1. Introduction
When cells are stimulated by growth factors and cytokines, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-3 (IL-3), hydrogen peroxide (H
2O
2) is produced through the activity of NADPH oxidases (NOXs) [
1]. The activation of NOXs results in the production of superoxide (O
2•−), which is subsequently transformed to H
2O
2 by the activity of superoxide dismutase (SOD) [
2]. This physiological H
2O
2 influences various intracellular signaling pathways. Its capacity to oxidize the cysteine residues of some proteins, such as protein tyrosine phosphatases (PTPs), results in a functional modification that notably impairs their activities [
3]. PTPs play pivotal roles in cellular processes involving cell growth, proliferation, and differentiation [
4]. Within their structural framework, all PTPs feature a cysteine residue in their active site [
5]. When exposed to H
2O
2, the cysteine residue undergoes oxidation, transforming into cysteine-sulfenic acid (Cys-SOH) and leading to the oxidative inhibition of PTP’s enzymatic activity [
6].
Phosphatase and tensin homolog (PTEN), a member of the PTP family, structurally comprises five functional domains: a short N-terminal phosphatidylinositol (PtdIns)(4,5)P2-binding domain (PBD), a catalytic phosphatase domain, a C2 lipid/membrane-binding domain, a C-terminal tail containing Pro, Glu, Ser, and Thr (PEST) sequences, and a class I PDZ-binding (PDZ-BD) motif. PTEN interacts with phospholipid membranes through the C2 domain located at the C-terminal end. The tight linkage between the C2 and phosphatase domains implies that the C2 domain not only aids in recruiting PTEN to the membrane but also optimizes the orientation of the catalytic domain associated with its membrane-bound substrate. Moreover, the phosphatase domain of PTEN has an enlarged active site to accommodate its phosphoinositide substrate, membrane-bound phosphatidylinositol-3,4,5-triphosphate (PIP3) [
7,
8]. Hence, PTEN is considered a tumor suppressor owing to its ability to dephosphorylate PIP3 and, consequently, negatively regulate the PI3K/AKT signaling pathway, which is pivotal in controlling cell survival, growth, and proliferation [
9,
10]. Besides cancer studies, substantial insights into PTEN’s role in physiological processes are available. Notably, PTEN inhibition has been demonstrated as a promising therapeutic intervention for neurodegenerative diseases, ischemia, infection, and insulin-resistant metabolic disorders [
11].
Similar to other members of the PTP family, PTEN contains a cysteine residue in the active site of its phosphatase domain, rendering it susceptible to oxidative inhibition by ROS, particularly H
2O
2 [
5]. Lee et al. were the first to demonstrate the reversible inactivation of PTEN by H
2O
2 through the oxidation of the Cys124 catalytic residue in the active site and the formation of an intramolecular disulfide bond with Cys71. This inactivation is reversible because oxidized PTEN can be converted back to the functional reduced form by cellular reducing agents, such as the Thioredoxin/Thioredoxin Reductase (Trx/TrxR) system [
12]. Additionally, in the cell, Peroxiredoxins (Prx), thiol-specific antioxidants of the peroxidase family, can function as modulators of H
2O
2-induced phosphorylation signaling due to their capacity to sense and eliminate peroxides [
13,
14,
15]. Thus, the mechanism of PTEN oxidation by transient H
2O
2, a signaling molecule produced in response to growth factor receptor stimulation, raises questions about how this small amount of physiological H
2O
2 can function to oxidize PTEN in a cellular environment rich in H
2O
2 scavengers such as catalase, glutathione peroxidase, and Prx, alongside the presence of ubiquitous Trx/TrxR reducing systems.
It has been known since 1980s that H
2O
2 can react with bicarbonate/carbon dioxide (HCO
3-/CO
2) to form peroxymonocarbonate (HCO
4-)
-: H
2O
2 + HCO
3-/CO
2 ⇌ HCO
4- + H
2O/H
+. In neutral pH aqueous solution, HCO
4- formation process occurs rapidly at 25 ℃ with half-life t
1/2 of 10 min or below. Carbonic anhydrase and a Zinc model complex can accelerate this reaction [
16,
17]. HCO
4- is a robust oxidant with a higher catalytic potency comparable to that of H
2O
2. Its interaction with target molecules proceeds at velocities ranging from 100 to 1000 times faster than those observed with H
2O
2 [
18]. Sulfide oxidation caused by HCO
4- surpasses that caused by H
2O
2 by approximately 300-fold [
19]. Notably, HCO
4- also serves as a potent two-electron oxidant primarily accountable for biothiol peroxidation [
20]. Hence, the presence of HCO
3- is demonstrated to be a pivotal factor in promoting the oxidative reactivity of H
2O
2 towards PTPs, such as PTP1B, SHP-2, and PTPN22, during signal transduction processes [
21,
22,
23]. When cells are stimulated by growth factors, HCO
3- is produced through the activation of a transmembrane enzyme, carbonic anhydrase IX, which catalyzes the hydration of CO
2 (CO
2 + H
2O → HCO
3- + H
+) [
24]. Dagnell et al. demonstrated that HCO
3- not only facilitates but also plays an essential role in H
2O
2-mediated inactivation of PTP1B, surpassing the protection provided by the Trx/TrxR reducing systems [
22]. Given that PTEN belongs to the PTP family, it is worth exploring whether HCO
3- may somehow affect the H
2O
2-mediated oxidative inactivation of PTEN. Based on the available pieces of information, we anticipated that the presence of HCO
3- in combination with H
2O
2, through the formation of HCO
4-, would augment oxidation of PTEN.
4. Discussion
The activation of receptor tyrosine kinases (RTK) is a pivotal event in transmitting phosphorylation signals upon stimulation by growth factors and, therefore, holds substantial importance in cell physiology [
26]. In this situation, a transient amount of H
2O
2 is generated by membrane NOXs [
27]. Subsequently, H
2O
2 causes reversible oxidative inhibition of PTEN, leading to an increase in PI3K/AKT signaling pathway and, consequently, eliciting cellular substantial effects [
28,
29]. The activation of AKT and its downstream cascades are proved to be related to variety of cancers [
30]. Besides, studies also indicate that the elevated AKT activity through oxidative inactivation of PTEN can yield benefits in particular physiological processes that require cell growth such as cardiac remodeling following ischemia [
31], neuronal regeneration [
32], immune response [
33], insulin-related metabolism [
34], and myogenesis [
35]. Within the cellular environment, H
2O
2 can be eliminated by thiol proteins from the Prx family. Furthermore, oxidized PTEN and Prx can be rapidly converted back to their active reduced forms via the Trx/TrxR/NADPH system [
36,
37]. Thus, PTEN phosphatase catalytic activity is conserved by Trx/TrxR and Prx systems [
14,
15,
36,
38]. However, PTEN is inhibited during PI3K/AKT signaling pathway activation. Through our experiment result, we presume the factor that facilitates PTEN oxidation by H
2O
2 in the presence of HCO
3- is the formation of HCO
4- (
Figure 5).
The addition of 44 mM sodium bicarbonate to DMEM D5648, which lacks HCO
3-, replicates the common condition of culture media. Previous investigations on NIH 3T3 cells exposed to H
2O
2 in standard DMEM revealed reversible PTEN oxidation in a time-dependent manner. Incubation with 0.5 mM H
2O
2 resulted in increased levels of oxidized PTEN, peaking at 10 min before declining gradually, suggesting that PTEN was initially inactivated by H
2O
2 and then reactivated by cellular reductants as the H
2O
2 declined. This reversible PTEN inactivation was also observed in HeLa cells exposed to H
2O
2 [
12]. In our recent studies, cells were treated with H
2O
2 for specific durations, extending the time points to 120 min to ensure the completion of the reduction process. Similarly, oxidized PTEN exhibited a time-dependent increase followed by a decline. The PTEN oxidation rate reached its peak after exposure for 10 min, and the oxidized PTEN was completely converted to the reduced form within 120 min of exposure. Based on these results, it is evident that treatments with H
2O
2 in standard HCO
3--containing media yield maximum PTEN oxidation after 10 min of exposure, followed by the conversion of oxidized PTEN to its reduced state by the cellular Trx/TrxR system within approximately 60 min after exposure [
25,
38]. Therefore, we initially tested the role of HCO
3- in PTEN oxidation by H
2O
2 through evaluating the proportions of oxidized PTEN following 10 min exposure to stimulation media. The pre-incubation period aimed to diminish the HCO
3- in both extracellular and intracellular environments. Our findings indicated HCO
3- alone did not cause any PTEN oxidation. Meanwhile, H
2O
2 alone has significant lower percentage than H
2O
2-HCO
3- combination. Additionally, there was no notable difference between HCO
3- concentration of 22 mM and 44 mM. This implies that the greater percentages observed in the H
2O
2-HCO
3- combination groups were not merely the cumulative effect of each H
2O
2 and HCO
3-. The plausible explanation is that HCO
3- can empower the catalytic activity of H
2O
2, supporting the hypothesis there might be a formation of HCO
4-, a more reactive oxidant.
Given that HCO
4- was demonstrated to exhibit a greater oxidation rate towards sulfide when compared to H
2O
2 [
19], we suppose that it may also influence PTEN oxidation. This implies that the peak of PTEN oxidation in each group may not be solely at the 10 min timepoint. Therefore, we extended experiments to additional time points to investigate the redox regulation of HCO
3- and H
2O
2. Additionally, we reduced the concentration of H
2O
2 to 0.5 mM and expect it could make the difference more visible. A similar pattern to results of our previous studies was observed this time on HepG2 cells in 44 mM HCO
3--supplemented media. PTEN was predominantly oxidized within 5–15 min, with the highest oxidation rate observed at 10 min, then reduced back to active form. Thus, the PTEN oxidation observed in the HCO
3--containing group in the present study follows a trend consistent with our former studies.
The exclusion of HCO
3- from the experimental conditions considerably prolonged the PTEN oxidation period, requiring up to 30 min to reach its peak. However, the H
2O
2-mediated oxidative inactivation of intracellular PTPs in signaling events typically happens swiftly within the timeframe of 5–15 minutes, typically peaking at 10 min [
12,
39,
40]. This disparity suggests that the absence of HCO
3- adversely affects the PTEN oxidation speed. Our result aligns with the findings of Dagnell et al., indicating that HCO
3- can potentiate the H
2O
2-dependent oxidative inactivation of PTP [
22]. Illustrative data analysis suggests that the combination of HCO
3- and H
2O
2 accelerated the rate of PTEN oxidation in the cellular environment by approximately 2.5-fold in a time-dependent manner. Notably, this was also strongly correlated to the similar decrease in the PTEN reduction speed in the HCO
3--free group. Based on growing evidence about the role of HCO
3- in H
2O
2-mediated oxidation under cell signaling processes, we suppose the formed HCO
4- promotes the PTEN redox regulation, therefore exerts function as a signaling molecule.
PTEN acts as a negative regulator of PI3K/AKT signaling pathway, which induces various cellular processes including protein synthesis, cell survival, proliferation, and migration. This mechanism plays important role in human physiological and pathological conditions [
7,
11,
41]. Following the result that H
2O
2-mediated oxidative inhibition of PTEN is potentiated and accelerated by the HCO
3-, we examined its subsequent impact on phosphorylation of AKT. The elevating trends of pAKT/AKT reflect similar patterns observed in PTEN oxidation, both in bicarbonate-containing and bicarbonate-free status. Our findings are consistent with the previous evidence suggesting that H
2O
2 can activate PI3K/AKT pathway by oxidizing PTEN [
10]. Additionally, the time-dependent increase of pAKT/AKT is equivalent to the rise of PTEN oxidation in the presence of bicarbonate, 2.7-fold and 2.5-fold, correspondingly. In another words, without bicarbonate, PTEN oxidation is impaired, leading to the delay in AKT phosphorylation. Thus, in this context, the increase in AKT activation definitely arises from the surge in PTEN oxidation. Considering HCO
4- is formed through the reaction between H
2O
2 and HCO
3-, we propose that HCO
4- can positively regulate PI3K/AKT signaling pathway via oxidative inactivation of PTEN.
In summary, the results of our experiment support the hypothesis that the presence of HCO3- promotes the H2O2-mediated inactivation of PTEN. This subsequently promotes the activation of PI3K/AKT signaling pathway. In addition, the reduction of oxidized PTEN by antioxidant systems is also be equivalently affected. The plausible explanation can be the formation of HCO4-. In the future, further experiments would be conducted to consolidate the role of HCO4- in the redox regulation of PTEN.
Figure 1.
Experimental scheme for detecting redox status of PTEN by mobility shift. Upon HCO4- exposure, a proportion of PTEN can be oxidized in the Cystein124 residue of their active site through creating a reversible intracellular disulfide bond with Cystein71. Subsequently, the supplement of NEM akylates PTEN reduced form into PTEN-NEM complex, increasing the molecular weight, thereby result in a lower shift on non-reducing SDS-PAGE.
Figure 1.
Experimental scheme for detecting redox status of PTEN by mobility shift. Upon HCO4- exposure, a proportion of PTEN can be oxidized in the Cystein124 residue of their active site through creating a reversible intracellular disulfide bond with Cystein71. Subsequently, the supplement of NEM akylates PTEN reduced form into PTEN-NEM complex, increasing the molecular weight, thereby result in a lower shift on non-reducing SDS-PAGE.
Figure 2.
HCO3- potentiates the PTEN oxidation by H2O2. HepG2 cells were cultured until they reached 90% confluency, then washed with PBS and transferred to DMEM (L-glutamine, 1X penicillin-streptomycin, 0.1% FBS, and 25 mM HEPES) without 44 mM sodium HCO3-, and the pH was neutral. Then, the cells were incubated at 37 °C with 0.1% CO2 for 4 h and, subsequently, treated with prepared stimulation media containing 44 mM HCO3- alone, or 1 mM H2O2 alone, or combination of 1 mM H2O2 and 22 mM HCO3-, or combination of 1 mM H2O2 and 44 mM HCO3-. After 10 min, the reaction was stopped by adding lysis buffer containing protease inhibitors and 10 mM NEM. Then, the cell lysates were collected and used for SDS-PAGE and western blotting analyses. The results obtained using PTEN antibody are presented in (A). The quantification result (B) indicated that with the presence of 22 mM or 44 mM HCO3-, PTEN oxidation is significantly higher than treatment with only H2O2 (P < 0.05). 44 mM HCO3- alone showed no oxidation effect.
Figure 2.
HCO3- potentiates the PTEN oxidation by H2O2. HepG2 cells were cultured until they reached 90% confluency, then washed with PBS and transferred to DMEM (L-glutamine, 1X penicillin-streptomycin, 0.1% FBS, and 25 mM HEPES) without 44 mM sodium HCO3-, and the pH was neutral. Then, the cells were incubated at 37 °C with 0.1% CO2 for 4 h and, subsequently, treated with prepared stimulation media containing 44 mM HCO3- alone, or 1 mM H2O2 alone, or combination of 1 mM H2O2 and 22 mM HCO3-, or combination of 1 mM H2O2 and 44 mM HCO3-. After 10 min, the reaction was stopped by adding lysis buffer containing protease inhibitors and 10 mM NEM. Then, the cell lysates were collected and used for SDS-PAGE and western blotting analyses. The results obtained using PTEN antibody are presented in (A). The quantification result (B) indicated that with the presence of 22 mM or 44 mM HCO3-, PTEN oxidation is significantly higher than treatment with only H2O2 (P < 0.05). 44 mM HCO3- alone showed no oxidation effect.

Figure 3.
Bicarbonate accelerates the redox regulation of PTEN by H2O2. HepG2 cells were cultured until they reached 90% confluency, then washed with PBS and transferred to DMEM (L-glutamine, 1X penicillin-streptomycin, 0.1% FBS, and 25 mM HEPES) with or without 44 mM sodium bicarbonate, and the pH was neutral. Then, the cells were incubated at 37 °C with 0.1% CO2 for 4 h and, subsequently, treated with prepared stimulation media containing 0.5 mM H2O2. After 5, 10, 15, 30, 60, and 120 min, the reaction was stopped by adding lysis buffer containing protease inhibitors and 10 mM NEM. Then, the cell lysates were collected and used for SDS-PAGE and western blotting analyses. The results obtained using PTEN and β-actin antibodies are presented in (A). The quantification results (B) indicate that in the absence of HCO3-, the rate of PTEN oxidation diminished, and the reduction period was prolonged. Data are presented as the mean ± SEM of three independent experiments. The linear regression trendlines of PTEN oxidation and reduction rates presented in (C) show that the presence of HCO3- accelerates the H2O2-mediated redox regulation of PTEN.
Figure 3.
Bicarbonate accelerates the redox regulation of PTEN by H2O2. HepG2 cells were cultured until they reached 90% confluency, then washed with PBS and transferred to DMEM (L-glutamine, 1X penicillin-streptomycin, 0.1% FBS, and 25 mM HEPES) with or without 44 mM sodium bicarbonate, and the pH was neutral. Then, the cells were incubated at 37 °C with 0.1% CO2 for 4 h and, subsequently, treated with prepared stimulation media containing 0.5 mM H2O2. After 5, 10, 15, 30, 60, and 120 min, the reaction was stopped by adding lysis buffer containing protease inhibitors and 10 mM NEM. Then, the cell lysates were collected and used for SDS-PAGE and western blotting analyses. The results obtained using PTEN and β-actin antibodies are presented in (A). The quantification results (B) indicate that in the absence of HCO3-, the rate of PTEN oxidation diminished, and the reduction period was prolonged. Data are presented as the mean ± SEM of three independent experiments. The linear regression trendlines of PTEN oxidation and reduction rates presented in (C) show that the presence of HCO3- accelerates the H2O2-mediated redox regulation of PTEN.

Figure 4.
HCO3- accelerates and potentiates the phosphorylation of AKT induced by H2O2. HepG2 cells were preincubated and exposed to 0.5 mM H2O2 in HCO3--supplemented or HCO3--free media. After 5, 10, 15, 30, 60 and 120 min, cell lysates were collected and subjected to Western blot using pAKT and total AKT antibody (A). Data are quantified by pAKT fold over total AKT and presented as mean ± SEM. The combination of HCO3- and H2O2 resulted in faster and higher AKT phosphorylation than H2O2 alone (B). The linear regression trendlines of pAKT/AKT presented in (C) show that the presence of HCO3- elevated the AKT phosphorylation rate.
Figure 4.
HCO3- accelerates and potentiates the phosphorylation of AKT induced by H2O2. HepG2 cells were preincubated and exposed to 0.5 mM H2O2 in HCO3--supplemented or HCO3--free media. After 5, 10, 15, 30, 60 and 120 min, cell lysates were collected and subjected to Western blot using pAKT and total AKT antibody (A). Data are quantified by pAKT fold over total AKT and presented as mean ± SEM. The combination of HCO3- and H2O2 resulted in faster and higher AKT phosphorylation than H2O2 alone (B). The linear regression trendlines of pAKT/AKT presented in (C) show that the presence of HCO3- elevated the AKT phosphorylation rate.
Figure 5.
Suggesting role of HCO4- in redox regulation of PTEN. Stimulation of RTK induces the assembly of PI3K to phosphorylate PIP2 to PIP3, subsequently activating AKT signaling cascade. PTEN can negatively modulate this pathway. During this condition, H2O2, produced through the activity of NOX2, oxidizes PTEN. In cellular environment, PTEN oxidation can be impaired and reversed by Trx/TrxR/NADPH reducing system. However, in the presence of HCO3- together with H2O2, a higher reactive oxidant HCO4- is formed. Hence, HCO4- is supposed to accelerate the oxidative inactivation of PTEN, consequently increase the PI3K/AKT signaling pathway, and as a result, promoting cell survival, proliferation, and differentiation in physiological processes or tumor growth.
Figure 5.
Suggesting role of HCO4- in redox regulation of PTEN. Stimulation of RTK induces the assembly of PI3K to phosphorylate PIP2 to PIP3, subsequently activating AKT signaling cascade. PTEN can negatively modulate this pathway. During this condition, H2O2, produced through the activity of NOX2, oxidizes PTEN. In cellular environment, PTEN oxidation can be impaired and reversed by Trx/TrxR/NADPH reducing system. However, in the presence of HCO3- together with H2O2, a higher reactive oxidant HCO4- is formed. Hence, HCO4- is supposed to accelerate the oxidative inactivation of PTEN, consequently increase the PI3K/AKT signaling pathway, and as a result, promoting cell survival, proliferation, and differentiation in physiological processes or tumor growth.