Hindawi Journal of Diabetes Research Volume 2020, Article ID 7814628, 12 pages https://doi.org/10.1155/2020/7814628

Review Article A Putative Prohibitin-Calcium Nexus in β-Cell Mitochondria and Diabetes

Gaurav Verma ,1,2 Aparna Dixit ,2 and Craig S. Nunemaker 3

1Molecular Metabolism, Lund University Diabetes Centre, Malmö -21428, Sweden 2School of Biotechnology, Jawaharlal Nehru University, -110067, New Delhi, India 3HCOM-Biomedical Sciences, Ohio University, Athens Camp, US-45701 Ohio, USA

Correspondence should be addressed to Gaurav Verma; [email protected]

Received 19 May 2020; Revised 1 August 2020; Accepted 5 August 2020; Published 8 October 2020

Academic Editor: Michaelangela Barbieri

Copyright © 2020 Gaurav Verma et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The role of mitochondria in apoptosis is well known; however, the mechanisms linking mitochondria to the proapoptotic effects of proinflammatory cytokines, hyperglycemia, and glucolipotoxicity are not completely understood. Complex Ca2+ signaling has emerged as a critical contributor to these proapoptotic effects and has gained significant attention in regulating the signaling processes of mitochondria. In pancreatic β-cells, Ca2+ plays an active role in β-cell function and survival. Prohibitin (PHB), a mitochondrial chaperone, is actively involved in maintaining the architecture of mitochondria. However, its possible interaction with Ca2+-activated signaling pathways has not been explored. The present review aims to examine potential crosstalk between Ca2+ signaling and PHB function in pancreatic β-cells. Moreover, this review will focus on the effects of cytokines and glucolipotoxicity on Ca2+ signaling and its possible interaction with PHB. Improved understanding of this important mitochondrial may aid in the design of more targeted drugs to identify specific pathways involved with stress-induced dysfunction in the β-cell.

1. Prohibitin: Role in Mitochondrial Function the ER Ca2+ leak in INS-1 β-cell that affects the mitochon- and β-Cell Physiology drial functions [9]. PHBs are mitochondrial chaperone recognized Mitochondria play a central role in pancreatic β-cell physiol- for their role in maintaining mitochondrial integrity [10]. ogy by coupling glucose to insulin exocytosis [1]. However, Located in the inner mitochondrial membrane, two PHB iso- mitochondria have emerged as critical players in Ca2+ forms, PHB1 and PHB2, form heterodimers to regulate mito- induced β-cell death in the context of diabetes [2–5]. Ca2+ chondrial structure and function [11–13]. Investigations with has a permissive role in apoptosis but a detailed mechanism PHBs have established that it plays a crucial role in mito- of Ca2+-induced apoptosis is not yet defined. Sequestration chondrial function in various complex disorders involving of Ca2+ by the endoplasmic reticulum (ER) prevents high β-cells dysfunction and diabetes [14, 15]. However, the 2+ 2+ 2+ ffl cytosolic Ca (Ca )c concentrations. Ca e ux out of the absence of PHBs could also induce the disintegration of ER can be induced by stresses such as cytokines and glucoli- mitochondria in nearly all kinds of cells that may lead to apo- – 2+ potoxicity [6 8]. High (Ca )c can cause mitochondrial dys- ptosis. The mechanism by which PHB protects against function, which may be mediated via prohibitin (PHB), a mitochondrial-mediated cell death is not yet understood. mitochondrial scaffold protein. Defects in ER Ca2+ storage As the role of PHB in Ca2+ homeostasis remains to be estab- and subsequent mitochondrial dysfunction are associated lished, it is still unknown whether PHB function is mediated with the development of diseases like diabetes. It has also by Ca2+ signaling which is central to both normal and path- been shown that glycogen synthase kinase 3 beta (GSK3β) ological cellular processes. In this review, we highlight the mediated Presenilin-1 phosphorylation is responsible for possible intricate tethering between PHB and Ca2+ signaling, 2 Journal of Diabetes Research and its role in deciding the fate of pancreatic β-cells. We dis- viding a scaffold that recruits membrane proteins to a desig- cuss different modern approaches that can be used to study nated lipid environment. the role of Ca2+ signaling in PHB function. We also discuss mitochondrial alterations attributed to the PHB complex with a focus on its implications in Ca2+ regulation and dis- 3. Diverse Role of Prohibitin in Transcriptional ease pathophysiology. Finally, we have proposed a hypothe- Regulation in Nucleus sized function of PHB in Ca2+ regulation and its involvement in disease pathophysiology. The factor from the E2F family is involved in various biological processes including cell differentiation, 2. A Multifunctional Mitochondrial Protein? proliferation, and apoptosis. It has been shown that PHBs inhibit the E2F transcriptional activity and regulate the cell The eukaryotic mitochondrial PHB complex consists of two cycle expression, transcription factors, and nuclear receptors heterodimer subunits, PHB1, and PHB2 [10, 12, 16]. PHB1, [26, 27]. However, a detailed mechanism by which these the principal mammalian PHB, is a potential tumor suppres- interactions bring about the transcriptional regulatory activ- sor due to its antiproliferative action, and therefore named ity is not yet elucidated. Interactions between PHBs and prohibitin [17]. Subsequently, this antiproliferative action minichromosome maintenance complex of proteins was attributed to the 3′-UTR of the mRNA encoding PHB, (MCM2-7) have been shown [28]. Wang et al. [26] have also [18]. PHB2 was characterized by virtue of its dimerization demonstrated that PHB1 interacts with the cell cycle retino- with PHB1. blastoma proteins (Rb), which are known to inhibit replica- PHB1 and PHB2, with molecular weights of 32 and tion in S phase through the attenuation of PCNA function. 34 kDa, respectively, participate to form a ring-like macro- The Rb-restricting region and another domain mapped to molecular structure of 1 MDa at the mitochondrial inner the C-terminal part of PHB are responsible for the suppres- membrane (IM). This high subatomic weight complex has sion of E2F action. PHB interacts with nuclear to mediate been found in yeast, C. elegans, and mammals [19]. Consid- transcription of p53 target [29]. In addition, PHB sup- ering the role of PHB in aging and cancer, assessing their pos- presses the activity of transcription factors like to E2F5, sible role in these processes would be beneficial [20]. The possibly via the Src activation, and thereby regulates cell pro- PHBs can form both homodimers as well as heterodimers liferation [29]. Mutations present in the 3′-untranslated [21]. The homodimeric interaction of PHBs was not revealed region of PHB in breast cancer cells suggest PHB transcrip- but recent work by Yoshinaka et al. has revealed the PHB2 tionally regulates genes involved in breast cancer develop- homodimer with the help of crystal structure that folds into ment [30]. In addition, PHB2 interacts with the an elongated shape with a highly charged surface. These family of MyoD and myocyte enhancer homodimers exhibited three acidic residues, namely gluta- element 2 (MEF2), potentially with the help of coactivators mates (E229,E231, and E233), and a glutamine (Q227) residue. such as histone deacetylases HDAC1 [31]. It has been shown Substitutions of these residues had no effect on the folding that PHB2 can inhibit muscle differentiation by repressing properties and physical hydrophobic interaction between the transcriptional activity of both MyoD and MEF2. Inter- PHBs homodimers but may be instrumental in the formation estingly, the coexpression of Akt and PHB2 has been shown of mitochondrial interactome [22]. Similar studies have to prevent the binding of PHB2 to MyoD. This stimulates shown evidence of the formation of PHB homodimer in muscle differentiation, suggesting that PHB2 may act as a HeLa cells; however, the functional validation of these topol- myogenic repressor. Furthermore, PHB specifically binds to ogy interactions demands further investigations [23]. On the estrogen receptor (ER) with the help of its ligand estra- contrary, the participation of the PHB heterodimers in vari- diol, resulting in suppression of ER transcriptional activity ous cellular processes is known, but the mechanistic insights [32]. In addition to the ER-binding motif LXXLL near the are not yet characterized. Having said that PHB1 and PHB2 N-terminus, amino acid residues spanning 175-198 in PHBs function as the same heterodimeric complex in mitochon- are required for its interaction with ER [33]. Knockdown of dria, it is intriguing that they have differences in their pheno- PHB by siRNA blocked the growth inhibitory effect of anties- types [24]. Depletion of either of the subunits results in trogens in MCF-7 breast cancer cells. PHB appeared to sup- dissociation of the complex [19]. Around 12 to 16 PHB het- press androgen receptor-mediated translation and erodimers assemble to form a ring-like structure (20-25 nm) androgen-dependent cell development. These data suggest in diameter at the mitochondrial (IM) [25]. Extensive that PHBs might play important role in determining the sen- research in the field of mitochondrial biology has revealed a sitivity of estrogen target cells including breast cancer cells. significant role of PHB in various cellular processes. The Finally, a binding domain site for the Vitamin D receptor PHB complex is integrated into the mitochondrial inner (VDR) has also been recognized in the promoter region of membrane matrix through N-terminal hydrophobic domain PHB1 [34]. VDR signaling is very important for the chemo- patches. The PHB complex controls mitochondrial mem- prevention of breast cancer cells using Vitamin D analogs. brane protein alteration by m-AAA protease, which works However, the antiproliferative effects of Vitamin D in breast as a holdase or unfoldase chaperone, and facilitates the fold- cancer cells are well recognized, but the target genes involved ing of unfolded membrane proteins. The complex may also in this process are yet to be identified. Recent reports have play a role in maintaining the mitochondrial genome. PHB suggested that PHB is a Vitamin D target and the Vita- complex also assists in mitochondrial morphogenesis by pro- min D treatment results in increased cellular PHB1 levels Journal of Diabetes Research 3

[35]. VDR/RXR binding sites are found in the promoter role in mitochondrial integrity and architecture, it is plausible region of PHB gene which makes it a novel Vitamin D target that PHB could also participate in mitochondrial DNA gene involved in the antiproliferative activity. The immense (mtDNA) maintenance. This notion is supported by the variety and diversity of important nuclear binding partners siRNA-mediated knockdown of PHB1 in HeLa cells resulting demonstrate the breadth of PHB function and highlight its in reduced mtDNA copy number, which is largely regulated action in essential cellular physiology. The main function of by mitochondrial transcription factor A (TFAM) [43, 44]. the PHB complex is to control proteins involved in the cell PHB also correlates with oxidative stress and affects electron proliferation and development, rather than binding to DNA transport chain complex activity. However, the role PHB plays as a transcription factor [36, 37]. All these reports suggest a in regulating ROS production is currently not understood. The critical function of PHBs in nuclear transcriptional regula- binding of PHB to mtDNA could explain these phenomena tion. Multiple roles of PHB in the nucleus are schematically given mtDNA encodes oxidative phosphorylation (OXPHOS) depicted in (Figure 1). subunits. PHBs play an important role in mitochondrial mem- brane integrity; therefore, it is possible that depletion of PHB 4. Multifaceted Role of Prohibitin in Cell could result in deficient OXPHOS activity. Survival and Apoptosis Oxidative stress has been the main cause in the manifes- tation of various complex disorders including myocardial Apoptosis, a controlled process of cell death, protects the injury, diabetes, and neurodegenerative disorders [45–47]. organism from malfunctioning cells. Oxidative stress in aging mitochondria also leads to the accu- Apoptosis may be triggered by different stimuli such as mulation of superoxide, which has been attributed as one of proinflammatory cytokines, UV irradiation, reactive oxygen the major causes of mitochondrial dysfunction. Generation species (ROS), hormones, and growth factors. Mitochondria of mitochondrial ROS (mtROS) in T2D could be manifested play an important role in mediating apoptosis through various by various mechanisms in which one of them is mitochon- signal transduction pathways. As previously mentioned, PHB drial membrane potential (ΔΨm). PHBs along with a mito- is a membrane protein with different cellular localizations, and chondrial anion carrier protein uncoupling protein 2 these differences in subcellular localization of PHB may direct (UCP2) reduces this ΔΨm and regulate the levels of mtROS what function it plays in the cell. A schematic diagram depict- and could be used as a biomarker surrogate for vascular ing the subcellular localization of PHB and its multiple func- health in patients with and without T2DM [48]. This PHB- tions in regulating cell fate is shown in (Figure 2). UCP2 axis could also be one of the potential biomarkers that Understressconditions,PHBtranslocatestothenucleus need to be investigated and further extrapolated as a disease and to the mitochondria, where it helps stabilizing the mito- biomarker. Having said that, PHB harbors diverse arrays of chondrial genome and controls mitochondrial morphology, signaling, and its potential as therapeutics could be attributed biogenesis, and the intrinsic apoptotic pathway [38]. Differen- by investigating its interacting partners like (Ca2+, ROS, and tial expression of PHB within cells has a protective mechanism, ATP) in a particular disease state. Furthermore, superoxide which prevents the cells from death induced by the chemother- activates UCP2 that leaks the apoptosis-triggering protein apeutic drug such as camptothecin [39]. Overexpression of cytochrome C from the inner mitochondrial membrane PHB in B-cell lymphoma Ramos cells showed a significant space when activated [49]. While the role of PHB as a mito- decrease in camptothecin-induced cell death compared to chondrial chaperone has been characterized [50, 51], its role parental cells. Levels of Rb family members that interact with in oxidative stress has not been elucidated. PHB levels are ele- E2F were decreased due to caspase-mediated degradation dur- vated during oxidative stress, and overexpression of PHB sig- ing camptothecin treatment. However, overexpression of PHB nificantly reverses this oxidative stress effect. Additionally, protected cells from cell death, suggesting an anti-apoptotic hydrogen peroxide (H2O2)-induced apoptosis was prevented role. In leukemic cell lines, PHB acts as a nuclear protein sub- in PHB-overexpressing cardiomyocytes [52]. strate [40]. Camptothecin treatment resulted in translocation It is possible that PHB promotes the mitochondrial mem- of PHB out of the nucleus to perinuclear regions [41]. The role brane permeability transition and inhibits the release of cyto- of PHBs in cancer is complex. Some types of tumor overexpress chrome c from mitochondria to trigger apoptosis. These PHB1 in the nucleus and some in mitochondria, whereas some studies reflect the protection conferred against oxidative of them do not express it at all. Given the existing evidence, stress-induced apoptosis that has been reported in various fine-tuning of the PHB localization may be beneficial in selec- mitochondrial complex disorders. These data indicate that tively inducing apoptosis in cancer cells. PHB protects from oxidative stress-induced damage, and fine PHB acts as a stabilizer of inner mitochondrial membrane tuning of PHB content in mitochondria could be a potential proteins. Therefore, loss of PHB severely affects the integrity of new therapeutic target for myocardial injury and diabetes. the mitochondria, as was shown for the first time in body wall Therefore, considering the PHB participation in integral sig- muscle cells of C. elegans. The mitochondrial PHB complex is naling of mitochondrial-mediated cellular disturbance, it is essential for embryonic viability and germline function in C. plausible that targeting PHB may inhibit the progression elegans. Loss of PHB results in mitochondria that appear dis- and pathology of diseases characterized by oxidative stress. integrated and fragmented [42]. These aspects of mitochon- However, it has also been reported that the treatment of pros- drial fragmentation in PHB deficient cells could be explained tate cancer cells with TGF-β results in translocation of PHB by the involvement of OPA1, which regulates the fusion pro- from the nucleus to the cytosol where it strongly associates cess in mitochondria [36]. Considering PHB plays a crucial with the mitochondrial apoptosis-suppressor Bcl-2 and 4 Journal of Diabetes Research

Estradiol Androgen Vitamin D

ER Prohibitin

AR VDR

p53 Rb E2F ER -estrogen receptor AR -androgen receptor VDR -vitamin D receptor Rb -retinoblastoma Nucleus

Cell proliferation

Figure 1: Prohibitins as regulators of the cell. Prohibitin complex interact with nuclear receptors, like ER, AR, VDR, and transcription factors p53, Rb, and E2F thereby regulate gene expression and cell proliferation of the target cell.

Prohibitin (i) Nuclear localization for gene regulation. (ii) Cellular protection from extracellular stress. (iii) Interaction with various cytosolic protein OPA1Bax/Bak regulating cell survival. Cytochrome c Caspase 9 Nucleus Caspase 3

Apoptosis Prohibitin

Figure 2: Putative role of prohibitin in mitochondria and nucleus. As mitochondrial chaperones, PHBs preserve the integrity of mitochondria. Their translocation to nucleus promotes binding of nuclear receptors and exhibits their action in the nucleus. PHBs interact with mitochondrial regulators including the key players of apoptotic pathways. PHBs interact with OPA1 complex (green and orange) which is located at the mitochondrial cristae junctional openings. Remodelling of these cristae majorly by OPA1 complex proteolysis or intermembrane proteases releases the cytochrome c in the cytosol. OPA1-mediated cristae opening is Bax/Bak dependent which is required for the cytochrome c release. Cytochrome c further activates the downstream effector molecules like caspase 9 and caspase 3 that culminates into apoptosis. Our hypothesized model proposes that overexpression of PHB could stabilize the mitochondrial structure and morphology that in turn may prevent the release of cytochrome c, activation of caspase, and apoptosis. represses its activity [53]. All these findings suggest that PHB cardiomyopathies, optic neuropathy, and inflammatory dis- has a diverse array of signaling, and its function may depend eases. During cellular stress, PHB can translocate to the on the type of stimulus the cells encounter. Mitochondrial nucleus or to the mitochondria and could possibly trigger dysfunction in these stressed cells can give rise to a broad apoptotic signals [54]. How a membrane protein such as spectrum of diseases such as neurodegenerative disorders, PHB that can control cell death requires future studies. That Journal of Diabetes Research 5 is to say, it is possible that PHB not only functions as a trans- chondrial membrane potential (ΔΨm) are depicted in membrane receptor in the cell but could also be acting as a (Figure 3). As evident from (Figure 3), IL-1β treatment 2+ regulator between cell survival and cell death; although, this results in a time-dependent increase in (Ca )c as measured is an area that requires further investigation. by Fluo-4 with a concomitant increase in ΔΨm by TMRM. Analysis of the ER in the IL-1β-treated cells shows an 5. Prohibitin-Ca2+ Interface: Possible ER- increase in ER size, suggesting ER attempts to mitigate the Mitochondria Ca2+ Juxtapose effect of IL-1β by increasing its biogenesis. It is important to further study Ca2+ signaling in these compartments, which Positively charged Ca2+ ions are one of the primary signal- could be a worthy area of investigation in both normal and ing elements found within cells. In almost all membrane pathological cellular processes. bound organelles, Ca2+ binds to hundreds of proteins to impact changes in localization, association, and function 6. Studying Prohibitin-Ca2+ Signaling with Live [55]. It is also an important second messenger participat- Cell Imaging: A Boon for Ca2+ Studies ing in many cellular activities including cell growth, differ- entiation, gene regulation, and cell survival [56, 57]. Ca2+ Using the live-cell imaging technology coupled with focused helps in reversing the damage elicited by cellular insults fluorescent dyes, the effect of proinflammatory cytokines on by both facilitating cell survival and mitigating cell death Ca2+ flux in pancreatic β-cells has been extensively studied responses. Interestingly, in the process of adaptive [61, 62]. Numerous reports have implicated Ca2+ oscillation responses, Ca2+ participates as a crucial second messenger as critical in ER and mitochondria to maintain pancreatic that decides whether a cell must survive or die. This dual- physiology [63, 64]. Extensive research demonstrates that ity of Ca2+ function makes it difficult to delineate mecha- Ca2+ is confined to particular regions of pancreatic β-cells, nisms required for Ca2+-mediated control of cell survival principally in the ER, and mitochondria [65, 66]. Likewise, and apoptosis. In the pancreatic β-cells, it is important observed Ca2+ movements from the ER to mitochondria are to determine the source of the disruption in Ca2+ signaling induced by cytokines that promote mitochondrial dysfunc- that ultimately affects the overall β-cell function. tion and cell death [67]. As stated earlier, the role of PHB Researchers have also proposed the role of mitochondria in controlling mitochondrial structural integrity, and subse- in such events; however, its involvement in this process quently β-cell capacity, has been studied extensively; how- remains poorly understood. One of the most important ever, there has been no experimental evidence that suggests questions to-date in the field is understanding the role that the possible role of PHB in the regulation of Ca2+ signaling PHBs might play with ER and mitochondrial Ca2+ storage. in β-cell mitochondria. Given that PHB is a mitochondrial However, untill now, PHBs are not known to regulate chaperone and could regulate Ca2+ in β-cell physiology, we Ca2+, but it has been shown that PHB2 regulates the hypothesize PHB-mediated regulation of Ca2+ could play a Mg2+ channel Transient Receptor Potential Melastatin 6 role in the β-cell function. (TRPM6) [58]. TRPM6 functions as the gatekeeper of transepithelial Mg2+ transport and regulation by PHB 7. Prohibitin-Ca2+ Signaling Crosstalk: hints the role of PHBs in the possible interaction with Potential Candidate in β-Cell Dysfunction the divalent cationic molecule like Ca2+. In this section, we will review the kinetics of Ca2+ as an indicator of β-cell Understanding the role of PHB in Ca2+ regulation will aid in function and explain the effect of proinflammatory cyto- the understanding of the mitochondrial capacity and dys- kines on Ca2+ oscillation in β-cells. Cytokines induce a function in metabolic pathologies like diabetes. Recent − − disruption in Ca2+ signaling that can impair insulin release reports suggest that β-cells from PHB2 / mice display a var- in response to glucose stimulation, and in chronic cases, ing degree of mitochondrial dysfunction immediately after can even lead to β-cell death. In particular, IL-1β can alter cell subculture [68]. Cytokine or glucolipotoxicity induced intracellular Ca2+ levels by depleting Ca2+ from the ER via changes in Ca2+ levels enhance Ca2+ flux out of the ER Ca2+ channels such as inositol 1,4,5-trisphosphate receptor through ER Ca2+ channels, thereby diminishing the Ca2+ (IP3R), Ryanodine receptor (RYR), and sarco/endoplasmic pool stored in the ER. Cellular stressors like cytokines, oxida- reticulum Ca2+-ATPase (SERCA) pump. tive stress, and hyperglycemia impair the storage capacity of 2+ 2+ These ER Ca channels can in turn facilitate the increase the ER, resulting in elevated (Ca )c and subsequent mito- in Ca2+ flux to the cytosol following its sequestration in cellu- chondrial dysfunction. Given that PHB is a mitochondrial lar organelles such as mitochondria [59]. Sudden elevation of chaperone, it is possible that PHB participates in Ca2+ buffer- 2+ 2+ 2+ 2+ mitochondrial Ca (Ca )m can lead to Ca toxicity and ing during (Ca )c elevation. Depletion of PHB could result ff 2+ trigger apoptosis. It is now widely accepted that depletion in adverse e ects associated with elevated (Ca )c levels. of Ca2+ levels leads to protein misfolding and activation of The outline of the current hypothesis is depicted in (Graph- the unfolded protein response (UPR) [60]. Organelles such ical Abstract) and in (Figure 4). However, the role of Ca2+ as the ER, mitochondria, and nucleus are also affected by in PHB-mediated maintenance of mitochondrial function cytokine like IL-1β which may lead to β-cell death in type 1 in pancreatic β-cells, and how PHB may mitigate future risk diabetes (T1D) and type 2 diabetes (T2D). IL-1β-induced of T2D is not yet explored; additional studies investigating changes in the level of the Ca2+ in the ER and cytosol, and this process are needed to aid in the development of thera- its resulting impact on mitochondrial physiology like mito- peutics for diabetes. 6 Journal of Diabetes Research

IL-1� (2 ng/ml), 0 hr IL-1� (2 ng/ml), 1 hr IL-1� (2 ng/ml), 2 hr

(a)

Ca2+ (fuo-4)

(b)

��m (TMRM)

(c)

ER tracker

(d)

� Merge 20 M

Figure 3: Fluorescent imaging of β-cells with Fluo-4, TMRM, and ER tracker. Mice islets were isolated and dissociated in β-cells. Further, β- cells were induced by IL-1β (2 ng/ml) for 0 hr, 1 hr, and 2 hr, respectively, and were processed for Ca2+ imaging with respective fluorescent dyes. Panel shows differential expression of Ca2+ level in different compartments of the cell. Panel (a) shows the level of cytosolic Ca2+as measured by Fluo-4 (green), panel (b) shows the (ΔΨm) with TMRM (red), panel (c) shows the endoplasmic reticulum with ER tracker (blue), and the merged image of all three fluorescence are represented in panel (d). Leica live cell imaging microscope was used to perform the imaging.

In the following section, experimental approaches to thereby promotes mitochondrial dysfunction and ultimately monitor Ca2+ flux in β-cells and the role PHB plays in regu- culminate in apoptosis. lating Ca2+ flux will be discussed. To date, confocal and live Despite the lack of evidence directly linking PHB to Ca2+ cell microscopes are the most commonly used approach to flux, PHB has been implicated in both β-cell and mitochon- evaluate Ca2+ oscillation in live cells. Cellular stressors (cyto- drial physiology. β-cell specific PHB2 knockout mice (β- − − kines and hyperglycemia) impair Ca2+ flux in living β-cells, PHB2 / ) have dysfunctional mitochondria, enhanced β-cell of which the immediate impact on PHB function can be eas- death, and diabetes [68]. However, it remains to be observed ily monitored with the help of genetically encoded Ca2+ indi- how this mitochondrial chaperone participates in handling cators (GECI), Ca2+ dyes and fluorescence microscopy. Ca2+ toxicity and to what extent the Ca2+ burden imposed These commercially available indicators are widely used to by the cellular insults is mitigated by PHB? PHB serves as a determine localization of Ca2+. To accurately measure the chaperone ensuring the correct order of the protein transla- Ca2+ flux changes in β-cells after stress induction, different tion machinery in the cell. It is therefore imperative to study combinations of fluorophores can be used to identify the sub- its role in this phenomenon. Given that the majority of chap- cellular region that exhibits fluorescent emission upon laser erones require Ca2+ for their function [72, 73], we hypothe- stimulations [69, 70]. Fluorescent markers like 4mtD3cpV size that PHB could be a plausible candidate in sequestering 2+ 2+ (mitochondrial FRET-based Ca sensor), D1ER [second- excess (Ca )m, inhibiting the execution of apoptosis by generation cameleon (calcium sensor) targeted to ER], and mitochondria. Primarily, mitochondria sequester the fl 2+ 2+ uo-4/FURA-2AM can be used to evaluate Ca oscillations (Ca )c by inner mitochondrial membrane (IMM) mito- in mitochondria, ER, and cytosol, respectively [71]. In most chondrial calcium uniporter (MCU) in cooperation with studies involving diabetes, either a combination of cytokines voltage dependent anion channel (VDAC) which is such as TNF-α, IL-1β, and IFNγ or 15-30 mM glucose mim- expressed at the outer mitochondrial membrane (OMM) icking hyperglycemia may be used to stimulate Ca2+ release surface (Figure 4). It is also interesting to note that PHBs from the ER. Cellular stress results in altered Ca2+ signaling interact with VDAC [74] and since VDAC is majorly Journal of Diabetes Research 7

Hyperglycemia Extracellular

GLUT2 Cytosol

PHB

2+ [Ca ]c VDAC

MCU [PHB]m mPTP 2+ [Ca ]m Decreased cellular �-cell loss Diabetes Cyt c functions Apoptosis

Figure 4: Plausible role of prohibitin in mitochondrial Ca2+ overload. During hyperglycemic condition, there is a remarkable increase in 2+ 2+ 2+ cytosolic Ca (Ca )c. Physiologically, the elevation of (Ca )c is required for maintaining the normal metabolic pathways; however, 2+ 2+ 2+ persistent elevation leads to the movement of the (Ca )c to the mitochondria and the subsequent rise in mitochondrial Ca (Ca )m ff 2+ concentration. Prohibitin may attempt to bu er this excess of (Ca )m to further prevent the mitochondrial-mediated apoptosis by preventing the release of cytochrome c (Cyt c). The consequence of this event may lead to decreased cellular function, β-cell loss, and diabetes. Overexpression of prohibitin may help in sequestering excess of the Ca2+ and prevent apoptosis. Mitochondrial calcium uniporter (MCU) and voltage-dependent anion channel (VDAC) are the channels on the inner and outer surface of mitochondria that 2+ sequesters the (Ca )m cooperatively. involved in the permeability pathways that control the flow lates Ca2+ exchange between the ER and mitochondria, par- of Ca2+, it is very likely that PHBs could regulate the Ca2+ sig- ticularly in β-cells. Alternatively, a more significant naling both dependent and independent of VDAC. The experiment would be to examine whether different types of mitochondrial architecture and its morphology are princi- PHB ligands regulate Ca2+ flux as described by Wang et al. pally maintained by the coordination of organellar and cyto- [76]. In the context of Ca2+-PHB orchestration, an optional solic communication. The involvement of Ca2+ signaling in methodology of cameleon test (genetically encoded fluores- the regulation of mitochondrial morphology in physiological cent markers for Ca2+) could also be utilized for monitoring and pathological conditions remains to be understood. Nev- of Ca2+ movement in different cell types [77]. Cameleon tests ertheless, there are reports demonstrating Ca2+ playing a like D1ER and 4mtD3cpV are very efficient to perform more central role in orchestrating these processes than previ- FRET-based imaging to monitor alterations in Ca2+ levels ously understood [75]. induced by cytokines or glucolipotoxicity challenges. Such We, therefore, postulate that excess Ca2+ in the mito- experiments would help in characterizing the phenomena chondria could be sequestered by overexpression of PHBs, involving Ca2+ levels and PHB capacity to prevent β-cell which in turn could prevent the induction of cell death or death. other pathological conditions. To better understand the role PHB plays in Ca2+ signaling, several approaches could be 8. Role of Prohibitin in Membrane Signaling employed. For example, studying ER Ca2+ channels in partial and Cellular Fate: A Binary Switch? knockdown of PHB could help distinguish the role of these channels in Ca2+ release. PHBs are located in various membranes where they play an Overexpression or knockdown of PHBs can reveal the instrumental role in cellular signaling. PHBs are engaged in degree to which PHB contributes to mitochondrial Ca2+ the tyrosine phosphorylation PI3K/Akt, MAPK/ERK path- overburden, and further, delineate the role of PHB in buffer- ways, and TGFβ signaling pathways, suggesting the impor- ing the elevated Ca2+. This will aid in understanding the link tance of PHB in processes such as metabolism, between PHB and Ca2+ homeostasis in cellular physiology. proliferation, and development [78]. PHB has also been Blocking Ca2+ channels using Ca2+ channel blockers and shown to be required for the Ras-mediated Raf-MEK-ERK monitoring the expression of PHBs would be a simple and activation, a critical process that occurs in conjunction with valuable experiment in testing the hypothesis that PHB regu- the plasma membrane [79]. PHB can also indirectly facilitate 8 Journal of Diabetes Research

Table 1

Subcellular PHB1 function Location Pathophysiology Reference localization Oxidative stress β-Cells Mitochondria Type 2 diabetes Lee et al., FEBBS J, 2010. regulation Oxidative stress Intestinal epithelial Inflammatory bowel Mitochondria Kathiria e al., Plos One, 2010. regulation cells disease Tubulointerstitial Zhou et al., J of Recep and Signal Trans, α-SMA regulation Renal cells Mitochondria lesions 2013. Oxidative stress Cardiomyocytes Mitochondria Myocardium damage Liu et al., Cell Stress Chaperone, 2009. regulation Oxidative stress Hepatocytes Mitochondria HCV Ivanov et al., Viruses, 2013. regulation Combating Lungs Mitochondria COPD Soulitzis et al., Resp. Med, 2012. inflammation P-53, Rb tumor Cancer cells Nucleus Cancer Fusaro et al., JBC, 2003. regulator Synaptic movement Neural cells Mitochondria/nucleus Schizophrenia Bemstein et al., Neuromol Med, 2012

2+ fi the crosstalk between the PI3K/Akt and Ras/MAPK/ERK elevated (Ca )c, possibly by ne-tuning PHB levels. Such pathways mediated by its signaling intermediates PtdIns fine-tuning by mitochondria may protect the cell from del- (3,4,5) P3, SH2-domain containing phosphatase (Shp)1 eterious events such as altered membrane potential, [80]. PHB has also been shown to be the intracellular effector impaired ATP production, deficiency in insulin secretion, molecule of TGF-β signaling in prostate cancer cells. Loss of and β-cell death. However, the exact role of PHB in han- PHB also culminates in apoptosis via the TGF-β-Smad axis dling excess Ca2+ in mitochondria is not known. Therefore, suggesting its role in cellular apoptosis [53]. PHB levels vary PHB may be a potential molecular target that may be under different stimuli, and it can be said that it acts as both a manipulated pharmacologically to treat obesity and diabe- proapoptotic as well as antiapoptotic molecule, depending tes. Recent reports involving the role of PHB in obesity upon the type of stress the cell has encountered with. It is draws attention to investigate this axis [89, 90]. Of note, reported that oxidative stress induces deleterious effects in PHB expression has to be modulated physiologically which pancreatic β-cells [81, 82]. is otherwise may lead to other serious consequences like However, PHB expression in pancreatic β-cells is pro- obesity and glucose homoeostasis imbalance. It is very tective against oxidative stress and apoptosis [83]. Further- interesting to note that almost 25 years ago, McClung more, we previously reported, IL-1β, a pro-inflammatory et al. [91] speculated prohibitin as an inner mitochondrial cytokine induces ER stress and mitochondrial-mediated membrane protein, which may control ion transport and apoptosis in pancreatic β-cells. This action is mediated by calcium-dependent ATP production, but surprisingly, there JNK, and in part, by Ca2+ [59]. In addition, mitochondrial has been no experimental evidence that demonstrates Ca2+ architecture is primarily governed by the integration of regulated PHB function in mitochondria. The possible role organellar and functional protein crosstalk. Mitochondria of PHB and its role in disease pathology is described in ff 2+ help in bu ering excess (Ca )c and maintain the appropri- (Table 1). ate Ca2+ level in the cells [84]. During stress, mitochondrial strength is compromised which in turn leads to mitochon- 9. Methods Involved in Calcium Signaling: An drial Ca2+ overload and apoptosis in pancreatic β-cells. Era of Modern Biology PHB stabilizes mitochondrial proteins by functioning as a chaperone. The majority of chaperones need Ca2+ to func- Genetically, manipulating PHB expression in both commer- tion [85, 86]. However, the role of Ca2+ in regulation of cially available pancreatic β-cells and human islets would mitochondrial PHB in both normal and pathological condi- provide a more robust model in which PHB function in tions is not known. Recent reports have suggested that Ca2+ Ca2+ homeostasis may be interrogated. Ca2+ signaling may plays a critical role in maintaining mitochondrial health be manipulated intracellularly via commercially available and proper function [87, 88]. Given both PHB and Ca2+ thapsigargin, an ER stress inducer used to discharge Ca2+ are critical players in mitochondrial dynamics, it is possible into the cytosol. To determine how PHBs contribute to that a crosstalk exists between PHB and Ca2+. PHB, a stabi- Ca2+ buffering, mitochondrial targeted GECI like lizer of inner mitochondrial membrane proteins, may par- 4mtD3cpV, mtGeCo1, and mt Pericam can be used to mon- 2+ 2+ ticipate in Ca handling in the mitochondria, thereby itor the continuous movement of (Ca )m. preventing mitochondrial Ca2+ overload which is a critical The respective fluorescence of these GECI in the mito- instigating event of pancreatic β-cell death. In an event of chondria will delineate the capacity of PHBs to buffer excess 2+ ff ff 2+ increased (Ca )c, the mitochondria make e orts to bu er (Ca )m. To fully understand these phenomena, experiments Journal of Diabetes Research 9 performed in β-cells PHB2-/- mice (and their control litter- designed to identify those mechanisms are worthy of inves- fl fl mate β-PHB2 / ) can be used to provide in vivo evidence. tigation and could suggest a role of PHB in maintaining Overexpression of PHBs in β-cells can aid in evaluating mitochondrial health. 2+ (Ca )m overload, a hallmark feature in mitochondrial- mediated pancreatic β-cell death. Additional analysis using 11. Conclusions patch-clamp electrophysiology (current and voltage clamps) can also be used to evaluate Ca2+ homeostasis as an alterna- The goal of this review is to better characterize Ca2+ effects on tive approach to investigate PHB capacity of buffering excess PHBs by directly examining how prohibitin handle Ca2+ in 2+ (Ca )c. the mitochondria during diabetic pathophysiology. We Proinflammatory cytokines, hyperglycemia, and glucoli- emphasized on the most significant differences observed potoxicity all alter Ca2+ levels such that the activated signal- between control and PHBs knockdown in β-cells that should ing cascades promote pancreatic β-cell death during be indicative of the target of Ca2+ action, thereby establishing diabetes. However, the mechanisms contributing to the proa- a biomarker for the detection of diabetic phenotype. This will poptotic effects of such stressors are not completely under- also deliver an additional mechanistic approach to identify stood. In addition to the seminal discovery of the core the most plausible targets for reversing Ca2+ induced damage molecular machinery involved in β-cell death, complex during diabetes progression. Finally, understanding of this 2+ fi ff Ca signaling has emerged as a signi cant contributor dur- study will determine most substantial e ects of PHBS in ing these events and has gained significant attention. Ca2+ mitochondrial health and will inform the design of more tar- actively participates in various physiological processes within geted future experiments to reverse diabetes. pancreatic β-cells and rapidly exchanges between the ER and mitochondria. The relationship between the changes in 2+ 2+ Conflicts of Interest (Ca )c and (Ca )m during glucose stimulus was not completely understood. Extensive work has been done in The authors (GV, AD, and CN) declare that there is no con- these areas from last two decades to understand this intricate flict of interest regarding the publication of this paper. tethering between ER and Mitochondrial Ca2+ and their sub- sequent involvement in ATP regulation and insulin secretion Acknowledgments [92–94]. Findings from these studies may provide a founda- tion upon which future experiments may be designed to The author is thankful to the Company of Biologist, Journal identify specific pathways involved in inflammatory or meta- of Cell Science, for providing travelling fellowship to perform bolic dysfunction in the β-cell. These discoveries illuminate a a part of this study in the University of Virginia, United more focused future on experiments designed to recognize States, with Dr. Craig Nunemaker, Associate Professor, Med- other novel channels involved in Ca2+ signaling and mito- icine: Endocrinology and Metabolism, USA. The author is chondrial dysfunction in β-cells. Such novel discoveries also also thankful to Harsh Kumar, PhD, Regional Centre for Bio- hold promise for unravelling a novel mechanism to aid in technology, Faridabad, India for his critical inputs during the the prevention of diabetes. preparation of the manuscript.

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