Disturbed magnesium (Mg2+) homoeostasis and increased levels of OS (oxidative stress) are associated with poor clinical outcomes in patients suffering from neurodegenerative, cardiovascular and metabolic diseases. Data from clinical and animal studies suggest that MD (Mg2+ deficiency) is correlated with increased production of ROS (reactive oxygen species) in cells, but a straightforward causal relationship (including molecular mechanisms) between the two conditions is lacking. The multifactorial protein PARK7/DJ-1 is a major antioxidant protein, playing a key role in cellular redox homoeostasis, and is a positive regulator of AR (androgen receptor)-dependent transcription. SLC41A1 (solute carrier family 41 member 1), the gene encoding a ubiquitous cellular Mg2+E (Mg2+efflux) system, has been shown to be regulated by activated AR. We hypothesize that overexpression/up-regulation of PARK7/DJ-1, attributable to OS and related activation of AR, is an important event regulating the expression of SLC41A1 and consequently, modulating the Mg2+E capacity. This would involve changes in the transcriptional activity of PARK7/DJ-1, AR and SLC41A1, which may serve as biomarkers of intracellular MD and may have clinical relevance. Imipramine, in use as an antidepressant, has been shown to reduce the Mg2+E activity of SLC41A1 and OS. We therefore hypothesize further that administration of imipramine or related drugs will be beneficial in MD- and OS-associated diseases, especially when combined with Mg2+ supplementation. If proved true, the OS-responsive functional axis, PARK7/DJ-1–AR–SLC41A1, may be a putative mechanism underlying intracellular MD secondary to OS caused by pro-oxidative stimuli, including extracellular MD. Furthermore, it will advance our understanding of the link between OS and MD.
Increased production of ROS (reactive oxygen species), also termed OS (oxidative stress), and systemic and intracellular MD (Mg2+ deficiency) have been shown to be involved in the pathophysiology of degenerative and chronic diseases [1–3]. Among these are AD (Alzheimer's disease), PD (Parkinson's disease), ALS (amyotrophic lateral sclerosis), schizophrenia, mitochondrial disorders, hypertension and diabetes [1–13].
Dickens et al.  reported that MD in vitro enhances intracellular ROS production leading to endogenous OS in endothelial cells, and Garcia et al.  showed that Mg2+ supplementation reduces ROS formation in mitochondria. Thus a link between extracellular MD and mitochondria-related endogenous OS was established. Currently, a large amount of circumstantial evidence suggests that MD is correlated with increased levels of ROS , but the underlying molecular mechanisms remain unclear.
Recent data from our group have suggested that SLC41A1 (solute carrier family 41 member 1) is an NME (Na+/Mg2+ exchanger) and the major cellular Mg2+E (Mg2+ efflux) system. It is integral to the cytoplasmic membrane and possesses ten transmembrane helices and inside-in configuration of termini [16–18]. SLC41A1 expression is regulated by the AR (androgen receptor) [19,20]. Upon binding to DHT (dihydrotestosterone), AR translocates to the nucleus, binds to AREs (androgen-responsive elements) in its target genes, and induces their expression [19–22]. AR can also be transactivated in the absence of or at low levels of DHT [21,22]. Such transactivation has been demonstrated for PARK7/DJ-1, a redox-sensitive antioxidant protein, which forms complexes with the AR . Moreover, Takahashi et al.  have demonstrated, in Cos7 cells, that PARK7/DJ-1–AR co-localization is essential for the transcriptional activity of AR in the presence of androgen antagonists. Two possible mechanisms have been suggested for AR transactivation: (i) a direct transactivation of AR by PARK7/DJ-1 through the formation of AR–PARK7/DJ-1 complexes , and (ii) an indirect transactivation of AR by PARK7/DJ-1 through extraction of the AR negative effector, PIAS [protein inhibitor of activated STAT (signal transducer and activator of transcription)] xα, from AR–PIASxα complexes .
PARK7/DJ-1 overexpression is known to result in a 20-fold increase in AR activity in LAPC4 cells without altering AR abundance, therefore AR function, and not its expression, is proposed to be regulated by PARK7/DJ-1 . OS is one of the major triggers leading to up-regulation of PARK7/DJ-1 expression . In addition, OS augments AR signalling .
We hypothesize that OS-induced overexpression/up-regulation of PARK7/DJ-1 initiates AR-dependent SLC41A1 transcription, which could explain, in part, MD in disease conditions associated with OS (Figure 1). This hypothesis implies (i) that the transcriptional activities of PARK7/DJ-1, AR and SLC41A1 serve as biomarkers of intracellular MD, and (ii) that pharmacological inhibition of SLC41A1 will improve the therapy of OS-associated diseases.
Putative mechanism describing involvement of PARK7/DJ-1 in the regulation of the Na+/Mg2+ exchanger SLC41A1 (Mg2+ efflux) under oxidative stress
MOLECULAR RATIONALE SUPPORTING THE HYPOTHESIS
AR, PARK7/ DJ-1 and SLC41A1 expression overlap
Activity of AR occurs primarily within tissues of the male urogenital system. Less well known, however, is that AR also regulates the expression of various target genes in a plethora of other cell types (http://www.genecards.org/cgi-bin/carddisp.pl?gene=AR) . Transcripts of PARK7/DJ-1 have been detected in all human tissues tested (http://www.genecards.org/cgi-bin/carddisp.pl?gene=PARK7) with the highest expression levels in liver, skeletal muscle and kidney . Finally, SLC41A1 has been identified as being ubiquitously expressed in various cell types of the human body (http://www.genecards.org/cgi-bin/carddisp.pl?gene=SLC41A1) . The ubiquitous expression of all three transcripts in a large variety of cell types underlines the functional importance of AR, PARK7/DJ-1 and SLC41A1 for normal cell physiology.
PARK7/DJ-1 and AR are known molecular targets of oxidative stress
PARK7/DJ-1 is a member of the DJ-1/Pfp1/YajL(Thij) protein family . It has an atypical peroxiredoxin-like peroxidase activity that is involved in scavenging mitochondrial ROS, and PARK7/DJ-1-knockout mice have increased mitochondrial hydrogen peroxide (H2O2) levels [31–33]. PARK7/DJ-1 accommodates three cysteine residues (Cys46, Cys53 and Cys106). The most sensitive to OS is Cys106. It is successively oxidized via sulfenic acid (SOH) and sulfinic acid (SO2H) to the inactive form of sulfonic acid (SO3H) [34,35]. Cys46 and Cys54 of PARK7/DJ-1 are oxidized or S-nitrosylated during excessive ROS production, rendering PARK7/DJ-1 dysfunctional . PARK7/DJ-1 has been localized in the cytoplasm, nucleus and mitochondria. Under basal conditions, PARK7/DJ-1 is present mostly in the cytoplasm and, to a lesser extent, in mitochondria and nucleus . PARK7/DJ-1 is believed to be functional as a dimer, but oxidative damage to the cysteine residues leads to the disruption of PARK7/DJ-1 dimerization [31,36]. The relocation of PARK7/DJ-1 from its default cytosolic localization to either mitochondria or the nucleus depends on the oxidative environment . PARK7/DJ-1 can translocate into the nucleus under normal physiological conditions; however, Björkblom et al.  have shown that cells exposed to nontoxic levels of H2O2 have a 2-fold increase in the total nuclear accumulation of PARK7/DJ-1. Furthermore, they have demonstrated that the monomeric form of PARK7/DJ-1 (present in response to high levels of ROS) interacts with KAPβ2 (karyopherin β2), increasing PARK7/DJ-1 translocation into the nucleus . This is in agreement with the study of Kim et al.  who have shown that the presence of PARK7/DJ-1 in the nucleus is increased in response to several oxidative stressors. Once in the nucleus, PARK7/DJ-1 acts as a co-activator of various pathways, including AR-activated pathways. PARK7/DJ-1 might interact directly with the AR-binding region of PIASxα . PARK7/DJ-1 subsequently absorbs PIASxα from the AR–PIASxα complexes and, as such, restores AR transcription activity . Niki et al.  suggested further that PARK7/DJ-1 antagonizes other negative regulators of AR activity. These include DJBP (DJ-1-binding protein), which is extracted from DJBP–HDAC (histone deacetylase) complexes in the presence of PARK7/DJ-1. Finally, Tillman et al.  demonstrated a direct interaction of PARK7/DJ-1 with AR in prostate cancer cells. The biological relevance of this direct interaction is not yet fully understood; however, Taira et al.  suggested that monomeric PARK7/DJ-1 is an essential factor for the AR to exert its full activity.
AR is a transcription factor with a large N-terminal transactivation domain (encoded by exon 1), a C-terminal ligand-binding domain (encoded by exons 4–8), a central DNA-binding domain (encoded by exons 2 and 3) and a hinge region between the DNA-binding domain and ligand-binding domain that contributes to nuclear localization and degradation . AR can also be transactivated in the absence or at low levels of DHT [21,22]. Recent discoveries of alternative transactivators have added to the understanding of the physiological/pathophysiological relevance of AR for the expression of AR-sensitive genes in tissues with lower response to androgens. Known transactivators include IGF-1 (insulin-like growth factor 1) and EGF (epidermal growth factor), both signalling through classic RTK (receptor tyrosine kinase) signalling cascades, and the IL-6 (interleukin 6) signalling pathway . These signalling pathways are redox-sensitive [41,42] (Figure 2). Modulation of AR activity evoked by ROS has been described in numerous reports mostly related to prostate cancer. For example, Sharifi et al.  have shown that the suppression of SOD2 (superoxide dismutase 2) results in an increased ROS production with the concurrent activation of AR signalling. ROS promotes the activation of AR signalling by both the overexpression  and transactivation of AR .
Known alternative transactivators of AR in tissues with lower response to androgens
The exact mechanism of AR translocation into the nucleus under OS is not fully understood, and the upstream molecular effectors might vary from cell to cell. Nevertheless, AR (trans)activation has to precede AR translocation. AR must be released from complexes with their chaperones, Hsp70/90 (heat-shock protein 70/90), to form complexes with transactivating androgenic or non-androgenic effectors [22,44]. Phosphorylation of AR seems to be an important event before or after effector binding, preceding the translocation of AR–effector complexes into the nucleus .
SLC41A1 plausibly links PARK7/DJ-1 and AR signalling to Mg2+ deficiency under oxidative stress conditions
The notion that exogenous (extracellular) MD contributes to increased production of ROS is supported by multiple studies [14,15,46–49]. However, it is not clear whether exogenous MD alone is a principal OS-triggering factor. According to Wolf et al. , conflicting results regarding the association between OS and MD might originate from the ‘hard to dissect’ experimental design as, in most of the studies, exogenous MD was combined with a strong pro-oxidant stimulus [e.g. the ROS-generating system (2E)-2,3,-dihydroxybut-2-enedioate/ADP-Fe3+, incubation in serum-free medium]. Furthermore, there is a paucity of information correlating the severity of endogenous (intracellular) OS with intracellular Mg2+ concentrations, thus leaving the interplay between extracellular MD, OS and intracellular MD unexplored. The status quo of the present knowledge allows for three causal scenarios: (1) extracellular MD triggers OS and intracellular MD simultaneously, but independently; (2) extracellular MD triggers (or facilitates) OS and OS consequently triggers intracellular MD; or (3) pro-oxidant factors other than extracellular MD promote OS and OS consequently induces intracellular Mg2+ wasting (with possible long-term consequences for extracellular Mg2+ availability).
SLC41A1 is a major Mg2+E system of the cell, operating as an NME [16,17]. In our previous in vitro studies, we showed that overexpression of SLC41A1 is correlated with an increased Mg2+E capacity of the cell [16,17]. Furthermore, we demonstrated that SLC41A1 is stimulated by PKA (protein kinase A) and inhibited by the antidepressant imipramine and the class I antiarrhythmic agent quinidine (Figure 3), thus conforming to the characteristics of the Na+-dependent Mg2+E system as defined by Günther and Vormann [1,16,17,51]. Romanuik et al.  identified SLC41A1 among novel androgen-responsive genes whose expression is increased after exposure to a synthetic androgen, R1881. Later, Myung et al.  demonstrated that the small molecule EPI-002 with 20S chlorohydrin blocked AR transcriptional activity that translated into a significant decrease in SLC41A1 expression. Androgen-responsivity of SLC41A1 is a key finding allowing for linking its expression with action of AR and its enhancer PARK7/DJ-1. The latter may functionally couple SLC41A1 to OS. Therefore the assumption that OS induces overexpression of SLC41A1 and consequently increases cellular Mg2+E capacity is certainly plausible in the case of scenarios (2) and (3).
Scheme depicting a putative mechanism of OS-evoked efflux of Mg2+ from intracellular stores (e.g. mitochondria and Golgi), fuelling Mg2+E from the cell via the Na+/Mg2+ exchanger SLC41A1
Physiological and pathophysiological relevance of the proposed functional axis: PARK7/DJ-1–AR–SLC41A1
The downstream effect of PARK7/DJ-1 on Mg2+E from the cell exposed to OS might contribute to the modulation of mitochondrial energetics. The decrease in extra-mitochondrial Mg2+ concentration has been shown to result in a fall (i.e. depolarization) in the mitochondrial membrane potential (ΔΨm) . This is in agreement with the reported important role of Mg2+ in the process of Ca2+-induced depolarization of ΔΨm through discrimination between low- and high-conductance modes of the mitochondrial permeability transition pore . Therefore excessive Mg2+E mediated by SLC41A1 might result in depolarization of the inner mitochondrial membrane and initiation of mitophagy (selective clearance of damaged mitochondria in cells) or pro-apoptotic molecular events [54,55]. Apart from acting via PARK7/DJ-1–AR–SLC41A1, an increased concentration of ROS has also been shown to contribute directly to the decrease in mitochondrial ΔΨm . Depolarization of the inner mitochondrial membrane leads to loss of Mg2+ (and other cations) from the matrix, which contributes further to a systemic failure . Hence the proposed mechanism might be a constituent of a more complex response of the cell to acute OS (toxic concentrations of ROS) or chronic OS (elevated subtoxic concentrations of ROS, characteristic of many degenerative diseases). Such complex responses may further involve perturbed insulin signalling. Deranged insulin signalling is implicated in the pathophysiology of numerous chronic and degenerative conditions . Insulin was reported to enhance ΔΨm and to reduce significantly several key mediators of oxidative, nitrosative and inflammatory stress [58,59]. In contrast, OS is considered to be a root cause of disturbed insulin signalling . Recently, we have reported that insulin signalling has an inhibitory effect on SLC41A1 NME activity through reduction of intracellular cAMP and consequent decrease in PKA activity in HEK (human embryonic kidney)-293 cells . Therefore OS could promote SLC41A1-mediated Mg2+E via at least two different pathways: (i) transcriptionally via PARK7/DJ-1–AR action, leading to increased cellular abundance of SLC41A1; and (ii) functionally by suppressing the inhibitory effect of insulin on SLC41A1.
Clinical relevance of the proposed hypothesis
Although there is good evidence for a functional link between cellular redox status and Mg2+ homoeostasis, the underlying processes are unclear, especially since there is a paucity of information on intracellular Mg2+ homoeostasis, especially in the clinical setting. Our hypothesis addresses a putative molecular mechanism linking OS and intracellular Mg2+ deficiency. Future research might establish the existence of the ROS-triggered PARK7/DJ-1–AR–SLC41A1 functional axis, which could open new possibilities for targeted therapies for intracellular MD and OS [1,62]. Mg2+ supplementation and/or clinically approved inhibitors of SLC41A1 (imipramine or quinidine) might be beneficial for the management of chronic MD and OS in degenerative diseases [16,17] (Figure 3). Moreover, because of the correlation between OS, MD and the increased transcriptional activities of the PARK7/DJ-1, AR and SLC41A1 genes, monitoring the expression of these genes might serve as a diagnostic tool permitting the stratification of patients that might benefit from the above-noted interventions. Furthermore, experimental confirmation of the existence of a PARK7/DJ-1–AR–SLC41A1 functional axis will allow tests as to whether particular mutations of individual components of this axis contribute to the development of pathological molecular mechanisms leading to diseases associated with MD and OS.
All three proteins of interest, namely AR, PARK7/DJ-1 and SLC41A1, respond at the functional level to OS. PARK7/DJ-1 is translocated into the nucleus at a higher rate when levels of ROS are high. Here it acts as a positive regulator of AR-binding AREs in the promotor region of SLC41A1 and initiating its transcription. Elevated expression of SLC41A1 results in its increased abundance and the consequent elevation of the Mg2+E capacity of the cell. The functional axis PARK7/DJ-1–AR–SLC41A1 might represent molecular mechanisms that, under acute OS, allow for significant Mg2+ wasting, resulting in decreased cellular metabolism (mitochondrial dysfunction) and pro-apoptotic responses. In the case of the chronic (mild) OS characteristic of most degenerative and chronic diseases, sustained stimulation of PARK7/DJ-1–AR–SLC41A1 might lead to chronic cellular energetic and metabolic imbalance (as Mg2+ is a major stabilizer of ATP and cofactor of numerous essential enzymes) and result in pathological phenotypes at the organism level. Testing our hypothesis (Figure 1) will support the beneficial effect of the Mg2+ supplementation of patients suffering from diseases in which OS and MD are important hallmarks. Furthermore, this might lead to the broader application of imipramine and quinidine, which are both inhibitors of SLC41A1, and which are clinically used drugs (however, with an MD-unrelated indication). Moreover, to our knowledge, our hypothesis is the first attempt to functionally link the key regulators of cellular redox and Mg2+ homoeostasis.
Our gratitude is due to Dr Theresa Jones for linguistic corrections before submission.
This work was supported by a research grant from the German Research Foundation [grant number KO-3586/3-2 (to M.K.)].
epidermal growth factor
insulin-like growth factor 1
protein inhibitor of activated STAT (signal transducer and activator of transcription)
protein kinase A
reactive oxygen species
receptor tyrosine kinase
solute carrier family 41 member 1
1Martin Kolisek is a co-discoverer on patent PCT/EP11/65979 ‘Na+/Mg2+ Exchanger’.