Galectin-1 Attenuates PDGF-Mediated AKT Signaling in Retinal Pigment Epithelial Cells

Galectins have the potential to interact with transmembrane glycoproteins to modulate their functions. Since galectin-1 interacts with PDGF-Rβ, we analyzed the effect of galectin-1 on PDGF-BB-mediated AKT signaling in primary human retinal pigment epithelial (RPE) cells and galectin-1-deficient immortalized human RPE cells (LGALS1−/−/ARPE-19) following incubation with PDGF-BB and galectin-1. Expression and localization of galectin-1, PDGF-Rβ and pAKT were investigated using western blot analysis and immunohistochemical staining. Cell proliferation of RPE cells was analyzed using BrdU ELISA. Following treatment of human RPE cells with human recombinant (hr)-galectin-1 and PDGF-BB, an intense clustering of PDGF-Rβ and colocalization with galectin-1 were detected. By Western blot analysis and immunocytochemistry of human RPE cells, an enhanced PDGF-BB-mediated expression of pAKT was observed, which was substantially reduced by additional incubation with hr-galectin-1. Vice versa, in LGALS1−/−/ARPE-19 cells, the PDGF-BB-induced pAKT signal was enhanced compared to wild-type cells. Furthermore, a decreased expression of PDGF-Rβ in human RPE cells was observed after treatment with PDGF-BB and hr-galectin-1, while in untreated LGALS1−/−/ARPE-19 cells, its constitutive expression was increased. In addition, after treatment of RPE cells with hr-galectin-1, the PDGF-BB-induced proliferation was markedly reduced. In summary, galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in human RPE cells—an effect that is most likely facilitated via a decreased expression of PDGF-Rβ.


Introduction
Galectins are endogenous lectins with highly conserved carbohydrate recognition domains that mediate specific binding to β-galactoside moieties [1].β-galactosylation is a common glycosylation motive of various intra-and extracellular as well as transmembrane proteins and receptors, which facilitates their potential interaction with galectins [2].In addition to their interaction with β-galactoside-binding sites, galectins can also undergo non-carbohydrate-dependent protein-protein interactions [1,3].
Galectin-1 has one carbohydrate recognition domain (CRD) and forms homodimers via sandwich binding of their two anti-parallel β-sheets, and it is found both intra-and extracellularly [4].In the cell, galectin-1 was observed in the cytoplasm and the nucleus, where it interacts with several proteins to modulate signaling pathways, transcription, apoptosis, actin polymerization or preRNA processing [4][5][6].Extracellularly, it is located in the outer plasma membrane, where it binds to glycosylated membrane proteins or receptors, or in the extracellular matrix [4].Via its two binding sites, galectin-1 homodimers can form an extracellular lattice of membrane proteins or receptors, which in turn can modulate their function, and subsequently, their intracellular signal pathways [5,7].Due to its various intra-and extracellular functions, galectin-1 is involved in several physiological and pathological processes, including fibrosis, diabetes and obesity, immune response, or tumor development and progression [8][9][10][11].
In the posterior eye segment, the retinal pigment epithelium is essential for the maintenance of the chorioretinal interface and plays a crucial role in various chorioretinal diseases.Intriguingly, a role for galectin-1 as a potential modulator is discussed in several chorioretinal pathologies, such as proliferative vitreoretinopathy (PVR).PVR is a severe complication following retinal detachment, leading to formation of epiretinal and subretinal membranes, and subsequently to recurrent retinal detachment.Intriguingly, within these membranes, which are predominantly formed by transdifferentiated RPE cells, a robust expression for galectin-1 was observed [12].In the pathogenesis of PVR RPE cell transdifferentiation, proliferation and subsequent membrane formation is driven and maintained by a variety of growth factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor−2, transforming growth factor-β or platelet-derived growth factor [13][14][15].
PDGF is a growth factor that is expressed in a wide range of cells.It consists of four isoforms, which are expressed from four different genes designated as PDGF-A to D [16].Apart from PDGF-AB, the biologically active forms are homodimers, which all have the potential to bind to PDGF receptor (PDGF-R)α and/or β [17].PDGF-Rα and β are classical receptor tyrosine kinases, with an extracellular ligand-binding domain and an intracellular tyrosine kinase domain [18].Following binding, PDGF forms a homoor heterodimer of its receptors, which in turn induce auto-phosphorylation of their intracellular domain and subsequent activation of different downstream pathways, such as mitogen-activated protein (MAP) kinase, phosphoinositide−3-kinase/protein kinase B (PI3-kinase/Akt) or phospholipase C (PLC)γ pathways, to promote cell biological effects like proliferation or migration [18].
Since retinal pigment epithelium (RPE) cells and PDGF play a critical role in most diseases of the chorioretinal interface, and galectins have the distinct potential to bind to PDGF-Rβ, thereby potentially modifying its intracellular shuttling and signaling [19][20][21], we investigated whether galectin-1 could modulate PDGF signaling in RPE cells.For this purpose, the potential interaction of galectin-1 and PDGF-Rβ was analyzed on isolated human RPE after PDGF-BB treatment via immunohistochemistry.To determine the downstream effects, human RPE cells and immortalized human RPE cells (ARPE-19) with and without a lack of galectin-1 expression (ARPE-19/LGALS1 −/− ) [22] were incubated with PDGF-BB in combination with human recombinant (hr)-galectin-1, and pAKT, pERK1/2 and β-catenin signaling, as well as the glycosylation-dependence of the observed downstream effects, were analyzed.Finally, their effect on cell proliferation was investigated.

Galectin-1 Colocalizes with PDGF-BB-Induced Clusters of PDGF-Rβ
To investigate whether exogenous galectin-1 interacts with PDGF-Rβ in human RPE cells, cultured human RPE cells were incubated with hr-galectin-1 and/or PDGF-BB and analyzed by immunohistochemistry.
Following treatment of human RPE cells with PGDF-BB for 30 min, a punctate staining for PDGF-Rβ was observed on the cell membrane, suggesting a PDGF-BB-induced clustering of this receptor (Figure 1A,C), whereas staining for endogenous galectin-1 was predominantly located in the nucleus (Figure 1B,C), as it was in untreated controls.No obvious colocalization between PDGF-Rβ and endogenous galectin-1 was observed following treatment with PDGF-BB alone.After incubation of human cultured RPE cells with hr-galectin-1 and PDGF-BB, the punctate staining for PDGF-Rβ was detected at the plasma membrane again, although it showed a more perinuclear localization (Figure 1D,F).
In addition, within the PDGF-BB-mediated clusters of PDGF-Rβ, an intense signal for galectin-1 was observed (arrow heads in Figure 1D,F), strongly suggesting an interaction of galectin-1 with PDGF-Rβ in cultured human RPE cells.
predominantly located in the nucleus (Figure 1B,C), as it was in untreated controls.No obvious colocalization between PDGF-Rβ and endogenous galectin-1 was observed following treatment with PDGF-BB alone.After incubation of human cultured RPE cells with hr-galectin-1 and PDGF-BB, the punctate staining for PDGF-R was detected at the plasma membrane again, although it showed a more perinuclear localization (Figure 1D,F).In addition, within the PDGF-BB-mediated clusters of PDGF-R, an intense signal for galectin-1 was observed (arrow heads in Figure 1D,F), strongly suggesting an interaction of galectin-1 with PDGF-R in cultured human RPE cells.

PDGF-BB-Induced AKT and ERK1/2 Signaling Is Reduced by hr-Galectin-1
Next, we investigated whether the interaction of galectin-1 with PDGF-R modifies the intracellular PDGF-BB-mediated signaling.To achieve this, the expression levels for pAKT, pERK1/2 and -catenin were analyzed on human cultured RPE cells via Western blot analyses and immunohistochemical staining after treatment with PDGF-BB, hr-galectin-1 or a combination thereof.
In untreated control cells, a weak but specific band for pAKT was observed, which was similar to that of hr-galectin-1-treated cells (Figure 2A,B).In contrast, following incubation with PDGF-BB for 30 min, a strong signal for pAKT was detected, which was 30.0  8.7-fold higher compared to untreated controls (Figure 2A,B).Intriguingly, the combined treatment with hr-galectin-1 and PDGF-BB reduced the PDGF-BB-mediated increase in pAKT to 18.1  5.2-fold compared to untreated controls (Figure 2A,B).Thus, the addition of hr-galectin-1 decreased the PDGF-mediated enhanced phosphorylation of AKT to 60.5  17.4%-a difference that was statistically significant (100  29.0%; n = 8; p = 0.033).Next, we investigated whether the interaction of galectin-1 with PDGF-Rβ modifies the intracellular PDGF-BB-mediated signaling.To achieve this, the expression levels for pAKT, pERK1/2 and β-catenin were analyzed on human cultured RPE cells via Western blot analyses and immunohistochemical staining after treatment with PDGF-BB, hr-galectin-1 or a combination thereof.
In untreated control cells, a weak but specific band for pAKT was observed, which was similar to that of hr-galectin-1-treated cells (Figure 2A,B).In contrast, following incubation with PDGF-BB for 30 min, a strong signal for pAKT was detected, which was 30.0 ± 8.7-fold higher compared to untreated controls (Figure 2A,B).Intriguingly, the combined treatment with hr-galectin-1 and PDGF-BB reduced the PDGF-BB-mediated increase in pAKT to 18.1 ± 5.2-fold compared to untreated controls (Figure 2A,B).Thus, the addition of hr-galectin-1 decreased the PDGF-mediated enhanced phosphorylation of AKT to 60.5 ± 17.4%-a difference that was statistically significant (100 ± 29.0%; n = 8; p = 0.033).
Homologous results, although to a lesser extent, were obtained for pERK1/2 signaling.Again, following the incubation of human RPE cells with PDGF-BB, a significant increase in pERK expression of 2.8 ± 0.4-fold was detected compared to untreated controls (Figure 2A,C).However, the additive treatment with hr-galectin-1 only led to a slight decline in PDGF-BB-mediated ERK phosphorylation (2.3 ± 0.7-fold) by approximately 18% (Figure 2A,C).Cultured human RPE cells that were only incubated with hr-galectin-1 showed a weak increase in ERK1/2 phosphorylation (1.55 ± 0.25-fold).
Furthermore, only minor effects on the expression of β-catenin in RPE cells were observed following PDGF-BB and/or hr-galectin-1 treatment (Figure 2A).
In line with this, immunohistochemical analysis revealed a marked increase in pAKT and pERK labeling in PDGF-BB incubated human cultured RPE cells when compared to untreated controls and hr-galectin-1 incubated cells (Figure 2D,E).Again, following the treatment with PDGF-BB and hr-galectin-1, the signal for pAKT and pERK was considerably lower compared to PDGF-BB incubated RPE cells (Figure 2D,E).Overall, our data strongly suggest that galectin-1 can reduce pAKT and, to a lesser extent, pERK signaling.Homologous results, although to a lesser extent, were obtained for pERK1/2 signaling.Again, following the incubation of human RPE cells with PDGF-BB, a significant increase in pERK expression of 2.8  0.4-fold was detected compared to untreated controls (Figure 2A,C).However, the additive treatment with hr-galectin-1 only led to a slight decline in PDGF-BB-mediated ERK phosphorylation (2.3  0.7-fold) by approximately 18% (Figure 2A,C).Cultured human RPE cells that were only incubated with hr-galectin-1 showed a weak increase in ERK1/2 phosphorylation (1.55  0.25-fold).
Furthermore, only minor effects on the expression of -catenin in RPE cells were observed following PDGF-BB and/or hr-galectin-1 treatment (Figure 2A).
In line with this, immunohistochemical analysis revealed a marked increase in pAKT and pERK labeling in PDGF-BB incubated human cultured RPE cells when compared to untreated controls and hr-galectin-1 incubated cells (Figure 2D,E).Again, following the treatment with PDGF-BB and hr-galectin-1, the signal for pAKT and pERK was considerably lower compared to PDGF-BB incubated RPE cells (Figure 3D,E).Overall, our data strongly suggest that galectin-1 can reduce pAKT and, to a lesser extent, pERK signaling.
As described before, galectin-1 is a carbohydrate-binding protein that mediates its function via galectin-carbohydrate but also galectin-protein interactions.To analyze whether hr-galectin-1 modulated pAKT signaling is glycosylation-dependent, human RPE cells were pretreated with kifunensine for 4 days.Kifunensine is a selective inhibitor of Golgi class I α-mannosidases [23-25], which prevents complex-type β1,6-N-glycosylation of proteins and thereby reduces the binding of galectin-1 to its binding partners [21].As observed before, pAKT signaling was substantially increased in human RPE cells following the treatment with PDGF-BB (38.9 ± 11.2-fold) compared to untreated controls-an effect that was reduced to approximately 60% when cells were additionally incubated with hrgalectin-1 (23.4 ± 8.8-fold; Figure 3A,B).When kifunesine-treated cells were incubated with PDGF-BB alone, the signal for pAKT was enhanced (20.0 ± 7.1-fold), but the increase was only 51.5 ± 18.2% of that of PDGF-BB incubated RPE cells without kifunensine pretreatment (100 ± 28.8%, p = 0.022; Figure 3A,B).
As described before, galectin-1 is a carbohydrate-binding protein that mediates its function via galectin-carbohydrate but also galectin-protein interactions.To analyze whether hr-galectin-1 modulated pAKT signaling is glycosylation-dependent, human RPE cells were pretreated with kifunensine for 4 days.Kifunensine is a selective inhibitor of Golgi class I α-mannosidases [23-25], which prevents complex-type 1,6-N-glycosylation of proteins and thereby reduces the binding of galectin-1 to its binding partners [21].As observed before, pAKT signaling was substantially increased in human RPE cells following the treatment with PDGF-BB (38.9  11.2-fold) compared to untreated controlsan effect that was reduced to approximately 60% when cells were additionally incubated with hr-galectin-1 (23.4  8.8-fold; Figure 3A,B).When kifunesine-treated cells were incubated with PDGF-BB alone, the signal for pAKT was enhanced (20.0  7.1-fold), but the increase was only 51.5  18.2% of that of PDGF-BB incubated RPE cells without kifunensine pretreatment (100  28.8%, p = 0.022; Figure 3A,B).
In line with this, the combined incubation of kifunensine-pretreated RPE cells with hr-galectin-1 and PDGF-BB also led to an increase in pAKT signaling, but again, this rise was not as pronounced as in cells without kifunensine treatment (14.5  4.2-fold with kifunensine vs. 23.4 8.8-fold without kifunensine; Figure 3A,B).Furthermore, the reduced 1,6-N-glycosylation lowered the impact of hr-galectin-1 on mitigating PDGF-BB-induced upregulation of pAKT, which was not as strong as in cells without prior kifunensine treatment (reduction in pAKT levels by 25.2% with kifunesine pretreatment vs. 38.7%without kifunensine pretreatment; Figure 3A,B).Thus, 1,6-N-glycosylation in human RPE cells is required for both for PDGF-BB-induced activation of the AKT pathway and for galectin-1-mediated modulation of pAKT signaling.In line with this, the combined incubation of kifunensine-pretreated RPE cells with hr-galectin-1 and PDGF-BB also led to an increase in pAKT signaling, but again, this rise was not as pronounced as in cells without kifunensine treatment (14.5 ± 4.2-fold with kifunensine vs. 23.4 ± 8.8-fold without kifunensine; Figure 3A,B).Furthermore, the reduced β1,6-N-glycosylation lowered the impact of hr-galectin-1 on mitigating PDGF-BB-induced upregulation of pAKT, which was not as strong as in cells without prior kifunensine treatment (reduction in pAKT levels by 25.2% with kifunesine pretreatment vs. 38.7%without kifunensine pretreatment; Figure 3A,B).Thus, β1,6-N-glycosylation in human RPE cells is required for both for PDGF-BB-induced activation of the AKT pathway and for galectin-1-mediated modulation of pAKT signaling.
In order to further confirm the relevance of carbohydrate binding in galectin-1-and PDGF-BB-mediated pAKT modulation in RPE cells, β-lactose was used as a competitive inhibitor for β-galactoside-dependent interactions.β-lactose can block the β-galactosidedependent protein interaction via binding to the carbohydrate recognition domain of galectins.Therefore, this assay is routinely used to demonstrate the carbohydrate dependence of galectin functions as opposed to protein-protein interactions, which would not be influenced by addition of β-lactose [12,26].
As described before, following the treatment of human RPE cells with PDGF-BB, a robust band for pAKT was detected compared to controls (Figure 3C,D).However, after the incubation of β-lactose-treated human RPE cells with PDGF-BB and/or hr-galectin-1, no enhanced signal for pAKT was detected, neither after the addition of PDGF-BB nor with PDGF-BB and hr-galectin-1 (Figure 3C,D), confirming that both PDGF-BB itself and hr-galectin-1 require β-galactoside moieties to mediate AKT signaling.

Galectin-1 Reduces PDGF-BB-Mediated Clustering of PDGF-Rβ
Since we could demonstrate that galectin-1 reduces PDGF-BB-mediated AKT signaling, we investigated whether galectin-1 mediates its effects via modulation of PDGF-BBinduced clustering of PDGF-R in cultured human RPE cells by immunohistochemistry.
In untreated human RPE cells, as well as following treatment with rh-galectin-1, only a weak and rather diffuse signal for PDGF-R could be detected (Figure 5A,B).As shown before, the treatment with PDGF-BB induced an enhanced, spotted fluorescent labeling for PDGF-R at the cell surface (Figure 5C), indicating clustering of PDGF-R.Intriguingly, after additional incubation of RPE cells with hr-galectin-1, the PDGF-BB-mediated clustering was markedly reduced when compared to cells treated with PDGF-BB alone (Figure 5D).

Galectin-1 Reduces PDGF-BB-Mediated Clustering of PDGF-Rβ
Since we could demonstrate that galectin-1 reduces PDGF-BB-mediated AKT signaling, we investigated whether galectin-1 mediates its effects via modulation of PDGF-BB-induced clustering of PDGF-Rβ in cultured human RPE cells by immunohistochemistry.
In untreated human RPE cells, as well as following treatment with rh-galectin-1, only a weak and rather diffuse signal for PDGF-Rβ could be detected (Figure 5A,B).As shown before, the treatment with PDGF-BB induced an enhanced, spotted fluorescent labeling for PDGF-Rβ at the cell surface (Figure 5C), indicating clustering of PDGF-Rβ.Intriguingly, after additional incubation of RPE cells with hr-galectin-1, the PDGF-BBmediated clustering was markedly reduced when compared to cells treated with PDGF-BB alone (Figure 5D).To further confirm our observations, the fluorescent signal for PDGF-R was quantified.The treatment of human RPE cells with hr-galectin-1 had only minor effects on the fluorescent signal (1.06  0.04-fold) compared to untreated controls (1.0  0.05-fold; Figure 5E).However, following incubation of the cells with PDGF-BB, the fluorescent signal for PDGF-R was significantly increased to 1.95  0.06-fold-an effect that was substantially blocked when hr-galectin-1 was additionally added (1.48  0.04-fold).Overall, our data strongly suggest that galectin-1 can attenuate PDGF-BB-mediated clustering.

Exogenous and Endogenous Galectin-1 Mitigates PDGF-BB-Mediated pAKT Signaling by Reducing PDGF-R Expression in RPE Cells
To provide further insight on the mechanism of galectin-1-mediated attenuation of PDGF-BB-induced AKT signaling and reduced PDGF-R clustering, the protein expression levels for PDGF-R were determined using Western blot analyses.
Vice versa, in immortalized galectin-1-deficient ARPE-19 cellsthe protein expression levels for PDGF-R were significantly higher (2.1- 0.5-fold, p < 0.032) compared to wildtype controls (Figure 6C,D).Overall, our data strongly suggest that both exogenously added and endogenously expressed galectin-1 has the distinct potential to downregulate the protein expression of PDGF-R.To further confirm our observations, the fluorescent signal for PDGF-Rβ was quantified.The treatment of human RPE cells with hr-galectin-1 had only minor effects on the fluorescent signal (1.06 ± 0.04-fold) compared to untreated controls (1.0 ± 0.05-fold; Figure 5E).However, following incubation of the cells with PDGF-BB, the fluorescent signal for PDGF-Rβ was significantly increased to 1.95 ± 0.06-fold-an effect that was substantially blocked when hr-galectin-1 was additionally added (1.48 ± 0.04-fold).Overall, our data strongly suggest that galectin-1 can attenuate PDGF-BB-mediated clustering.

Exogenous and Endogenous Galectin-1 Mitigates PDGF-BB-Mediated pAKT Signaling by Reducing PDGF-Rβ Expression in RPE Cells
To provide further insight on the mechanism of galectin-1-mediated attenuation of PDGF-BB-induced AKT signaling and reduced PDGF-Rβ clustering, the protein expression levels for PDGF-Rβ were determined using Western blot analyses.

Galectin-1 Attenuates PDGF-Induced RPE Cell Proliferation
To investigate whether the interaction of PDGF-R and galectin-1 and its influence on PDGF-BB-induced pAKT signaling result in a downstream functional effect, cells were treated with PDGF-BB with or without the addition of hr-galectin-1, and the proliferation of cultured human RPE cells was assessed by BrdU ELISA.
Following incubation of cells with hr-galectin-1 alone, a slight increase in cell proliferation was observed, while the treatment of RPE cells with PDGF-BB for 24 h led to an increased proliferation of 62.7  7.4% (p < 0.001) compared to untreated controls (Figure 7).However, the PDGF-BB-mediated proliferative effect was reduced to 32.2  4.6% when cells were additionally incubated with hr-galectin-1-an effect that was statistically significant (p < 0.01; Figure 7).Overall, our data strongly suggest that galectin-1-induced inhibition of PDGF-BB signaling leads to cell biological effects, such as reduced proliferation of RPE cells.

Galectin-1 Attenuates PDGF-Induced RPE Cell Proliferation
To investigate whether the interaction of PDGF-Rβ and galectin-1 and its influence on PDGF-BB-induced pAKT signaling result in a downstream functional effect, cells were treated with PDGF-BB with or without the addition of hr-galectin-1, and the proliferation of cultured human RPE cells was assessed by BrdU ELISA.
Following incubation of cells with hr-galectin-1 alone, a slight increase in cell proliferation was observed, while the treatment of RPE cells with PDGF-BB for 24 h led to an increased proliferation of 62.7 ± 7.4% (p < 0.001) compared to untreated controls (Figure 7).However, the PDGF-BB-mediated proliferative effect was reduced to 32.2 ± 4.6% when cells were additionally incubated with hr-galectin-1-an effect that was statistically significant (p < 0.01; Figure 7).Overall, our data strongly suggest that galectin-1-induced inhibition of PDGF-BB signaling leads to cell biological effects, such as reduced proliferation of RPE cells.

Discussion
In our present study, we showed that both hr-galectin-1 and endogenous galectin-1 have the potential to attenuate PDGF-BB-mediated pAKT signaling in human RPE cells via a reduced expression of PDGF-R.Our conclusions rest upon (1) the observation that the interaction of PDGF-R with hr-galectin-1 leads to decreased pAKT signaling; (2) the finding that in galectin-1-deficient immortalized RPE cells, the pAKT signal was significantly enhanced following PDGF-BB treatment; (3) the potential of rh-galectin-1 to reduce PDGF-BB-induced receptor clustering and expression of PDGF-R; (4) the observation of an enhanced expression of PDGF-R in galectin-1-deficient RPE; and finally, (5) the ability of hr-galectin-1 to reduce PDGF-BB-induced proliferation of RPE cells.
Galectin-1 is a protein that interacts with several intra-and extracellular proteins to modulate apoptotic processes, preRNA processing, transcription or signaling pathways [4][5][6][7].In previous works, a glycosylation-dependent interaction between PDGF-R and galectin−3 has been reported, which in turn promoted the clustering of both proteins and a moderate phosphorylation of ERK1/2, AKT and GSK−3/ [21].In line with this, by employing immunohistochemical analysis, we observed a colocalization of PDGF-R with galectin-1 in RPE cells following the treatment with PDGF-BB and hr-galectin-1, strongly suggesting an interaction of both proteins at the cell surface.
PDGF-R belongs to class III receptor tyrosine kinases that activate several intracellular pathways, including MAP kinase, AKT, PLC, -catenin or signal transducer and activator of transcription (STAT) following the binding of PDGF-BB [27][28][29].After treatment of human RPE cells with PDGF-BB, we observed a significant increase in phosphorylated AKT and ERK1/2, while the expression of -catenin remained unchanged, indicating a selective effect of PDGF-BB on these signaling pathways in RPE cells, which is in agreement with previous observations [30].Intriguingly, the additional incubation of cells with hr-galectin-1 led to a reduction in PDGF-BB-induced pAKT and pERK levels.Vice versa, in immortalized RPE cells with targeted deletion of the galectin-1 gene, the pAKT signal was significantly enhanced following PDGF-BB treatment, strongly suggesting that galectin-1 can modulate the intensity of this pathway, irrespective of whether it is added exogenously or endogenously expressed.A comparable influence of galectin-1 was found in PDGF-BB-treated airway smooth muscle cells, where galectin-1 reduced pAKT

Discussion
In our present study, we showed that both hr-galectin-1 and endogenous galectin-1 have the potential to attenuate PDGF-BB-mediated pAKT signaling in human RPE cells via a reduced expression of PDGF-Rβ.Our conclusions rest upon (1) the observation that the interaction of PDGF-Rβ with hr-galectin-1 leads to decreased pAKT signaling; (2) the finding that in galectin-1-deficient immortalized RPE cells, the pAKT signal was significantly enhanced following PDGF-BB treatment; (3) the potential of rh-galectin-1 to reduce PDGF-BB-induced receptor clustering and expression of PDGF-Rβ; (4) the observation of an enhanced expression of PDGF-Rβ in galectin-1-deficient RPE; and finally, (5) the ability of hr-galectin-1 to reduce PDGF-BB-induced proliferation of RPE cells.
Galectin-1 is a protein that interacts with several intra-and extracellular proteins to modulate apoptotic processes, preRNA processing, transcription or signaling pathways [4][5][6][7].In previous works, a glycosylation-dependent interaction between PDGF-Rβ and galectin−3 has been reported, which in turn promoted the clustering of both proteins and a moderate phosphorylation of ERK1/2, AKT and GSK−3α/β [21].In line with this, by employing immunohistochemical analysis, we observed a colocalization of PDGF-Rβ with galectin-1 in RPE cells following the treatment with PDGF-BB and hr-galectin-1, strongly suggesting an interaction of both proteins at the cell surface.
PDGF-Rβ belongs to class III receptor tyrosine kinases that activate several intracellular pathways, including MAP kinase, AKT, PLCγ, β-catenin or signal transducer and activator of transcription (STAT) following the binding of PDGF-BB [27][28][29].After treatment of human RPE cells with PDGF-BB, we observed a significant increase in phosphorylated AKT and ERK1/2, while the expression of β-catenin remained unchanged, indicating a selective effect of PDGF-BB on these signaling pathways in RPE cells, which is in agreement with previous observations [30].Intriguingly, the additional incubation of cells with hrgalectin-1 led to a reduction in PDGF-BB-induced pAKT and pERK levels.Vice versa, in immortalized RPE cells with targeted deletion of the galectin-1 gene, the pAKT signal was significantly enhanced following PDGF-BB treatment, strongly suggesting that galectin-1 can modulate the intensity of this pathway, irrespective of whether it is added exogenously or endogenously expressed.A comparable influence of galectin-1 was found in PDGF-BBtreated airway smooth muscle cells, where galectin-1 reduced pAKT expression, which in turn led to reduced migration and proliferation [20].Nevertheless, the influence of galectin-1 on AKT and ERK signaling appears to vary and depend on several factors, including cell type, treatment, glycosylation, pathological conditions and other biological contexts [31][32][33].For instance, in myeloma cell lines, but not in B-cell lines, galectin-1 promotes survival via an enhanced signaling of pAKT and pERK1/2-an effect that depends on the expression of a specific isoform of CD45 [31].Furthermore, we found that the potential of recombinant galectin-1 to reduce PDGF-BB-mediated pAKT signaling is most likely mediated by a carbohydrate-dependent binding to PDGF-Rβ.
Pretreatment with two inhibitors of the β-galactoside-dependent carbohydrate-protein interaction markedly reduced PDGF-BB-mediated pAKT signaling in RPE cells.This finding provides evidence that the state of glycosylation of PDGF-Rβ itself can modulate PDGF-BB-induced AKT signaling, which is in line with a previous report on tenon-derived fibroblasts [34].In addition, both kifunensine and β-lactose substantially mitigated the capacity of hr-galectin-1 to attenuate PDGF-BB-induced AKT phosphorylation in RPE cells, strongly supporting our hypothesis of a specific glycosylation-dependent effect of galectin-1 on the downstream action of PDGF-Rβ.
Following internalization via clathrin-mediated or clathrin-independent endocytosis, the receptor tyrosine kinases are shuttled to early endosomes from where they are either transferred back to the plasma membrane or tagged for lysosomal degradation [35].For PDGF-Rβ, both endocytic pathways were detected, leading to cell-type-specific biological effects after stimulation with PDGF-BB [19].Intriguingly, galectin−3 has been found to be involved in clathrin-independent endocytosis of PDGF-Rβ in fibroblasts, although it is unclear whether it affects intracellular shuttling of the endosomes [19].Furthermore, galectin−3 and −9 can accumulate in the lysosomal compartment of shigella infected Hela and prostate cancer cells and have been associated with ubiquitinated proteins [36].Following the incubation of RPE cells with PDGF-BB together with galectin-1, we observed a reduced clustering and lower protein expression of PDGF-Rβ compared to the treatment with PDGF-BB alone.Vice versa, in immortalized ARPE−19/LGALS1 −/− cells, the expression level of PDGF-Rβ was increased.Although not investigated in this study, it is tempting to speculate whether galectin-1 may promote enhanced lysosomal degradation of PDGF-Rβ following PDGF-BB stimulation in human RPE cells-an effect of galectins, which has been observed before for other glycoproteins and cell types [37,38].
Proliferative vitreoretinopathy (PVR) is a devastating vision-threatening complication following retinal detachment that is characterized by the formation of cell-rich, contractile epiretinal membranes composed of various transdifferentiated retinal cell types in a proliferative state [39].Proliferation, migration and attachment of detached RPE cells are key cellular events in the pathogenesis of PVR.Intriguingly, galectin-1 is upregulated in transdifferentiated RPE cells and expressed in PVR membranes [12].Furthermore, immunohistochemical studies evidenced an expression of PDGF-Rβ and PDGF-BB in these membranes, together with several other growth factors and their receptors [12,40].The evidence for a pathogenic relevance of PDGF-BB in PVR is derived from our own studies on RPE cells from epiretinal membranes.In these cells, PDGF-BB activated AKT signaling, while depletion of PDGF-Rβ led to attenuated signaling and reduced proliferation, migration and contraction of these cells following incubation with the vitreous of patients suffering from PVR [41].Since we observed an inhibition of attachment, spreading and migration of transdifferentiated RPE cells by hr-galectin-1 in a previous work [42], and we now demonstrated that galectin-1 attenuates PDGF-BB-mediated proliferation of RPE cells via attenuation of PDGF-BB-induced AKT signaling, it is tempting to speculate that in vitreo-retinal diseases involving transdifferentiated RPE cells, galectin-1 may function to block an exaggerated wound-healing response, as observed in severe PVR.
Overall, we conclude that galectin-1 has the distinct potential to reduce PDGF-mediated pAKT signaling and proliferation in retinal pigment epithelial cells in a glycosylationdependent manner-an effect that is most likely facilitated by a decreased expression of PDGF-Rβ.24 h under standard conditions to ensure complete cell adherence.Following an additional incubation with 20 ng/mL human recombinant PDGF-BB (Bio-Techne, Minneapolis, MN, USA) and/or 10 µg/mL hr-galectin-1 in cell culture medium without supplements containing BrdU labeling solution for 24 h, cells were fixed and incubated with anti-BrdU antibodies.After colorimetric development, the amount of BrdU incorporation into the DNA was quantified by absorbance measurement at a wavelength of 450 nm and a reference at 690 nm on the SpectraMax 190 ELISA reader (Molecular Devices, San Jose, CA, USA).
For visualization, membranes were washed twice with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5) for 15 min and once with detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5) for 5 min.Following incubation with CDP-Star substrate for 5 min, in accordance with the manufacturer's recommendations (CDP-Star, Thermo Fisher Scientific, Waltham, MA, USA), membranes were documented with an iBrightCL1000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis
All calculations and statistical analyses were performed using EXCEL for MAC version 16.85 (Microsoft, Redmond, WA, USA) and SPSS version 29.0 (IBM, Armonk, NY, USA).For data presentation, the mean and standard error of the mean (SEM) were calculated and shown as indicated.For comparison of two groups of mean variables, Student's t-test was used, and for comparison of more than two groups, one-way ANOVA was used.For data that met the assumption of homogeneity of variances, a least significant difference (LSD) post hoc test was employed, and for data not meeting the criteria, a Games-Howell post hoc test was performed.p-values of less than 0.05 were considered statistically significant.

Figure 3 .
Figure 3. Galectin-1 reduces PDGF-BB-induced AKT signaling in a glycosylation-dependent manner.Western blot analysis (A,C) and densitometry (B,D) for pAKT of human RPE cells after pretreatment with 10 µM kifunensine for 4 d (A,B) or -lactose immediately before the experiment (C,D) and incubation with 10 µg/mL hr-galectin-1 and/or 20 ng/mL PDGF-BB for 30 min.Mean  SEM; n = 6 for B, n = 3 for D.