Calcium alterations signal either to senescence or to autophagy induction in stem cells upon oxidative stress

Results

Oxidative stress induces a rapid [Ca²⁺]i increase in hMESCs

We first investigated the effects of the sublethal oxidative stress on the intracellular calcium levels in hMESCs. Using Fluo-3 imaging techniques we revealed a rapid elevation of [Ca²⁺]_i in response to H₂O₂ addition that gradually increased during 1 h treatment (Fig. 1a, 1b and 1e). It is well known that cytosolic calcium influx may originate from both intra- and extracellular sources [30]. Thus, to discriminate the wellspring of the observed [Ca²⁺]_i elevation in H₂O₂-treated hMESCs, we performed assays in the absence of extracellular calcium. The results presented in Fig. 1c, 1d and 1e show that omitting Ca²⁺ from bathing solution had no dramatic suppressive effect on [Ca²⁺]_i increase in response to H₂O₂ stimulation. Therefore, Ca²⁺ release from intracellular stores rather than Ca²⁺ entry across the plasma membrane might be responsible for the [Ca²⁺]_i elevation in hMESCs during H₂O₂ treatment.

Figure 1. Oxidative stress induces intracellular calcium increase. hMESCs were treated with 200 µM H₂O₂ for 1 h and intracellular calcium levels were determined using Fluo-3 imaging techniques. (a) Time course of the relevant increase of [Ca²⁺]_i during 1 h H₂O₂ treatment in Ca²⁺-containing solution. Number of cells = 24. (b) Histogram, based on the data from (a), reflecting the relevant values of [Ca²⁺]_i on 2 min in control and on 60 min after H₂O₂ addition. (c) Time course of the relevant increase of [Ca²⁺]_i during 1 h H₂O₂ treatment in Ca²⁺-free solution containing 4mM EGTA. Number of cells = 28. (d) Histogram, based on the data from (c), reflecting relevant values of [Ca²⁺]_i in Ca²⁺-free basic solution on 2 min in control, on 5 min of EGTA action and on 60 min of H₂O₂ action. (e) Confocal images of hMESCs loaded with Fluo-3AM on 2 min of control, on 60 min of H₂O₂ action in Ca²⁺-containing solution and on 60 min of H₂O₂ action in Ca²⁺-free solution with EGTA. Scale bar is 100 µm and valid for all images. Values are M ± Std.Er. *** p<0.0001 by Mann-Whitney test. Application intervals and duration are marked with black lines above the graphs. Ctr – untreated cells. Representative results of three independent experiments are shown.

PLC/IP3/IP3R pathway orchestrates the prompt cytosolic Ca²⁺ response in H₂O₂-treated hMESCs

Having established the fact of the [Ca²⁺]_i increase in hMESCs during 1 h H₂O₂ treatment, we next explored what signaling pathway might be responsible for it. As a matter of fact Ca²⁺ release from internal stores occurs primarily from the endoplasmic reticulum (ER) [30–32]. Thus, we speculate that inositol trisphosphate (IP3), known to mediate a rapid calcium store release through the activation of IP3 receptors (IP3R) in the ER membrane, might be a good candidate for generating calcium increase in H₂O₂-treated hMESCs. In order to check this hypothesis we applied various drugs to modulate IP3 synthesis and determined cytosolic Ca²⁺ signals with Fluo-3 in H₂O₂-treated cells. The phospholipase C (PLC) is the up-stream regulator of the intracellular IP3 generation [33]. Therefore, to investigate whether PLC-dependent IP3 production could contribute to the revealed [Ca²⁺]_i increase in response to H₂O₂, we examined the effects of its inhibitor U-73122 in the Ca²⁺-free external solution containing EGTA. Interestingly, blocking IP3 synthesis with U-73122 distinctly suppressed H₂O₂-induced Ca²⁺ increase (Fig. 2a and 2b).

Figure 2. PLC/IP3/IP3R pathway mediates H₂O₂-induced intracellular calcium elevation in hMESCs. All measurements were done in Ca²⁺-free basic solution supplemented with 4 mM EGTA in 2 min after the beginning of the imaging, which further was present throughout the whole experiment. (a) Time course of the relevant [Ca²⁺]_i increase during 5 min of 1 µM U73122 pretreatment followed by 60 min of 200 µM H₂O₂ stimulation. (b) Histogram based on the data from (a), reflecting the relevant values of [Ca²⁺]_i on 2 min in control, on 5 min of EGTA alone action, on 5 min of U73122 action, on 60 min of U73122 + H₂O₂ action, and on 60 min of H₂O₂ alone action. Number of cells = 24. (c) Time course of the relevant [Ca²⁺]_i increase in H₂O₂-stimulated hMESCs pretreated with 100 µM UTP for 10 min. (d) Histogram, based on the data from (c), reflecting the relevant values of [Ca²⁺]_i on 2 min in control, on 5 min of EGTA alone action, on 10 min of UTP action, on 60 min UTP + H₂O₂ action, and on 60 min of H₂O₂ alone action. Number of cells = 29. (e) Time course of the relevant [Ca²⁺]_i increase during 20 min of 1 µM TG pretreatment followed by 60 min of 200 µM H₂O₂ stimulation. (f) Histogram based on the data from (e), reflecting the relevant values of [Ca²⁺]_i on 2 min in control, on 5 min of EGTA alone action, on 20 min of TG action, on 60 min TG + H₂O₂ action, and on 60 min of H₂O₂ alone action. Number of cells = 31. (g) Time course of the relevant [Ca²⁺]_i increase in H₂O₂-stimulated hMESCs pretreated with 50 µM 2-APB for 5 min. (h) Histogram based on the data from (g), reflecting the with relevant values of [Ca²⁺]_i on 2 min in control, on 5 min of EGTA alone action, on 5 min of 2-APB action, on 60 min 2-APB + H₂O₂ action, and on 60 min of H₂O₂ alone action. Number of cells = 28. Results are shown as M ± Std.Er. *p<0.05, **p<0.001, ***p<0.0001 by Mann-Whitney test. Application intervals and duration are marked with black lines above the graphs. Representative results of three independent experiments are shown.

To further verify the involvement of PLC/IP3 pathway in H₂O₂-induced [Ca²⁺]_i elevation, we utilized the uridine triphosphate (UTP) as the modulator of PLC activation. In various cells UTP was shown to elicit initial release of Ca²⁺ from IP3-sensitive stores, leading to ER pool depletion [22,34]. As expected, hMESCs pretreatment with UTP in Ca²⁺-free external solution supplied with EGTA resulted in a rapid release of Ca²⁺ into cytosole. In this case subsequent H₂O₂ addition had a lesser effect on [Ca²⁺]_i levels compared to H₂O₂-stimulated cells non-pretreated with UTP, indicating the essential role of PLC-mediated store depletion in response to H₂O₂ (Fig. 2c and 2d).

As an additional confirmation of the ER stored calcium contribution to the hMESCs oxidative stress response, we next applied a non-competitive inhibitor of the Ca²⁺ ATPase (SERCA) localized in the ER – thapsigargin (TG) [22,35]. By inhibiting SERCA, TG blocks the ability of the cell to pump calcium into the ER store, what leads to gradual calcium outflow from the cell. Thus, by utilizing very different mechanisms of action both compounds TG and UTP ultimately lead to the ER pool depletion. However, in contrast to an immediate calcium outflow caused by UTP, TG induced a comparatively slow calcium release. Results presented in Fig. 2e and 2f generally are in accordance with the data obtained in presence of UTP: cell pretreatment with TG in Ca²⁺-free external solution supplied with EGTA slightly attenuated H₂O₂-induced calcium increase. This is the extra evidence in support of the ER-dependent calcium release in hMESCs upon the oxidative stress.

It is well documented that IP3 causes an immediate calcium release through the activation of IP3R in the ER membrane. Thus, to confirm that IP3R might be responsible for the mobilization of intracellular Ca²⁺ stores, we next blocked the activation of IP3R using antagonist of these receptors 2-aminoethoxydiphenyl borate (2-APB) to reduce Ca²⁺ release from the intracellular stores [36]. Interestingly, 2-APB significantly diminished H₂O₂-induced [Ca²⁺]_i rise (Fig. 2g and 2h). The obtained data clearly indicate that observed intracellular calcium elevation in H₂O₂-stimulated cells at least in part is mediated by the activation of PLC/IP3/IP3R pathway.

Intracellular calcium accumulation mediates H₂O₂-induced senescence of hMESCs

We next wanted to know whether the observed increase in [Ca²⁺]_i has an impact on H₂O₂-induced senescence development. To this end, firstly we measured intracellular calcium level at day 6 after senescence induction. As shown in Fig. 3a senescent cells displayed about 2 times higher basal Ca²⁺ as compared to control cells, suggesting that Ca²⁺ might be involved in senescence progression. In order to confirm this assumption we applied widely used intracellular calcium chelator – BAPTA-AM. Loading cells with 10 µM BAPTA-AM had no effect on cell viability (Fig. 3b), but effectively reduced [Ca²⁺]_i in H₂O₂-treated cells up to the control levels at day 6 (Fig. 3a).

Figure 3. Loading cells with BAPTA effectively decreases H₂O₂-induced [Ca²⁺]_i elevation, and has no effect on cell viability. Cells were either pretreated or not with 10 µM BAPTA (loading procedure is described in “Materials and Methods” section), then were subjected to 200 µM H₂O₂ for 1 h with the following H₂O₂ replacement and cell cultivation under normal conditions for the indicated time. (a) Intracellular calcium levels measured by FACS after staining hMESCs with the fluorescent probe Fluo-3AM at day 6 after the oxidative stress. (b) Application of 10 µM BAPTA had no effect on viability of H₂O₂-treated hMESCs. The percentage of viable cells was evaluated in 24 h after H₂O₂ treatment by FACS analysis as described in “Materials and Methods” section. M ± Std.dev., N=3. **p˂0.005, versus control; §§p˂0.005, versus H₂O₂-treated cells by Student’s t-test. Ctr – untreated cells.

Earlier we have convincingly shown that hMESCs treated with 200 µM H₂O₂ enter the premature senescence accompanied by the irreversible cell cycle arrest, cell hypertrophy and enhanced SA-β-Gal staining [37]. First of all we checked whether calcium chelation affected oxidative stress-induced senescence. As presented in Fig. 4a, 4b and 4c BAPTA application impeded H₂O₂-induced increase of the cell size as well as SA-β-Gal activity, indicating some modulation of the senescence phenotype. Moreover, BAPTA treatment led to marked increase in the number of proliferating cells compared to H₂O₂-stimulated cells, indicating that Ca²⁺ chelation overcame the growth arrest induced by H₂O₂ (Fig. 4d and 4e). Additional evidence in favor of growth arrest escape in BAPTA-loaded cells was obtained by the analysis of the cell cycle phase distribution. In our previous findings we clearly showed that hMESCs treatment with 200 μM H₂O₂ led to the prolonged irreversible cell cycle arrest in all phases [37]. Loading cells with BAPTA resulted in a distinct phase redistribution and enhanced amount of cells in S-phase, as can be seen in two-parametric histogram based on the distribution analysis with using light scattering (Fig. 4f).

Figure 4. Intracellular calcium chelation by BAPTA prevents oxidative stress-induced senescence of hMESCs. Cells were treated as indicated in the legend to Figure 3. (a) BAPTA partially prevented H₂O₂-induced increase of cell size. Cell size was determined at day 6 after the oxidative stress. Forward scatter (FS) reflects the average cell size. (b) SA-β-Gal staining of untreated, H₂O₂-treated and (BAPTA+H₂O₂)-treated hMESCs. In 5 days after the oxidative stress cells were harvested by trypsinization and plated at a density of 4.5*10³ cells per cm² and additionally cultured for 5 days, in order to perform staining in non-confluent cultures. Scale bar is 500 µm and valid for all images. (c) Quantification of β-galactosidase activity values in control, H₂O₂-treated and (BAPTA+H₂O₂)-treated hMESCs. (d) BAPTA retained cell proliferation as compared to H₂O₂-treated cells. Cell number was determined by FACS at indicated time points. (e) BAPTA pretreated hMESCs maintained the colony forming ability. In 14 days after oxidative stress cells were fixed and stained to monitor cell growth. (f) [Ca²⁺]_i chelation in H₂O₂-treated resulted in cell cycle phase re-distribution. Flow cytometric analysis of cell cycle phase distribution: the percentage of cells in the G0/G1, S, and G2/M phases, visualization of phase distribution is based on light-scattering analysis. SS log - side scattering. (g) Intracellular ROS levels detected at day 6 after H₂O₂ stimulation by FACS analysis after staining with H₂DCFDA. Images shown are representative of experiments performed at least three times. Graphs are presented as M ± Std.dev., and the Student’s t-test was used to determine p-value. *p˂0.05, **p˂0.005, ***p˂0.001, versus control; §p˂0.05, §§§p˂0.001, versus H₂O₂-treated cells. Ctr – untreated cells.

Moreover, the above results were verified by two extra sets of the experiments. First set was performed on H₂O₂-treated hMESCs preloaded with another well-known [Ca²⁺]_i chelator – Quin2-AM. The second one was directed to prevent H₂O₂-induced senescence of human embryonic fibroblasts pretreated with BAPTA. In both cases [Ca²⁺]_i chelation resulted in partial prevention of oxidative stress-induced senescence, strongly confirming calcium implication in senescence progression (Suppl. Fig. 1a, 1b, 1c and Suppl. Fig. 2a, 2b, 2c, 2d).

As we described earlier, senescent hMESCs are characterized by persistently elevated ROS levels [27]. To test whether calcium chelation could alter ROS production, we evaluated intracellular ROS levels in BAPTA-loaded and unloaded senescent cells, using H₂DCFDA staining. Loading of H₂O₂-stimulated cells with BAPTA led to a noticeable decrease in the intracellular ROS levels (Fig. 4g).

Summarizing all described above results, we can conclude that H₂O₂-induced increase of intracellular calcium levels is implied in hMESCs senescence progression, whereas calcium chelation may protect the cells from premature senescence.

Loading with BAPTA attenuates DNA damage response activation in H₂O₂-treated cells

Our previous data clearly state that premature senescence in H₂O₂-treated hMESCs develops by the following scenario: added H₂O₂ rapidly penetrates into the cells and causes DNA damage as detected by the activation of the main DNA damage response (DDR) members, including ATM, 53BP1 and H2A.X. DDR activation, in turn, leads to the initiation of the p53/p21/Rb pathway, cell cycle arrest and further senescence progression [27]. Taking into account the prompt intracellular calcium response to H₂O₂ stimulation, further we speculated that buffering of [Ca²⁺]_i by BAPTA might affect an early DDR. Indeed, loading with BAPTA dramatically reduced phosphorylation of each DDR participant as compared to H₂O₂-treated cells over the entire observation period (Fig. 5a). Accordingly, in H₂O₂-treated hMESCs, calcium chelation significantly attenuated phosphorylation of p53, prevented enhanced p21 protein expression and elevated the Rb phosphorylation levels long after senescence induction (Fig. 5b). These findings correlate well with the partial proliferation retaining of (BAPTA+H₂O₂)-treated hMESCs (Fig. 4d and 4e). These results were confirmed by applying Quin2-AM in hMESCs (Suppl. Fig. 1e, 1e). Interestingly, in BAPTA-pretreated fibroblasts we also detected reduction of ATM and H2A.X prosphoryation as well as decreased activity of p53/p21 pathway, but the observed effects were less pronounced, what might be due to cell specificity (Suppl. Fig. 2e, 2f). Taken together, these data suggest that BAPTA-induced DDR down-regulation is implicated in the senescence prevention in stressed hMESCs.

Figure 5. BAPTA attenuates activation of both DNA damage response and p53/p21/Rb pathway in H₂O₂-treated hMESCs. Cells were treated as indicated in the legend to Figure 3 and were subsequently analyzed by western blotting with the indicated antibodies at the various time points. (a) Phosphorylation levels of the main DDR members: ATM, H2A.X, 53BP1, as well as p53. (b) Western blot analysis of p53 and Rb phosphorylation, and p21 protein expression performed at indicated time points. Representative results of the three experiments are shown in the Figure. GAPDH was used as loading control.

BAPTA triggers autophagy in hMESCs under the oxidative stress

Recent reports revealed that AMPK-dependent autophagy may protect cells from oxidative stress-induced senescence [38]. We next explored whether the observed protective effect of calcium chelation from H₂O₂-induced senescence might be interlinked with autophagy process. The most common mechanism underlying the AMP-activated protein kinase (AMPK)-mediated autophagy progression is suppression of the mTORC1 pathway: activated AMPK phosphorylates TSC2, what increases the GAP activity of TSC2 and, thus reduces mTORC1 activation [39]. As it turned out, BAPTA pretreatment caused a short-term increase in AMPK activation in 30 min after H₂O₂ stimulation (Fig. 6a). Accordingly, loading cells with BAPTA promoted a rapid increase in pTSC2 and a reduction in phosphorylation levels of the main mTORC1 targets – p70S6K and 4E-BP1 as compared to H₂O₂-treated cells (Fig. 6a). Another important protein involved in the initiation of autophagosome formation is Unc-51-like kinase 1 (ULK1) [40]. Notably, ULK1 is a down-stream target for both AMPK and mTOR, however, they oppositely regulate its activity: AMPK phosphorylates ULK1 at Ser555, thus activating it, whereas mTOR is responsible for ULK1 downregulation by phosphorylating it at Ser757 [40]. Fig. 6b displays enhanced phosphorylation of “pro-autophagic” Ser555 of ULK1 and attenuated “anti-autophagic” phosphorylation of ULK1 at Ser757 in H₂O₂-treated hMESCs in presence of BAPTA. Finally, we were able to detect the appearance of a lipidated form of LC3 (LC3-II) and its further turnover (Fig. 6b), what serves as a crucial evidence in favor of autophagy process in BAPTA-loaded hMESCs under the oxidative stress. It should be noted that activation kinetics of mTOR signaling and ULK1 protein in (Quin2-AM)-pretreated hMESCs was similar to those of BAPTA-pretreated but was delayed in time (Suppl. Fig. 1f). Minor differences in effects of BAPTA and Quin2-AM are possibly connected with the fact that BAPTA more rapidly binds intracellular calcium ions as compared to other known chelators. However, the data obtained on fibroblasts were rather contradictory: loading H₂O₂-stimulated fibroblasts with BAPTA simultaneously led to the opposite results in mTOR signaling (enhanced phosphorylation of 4E-BP1 and altered phosphorylation of p70S6K) and in signaling for autophagy (increased phosphorylation of both pro- and anti-autophagic sites of ULK1) (Suppl. Fig. 2g). These differences between hMESCs and fibroblasts might be connected with cell specificity. Overall, we can assume that [Ca²⁺]_i chelation by BAPTA partially prevents oxidative stress-induced senescence of hMESCs via autophagy initiation.

Figure 6. BAPTA induces an AMPK-dependent autophagy in H₂O₂-treated hMESCs. Cells were treated as indicated in the legend to Figure 3 and were subsequently analyzed by western blotting with the indicated antibodies. (a) Western blot analysis of pAMPK, pTSC2, p70S6K and p4E-BP1 at the various time points after H₂O₂ addition. (b) Alterations in ULK1 phosphorylation at Ser757 and Ser555 as well as LC3 lipidization induced by BAPTA pretreatment in H₂O₂-stimulated cells. Representative results of the three experiments are shown in the Figure. GAPDH was used as loading control.

Discussion

In keeping with the latter observations oxidative stress may induce DNA damage, autophagy, premature senescence and Ca²⁺ mobilization, however, yet there is no common opinion whether and how these responses are interrelated [27,37,41–43]. In the present study we investigated the role of intracellular calcium in oxidative stress-induced senescence progression in hMESCs.

Various oxidants, including t-butylhydroperoxide, menadione, H₂O₂, thimerosal, and superoxide generating systems such as hypoxanthine/xanthine oxidase, are shown to cause an elevation of cytosolic Ca²⁺ in intact cells [1,44–48]. Our findings strongly support these observations: in hMESCs we revealed an increase in intracellular calcium levels in response to sublethal H₂O₂ treatment. Depending on the concentration and type of oxidant used, intracellular calcium rise can be mediated either by Ca²⁺ import from the extracellular spaces and/or calcium release from internal stores, such as ER [1,41,49]. For example, high H₂O₂ concentrations lead to a sustained elevation of cytosolic calcium, which was not observed in Ca²⁺-free medium, suggesting that severe oxidative stress evoked Ca²⁺ uptake from extracellular spaces [50]. On the contrary, mild oxidative stress caused by moderate H₂O₂ levels increased cytoplasmic Ca²⁺ in smooth muscle and endothelial cells even in calcium-free medium, but it failed to do so if Ca²⁺ was first depleted from the ER [50,51]. Results presented in our study are in line with the last notion: (1) H₂O₂-induced mobilization of intracellular calcium persisted even in Ca²⁺-free bathing solution, (2) application of either UTP or TG, known to deplete ER store, greatly attenuated observed Ca²⁺ increase; together indicating that in hMESCs sublethal oxidative stress triggered calcium release predominantly from internal stores.

According to the literary data, calcium outflow from intracellular pools occurs principally from ER wherein one of the most important Ca²⁺ release channels – IP3R – has been identified [30–32]. The ligand for IP3R is IP3 that binds to the receptor, thus leading to calcium release from ER to cytoplasm [52]. Actually, different exogenously added oxidants can stimulate IP3R-mediated Ca²⁺ mobilization [53–56]. Indeed, we revealed that in H₂O₂-treated hMESCs intracellular calcium release was provided through IP3R. Typically, IP3R is regulated by the signaling pathways mediating IP3 generation [30–32,57], the most common is initiated by PLC that catalyzes the hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and IP3 at the plasma membrane [33]. In context of hMESCs, sublethal H₂O₂ treatment led to an increased intracellular calcium levels namely via PLC/IP3/IP3R pathway. In contrast, in intraneurons of the spinal cord H₂O₂ provoked calcium outflow through IP3R, but application of the phospholipase C (PLC) inhibitor was not able to reduce elevation of cytoplasmic calcium [58]. This observation allowed authors to suggest that the targets of H₂O₂ action lie downstream in the IP3 signaling cascade.

To date, only a few studies elucidating calcium implication in cellular senescence have been published [8,9,12,38]. The common notion is that intracellular calcium increases during senescence progression. For example, McCarthy and coauthors reported a higher basal calcium levels in replicatively senescent fibroblasts as compared to non senescent cells [8]. Another group evidenced that cytosolic Ca²⁺ rise is important for rotenone-induced senescence in human neuroblastoma cells [9]. Our observations are in accordance with the described above, as we also revealed an elevation of cytoplasmic calcium in H₂O₂-stimulated hMESCs, suggesting that Ca²⁺ might be involved in senescence progression. In favor of this hypothesis calcium chelation by BAPTA partially prevented senescence in hMESCs, as detected by both the proliferation maintaining and partial loss of the senescence phenotype.

Earlier we have shown that permanently enhanced ROS are typical for the senescent hMESCs [27]. Remarkably, calcium chelation was able to decline endogenous ROS levels long after hMESCs senescence induction. For now, it is well established that calcium released from the ER through IP3R is then taken up by mitochondria [30,59]. Thereby, sustained intracellular calcium increase during senescence may lead to mitochondrial calcium accumulation and altered mitochondrial activity, what mediates elevated ROS generation [12]. This may have sense in context of senescent hMESCs, as previously we have displayed that an increased mitochondrial activity is responsible for long-term ROS production [27]. In line with this assumption, french research group recently postulated implication of the mitochondrial calcium accumulation in both replicative and oncogene-induced senescence [12]. Moreover, decreasing calcium levels by mitochondrial calcium uniporter knockdown allowed cells to escape senescence.

It is generally accepted that the most prominent feature of senescent cells is irreversible cell cycle arrest, resulting in proliferation block [13–15]. Notably, BAPTA pretreatment decreased the activity of p53/p21/Rb pathway and thus prevented proliferation loss in H₂O₂-treated hMESCs. There are some data suggesting that intracellular Ca²⁺ fluctuations may modulate the functioning of this signaling cascade [60]. For instance, Ca²⁺ can regulate the ability of Ca²⁺-binding proteins to interact with p53, leading to its enhanced transcriptional activity or protein stability [11]. It was also demonstrated, that Ca²⁺/calmodulin may influence cell cycle progression via cdc2 kinase activation and Rb phosphorylation [61].

Our previous findings indicate that functional activation of the p53/p21/Rb pathway in hMESCs occurs as a result of DNA damage caused by H₂O₂ treatment [27]. Here we revealed that calcium chelation significantly decreased phosphorylation of the ATM, H2A.X and 53BP1 proteins in H₂O₂-treated hMESCs. Similar data were obtained by other researchers: intracellular calcium chelation by Quin2-AM suppressed DNA damage induced by either H₂O₂ or ROS produced by xanthine/xanthine oxidase [62,63]. The initial studies suggested that the protective effects of calcium chelators against oxidative stress-initiated DNA damage might be associated with their ability to chelate iron ions as well [63,64]. Iron ions are known to catalyze Fenton reaction with production of highly reactive OH^. radicals which enhance DNA damage [65]. Worth mentioning that in the cited articles authors applied rather high concentrations of calcium chelators as well as comparatively long pretreatment time. However, the more recent observations pointed out that even a very high concentration of BAPTA failed to chelate iron in intramitochondrial pool [66]. Moreover, it was reported that BAPTA retained the protective effect even being added after the stress [67]. Although our results do not prove or disapprove the capacity of BAPTA to chelate iron ions, it definitely should be taken into consideration. According to the modern point of view, a so-called “connecting link” between stress-induced Ca²⁺ mobilization and DNA damage might be PARP1 activation [67]. On the one hand, PARP1 functions as a DNA damage sensor that binds to both single and double strand breaks, and serves as a platform for the recruitment of proteins associated with the DDR [68]. Thereby, PARP1 participates in a variety of DNA damage consequences, including chromatin remodeling, transcriptional regulation, DNA repair, cell cycle arrest, senescence, or cell death. On the other hand, it was shown that PARP1 activation was mediated by an increase in intracellular Ca²⁺ caused either by β-lapachone-, H₂O₂-, or peroxynitrite-treatment [67,69]. Therefore, we can speculate that calcium chelation by BAPTA may prevent PARP1 activation and decrease DDR initiation, thereby averting cell cycle arrest and senescence progression in H₂O₂-treated hMESCs. In support of this hypothesis it was shown that intracellular calcium chelation was able to preclude both PARP1 activation and γH2A.X foci formation and a subsequent oxidative stress-induced cell death [67].

A growing number of studies are focused on the investigation of the interplay between senescence and autophagy [38,70–72]. For example, genetic impairment of autophagy in young satellite cells caused entry into senescence, whereas re-establishment of the process reversed senescence and recovered regenerative functions of these cells [72]. The same is true for the oxidative stress conditions: autophagy restoration averted H₂O₂-induced senescence in fibroblasts [38]. Based on the described above, we proposed that BAPTA-induced senescence prevention in H₂O₂-treated hMESCs might somehow be connected to the autophagic process.

It is well known that Ca²⁺ homeostasis is tightly linked to the autophagy, but depending on the cellular state, Ca²⁺ alterations may either inhibit or stimulate autophagy [22,23,73]. Interestingly, various Ca²⁺-mobilizing agents (thapsigargin, ATP and ionomycin) were clearly shown to induce autophagy, indicating pro-autophagic role of increased cytosolic calcium levels [74,75]. However, it should be noted that Ca²⁺ ionophores as well as TG-induced depletion of ER, described in the above articles, elevate Ca²⁺ to a non-physiological means and for prolonged periods of time, whereas physiological [Ca²⁺]_i signals typically have shorter duration and smaller amplitudes [23]. Another evidence considering calcium as an activator of autophagy is based on the observations that calcium chelation decreased stress-induced autophagy in various cells [22,76,77].

Despite of the intracellular calcium mobilization detected in our stress conditions sublethal H₂O₂ treatment did not cause any detectable autophagy in hMESCs. Contrarily, calcium chelation by BAPTA resulted in rapid short-term autophagy stimulation, as indicated by the appearance of the lipidated LC3 form. Similar inhibitory role of Ca²⁺ towards autophagy was described for TKO cells, where Ca²⁺ signals were abolished by either IP3R knocking down or its suppression [32]. Furthermore, we revealed that BAPTA-triggered autophagy was AMPK-dependent. AMPK – a major inducer of autophagy – is activated in response to decrease in ATP and a following increase in AMP or ADP [78,79]. As it was mentioned above, calcium outflowing from ER is then uptaken by mitochondria, where it can drive ATP production via regulation of mitochondrial dehydrogenases and ATP synthase [59,23]. Decreased calcium level may impair mitochondrial bioenergetics thus elevating AMP/ATP ratio and leading to AMPK activation [23]. Accordingly, it was shown that different manipulations, including inhibition of IP3R-mediated Ca²⁺ release or IP3R activity (by xestospongin B), inhibition PLC (by U73122), attenuation of mitochondrial Ca²⁺ uptake (Ru360 or mitochondrial calcium uniporter RNAi) etc., resulted in reduced ATP production followed by AMPK activation and the induction of pro-survival autophagy [23,32]. These data allow assuming that in H₂O₂-treated hMESCs BAPTA-induced senescence prevention might be linked with the disturbance in Ca²⁺ transfer from ER to mitochondria, leading to activation of AMPK, which in turn activates autophagy as a survival mechanism.

In conclusion, the present study is the first to elucidate the impact of intracellular calcium mobilization in oxidative stress-induced senescence of hMESCs. Here we revealed that calcium chelation can protect cells from premature senescence by both the reduction of DDR activation with a subsequent decrease in p53/p21/Rb pathway functioning and the autophagy stimulation (Fig. 7).

Figure 7. An interaction scheme displays the proposed molecular mechanism of intracellular calcium involvement in the premature senescence and autophagy of hMESCs under oxidative stress.

Acknowledgments

The authors are thankful to Dr. Alexey Shalygin and Dr. Vladimir Vigont (Institute of Cytology, RAS) for providing TG, UTP, 2-APB.

Conflicts of Interest

The authors have no conflict of interests to declare.

Funding

A.V. Borodkina, E.B. Burova, N.N. Nikolsky, A. N. Shatrova, A.A. Grukova and P.I. Deryabin acknowledge the support from the Russian Science Foundation (project #14-50-00068) with the financial support from the Federal Agency of Scientific Organizations (Russia) ensured cultivation of human endometrial mesenchymal stem cells. A.V. Borodkina and A.A. Grukova acknowledge the support the Russian Foundation for Basic Research (project #16-34-00081mol_a) allowed performing FACS analysis. E.B. Burova and P.I. Deryabin acknowledge the support from the Russian Foundation for Basic Research (project #14-04-01720), due to which electrophoresis, immunoblotting were performed. P.A. Abushik and S.M. Antonov acknowledge the support from the Russian Foundation for Basic Research (project #14-04-00227) allowed to perform confocal microscopy Ca²⁺-imaging and BAPTA experiments.

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