glucose oxidase treatment (F). Green fluorescence represents live cells as detected by Calcein-AM, and red fluorescence detected by EthD-1 refers to dead cells. (D) Dynamic H₂O₂ concentration generated from 40mU glucose oxidase (GO) in the DMEM medium. (E) Dynamic changes of free thiol levels in mouse lens epithelial cells cultured in the DMEM medium under 40 mU GO treatment. All experiments were repeated three times. Error bar represents standard deviation, N=3. ** p<0.01.

Next, we treated different lines of lens epithelial cells, αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 with 40 mU glucose oxidase (GO) for 6 hours (Figure 3C and 3F). Hydrogen peroxide was consistently generated from 3 to 6 hours (Figure 3D). At the same time, the free thiol levels in these cells were significantly downregulated (Figure 3E). Live/dead viability/cytotoxicity assay and ATP loss analysis [89] revealed that cells expressing wild type CREB were most sensitive to GO-induced apoptosis (Figure 3C and 3F). A 6-hour treatment with 40 mU GO caused ATP loss in more than 90% cells expressing wild type CREB (Figure 3F). Thus, our results revealed that expression of exogenous wild type CREB sensitizes lens epithelial cells to oxidative stress-induced apoptosis.

RNAseq analysis revealed that expression of exogenous CREB significantly downregulates αB-crystallin gene in lens epithelial cells

To understand why CREB-expressing cells displayed strong sensitivity to oxidative stress insult, we conducted RNAseq analysis between wild type (WT) CREB transfected cell and vector-transfected cells. As shown in Figure 4A (SRA accession: PRJNA566306), overexpression of WT CREB altered expression patterns of 1916 genes among which 872 were upregulated, and 1044 were downregulated. These genes belong to various signaling pathways (Supplementary Figure 2). Among these genes, we noticed that the most striking gene with consistently changed expression pattern is the one coding for αB-crystallin (Figure 4B, Cryab). QRT-PCR analysis confirmed the RNAseq result about αB-crystallin expression in vector and CREB-expressing cells (Figure 4C). Thus, our results revealed that expression of exogenous CREB suppresses αB-crystallin expression to confer its hypersensitivity to stress response.

Figure 4. Comparative transcriptome analysis. (A–C) Both pCI-αTN4-1 and pCI-CREB-αTN4-1 cells were grown to 90% confluence and then harvested for RNAseq analysis. The gene expression patterns between vector-transfected cells and wild type CREB-transfected cells were compared (SRA accession: PRJNA566306). Compared to pCI-vector, expression of the exogenous WT-CREB caused changes in the expression patterns of 1916 genes, 872 genes were upregulated and 1044 genes were downregulated (A). (B) Hierarchical cluster analysis of apoptosis-associated genes. (C) The expression levels of the anti-apoptotic gene αB-crystallin in pCI-αTN4-1 and pCI-CREB-αTN4-1 cells (B) were further verified by qRT-PCR. Note that the expression of anti-apoptotic gene coding for αB-crystallin was significantly downregulated in pCI-CREB-αTN4-1 cell. (D–F) CREB downregulates expression of αB-crystallin during H₂O₂-induced apoptosis of CREB-expressing cells. (D) Apoptosis rate changes in αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 cells under treatment of 40 mU GO from 0 to 6 hours were measured by CellTiter-Glo® Luminescent Cell Viability Assay analysis [89]. (E) Western blot analysis of the expression levels of αB-crystallin in αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 cells under 40 mU GO. Note that the expression level of αB-crystallin was significantly downregulated in pCI-CREB-αTN4-1 cell. In addition, 40 mU GO treatment downregulated expression level of αB-crystallin in αTN4-1, pCI-αTN4-1 and pCI-CREB-αTN4-1 cells. (F) Semi-quantification of the western blot results in E. All experiments were repeated three times. Error bar represents standard deviation, N=3. ** p<0.01.

Overexpression of CREB in mouse lens epithelial cells dramatically down-regulates endogenous αB-crystallin

To confirm the RNAseq analysis data and demonstrate that the αB-crystallin downregulation by CREB indeed accounts for the hypersensitivity of pCI-CREB-αTN4-1 cells to stress-induced apoptosis (Figure 4D), we conducted western blot analysis and examined the relative levels of αB-crystallin in all 3 types of cells under treatment by 40 mU GO for 3 or 6 hours (Figure 4E and 4F). As shown in Figure 4D, cells expressing CREB displayed quick ATP loss under 40 mU GO treatment. Consistent with quick loss of ATP, the expression of αB-crystallin in cells expressing CREB remained constantly very lower level, and 40 mU GO treatment further downregulated it to barely detectable level (Figure 4E and 4F). In αTN4-1 and pCI-αTN4-1cells, 40 mU GO also down-regulated the expression of αB-crystallin to certain degree. Thus, in CREB-expressing cells, loss of cell viability is closely linked to the suppression of αB-crystallin expression by CREB.

Knockdown of endogenous CREB in lens epithelial cells upregulates αB-crystallin expression and confers resistance to oxidative stress-induced apoptosis

To confirm that CREB suppresses αB-crystallin expression, which affects the sensitivity of mouse lens epithelial cells to stress response, we used CRISPR/CAS9 technology to knockout expression of CREB in αTN4-1 cell (Figure 5A). The deletion of a single nucleotide in exon 5 was confirmed with DNA sequencing (Figure 5A) and the absence of CREB protein expression was verified by western blot analysis (Figure 5B, 5C). When CREB expression was significantly knocked down, the expression level of αB-crystallin gene was distinctly upregulated (Figure 5B–5D). Next, we treated Mock-KO-αTN4-1 and CREB-KO-αTN4-1 cells with 40 mU GO, and the cell viability was measured by ATP loss. As shown in Figure 5E, cells with knockdown of endogenous CREB expression and upregulation of endogenous αB-crystallin expression displayed much stronger resistance to oxidative stress-induced apoptosis than mock knockdown cells. Together, these results confirm that CREB is a negative regulator of αB-crystallin gene and it promotes oxidative stress-induced apoptosis of mouse lens epithelial cell by suppressing αB-crystallin expression.

Figure 5. Silence of CREB activates expression of αB-crystallin in αTN4-1 cells. (A). CREB knockout strategy in αTN4-1 cells. The homozygous point deletion was created by CRISPR/CAS9 technology. A single nucleotide was deleted in exon 5, and the deletion mutation was verified by DNA sequencing. (B) Western blot analysis of the expression levels of CREB and αB-crystallin in αTN4-1 mock knockdown and CREB knockdown cells. (C, D) Semi-quantification of the western blot results in (B). (E) Apoptosis rate in, mock KO and αTN4-1 CREB KO cells under treatment of 40 mU GO for 6 hours measured by CellTiter-Glo® Luminescent Cell Viability Assay analysis [89]. All experiments were repeated three times. Error bar represents standard deviation, N=3. * p<0.05, *** p<0.005.

Overexpression of exogenous human αB-crystallin partially restores the resistance against stress-induced apoptosis of mouse lens epithelial cells expressing CREB

To further confirm that CREB-induced down-regulation of αB-crystallin was indeed the main reason for the enhanced apoptosis of the CREB-transfected cells under GO treatment, we next overexpressed human αB-crystallin (HαB) cDNA in pCI-CREB-αTN4-1 cells using pEGFPC3-HαB with the vector pEGFPC3 as control. Expression of EGFP or EGFP-HαB fusion protein can be distinguished by their localization (Figure 6A-b/c). While EGFP alone was homogenously expressed within the cells (Figure 6A-b), expression of EGFP-HαB was largely restrained in the cytoplasm (Figure 6A-c). The expression of exogenous EGFP and the fusion protein, EGFP-αB-crystallin were further verified by western blot analysis using antibodies against αB-crystallin (Figure 6B) and GFP (Figure 6C). Next, we compared the sensitivity of the 3 types of cells to 40 mU GO-induced apoptosis. As shown in Figure 6D, expression of human αB-crystallin cDNA in pCI-CREB-αTN4-1 cells decreased more than 50% of 40 mU GO-induced apoptosis. Together, these results further demonstrated that CREB sensitizes lens epithelial cells to stress-induced apoptosis mainly through suppression of αB-crystallin expression.

Figure 6. Exogenous human αB-crystallin restores the ability of pCI-CREB-αTN4-1 cells against hydrogen peroxide-induced apoptosis. (A) The pCI-CREB-αTN4-1 cells were either untransfected (A-a), or transfected with pEGFPC3 vector (A-b), or pEGFPC3-HαB (A-c) transiently. Transfection was confirmed by fluorescence microscopy. The pEGFPC3 vector-transfected pCI-CREB-αTN4-1 cells displayed homogenous distribution of green fluorescence protein in the whole cells (A-b). In contrast, in the pEGFPC3-HαB-transfected pCI-CREB-αTN4-1 cells, the green fluorescence fusion protein was largely restricted in the cytoplasm (A-c). (B) Western blot analysis of the expression level of endogenous αB-crystallin and GFP-αB fusion protein in pCI-CREB-αTN4-1 cells (a), pCI-CREB-αTN4-1/pEGFPC3-αTN4-1 cells (b) and pCI-CREB-αTN4-1/pEGFPC3-HαB-αTN4-1 cells (c) detected with anti-αB antibody. (C) Western blot analysis of the expression level of GFP and GFP-αB fusion protein in pCI-CREB-αTN4-1 cells (a), pCI-CREB-αTN4-1/pEGFPC3-αTN4-1 cells (b) and pCI-CREB-αTN4-1/pEGFPC3-HαB-αTN4-1 cells (c) detected with anti-EGFP antibody. (D) After treatment by 40 mU GO for 6 hours, apoptosis in the 3 types of cells as indicated were analyzed. Note that pCI-CREB-αTN4-1/pEGFPC3-HαB-αTN4-1 cells expressing exogenous HαB displayed over 50% less apoptosis than pCI-CREB-αTN4-1 cells and pCI-CREB-αTN4-1/pEGFPC3-αTN4-1 cells. All experiments were repeated three times. Error bar represents standard deviation, N=3. * p<0.05; NS, statistically not significant.

CREB directly regulates αB-crystallin gene

Next, we determined if CREB can directly regulate αB-crystallin gene. First, we used bioinformatics to search the CREB binding sites in αB-crystallin gene promoter. As shown in Supplementary Figure 3, the mouse αB-crystallin gene contains multiple copies of either well-conserved full CREB binding site such as M8 or the variated CREB full binding sites like M10 within the 250 kb sequences examined. Next, we tested if CREB can bind to these putative sites. We chose M8, the well conserved full CREB binding site as well as M10, the less conserved variant CREB binding sites (it has one nucleotide variation) as oligo probes to conduct gel mobility shifting assay. As shown in Figure 7A, 7B, nuclear extracts from pCI-CREB-αTN4-1 cells displayed strong binding to the M8 sequences, which can only be competed off by wild type but not mutant oligos. A much-reduced binding was observed when probe was derived from M10 site region. The authenticity of the CREB binding was confirmed by the formation of the supershifting bands after incubation with anti-CREB antibody (Figure 7A, 7B). Interestingly, we did not observe the supershifting band formation with the M1 oligos (Supplementary Figure 4B). Lack of the supershifting band may be due to the formation of heterodimers (see discussion). Together, our results suggest that different CREB binding sites in the αB-crystallin gene promoter and enhancer regions display differential affinities with CREB in the in vitro binding assays.

Figure 7. Electrophoretic mobility shifting assays (EMSA) demonstrated that CREB directly binds to the promoter enhancer sequences of the αB-crystallin gene to control its expression. Bioinformatics analysis revealed that the mouse αB-crystallin gene contains many half CREB binding sites in the proximal promoter, and completely conserved CREB sites in the upstream enhancer or downstream enhancer regions (Supplementary Figure 3). EMSA revealed that CREB can directly bind to the conserved CREB binding sites in both upstream (M8) or downstream (M10). A-a, diagram of the two oligos containing a well-conserved CREB binding site (WT-M8, top) or mutant CREB binding site (MT-M8, bottom), which were used for gel mobility shifting assays described in A-b. A-b, gel mobility shifting assays. Nuclear extracts prepared from pCI-CREB-αTN4-1 cells were incubated with γ-32P-ATP-labeled oligo-nucleotide containing wild-type CREB binding site (A-a, top) under various conditions shown in the figure. B-a, diagram of the two oligos containing a less conserved CREB binding site (WT-M10, top) or mutant CREB binding site (MT-M10, bottom), which were used for gel mobility shifting assays described in B-b. B-b, gel mobility shifting assays. Nuclear extracts prepared from pCI-CREB-αTN4-1 cells were incubated with γ-³²P-ATP-labeled oligo-nucleotide containing wild-type M10 CREB binding site (B-a, top) under various conditions shown in the figure.

CREB regulates αB-crystallin gene in vivo.

We next determined if CREB can regulate αB-crystallin gene in vivo. To do so, we conducted ChIP assays using the oligos (Supplementary Table 1) from the M8 and M10 regions. The nuclear extracts isolated from vector- or CREB-transfected cells were immuno-precipitated with mock IgG or anti-CREB antibody. The precipitated complexes were used for extraction of template DNAs, which were then amplified in QPCR analysis. As shown in Figure 8, both M8 and M10 cis-elements can be bound by CREB. Moreover, the M10 region seems to be bound by CREB more tightly in the exo vivo condition. ChIP assay also confirmed that CREB can bind to M1 region (Supplementary Figure 4C). These results demonstrated that CREB can bind to multiple sites of the enhancer regions of the αB-crystallin gene in vivo to suppress expression of the later.

Figure 8. ChIP assays to demonstrate that CREB binds to the promoter of αB-crystallin gene in vivo. qChIP experiments revealed that in the in vivo condition, CREB displayed stronger affinity with M10 site (B), suggesting that CREB may interact with its partner to bind to the M10 site, and to a less degree, to the M8 site (A). All experiments were repeated three times. Error bar represents standard deviation, N=3. * p<0.05; ** p<0.01; *** p<0.005; NS, statistically not significant.

Abbreviations

BDGF: brain-derived neurotrophic factor; CREB: cAMP response element binding protein; EGFP: enhanced green fluorescence protein; αB: αB-crystallin; EGFP-αB: enhanced green fluorescence and αB-crystallin fusion protein; DMEM: Dulbecco's modified eagle medium; GO: glucose oxidase; NGF: nerve growth factor; OS: oxidative Stress; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; αTN4-1: mouse lens epithelial cells; SDS: sodium dodecylsulfate; TBS: tris-buffered saline; TBS-T: tris-buffered saline with tween-20.

Acknowledgments

We thank Dr. Paul Russel for the αTN4-1 mouse lens epithelial cell line, Dr. Joseph Horwitz for the anti-αB-crystallin antibody, and all members of David Li’s Laboratory in the State Key Laboratory of Ophthalmology in Zhongshan Ophthalmic Center of Sun Yat-sen University.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

This study was supported in part by the Grants from the National Natural Science Foundation of China (81770910; 81900842; 81970787 and 81970784), the Fundamental Research Funds of the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University.

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