A fine-tuned β-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo—G0/G1) and proliferating (in vitro—G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed β-catenin and transforming growth factor (TGF-β) pathways to be correlated with the identified proteins. Treatment of rCEnC with a β-catenin activator and inhibitor showed that β-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-β were regulated through β-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of β-catenin and TGF-β.

Gene ontology and literature analysis. Gene ontology analysis (Table 3) of the proteomic results revealed that some specific biological processes were significantly dysregulated, especially those involved in the formation of the proteasome complex, in the negative regulation of canonical Wnt signalling pathway, in the endoplasmic reticulum function, and in cellular trafficking (GO:0000502, GO:0090090, GO:0005789, GO:0006888, GO:0031201, GO:0031032 and KEGG pathway 04141). Other processes found in the gene ontology analysis, listed in Table 3, were not significantly modulated (p > 0.05). Literature analysis showed that some of these proteins have been previously associated with CE function and dysfunction (Supplementary Table S2) and that several of them have a role in multiple fundamental intracellular pathways (Table 4). More specifically, based on the proteomic results, we found numerous proteins differentially expressed in proliferating in vitro versus ex vivo cells, which were involved in β-catenin and TGF-β pathways.
Several proteins involved in promoting the Wnt/β-catenin pathway were significantly downregulated in cultured rCEnC when compared with the tissue-derived counterpart. For instance, HIPK1 was previously shown to induce Wnt/β-catenin signalling 31 . KHDRBS1 promotes proliferation through the same pathway 32 , while ITGB5 inhibits β-catenin degradation by the proteasome, leading to Wnt/β-catenin pathway activation as well 33 . Similarly, Endoglin (ENG) 34 , CUX 35 , CARF 36 , SPARCL1 37 , and SPARC 38 were proved to trigger the Wnt/βcatenin pathway. Downregulation of proteins 4.1R (EPB41) and 4.1G (EPB41L2), which associates β-catenin to the cell membrane in gastric epithelium 39 , may play a role in cytoplasmic β-catenin translocation. Moreover, DAVID analysis predicted a negative regulation of the canonical Wnt signalling pathway (Table 3), identifying some proteasome proteins that were upregulated in rCEnC (PSMD7, PSMD2, PSMD12, PSMD3, PSMD6, and PSMB7). The PSMD are components of the 19S subcomplex, known to be involved in β-catenin/Wnt signalling; PSMD7 and PSMD2 in particular are found to be directly associated with the degradation of β-catenin from the proteasome 40 . In (a), the graph shows rabbit CEnC from the ex vivo tissue, the peak represents the G0/G1 phase, while in (b), the graph identifies three different peaks for the cultured rCEnC, representing the G0/G1, S, and G2/M phases, from left to right. In (c), we can observe how confluent rCEnC decrease their proliferating component (G2/M). On the right, a representative image of a confluent rCEnC culture, obtained with the Axiovert 40C inverted microscope (Zeiss), objective 5x. Similarly, in (d), the graph shows a unique peak for the ex vivo human CE cells (G0/G1), while the graph (e) exhibits the distribution of the human CEnC in all three different phases of the cell cycle. Panel (f) represents a bar chart of Propidium Iodide flow cytofluorimetric analysis comparing human and rabbit CEnC at 60% confluence with confluent rabbit CEnC. Experiments were performed n = 3. Results are presented as mean ± Standard Error (SE). T-test was performed *p < 0.05, **p < 0.01. Scientific RepoRtS | (2020) 10:13841 | https://doi.org/10.1038/s41598-020-70800-w www.nature.com/scientificreports/ Although Wnt/β-catenin is involved in CE development 41 and in CE mesenchymal transformation 26,28,42 , the mechanism of β-catenin pathway downregulation observed here is not completely clear and it will be further dissected in the next sections.
Multiple proteins that were found downregulated in cultured rCEnC are also involved in TGF-β pathway regulation. Endoglin, previously shown to be expressed in CEnC 43 , is an auxiliary receptor for TGF-β, controlling proliferation and quiescence 44 , in particular in the endothelium 45 , where it also regulates the EMT 46 . CUX1, a protein with a role in regulating cell cycle progression 47 , EMT 48 , and repressing E-cadherin 49 , is proved to be a target of TGF-β pathway 47 and PSPC1 potentiates TGF-β autocrine signalling 50 . Similarly, expression of LRP1, a cell receptor involved in the clearance of growth factors, including TGF-β 51 is increased upon stimulation of TGF-β1 51 , and HPIP is a downstream factor of TGF-β1 stimulation, promoting EMT in A549 cells 52 .
These results show that Wnt/β-catenin modulators and TGF-β effectors are downregulated in culture-derived proliferating rCEnC and, in accordance with previous reports, demonstrate a fine-tuned regulation involved in promoting CEnC proliferation while still suppressing the EnMT.
β-Catenin cellular localization. Staining of rCEnC showed that β-catenin is mainly located in the plasma membrane when cultured cells are confluent and reconstitute the tissue-like alveolar structure (Fig. 2a, panel 1). Nevertheless, the external cells of the colonies, not presenting any cell-cell contact, do not show β-catenin on the plasma membrane (Fig. 2a, panel 2). Most importantly, cytoplasmic and nuclear translocation of β-catenin from the plasma membrane was observed in the dividing or in some isolated cells (Fig. 2a, panels 3 -5). These data, together with available literature, suggest a complex mechanism containing two phases. In the first phase, β-catenin is released from the cell-junctions, moving eventually to the nucleus for regulation of the gene expression. In the second phase, it is degraded or transferred back to the membrane, while its activators are downregulated, as observed in the proteomic results (Fig. 2b,c).
We suggest this hypothesis based on the observation of β-catenin translocation in the first phase, which can also explain why it was quickly degraded and subsequently downregulated.
Variability in the cell cycle and β-catenin distribution after treatments with bFGF and TGF-β. In order to increase or diminish the number of cells in the G2/M phase of the cell cycle and analyse in parallel the effect on β-catenin nuclear translocation as well as the maintenance of a corneal endothelial phenotype, we treated rCEnC with different compounds.
Initially, bFGF was used as a positive control for proliferation induction in CEnC 19 and its cross-regulations with the Wnt pathway, as observed in other cellular models 53 and in CEnC 26 . Moreover, bFGF signalling was shown to downregulate GSK-3β activity through a mechanism involving Akt, which in turn activates β-catenin response 54 . As expected, rCEnC treated with 20 ng/mL of bFGF showed a significant reduction in the number of non-proliferating cells and a consequent increase in the G2/M population (Fig. 3a).
In parallel, TGF-β was tested on rCEnC as it correlates with multiple proteins that emerged from the proteomic analysis and has an ascertained role in reducing CEnC proliferation 17,18,[55][56][57][58] . Herein, TGF-β-treated cells presented a significant decrease in G2/M phase of the cell cycle as it was detected by using cytofluorimetric analysis, confirming the anti-proliferative effect of TGF-β on CEnC. When TGF-β was added to bFGF-stimulated cells, we observed a distribution comparable to the TGF-β alone treatment, proving the TGF-β interference with the bFGF-activated pathway (Fig. 3a). Table 1. List of proteins from proteomic analysis which were over-expressed in rCEnC isolated and cultured in vitro if compared with the rCEnC isolated from the tissue, with a p value < 0.005 and < 0.001. www.nature.com/scientificreports/ The same treatments were analysed for β-catenin expression by immunocytochemistry. We detected a significant increase of β-catenin nuclear translocation in bFGF-treated cells, while the cytoplasmic signal was significantly reduced both in TGF-β and TGF-β + bFGF-treated rCEnC when compared to the control (Fig. 3b). These data collectively proved that the activity of bFGF was mediated by an increased translocation of β-catenin to the nucleus, which is inhibited by TGF-β, in accordance with the previous literature 19 .
The studies by immunofluorescence on α-SMA (Fig. 4), a marker of EMT, did not show any significant evidence of mesenchymal transformation. The amount of α-SMA-positive cells was not modified by bFGF treatment, as similarly assessed by Tseng and Heur groups 26,59 . Although TGF-β can induce EnMT 42,60 and differently from what observed by Tseng et al. 26 , the treatment of rCEnC with either TGF-β and TGF-β + bFGF did not show any significant increase in α-SMA expression. Nevertheless, in the Tseng et al. study, the treatment lasted 3 days and was followed by EDTA dissociation, likely provoking a more sustained stimulation of β-catenin as well as other cellular responses.
The data obtained with α-SMA were corroborated by immunostaining with an early marker of EMT, S100A4 61,62 , expressed within the cytoplasm by human adult CEnC in vivo 63 . Conversely, S100A4 expression was observed in the nucleus when CEnC underwent EnMT 26 . Similarly to what observed for α-SMA, we did not detect any significant variation in S100A4 expression between the treatments with bFGF, TGF-β, TGF-β + bFGF and the Mock control ( Fig. 4a,b). In each treatment tested S100A4 presented as mainly cytoplasmic and/or perinuclear. As a positive control we used a rCEnC strain with a high number of passages (P10) which showed an elongated phenotype. In this condition S100A4 was localized in the nuclei of the majority of rCEnC, which were also showing a high α-SMA positivity (Fig. 4c). This result confirm that both proteins may be considered as valid markers for EnMT. Table 2. List of proteins from proteomic analysis which were down-regulated in rCEnC isolated and cultured in vitro if compared with the rCEnC isolated from the tissue, with a p value < 0.005 and < 0.001.  www.nature.com/scientificreports/ Altogether, these results showed that bFGF and TGF-β treatments did not cause any mesenchymal transformation on rCEnC at 24 h, although they were able to interfere with β-catenin and activate or inhibit proliferation. Further experiments in the following section, using small molecules targeting β-catenin pathways, helped to reveal a possible role of this crosstalk in rCEnC maintenance and propagation.
Variation in the cell cycle phases and β-catenin distribution after treatments with Wnt activators/inhibitors. CHIR99021 was previously described to inhibit GSK-3β 64 , thereby stabilizing cytoplasmic β-catenin and eventually promoting its nuclear translocation. On the basis of CHIR99021 IC50 (0.04 µM) 64 , the treatment was initially tested in a range of concentrations between 0.05 and 10 µM. The distribution in the cell cycle phases was not statistically different for all the CHIR99021 concentrations tested except for 10 µM. This concentration produced a significant decrease of cells in the G2/M phase of the cell cycle in comparison with untreated cells (Fig. 5a), although promoting a consistent β-catenin nuclear translocation (Fig. 5b,c). Interestingly, CHIR99021 at 0.5 µM, despite not showing any significant difference in cell phases distribution, revealed an increase in cytoplasmic and nuclear β-catenin if compared with the untreated cells (Fig. 5b,c). Collectively these results suggest that CHIR99021, although able to promote β-catenin migration to the nuclei, did not cause an increase in rCEnC proliferation. Conversely, at high concentration (10 µM), CHIR99021 decreased rCEnC proliferation. This unexpected effect might be due to a negative feedback regulation of β-catenin, once overactivated. The possibility of β-catenin feedback regulation was also previously proposed by Hirata-Tominaga et al. 28 . CHIR99021 concentrations of 0.5 and 10 µM were also tested at 12 and 48 h: while at 12 h both concentrations did not show any effect on the cell cycle of rCEnC, at 48 h only 10 µM CHIR99021 elicits an effect not significantly different to what observed at 24 h (data not shown). Moreover, a vitality assay using calcein AM/Propidium Iodide staining was performed to assess if CHIR99021, at the concentrations used for the experiments on rCEnC, demonstrated a cytotoxic effect. However, we could not observe any Propidium Iodide positive cell nuclei, confirming that CHIR99021 and DMSO, at all the concentration tested under the same experimental conditions used in this study, did not induce cell death (Supplementary Figure S3).
In addition, we observed an increased amount of α-SMA positive cells and nuclear S100A4 at 10 µM CHIR99021 (Fig. 5b,c), suggesting that activation of β-catenin nuclear translocation over a certain limit may induce EnMT. Similar results were previously obtained via disrupting CEnC junctions with EDTA and with prolonged addition of TGF-β (2 days) 26 . Under these conditions, rCEnC might thus interfere with the β-catenin pathway to avoid the loss of hexagonal morphology and the mesenchymal transformation.
Next, we tested an inhibitor of β-catenin pathway: quercetin 65,66 , previously proven to inhibit the Wnt/βcatenin signalling by interfering with β-catenin nuclear translocation 67 . The 25 µM concentration was selected for our study. Quercetin was shown here to completely abolish the rCEnC populations both in S and G2/M phases, maintaining the cells in the G0/G1 phase of the cell cycle (Fig. 5d). Similar results were obtained by treating rCEnC with both bFGF and quercetin (Fig. 5d), highlighting how the latter was also able to knockdown the proliferative effect of bFGF. These data suggest that quercetin may act, at least in part, via the same pathway activated by bFGF. Table 4. List of proteins from the proteomic analysis found to be involved, as an effector or as a regulator, in different cellular pathways: Wnt/β-catenin, AKT, TGF-β and NF-kB. www.nature.com/scientificreports/ Overall, the presented results show how β-catenin appears necessary but not sufficient to promote proliferation of rCEnC. The β-catenin pathway is activated by the disruption of cell-cell junction and β-catenin expression is increased by bFGF, while it is inhibited by the presence of TGF-β. However, β-catenin is quickly degraded by cells, underlining the sophisticated regulation of this specific pathway in the corneal endothelium, with the likely effect to avoid EnMT.
Discussion. Several studies have been carried out with the aim to characterize how CEnC regulate their proliferation, mainly focusing on the role of specific growth factors (EGF 68 , bFGF 19 , and TGF-β 55,56 ) and the final downstream effectors, in particular those involved in the regulation of the cell cycle (cyclins, p16, p21, and p27 2,57 ). Herein we propose a hypothesis-free approach, starting directly from the analysis of the CE proteome through a correlation between ex vivo non-proliferating rCEnC and in vitro proliferating rCEnC. Using rabbits farmed in standard conditions (controlled environment, slaughtered at the same age) and correlating the two eyes of the same individual, we were able to minimize the inter-individual variations. Moreover, although cellular division was observed in rabbit CE in vivo 69 , human and rabbit CEnC showed a similar cell cycle distribution, either when isolated from the tissue or when cultured in vitro. For this reason, rCEnC were selected as a model to study proliferative mechanisms that are utilised by the cells when expanded in vitro. The proteomic analysis allowed the selection of 77 proteins, out of the 3,328 identified, with a significant differential expression between the two groups in comparison (p < 0.005). A targeted dissection of each identified protein and their related intracellular routes, enabled to propose a correlation between specific pathways and rCEnC expansion in vitro.
Beside Wnt/β-catenin, multiple proteins involved in the TGF-β signalling were found to be downregulated. In particular, the expression of TGF-β effectors like Endoglin 44-46 , CUX1 47-49 , LRP1 51 , HPIP 52 , and PSPC1 50 was significantly reduced in rCEnC.  (Table 4). (b) Hypothetic representation of the initial phase of CEnC proliferation in which β-catenin is released from the membrane (where it interacts with N-cadherin) and moves to the nucleus in order to promote proliferation. (c) A second phase, when β-catenin is no more promoting cellular proliferation and is (i) degraded from the proteasome, or (ii) Inactivated through the downregulation of β-catenin effectors, or (iii) moved back to the membrane (once the cell-cell junctions are re-established).
Proteins of the TGF-β family are cytokines activated upon different stimulation (i.e., injury or mechanical stress) 5 . In the cornea, TGF-βs (1, -2, and -3) are present in the aqueous humor 71 and, once released, trigger multiple downstream processes 72 . A double role was recently described in CEnC for TGF-β: it induces the correct morphology and formation of novel cell junctions during the maturation phase, while it promotes a fibroblastic phenotype during proliferation 23 . This double role reflects its already observed activity in other studies, where TGF-βs is involved in the correct maturation of CE 41 , in stimulating CEnC migration during wound healing 26 , in promoting EnMT 42,60 and, most importantly, in suppressing CEnC entry into the S-phase 17,18,55-58 .
Following the proteomic results, downregulation of some TGF-β effectors observed in dividing rCEnC may be consequent to the absence of a TGF-β stimulus in vitro: cultured rCEnC, no longer blocked in a non-proliferative state by TGF-β, acquire in this manner the ability to proliferate.
The other pathway arising from the proteomic analysis involves Wnt/β-catenin signalling. When the cells are in contact with each other, β-catenin is usually bound to the cell membrane in complex with cadherin (N-cadherin in CE 73 ) while it is released when cells lose their cell-cell junctions 74 . Once in the cytoplasm, free β-catenin is targeted for degradation by GSK-3β phosphorylation. However, when Wnt activates the canonical pathway, it promotes the stabilisation of the cytoplasmic β-catenin and eventually, its translocation into the nucleus 5 , where it drives transcription of target genes, in particular of those that are critical in promoting cell proliferation, such as c-myc and cyclin D1 75 . Proliferation is known to be induced by β-catenin activation in different cell types, including cancer 76 and stem cells 74,77 . In CEnC, β-catenin acts as a key regulator of two crucial mechanisms: proliferation and cellular morphology 26,27 but also in expressing CE markers, as observed following treatment with a GSK-3β inhibitor (6-bromoindirubin-3′-oxime, BIO) which increases β-catenin levels 41 .
Herein we attempt to explain the role of β-catenin during rCEnC proliferation and uncover the reasons of its rapid degradation in the cytoplasm via cellular machinery. In fact, we observed binding of β-catenin to the cell membrane in confluent cells in vitro, while it disappeared from the membrane when the cell-cell junctions were www.nature.com/scientificreports/ lost (Fig. 1). This result was confirmed by the proteomic characterization that indicated activation of proteasome components, previously linked to β-catenin degradation 40 (Tables 1, 2, 3). Moreover, the majority of cells in culture were shown to downregulate various proteins involved in β-catenin activation. Of those proteins, Endoglin 43 , ITGB5 78 , and SPARC 79 were previously found to be expressed in CEnC (Supplementary Table S2). β-catenin is indeed generally fast degraded once in the cytoplasm 75 , and the condition captured by the proteomic analysis showed how β-catenin may have been already digested by the proteasome or moving back to the cell membrane (Fig. 2b,c). At the same experimental condition, half of rCEnC population (60% confluence) was in the G0/G1 phase, while only a minority of them were actively proliferating in the G2/M phase of the cell cycle (Fig. 1b). However, within this population, we could observe a nuclear β-catenin localisation in isolated or mitotic CEnC (Fig. 2a), suggesting the importance of this translocation during cell division. Collectively, these results raised questions regarding the possible role of β-catenin in rCEnC expansion that we tackled using a GSK-3 inhibitor, CHIR99021, to induce β-catenin stability, and quercetin as a counteracting inhibitor of β-catenin nuclear translocation 67 . Treating rCEnC with quercetin, we observed a substantial decrease in the cells in the G2/M phase of the cell cycle (Fig. 5d), while we never found a corresponding increased amount of cells in the G2/M phase at all tested CHIR99021 concentrations (Fig. 5a). Conversely, CHIR99021 (at 10 and 0.5 µM) was able to raise drastically the number of cells where β-catenin had translocated to the nucleus (Fig. 5b,c). Taken together, these data confirm how β-catenin expression and localization are fundamental during cell proliferation process, in accordance with previous reports 26, 27 , but probably not sufficient to promote it.
Based on the described role of β-catenin in dividing rCEnC and on the effect of growth factors in eliciting in vitro CEnC proliferation 19 , we sought to understand whether specific growth factors would act via β-catenin activation. β-catenin was shown to be involved in orchestrating the downstream activity of FGF in various cell types 54,80 , and, as previously introduced, bFGF stimulates CEnC proliferation through PI3K/Akt activation 4,19-21 , while TGF-β 19,22 inhibits cell proliferation elicited by bFGF 54 . Here we investigated how the pro-and antiproliferative activities of these two specific growth factors might crosstalk through β-catenin signalling. Consistently with previous reports, we confirmed the role of bFGF in promoting CEnC proliferation 19,26 showing how bFGF mediates an increase in the G2/M phase of the cell cycle and in β-catenin nuclear translocation (Fig. 3). Moreover, bFGF pro-proliferative effect was completely abolished by the quercetin-mediated inhibition of the (a) The panel shows representative immunofluorescence images of α-SMA (red, first row) and S100A4 (green, second row) in rCEnC treated with Mock control, bFGF, TGF-β and bFGF + TGF-β, respectively. White arrows indicate the cells positive for α-SMA. In blue DAPI, scale bar 50 µM for all the images. (b) The bar chart on the right shows the percentage of cells positive for α-SMA and the percentage of cells in which S100A4 moved to the nuclei as a mean of 12 fields (n = 3 biological replicates) for each condition. Results are presented as mean ± SE. T-test was performed n.s. non-significant. (c) The panel illustrates a representative image of a double immunostaining with S100A4 in green, DAPI in blue and α-SMA in red of rCEnC at a high passage number (P10). Letters P, perinuclear, and N, nuclear, underlie the different localization of S100A4 staining, corresponding to a low and high α-SMA positivity, respectively. (d) The panel shows a secondary only control on Mock rCEnC, used as a negative control with DAPI in blue.
Scientific RepoRtS | (2020) 10:13841 | https://doi.org/10.1038/s41598-020-70800-w www.nature.com/scientificreports/ β-catenin pathway (Fig. 5d). Similarly, we assessed the anti-proliferative effect of TGF-β 17,18,55,56 , which exerted an analogous decrease in the number of cells in the G2/M phase either alone or in the presence of bFGF, thus abolishing any pro-proliferative effect of the latter (Fig. 3a). We proved for the first time here that this effect, described also by Lu and collaborators 19 , is mediated by the inhibition of β-catenin translocation to the nucleus (Fig. 3b). Collectively, the results reported in our study confirmed the importance of β-catenin during CEnC proliferation process both after cell-cell disruption and bFGF stimulation. Consistent with this finding and with previous reports, TGF-β inhibits proliferation by blocking β-catenin nuclear translocation. As introduced earlier, the proliferation process is often balanced in a delicate equilibrium with EnMT. Although more specific experiments are necessary to clarify the role of β-catenin in EnMT, we measured a significant increase in the number of α-SMA and S100A4 (markers of EMT) positive CEnC when treating cells with 10 µM CHIR99021 (Fig. 5b,c). Such evidence suggests that a sustained stimulation with CHIR99021 may induce β-catenin nuclear translocation and that EnMT ensues when β-catenin is activated over a certain threshold. Similarly, Zhu et al. confirmed how CEnC loosing cell-cell junctions after treatment with EDTA exhibit a defined nuclear localization of β-catenin, promoting proliferation but also EnMT 26 , whereas Kinoshita et al. observed that CEnC attempt to block EnMT by deregulating β-catenin 28 . Altogether these results confirmed that β-catenin expression requires fine regulation since a sustained stimulus towards β-catenin activation might induce CEnC transformation into a mesenchymal phenotype, similar to cancer progression 75 . This observation may also explain why CEnC degrade β-catenin in the cytoplasm and deregulate β-catenin-activating pathways immediately after cell division, enabling thereby a putative CEnC mechanism of protection from β-catenin induced EnMT.
The data presented here suggest a scenario where β-catenin is necessary for CEnC proliferation, but its overactivation drives cells to EnMT. However, β-catenin alone is not sufficient to unlock the mitotic block requiring other cellular effectors, as those activated by the disruption of cell-cell membranes and stimulated by growth , the second row is α-SMA (red), the third is S100A4 (green) while blue is DAPI. White arrows indicate cells in which β-catenin translocated to the nuclei and cells that express α-SMA. Scale bar 50 µM. The panel (c) on the right shows a bar chart for the quantification of the immunofluorescence analysis (in % of cells) of cells having β-catenin in the nuclei, expressing α-SMA or presenting S100A4 in the nuclei (12 fields and n = 3 replicates). Results are presented as mean ± Standard Error (SE). T-test was performed *p < 0.05, **p < 0.01, ***p < 0.001. (d) Represents a bar chart of Propidium Iodide flow cytofluorimetric analysis for quercetin treatment of rCEnC. Experiments were performed n = 3. Results are presented as mean ± SE. T-test was performed *p < 0.05, **p < 0.01, ***p < 0.001. Scientific RepoRtS | (2020) 10:13841 | https://doi.org/10.1038/s41598-020-70800-w www.nature.com/scientificreports/ factors. For instance, RAC1 has been proposed to act in concert with β-catenin in inducing CEnC proliferation through Cyclin D1 transcriptional activation 27 .
In conclusion, herein we present a study that, starting from a proteomic analysis, provides insights into the major intracellular pathways that CEnC activate in culture. Further studies of these mechanisms are fundamental to understand how to unlock the CEnC mitotic block and improve their regenerative capacity in order to develop a localised therapy that would overcome the need for corneal transplantation.

Materials and methods
Corneal endothelial cell harvesting and culture. Human corneas, preserved in Eusol at 4 °C, were selected for experiments with the following criteria: age ranging from 49 to 78 years old, no history of corneal diseases, CEnC density greater than 1,800 cells/mm 2 , death to preservation interval lower than 15 h and used for experiments within 10 days from death. CEnC isolation was performed following washing in Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific, USA), Descemet's stripping and subsequent digestion with 1 mg/ml Collagenase A (Roche, USA) in DMEM (Thermo Fisher Scientific, USA) for 3 h at 37 °C. Isolated cell tangles were then further dissociated with TrypLE (Thermo Fisher Scientific, USA) for 5 min at 37 °C. After that, the cells were pelleted at 1,200 rpm for 3 min and harvested for direct cytofluorimetric analysis or plated after coating the wells with FNC Coating mix (AthenaES, USA). Growth medium was composed of Opti-MEM-I (Thermo Fisher Scientific, USA), 8% HyClone fetal bovine serum (FBS; FisherScientific, USA), 5 ng/ mL epidermal growth factor (EGF; Thermo Fisher Scientific, USA), 20 μg/mL ascorbic acid (Sigma-Aldrich, USA), 200 mg/L calcium chloride (Sigma-Aldrich, USA), 0.08% chondroitin sulphate (C4384, Sigma-Aldrich, USA), and penicillin/streptomycin (Euroclone, Italy). Human CEnC were cultured at 37 °C in 5% CO 2, and the medium was changed every 2 days.
Corneas from white New Zealand rabbits (3 months old, equivalent to a human age of 10 years), obtained from a local slaughterhouse (Maini SRL Modena), were used for proteomic purposes and subsequent analyses.
Corneas were harvested the same day of euthanization and processed within 24 h. Intact Descemet's membrane was stripped off the corneas and transferred to Accutase (ECB3056D, Euroclone, Italy) for 20 min at 37 °C. Isolated corneal endothelial cells were then pelleted at 1,200 rpm for 3 min. The fifteen pellets obtained from the right eyes of each rabbit were washed in DPBS, the Descemet's nude tissue was removed, and the cells were immediately frozen at − 80 °C after a second centrifugation. The fifteen corresponding left eyes were used for cultures, expanding the cells in 6 well plates coated with FNC Coating Mix with using the same medium as for human CEnC, changing it every 2 days, at 37 °C in 5% CO 2 . Upon confluence, the cells were rinsed in DPBS and passaged at ratio of 1:2 or 1:3 with TrypLE for 10-15 min at 37 °C in 5% CO 2 . Cells at 60% confluence at passage 1 were harvested, washed in DPBS, and pellets were frozen at − 80 °C for the proteomic analysis. In all other experiments cells were used between the first and the third passages, when the morphology was maintained perfectly polygonal, and always compared with their internal control.
Cell cycle analysis by flow cytometry. The cell cycle distribution was studied using Propidium Iodide (PI) staining (Sigma-Aldrich). rCEnC from tissue or cell culture were washed with DPBS and incubated in 300 μL of a PBS solution containing PI 50 µg/mL, Triton X-100 (Bio-Rad, USA) 0.1% for 1 h at 4 °C in the dark. After staining, cells were analysed using BD FACSCanto II (BD BIOSCIENCES; San Jose, CA USA). For each sample, 20,000 events were counted and considered for the analysis to ensure statistical relevance. Results were analysed with a ModFit 3.0 software. Proteomic analysis. Cell pellets were defrosted from − 80 °C and proteins were isolated using RIPA buffer, supplemented with phosphatase I, protease I, and EDTA (all from Thermo Fisher Scientific, USA), following the manufacturer's instructions.
Total protein (20 µg) was reduced with DTE, alkylated with iodoacetamide (Sigma-Aldrich), and digested with trypsin. Aliquots of the sample containing tryptic peptides were desalted using StageTip C18 (Merck Millipore, Italy), injected into and separated by UPLC, and analysed using nLC-MS/MS UPLC in line with a mass spectrometer Q-Exactive (Thermo Fisher Scientific, Germany). Peptide separations occurred on a reverse-phase silica capillary column (75 μm i.d., 15 cm long), packed with 1.9-μm ReproSil-Pur 120 C18-AQ (Dr. Maisch GmbH, Germany). A gradient of eluents A (LC-MS grade water containing 0.1% v/v formic acid) and B (acetonitrile containing 0.1% v/v formic acid) was used to achieve separation of peptides (300 nL/min flow rate), from 2 to 40% B in 88 min. Full scan spectra were acquired with the lock-mass option, resolution set to 70,000, and mass range from m/z 300 to 20,000 Da. The ten most intense doubly and triply charged ions were selected and fragmented in the ion trap, using a resolution of 17,500. All samples were analysed in technical replicates. MaxQuant software (v 1.6.1.0) was used in order to perform a label-free quantification, based on the intensity of the precursors, to identify the proteins in the complete rabbit proteome 20,190,508. Statistically significant differences between the two sets of samples were identified using the software for statistical analysis MeV v. 4.9.0. T-test was selected for the comparison and proteins differently represented in the two conditions, with p-values lower than 0.001 and 0.005, were screened for further analysis.
Gene ontology analysis. Gene Ontology (GO) analysis was performed in order to identify enriched biological themes on the toweb-based DAVID Bioinformatics Resources v6.8 (NIAID, NIH, USA). Adjusted Benjamini P values, calculated by the software using the Modified Fisher Exact test, were measured for each theme. Smaller P values represent higher significance of enrichment.
Growth factors and β-catenin Inhibitor/activator cell treatment. Rabbit CEnC (2.5 × 10 5 cells) were seeded on an FNC-coated 6 well, 24 h prior to harvesting for the cytofluorimetric analysis. bFGF (Thermo Fisher Scientific, USA) and TGFβ (Miltenyi, Germany) were resuspended in MilliQ H 2 0 and then used at a final concentration of 20 ng/mL and 10 ng/mL, respectively. Cells were treated at different concentrations of Quercetin (Q4951, Sigma-Aldrich, USA) and CHIR 99,021 (SML1046, Sigma-Aldrich, USA). The treatments were performed at 24 h as both drugs were previously shown to elicit their effect at this time point 81,82 . CHIR99021 was tested at 50, 500 nM, 1, 3, 6 and 10 µM, Quercetin at 10 and 25 µM. Both compounds were dissolved in DMSO (Sigma-Aldrich, USA) and used 0.1% v/v in culture medium. Cytofluorimetric analysis of treated and untreated cells (DMSO as a vehicle control) was performed as described in the flow cytometry section. All the treatments described were performed 3 h after plating and the cells harvested 24 h after the treatment.
Cell vitality assay. Viability of rCEnC upon CHIR99021 treatment was evaluated with calcein-AM (Thermo Fisher Scientific, USA), and propidium iodide (P3566_Invitrogen, Thermo Fisher Scientific, USA) staining. After 24 h from CHIR99021 treatment, rCEnC were incubated with 4 μM Calcein AM and 5 μg/ml propidium iodide for 30 min at 37 °C in 5% of CO 2 , then stained with DAPI 1:40,000, mounted on a glass slide and imaged with fluorescent microscope, as previously described. Cells were treated with 10 mM H 2 O 2 for 2 h before vitality assay as a positive control for cell death.
Ethical statement. Human corneas, non-suitable for transplantation, were obtained from Monza Eye Bank with written informed consent from donor's next of kin. Experimental protocol was approved by ISS-CNT (Italian National Transplant Centre): a national health authority managing the national procedures and rules regarding all Italian transplants and delegating the Tissue Banks to collect the written informed consents. The research protocol on human corneal tissues was approved by the local ethical committee (Comitato Etico dell' Area Vasta Emilia Nord, p. 0002956/20). The tissues were handled in accordance with the declaration of Helsinki.