Intracellular Mg2+ protects mitochondria from oxidative stress in human keratinocytes

Fujita, Keigo; Shindo, Yutaka; Katsuta, Yuji; Goto, Makiko; Hotta, Kohji; Oka, Kotaro

doi:10.1038/s42003-023-05247-6

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Published: 24 August 2023

Intracellular Mg²⁺ protects mitochondria from oxidative stress in human keratinocytes

Keigo Fujita¹^na1,
Yutaka Shindo^1,2^na1,
Yuji Katsuta³,
Makiko Goto³,
Kohji Hotta¹ &
…
Kotaro Oka ORCID: orcid.org/0000-0001-5358-1003^1,2,4,5

Communications Biology volume 6, Article number: 868 (2023) Cite this article

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Abstract

Reactive oxygen species (ROS) are harmful for the human body, and exposure to ultraviolet irradiation triggers ROS generation. Previous studies have demonstrated that ROS decrease mitochondrial membrane potential (MMP) and that Mg²⁺ protects mitochondria from oxidative stress. Therefore, we visualized the spatio-temporal dynamics of Mg²⁺ in keratinocytes (a skin component) in response to H₂O₂ (a type of ROS) and found that it increased cytosolic Mg²⁺ levels. H₂O₂-induced responses in both Mg²⁺ and ATP were larger in keratinocytes derived from adults than in keratinocytes derived from newborns, and inhibition of mitochondrial ATP synthesis enhanced the H₂O₂-induced Mg²⁺ response, indicating that a major source of Mg²⁺ was dissociation from ATP. Simultaneous imaging of Mg²⁺ and MMP revealed that larger Mg²⁺ responses corresponded to lower decreases in MMP in response to H₂O₂. Moreover, Mg²⁺ supplementation attenuated H₂O₂-induced cell death. These suggest the potential of Mg²⁺ as an active ingredient to protect skin from oxidative stress.

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Introduction

Reactive oxygen species (ROS) are constantly produced in the human body and have harmful effects. Exposure to ultraviolet (UV) irradiation in particular is a notable trigger for ROS generation¹. ROS generation is attributed to several factors represented by enzyme activities of the electron transport chain in mitochondria². Oxidative stress induced by ROS constitutes a harmful condition because ROS have high reactivity and cause DNA damage, lipid peroxidation, and protein carbonylation. These forms of oxidative damage increase with age^3,4. Excessive ROS generation also causes mitochondrial dysfunction. In cellular-level experiments, the addition of H₂O₂ (a type of ROS) decreased mitochondrial membrane potential (MMP)^5,6. Moreover, it has been reported that ROS-induced mitochondrial dysfunction contributes to various diseases, including Alzheimer’s disease, type 1 diabetes, atherosclerosis, and cancer⁷. Skin in particular is frequently exposed to ROS stress since UV irradiation from sunlight is the main generator of ROS¹; ROS stress has been suggested to be involved in aging, inflammation, and pathogenesis of skin cancer, among other conditions^8,9. Therefore, protecting biomolecules and mitochondria from ROS is critical for maintaining normal cellular functions, and cells express antioxidants and several mechanisms to avoid oxidative stress¹⁰. Skin cells, such as the keratinocytes and fibroblasts which constitute the epidermis, also have ROS clearance mechanisms¹¹. In our previous study, we suggested that Mg²⁺ was involved in mechanisms to help cells avoid oxidative stress¹².

Mg²⁺ is the most abundant divalent cation in living cells and is related to more than 600 enzymes as a cofactor^13,14,15. Recent studies have demonstrated that small changes in intracellular Mg²⁺ can have large impacts on cellular events, such as cell division, maturation of neurons, and neurodegeneration^16,17,18. In neurons and cancer cells, intracellular Mg²⁺ is stored in the mitochondria and constitutes Mg²⁺ an important factor for sustainable mitochondrial functioning^12,19. The impact of Mg²⁺ in relation to retaining MMP has been reported at the cellular and isolated mitochondrial levels^20,21,22. The protective effects of Mg²⁺ toward oxidative stress have also been reported in various types of cells, such as endothelial cells^23,24, bone marrow mesenchymal stem cells^25,26, and chick embryo hepatocytes²⁷. On the other hand, in monocytes the environment is the determining factor in whether high levels of Mg²⁺ will increase or decrease ROS levels²⁸. These findings clearly indicate the important role of Mg²⁺ in protecting cells from oxidative stress. However, it has not been well understood whether cells change intracellular Mg²⁺ concentrations in response to oxidative stress, nor exactly how Mg²⁺ protects cells from oxidative stress.

In this study, we examined changes in Mg²⁺ concentration in response to ROS in keratinocytes. Keratinocytes were chosen because they are one of the most ROS-exposed cells in the human body, as ROS are generated by UV from sunlight. The dynamics of cytosolic Mg²⁺ were visualized using fluorescence imaging under H₂O₂ stress to find that H₂O₂ induced an increase in cytosolic Mg²⁺ concentration ([Mg²⁺]_cyto) due to the release of Mg²⁺ bound to ATP upon the H₂O₂-induced decrease in ATP level. The effects of Mg²⁺ on mitochondrial functions were investigated by comparison to changes in MMP to find that the increased Mg²⁺ attenuated the decrease in MMP. Interestingly, Mg²⁺ supplementation further suppressed the decrease in MMP and H₂O₂-induced cell death. Our results suggest that Mg²⁺ provides robustness to intracellular ATP levels under oxidative stress.

Results

H₂O₂-induced cytosolic Mg²⁺ increase

Spatio-temporal dynamics of Mg²⁺ in keratinocytes derived from adults (adult keratinocytes) were visualized with an Mg²⁺ selective indicator, KMG-104. It was confirmed that H₂O₂ did not directly react with KMG-104 (Supplementary Fig. 1a). H₂O₂ caused the [Mg²⁺]_cyto to increase in keratinocytes from 40-year-old donor (Fig. 1a–c). The average time-course of [Mg²⁺]_cyto increased immediately after application of 1 mM H₂O₂ and reached a plateau at 5 min (Fig. 1a). The spatial distribution of the changes in [Mg²⁺]_cyto was almost uniform within the cells, whereas the amplitude of the change in each cell varied (Fig. 1c). To examine whether the H₂O₂-induced Mg²⁺ increase constituted a common phenomenon in keratinocytes across individual differences and age, the responses were compared in five keratinocyte cell lines: three from newborns (three different 0 years old donors: newborn keratinocytes) and two from adults (40 and 57 years old donors: adult keratinocytes). There was large variability in the amplitudes of the H₂O₂-induced Mg²⁺ response in each cell, regardless of age (Fig. 1d and Supplementary Fig. 2a). While some newborn keratinocytes increased and others decreased in [Mg²⁺]_cyto in response to H₂O₂ for all three cell lines, most of the adult keratinocytes showed increases in [Mg²⁺]_cyto. As a result, all newborn keratinocyte lines showed no or slightly decreased [Mg²⁺]_cyto responses on average, whereas adult keratinocytes lines exhibited increased [Mg²⁺]_cyto on average (Supplementary Fig. 2b). Therefore, the data were divided into two groups, newborn and adult, and the difference in these two groups were compared. The amplitude of H₂O₂-induced Mg²⁺ responses was significantly larger in adult keratinocytes than in newborn keratinocytes (Fig. 1e). A higher concentration of H₂O₂ (10 mM) elicited increases in [Mg²⁺]_cyto, even in newborn keratinocytes, indicating that this is not a phenomenon specific to adult keratinocytes, but simply that adult keratinocytes are more sensitive to H₂O₂ than newborn keratinocytes (Supplementary Fig. 2c, d). The responses to 10 mM H₂O₂ were also larger in adult keratinocytes than in newborn keratinocytes (Supplementary Fig. 2e, f). To examine underlying mechanism of the increase in [Mg²⁺]_cyto and role of the Mg²⁺, the following experiments were performed in adult keratinocytes from a 40-year-old donor.

**Fig. 1: [Mg²⁺]_cyto increase in response to H₂O₂ in keratinocytes.**

A major source of Mg²⁺ is Mg²⁺ dissociation from ATP in the process of ATP consumption

To identify the Mg²⁺ source that was responding to H₂O₂, we examined Mg²⁺ entry from an extracellular medium, and fluorescence imaging was performed in the medium without Mg²⁺. An H₂O₂-induced Mg²⁺ increase was still observed in this condition (Fig. 2a), which indicates that H₂O₂ induced the release of Mg²⁺ from an intracellular Mg²⁺ source. In other cell types, mitochondria were identified as intracellular Mg²⁺ storage sites and released Mg²⁺ into cytosol upon a depolarization of MMP^19,29,30. Simultaneous imaging of [Mg²⁺]_cyto and MMP revealed that FCCP, an uncoupler of mitochondria, rapidly decreased MMP; however, FCCP did not increase [Mg²⁺]_cyto in keratinocytes, but rather deceased it (Fig. 2b). This indicates that depolarization of the mitochondria did not cause Mg²⁺ release from mitochondria in keratinocytes.

**Fig. 2: Examination of Mg²⁺ source in response to H₂O₂.**

Next, the involvement of Mg²⁺ transporters was investigated. It has been reported that Na⁺/Mg²⁺ exchangers, one of which is SLC41A1, mediate Mg²⁺ efflux from the cells and are inhibited by quinidine^14,22,31. Therefore, if one of the targets of H₂O₂ is the Na⁺/Mg²⁺ exchanger, which leads to the increase in [Mg²⁺]_cyto, it is expected that quinidine also increases [Mg²⁺]_cyto and that prior application of quinidine abolishes the H₂O₂-induced increase in [Mg²⁺]_cyto. Quinidine alone induced increase in [Mg²⁺]_cyto in both normal and Mg²⁺-free conditions, whereas vehicle (DMSO, final concentration 0.5%) decreased [Mg²⁺]_cyto, indicating that inhibition of Mg²⁺ efflux leads to increase in [Mg²⁺]_cyto in keratinocytes (Supplementary Fig. 3a). The amplitude of quinidine-induced Mg²⁺ increase was greater in normal condition than Mg²⁺-free condition (Supplementary Fig. 3b). These results suggest that [Mg²⁺]_cyto is normally balanced by Mg²⁺ influx and efflux. The effect of quinidine on H₂O₂-induced Mg²⁺ responses was also investigated. The responses were greater in the presence of quinidine than that in the presence of vehicle both in normal and Mg²⁺-free medium (Fig. 2c, d), while those were slightly smaller in the presence of vehicle (DMSO, final concentration 0.5%) than in the absence of vehicle (compare Fig. 2a and c). This result indicates that the Na⁺/Mg²⁺ exchanger does not mediate H₂O₂-induced Mg²⁺ responses and that inhibition of Mg²⁺ efflux retains Mg²⁺ released from intracellular Mg²⁺ sources.

Most of the intracellular Mg²⁺ binds to various biomolecules, and a major binding partner of Mg²⁺ in the cytoplasm is ATP. ATP normally binds to Mg²⁺ in the form of an Mg–ATP complex, but the Mg²⁺ is dissociated when ATP is consumed and degraded to ADP, leading to an increase in free Mg²⁺. Mg²⁺ dissociation from ATP due to ATP consumption has been reported as the cause of increases in [Mg²⁺]_cyto during mitosis and apoptosis^18,32. To confirm the dissociation of Mg²⁺ from ATP, the genetically encoded ATP sensor ATeam³³ was expressed in keratinocytes and the intracellular ATP level was visualized (Fig. 3a). It was confirmed that H₂O₂ does not directly affect ATeam signals independently of ATP (Supplementary Fig. 1b). Upon an application of H₂O₂, ATP concentration decreased in keratinocytes (Fig. 3b blue line). To examine the relationship between the decrease in ATP and increase in [Mg²⁺]_cyto, H₂O₂-induced change in ATP concentration was also examined in the newborn keratinocytes. These cells showed smaller increases in H₂O₂-induced [Mg²⁺]_cyto (Fig. 1e) and relatively smaller decreases in ATP compared to adult keratinocytes (Fig. 3b red line and c), whereas there were no significant difference in cellular ATP contents between those cells (Supplementary Fig. 4). We also examined whether the cells with large ATP decreases showed larger increases in [Mg²⁺]_cyto. Mitochondria are a major source of cellular ATP, and their inhibition affects cellular ATP production. Therefore, the effects of prior inhibition of ATP production in the mitochondria on the H₂O₂-induced responses in ATP and Mg²⁺ were investigated. Neither oligomycin, which is an inhibitor of F_oF₁ ATP synthase, nor FCCP alone elicited decreases in ATP levels at least within minutes of the application (Supplementary Fig. 5a, b). The efficacy of oligomycin and FCCP was confirmed by the results that when combined with the glycolysis inhibitor 2-deoxy-d-glucose (2DG), those induced greater decreases in cellular ATP levels than 2DG alone (Supplementary Fig. 5c, d). These results also indicate that mitochondria and glycolysis complement each other to maintain ATP concentration in keratinocytes. Interestingly, pretreatment with oligomycin or FCCP significantly enhanced H₂O₂-induced decreases in ATP (Fig. 3d, e). The inhibition of ATP synthesis in the mitochondria also enhanced the H₂O₂-induced increase in [Mg²⁺]_cyto, although neither oligomycin alone nor FCCP alone induced increases in [Mg²⁺]_cyto (Fig. 3f, g), and Mg²⁺ and ATP dynamics in response to H₂O₂ were mirror images of each other (compare Fig. 3d and f). These results indicate that Mg²⁺ dissociation from ATP was the major Mg²⁺ source in response to H₂O₂ in keratinocytes.

**Fig. 3: Inhibition of oxidative phosphorylation enhances H₂O₂-induced increases in Mg²⁺ and decreases in ATP.**

Mg²⁺ suppresses the H₂O₂-induced decrease in MMP in a concentration-dependent manner

Our next question was whether increased [Mg²⁺]_cyto protected cells from oxidative stress and via what mechanism this occurred. It has been reported that H₂O₂ suppresses mitochondrial function by decreasing MMP^5,6. On the other hand, previous studies show that Mg²⁺ contributes to the maintenance of MMP and that increased Mg²⁺ by Mg²⁺ supplementation or inhibition of Mg²⁺ efflux attenuates decreases in MMP under cellular stress conditions^20,21,22. Therefore, the relationship between [Mg²⁺]_cyto and MMP was examined using simultaneous imaging with KMG-104 and TMRE (Fig. 4a). H₂O₂ caused an increase in [Mg²⁺]_cyto and a gradual decrease in MMP within 10 min (Fig. 4b). Keratinocytes that showed large Mg²⁺ increases exhibited lower decreases in MMP (thin solid lines in Fig. 4b), and conversely, cells with small increases in [Mg²⁺]_cyto showed large decreases in MMP (thin dotted lines in Fig. 4b). To prove that Mg²⁺ had a direct effect on H₂O₂-induced decreases in MMP, the cytosolic level of Mg²⁺ was increased by adding Mg²⁺ to the extracellular medium before H₂O₂ stimulation. Supplementation of Mg²⁺ to the extracellular medium increased the Mg²⁺ concentration in the medium from 0.9 to 5 mM and led to a steep increase in [Mg²⁺]_cyto. The subsequent application of H₂O₂ did not induce significant changes in averaged [Mg²⁺]_cyto, while some cells showed an increase or decrease in [Mg²⁺]_cyto (green line in Fig. 4c). Interestingly, H₂O₂-induced decreases in MMP were significantly prevented by the prior addition of Mg²⁺ (Fig. 4d, e), whereas Mg²⁺ supplementation itself had little effect on MMP (red line in Fig. 4c). These findings suggest that [Mg²⁺]_cyto affects MMP primarily under stress conditions. To estimate the relationship between [Mg²⁺]_cyto and H₂O₂-induced decreases in MMP, the changes in [Mg²⁺]_cyto from initial levels (shown as green lines in Fig. 4b, c) and the H₂O₂-induced changes in MMP that were normalized at 1 min before H₂O₂ application (from the data in Fig. 4d) in each cell were plotted (Fig. 4f). In both the normal condition (gray dots) and the high Mg²⁺ condition (orange dots), strong correlations between Mg²⁺ increases and MMP were observed; higher levels of cytosolic Mg²⁺ were correlated with lower decreases in MMP. Interestingly, these two plots appear to line up on the same line, indicating that [Mg²⁺]_cyto is one of the key determinants for protecting the mitochondria from H₂O₂ damage (Fig. 4f).

**Fig. 4: H₂O₂-induced decreases in MMP were suppressed by increases in Mg²⁺.**

Finally, we confirmed that the Mg²⁺ supplementation suppresses the toxicity of H₂O₂. Exposure to 1 mM H₂O₂ for 24 h induced ~40% cell death in keratinocytes in a normal culture medium. Supplementation of additional 5 mM Mg²⁺ to the culture medium suppressed the toxicity (Fig. 5). Our results indicate that Mg²⁺ supplementation is effective in protecting keratinocytes from H₂O₂ toxicity.

**Fig. 5: Mg²⁺ supplementation attenuated H₂O₂-induced cell death.**

Discussion

The skin is continuously exposed to oxidative stress. This study revealed that H₂O₂, which is a kind of ROS, causes increases in [Mg²⁺]_cyto in human keratinocytes. This H₂O₂-induced Mg²⁺ response was higher in adult keratinocytes than in newborn keratinocytes. Our findings indicate that the source of Mg²⁺ was dissociation from ATP in the process of ATP consumption. Upon the addition of H₂O₂, decreases in MMP were also observed, and the change in MMP was strongly correlated with increases in [Mg²⁺]_cyto. In other words, higher levels of cytosolic Mg²⁺ were connected to lower MMP decreases. Furthermore, H₂O₂-induced decreases in MMP were significantly prevented by prior addition of Mg²⁺, suggesting direct effects of Mg²⁺ on the mitochondria. Moreover, Mg²⁺ supplementation also suppressed H₂O₂-induced cell death. In summary, this study revealed that H₂O₂-induced Mg²⁺ dissociation from ATP and that the resulting increase in [Mg²⁺]_cyto prevented MMP depolarization in keratinocytes. Our results suggest that Mg²⁺ that has dissociated from ATP is not merely a byproduct, but functions as a cytoprotective mechanism against oxidative stress and that Mg²⁺ supplementation is effective in protection against oxidative stress.

Mg²⁺ mobilization from intracellular storage in keratinocytes has not yet been thoroughly investigated, while Mg²⁺ influx via NIPAL4, an Mg²⁺ transporter, was reported previously^34,35,36. In the present study, we demonstrated that H₂O₂ increases cytosolic free Mg²⁺ by dissociation from ATP due to a decrease in cellular ATP level, although neither Mg²⁺ influx, Mg²⁺ transport via Na⁺/Mg²⁺ exchanger nor Mg²⁺ release from the mitochondria was involved in this response. It has been reported that mitochondria are intracellular Mg²⁺ storage sites and that depolarization of MMP induces Mg²⁺ release from the mitochondria into the cytosol in other cell types^16,19,29,30. In contrast, [Mg²⁺]_cyto instead decreased in response to FCCP in keratinocytes. This finding suggests that mitochondria do not act as Mg²⁺ sources in keratinocytes, although they probably contain Mg²⁺ in their matrix, similarly to other cell types, since some enzymes in the mitochondria require Mg²⁺ for their activity^37,38. ATP is known to be a major intracellular Mg²⁺ binding partner: hundreds of enzymes utilize ATP in the form of Mg-ATP, and Mg²⁺ is dissociated upon the degradation of ATP into ADP, increasing intracellular free Mg²⁺^13,32,39. Therefore, inhibition of mitochondrial ATP synthesis enhanced not only the decrease in ATP level but also the increase in [Mg²⁺]_cyto that was induced by H₂O₂. In contrast, newborn keratinocytes, which showed relatively smaller decreases in ATP compared to adult keratinocytes, had smaller Mg²⁺ responses to H₂O₂ than adult keratinocytes. In the process of aging, the contribution of anaerobic respiration to the energy metabolism becomes substantial in keratinocytes⁴⁰. This difference in the ATP production process between adult and newborn keratinocytes may cause larger ATP decreases and result in greater increases in Mg²⁺ in response to H₂O₂ in adult keratinocytes compared to newborn keratinocytes. Our results show that keratinocytes that exhibited large decreases in ATP showed large Mg²⁺ reactions in response to H₂O₂ and vice versa, strongly indicating that a major source of Mg²⁺ under oxidative stress conditions is the dissociation of Mg²⁺ from ATP.

In keratinocytes, oxidative stress changes metabolism drastically and acutely. Glucose usage changes from glycolysis to the pentose phosphate pathway within seconds of oxidative stress and decreased ATP levels occurrence¹¹. Our study also focused on the acute response of keratinocytes to oxidative stress and demonstrated that reduced ATP levels lead to an increase in Mg²⁺ levels. Mg²⁺ has large effects on the cellular metabolism and also on mitochondrial functions. The positive effect of Mg²⁺ on MMP under mitochondrial stress conditions has been demonstrated not only in isolated mitochondria but also in cells^20,21. The contributions of Mg²⁺ on MMP retention via inhibiting K⁺/H⁺ exchangers, preventing mitochondrial permeability transition pores (mPTP) from opening, and activating the tricarboxylic acid (TCA) cycle, has been reported previously⁴¹. In the present study, MMP levels under oxidative stress were also determined by [Mg²⁺]_cyto, which was perturbed by oxidative stress. This result suggests that the change in [Mg²⁺]_cyto is a mitochondria-protective mechanism under oxidative stress conditions. Previous studies have also demonstrated the long-term effects of Mg²⁺ on cell protection in keratinocytes⁴² and other cells^22,24. [Mg²⁺]_cyto changes the activity of phosphatases, such as mTOR, CREB, and ERK¹⁶, and leads to cell protection under stress conditions in keratinocytes⁴² and neurons²². Therefore, Mg²⁺ has both acute and long-term protective effects for cells.

Dissociation from ATP is a well-known source of Mg²⁺. However, little has previously been known about the role of the dissociated Mg²⁺. It has been reported that an increase in [Mg²⁺]_cyto, which is probably dissociated from Mg-ATP, is required for DNA condensation during mitosis in HeLa cells¹⁸, indicating that increased [Mg²⁺]_cyto resulting from ATP consumption is not a byproduct but instead plays an important role in cellular events. In the present study, we demonstrated that increasing levels of Mg²⁺ during the process of ATP level decrease protected the mitochondria, an organelle that is responsible for ATP generation, under oxidative stress in keratinocytes. This suggest that Mg²⁺ acts as a negative feedback signal to maintain ATP level against stress on cells. Some studies have already demonstrated that mitochondria was protected from oxidative stress under Mg²⁺ rich conditions, but these studies have not referred to changes in [Mg²⁺]_cyto levels^24,27,42. Our data reveal that [Mg²⁺]_cyto increases in response to oxidative stress and that Mg²⁺ that dissociated from Mg-ATP is not a byproduct but instead acts as a mechanism to protect the mitochondria. Since the protective effect of Mg²⁺ supplementation against oxidative stress has been reported in other cell types^{23,24,25,26,27}, the mechanism revealed here might be common in mammalian cells to protect cells against cellular stress.

In conclusion, we demonstrated that H₂O₂ induced an increase in [Mg²⁺]_cyto due to dissociation from Mg-ATP, and the increased [Mg²⁺]_cyto protected mitochondria from ROS damage. Moreover, supplementation of Mg²⁺ to extracellular medium further suppressed the decrease in MMP and attenuated H₂O₂ toxicity. The skin is always exposed to oxidative stress from UV, so Mg²⁺ would play an important role in maintaining the robustness of energy metabolism and protecting the skin from oxidative stress. Moreover, the addition of Mg²⁺ from an external source showed an additive effect for cell protection, suggesting that Mg²⁺ is a candidate active ingredient to protect skin from oxidative stress.

Methods

Cell cultures

Normal human epidermal keratinocytes were purchased from Kurabo (Osaka, Japan). To compare the response of keratinocytes from newborn babies and adults, four different batches of newborn keratinocytes vials from different donors (0 years–1, 2, 3 and 4) and two different batches of adult keratinocytes vials (donors aged 40 years and 57 years) were used (details are summarized in Supplementary Table 1). Cells were cultured in EPILIFE^TM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with insulin (10 μg mL⁻¹), human recombinant epidermal growth factor (0.1 ng mL⁻¹), hydrocortisone (0.67 μg mL⁻¹), gentamicin (50 μg mL⁻¹), amphotericin B (50 ng mL⁻¹), and bovine pituitary extract (0.4%, v/v), all of which were sourced from Kurabo, at 37 °C in a CO₂ incubator. Undifferentiated keratinocytes between passage 2 and passage 6 were used for experiments. EPILIFE^TM medium contains Mg²⁺, but the concentration is not disclosed.

For fluorescence imaging, keratinocytes were seeded on glass bottom dishes (IWAKI, Shizuoka, Japan) coated with 5 μg mL⁻¹ collagen (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 6–8 × 10⁴ cells mL⁻¹.

Dye loading and fluorescence imaging

For Mg²⁺ imaging, cells were stained with an Mg²⁺-selective fluorescent probe, KMG-104⁴³. Keratinocytes were incubated with 20 μM KMG-104-AM and 200 μg mL⁻¹ Pluronic F-127 (Thermo Fisher Scientific) at 37 °C. After 30 min, the keratinocytes were washed twice with Ca²⁺-free HBSS (Thermo Fisher Scientific; the pH was buffered using 10 mM HEPES and adjusted to 7.4 with NaOH, HBSS contains 0.9 mM Mg²⁺) and incubated for a further 15 min in Ca²⁺-free HBSS to allow the complete hydrolysis of acetoxy methyl (AM) groups. To avoid differentiation of keratinocytes, all experimental procedures were performed in Ca²⁺-free medium.

For simultaneous imaging of cytosolic Mg²⁺ and MMP, keratinocytes that had been loaded with KMG-104 were then incubated in Ca²⁺-free HBSS containing 25 nM TMRE (Thermo Fisher Scientific) for 15 min at 37 °C. Fluorescence imaging was performed in Ca²⁺-free HBSS with 2.5 nM TMRE.

A confocal laser scanning microscope system, FluoView FV1000 (Olympus, Tokyo, Japan), was used for the measurement of fluorescence. For the imaging of Mg²⁺ alone, KMG-104 was excited at 488 nm using an Ar laser through a dichroic mirror (DM405/488, Olympus), and fluorescence at 510–610 nm was detected with a photomultiplier. Images were acquired every 4–6 s. For simultaneous imaging of cytosolic Mg²⁺ and MMP, KMG-104 and TMRE were simultaneously excited at 488 nm using an Ar laser and 559 nm from a laser diode, respectively, through a dichroic mirror (DM405/488/559, Olympus). The emitted fluorescence was separated at 560 nm (SDM560, Olympus) and observed at 505–545 nm for KMG-104 and 570–670 nm for TMRE.

Fluorescence imaging of intracellular ATP

A fluorescence resonance energy transfer (FRET)-type ATP sensor, ATeam1.03³³, was kindly gifted from Dr. Imamura and was used to measure intracellular ATP levels. The plasmids that encoded ATeam were transfected into keratinocytes using Lipofectamine LTX (Thermo Fisher Scientific). These keratinocytes were cultured for 1–2 days after transfection to express the sensor proteins. Before observation, the cells were rinsed with and placed in Ca²⁺-free HBSS.

The cells were observed on the confocal laser scanning microscope system Fluoview FV1000. ATeam was excited at 440 nm using a laser diode through a dichroic mirror (DM405–440/515, Olympus), and the emitted fluorescence was separated by a dichroic mirror (SDM515, Olympus) and observed at 460–500 nm for CFP and at 515–615 nm for YFP.

For the negative control experiments, an ATP-insensitive variant of ATeam was constructed by inducing mutations of R122K and R126K in the ATP-sensing domain, following previous work³³. The ATP-insensitive ATeam was expressed in the keratinocytes, and it was confirmed that H₂O₂ at a concentration below 10 mM had no impact on the fluorescent proteins.

Image analysis

The acquired images were analyzed using the software packages FluoView (Olympus), Aquacosmos (Hamamatsu Photonics, Shizuoka, Japan), and ImageJ. A region of interest (ROI) was assigned to the whole cell body of each cell, and the average fluorescence intensity in each ROI was calculated respectively. After subtracting the background, the time-course of fluorescence intensity for each cell (F) was normalized by the initial value (F₀), and the resulting F/F₀ values were compared between KMG-104 and TMRE. For ATeam, the ratio (R) of the fluorescence of cyan and yellow fluorescent protein (YFP/CFP) was calculated after subtracting background. The time-course of R was normalized by the initial value (R₀), and the resulting R/R₀ values were compared.

Two groups of data were compared using Student’s t-test. To compare multiple data sets, Dunnett’s test or Tukey’s tests were used. P < 0.05 was used to indicate significant differences.

Measurement of H₂O₂ sensitivity of KMG-104 in vitro

KMG-104 and several concentrations of H₂O₂ were mixed in 96-well plate and the fluorescence of these mixtures was measured using a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific). KMG-104 was excited at 500 nm and fluorescence intensity at 530 nm was measured and compared. H₂O₂ in the concentration range of 0–100 mM had no effect on the fluorescence of KMG-104 (Supplementary Fig. 1a).

MTT assay

Keratinocytes were plated at a density of 8.0 × 10³ cells par well in a 96-well plate and incubated at 37 °C for more than 24 h. Medium was replaced to Mg²⁺ normal medium (normal EPILIFE with supplements) or Mg²⁺ +5 mM medium (EPILIFE with additional 5 mM Mg²⁺ and supplements) 10 min prior to H₂O₂ application. The medium was replaced to Mg²⁺ normal medium or Mg²⁺ +5 mM medium containing 0 or 1 mM H₂O₂, and cells were incubated for 24 h in the incubator. Then, the medium was replaced to 0.5 mg mL⁻¹ MTT containing culture medium, and the cells were incubated for 2 h. The medium was discarded, and DMSO was then added to each well to dissolve the precipitate. The absorption at 575 nm was measured on a microplate reader, Valioscan (Thermo Fisher Scientific). The viabilities were calculated as a ratio to the average of H₂O₂ 0 mM condition for each Mg²⁺ concentration.

Statistics and reproducibility

Fluorescence imaging experiments were repeated for 3–4 times for each experiment, and response of all the cells emitting sensor fluorescence in the field of view were analyzed. MTT assay was repeated for six times. Student’s t-test was used for comparison of a pair of data. Dannett’s test was used to compare multiple groups to control group. Tukey’s test was used to compare all differences among data group more than three groups.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data used in this study have been uploaded as a supplementary file named “Supplementary data 1”.

Code availability

FluoView (Olympus), Aquacosmos (Hamamatsu Photonics, Shizuoka, Japan), and ImageJ were used for process and analyze image data.

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Acknowledgements

The plasmid encoding ATeam was kindly gifted from Dr. Imamura (Kyoto University).

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These authors contributed equally: Keigo Fujita, Yutaka Shindo.

Authors and Affiliations

Department of Bioscience and Informatics, Faculty of Science and Technology, Keio University, Yokohama, Japan
Keigo Fujita, Yutaka Shindo, Kohji Hotta & Kotaro Oka
School of Frontier Engineering, Kitasato University, Sagamihara, Japan
Yutaka Shindo & Kotaro Oka
MIRAI Technology Institute, Shiseido Co. Ltd., Yokohama, Japan
Yuji Katsuta & Makiko Goto
Waseda Research Institute for Science and Engineering, Waseda University, Tokyo, Japan
Kotaro Oka
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung City, Taiwan
Kotaro Oka

Authors

Keigo Fujita
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Yutaka Shindo
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Yuji Katsuta
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Makiko Goto
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Kohji Hotta
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Kotaro Oka
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Conceptualization, K.O. and Y.K.; Investigation, K.F., Y.S., and M.G.; Writing—original draft, K.F. and Y.S.; Writing—review & editing, Y.K., M.G., K.H., and K.O.; Supervision, Y.K. and K.O.

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Correspondence to Kotaro Oka.

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Fujita, K., Shindo, Y., Katsuta, Y. et al. Intracellular Mg²⁺ protects mitochondria from oxidative stress in human keratinocytes. Commun Biol 6, 868 (2023). https://doi.org/10.1038/s42003-023-05247-6

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Received: 05 March 2023
Accepted: 15 August 2023
Published: 24 August 2023
DOI: https://doi.org/10.1038/s42003-023-05247-6

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