Mitochondrial calcium signaling mediated transcriptional regulation of keratin filaments is a critical determinant of melanogenesis.

Mitochondria are versatile organelles that regulate several physiological functions. Many mitochondria-controlled processes are driven by mitochondrial Ca2+ signaling. However, role of mitochondrial Ca2+ signaling in melanosome biology remains unknown. Here, we show that pigmentation requires mitochondrial Ca2+ uptake. In vitro gain and loss of function studies demonstrated that Mitochondrial Ca2+ Uniporter (MCU) is crucial for melanogenesis while the MCU rheostats, MCUb and MICU1 negatively control melanogenesis. Zebrafish and mouse models showed that MCU plays a vital role in pigmentation in vivo. Mechanistically, MCU controls activation of transcription factor NFAT2 to induce expression of three keratins (keratin 5, 7 and 8), which we report as positive regulators of melanogenesis. Interestingly, keratin 5 in turn modulates mitochondrial Ca2+ uptake thereby this signaling module acts as a negative feedback loop that fine-tunes both mitochondrial Ca2+ signaling and melanogenesis. Mitoxantrone, an FDA approved drug that inhibits MCU, decreases physiological melanogenesis. Collectively, our data demonstrates a critical role for mitochondrial Ca2+ signaling in vertebrate pigmentation and reveal the therapeutic potential of targeting MCU for clinical management of pigmentary disorders. Given the centrality of mitochondrial Ca2+ signaling and keratin filaments in cellular physiology, this feedback loop may be functional in a variety of other pathophysiological conditions.

MITF is a novel transcriptional regulator of the calcium sensor STIM1: Significance in physiological melanogenesis.

Stromal Interaction Molecule1 (STIM1) is an endoplasmic reticulum membrane-localized calcium (Ca2+) sensor that plays a critical role in the store-operated Ca2+ entry (SOCE) pathway. STIM1 regulates a variety of physiological processes and contributes to a plethora of pathophysiological conditions. Several disease states and enhanced biological phenomena are associated with increased STIM1 levels and activity. However, molecular mechanisms driving STIM1 expression remain largely unappreciated. We recently reported that STIM1 expression augments during pigmentation. Nonetheless, the molecular choreography regulating STIM1 expression in melanocytes is completely unexplored. Here, we characterized the molecular events that regulate STIM1 expression during pigmentation. We demonstrate that physiological melanogenic stimuli α-melanocyte stimulating hormone (αMSH) increases STIM1 mRNA and protein levels. Further, αMSH stimulates STIM1 promoter-driven luciferase activity, thereby suggesting transcriptional upregulation of STIM1. We show that downstream of αMSH, microphthalmia-associated transcription factor (MITF) drives STIM1 expression. By performing knockdown and overexpression studies, we corroborated that MITF regulates STIM1 expression and SOCE. Next, we conducted extensive bioinformatics analysis and identified MITF-binding sites on the STIM1 promoter. We validated significance of the MITF-binding sites in controlling STIM1 expression by performing ChIP and luciferase assays with truncated STIM1 promoters. Moreover, we confirmed MITF's role in regulating STIM1 expression and SOCE in primary human melanocytes. Importantly, analysis of publicly available datasets substantiates a positive correlation between STIM1 and MITF expression in sun-exposed tanned human skin, thereby highlighting physiological relevance of this regulation. Taken together, we have identified a novel physiologically relevant molecular pathway that transcriptionally enhances STIM1 expression.

Temporal analysis of melanogenesis identifies fatty acid metabolism as key skin pigment regulator.

Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating cutaneous hyperpigmentary conditions. Towards defining new potential targets, we followed temporal dynamics of melanogenesis using a cell-autonomous pigmentation model. Our study elucidates 3 dominant phases of synchronized metabolic and transcriptional reprogramming. The melanogenic trigger is associated with high MITF levels along with rapid uptake of glucose. The transition to pigmented state is accompanied by increased glucose channelisation to anabolic pathways that support melanosome biogenesis. SREBF1-mediated up-regulation of fatty acid synthesis results in a transient accumulation of lipid droplets and enhancement of fatty acids oxidation through mitochondrial respiration. While this heightened bioenergetic activity is important to sustain melanogenesis, it impairs mitochondria lately, shifting the metabolism towards glycolysis. This recovery phase is accompanied by activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism can resolve hyperpigmentary conditions in a guinea pig UV-tanning model. Our study reveals rewiring of the metabolic circuit during melanogenesis, and fatty acid metabolism as a potential therapeutic target in a variety of cutaneous diseases manifesting hyperpigmentary phenotype.

Mitofusin-2 Negatively Regulates Melanogenesis by Modulating Mitochondrial ROS Generation.

Inter-organellar communication is emerging as one of the most crucial regulators of cellular physiology. One of the key regulators of inter-organellar communication is Mitofusin-2 (MFN2). MFN2 is also involved in mediating mitochondrial fusion-fission dynamics. Further, it facilitates mitochondrial crosstalk with the endoplasmic reticulum, lysosomes and melanosomes, which are lysosome-related organelles specialized in melanin synthesis within melanocytes. However, the role of MFN2 in regulating melanocyte-specific cellular function, i.e., melanogenesis, remains poorly understood. Here, using a B16 mouse melanoma cell line and primary human melanocytes, we report that MFN2 negatively regulates melanogenesis. Both the transient and stable knockdown of MFN2 leads to enhanced melanogenesis, which is associated with an increase in the number of mature (stage III and IV) melanosomes and the augmented expression of key melanogenic enzymes. Further, the ectopic expression of MFN2 in MFN2-silenced cells leads to the complete rescue of the phenotype at the cellular and molecular levels. Mechanistically, MFN2-silencing elevates mitochondrial reactive-oxygen-species (ROS) levels which in turn increases melanogenesis. ROS quenching with the antioxidant N-acetyl cysteine (NAC) reverses the MFN2-knockdown-mediated increase in melanogenesis. Moreover, MFN2 expression is significantly lower in the darkly pigmented primary human melanocytes in comparison to lightly pigmented melanocytes, highlighting a potential contribution of lower MFN2 levels to higher physiological pigmentation. Taken together, our work establishes MFN2 as a novel negative regulator of melanogenesis.

Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel.

Store operated Ca2+ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous Ca2+ influx pathway. Although the role of Orai1 channels is well studied, the significance of Orai2/3 channels is still emerging in nature. In this study, we performed extensive bioinformatic analysis of publicly available datasets and observed that Orai3 expression is inversely associated with the mean survival time of PC patients. Orai3 expression analysis in a battery of PC cell lines corroborated its differential expression profile. We then carried out thorough Ca2+ imaging experiments in six PC cell lines and found that Orai3 forms a functional SOCE channel in PC cells. Our in vitro functional assays show that Orai3 regulates PC cell cycle progression, apoptosis and migration. Most importantly, our in vivo xenograft studies demonstrate a critical role of Orai3 in PC tumor growth and secondary metastasis. Mechanistically, Orai3 controls G1 phase progression, matrix metalloproteinase expression and epithelial-mesenchymal transition in PC cells. Taken together, this study for the first-time reports that Orai3 drives aggressive phenotypes of PC cells, i.e., migration in vitro and metastasis in vivo. Considering that Orai3 overexpression leads to poor prognosis in PC patients, it appears to be a highly attractive therapeutic target.

Dysregulation of host cell calcium signaling during viral infections: Emerging paradigm with high clinical relevance.

Viral infections are one of the leading causes of human illness. Viruses take over host cell signaling cascades for their replication and infection. Calcium (Ca2+) is a versatile and ubiquitous second messenger that modulates plethora of cellular functions. In last two decades, a critical role of host cell Ca2+ signaling in modulating viral infections has emerged. Furthermore, recent literature clearly implicates a vital role for the organellar Ca2+ dynamics (influx and efflux across organelles) in regulating virus entry, replication and severity of the infection. Therefore, it is not surprising that a number of viral infections including current SARS-CoV-2 driven COVID-19 pandemic are associated with dysregulated Ca2+ homeostasis. The focus of this review is to first discuss the role of host cell Ca2+ signaling in viral entry, replication and egress. We further deliberate on emerging literature demonstrating hijacking of the host cell Ca2+ dynamics by viruses. In particular, a variety of viruses including SARS-CoV-2 modulate lysosomal and cytosolic Ca2+ signaling for host cell entry and replication. Moreover, we delve into the recent studies, which have demonstrated the potential of several FDA-approved drugs targeting Ca2+ handling machinery in inhibiting viral infections. Importantly, we discuss the prospective of targeting intracellular Ca2+ signaling for better management and treatment of viral pathogenesis including COVID-19. Finally, we highlight the key outstanding questions in the field that demand critical and timely attention.

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy.

Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial-associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2gib005Δ8/+ ) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta-b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2-mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA-mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.

Molecular machinery regulating mitochondrial calcium levels: The nuts and bolts of mitochondrial calcium dynamics.

Mitochondria play vital role in regulating the cellular energetics and metabolism. Further, it is a signaling hub for cell survival and apoptotic pathways. One of the key determinants that calibrate both cellular energetics and survival functions is mitochondrial calcium (Ca2+) dynamics. Mitochondrial Ca2+ regulates three Ca2+-sensitive dehydrogenase enzymes involved in tricarboxylic acid cycle (TCA) cycle thereby directly controlling ATP synthesis. On the other hand, excessive Ca2+ concentration within the mitochondrial matrix elevates mitochondrial reactive oxygen species (mROS) levels and causes mitochondrial membrane depolarization. This leads to opening of the mitochondrial permeability transition pore (mPTP) and release of cytochrome c into cytosol eventually triggering apoptosis. Therefore, it is critical for cell to maintain mitochondrial Ca2+ concentration. Since cells can neither synthesize nor metabolize Ca2+, it is the dynamic interplay of Ca2+ handling proteins involved in mitochondrial Ca2+ influx and efflux that take the center stage. In this review we would discuss the key molecular machinery regulating mitochondrial Ca2+ concentration. We would focus on the channel complex involved in bringing Ca2+ into mitochondrial matrix i.e. Mitochondrial Ca2+ Uniporter (MCU) and its key regulators Mitochondrial Ca2+ Uptake proteins (MICU1, 2 and 3), MCU regulatory subunit b (MCUb), Essential MCU Regulator (EMRE) and Mitochondrial Ca2+ Uniporter Regulator 1 (MCUR1). Further, we would deliberate on major mitochondrial Ca2+ efflux proteins i.e. Mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) and Leucine zipper EF hand-containing transmembrane1 (Letm1). Moreover, we would highlight the physiological functions of these proteins and discuss their relevance in human pathophysiology. Finally, we would highlight key outstanding questions in the field.

Orai3: Oncochannel with therapeutic potential.

Ion channels in particular Calcium (Ca2+) channels play a critical role in physiology by regulating plethora of cellular processes ranging from cell proliferation, differentiation, transcriptional regulation and programmed cell death. One such physiologically important and highly Ca2+ selective channel family is Orai channels consisting of three homologs Orai1, Orai2 and Orai3. Orai channels are responsible for Ca2+ influx across the plasma membrane in response to decrease in Endoplasmic Reticulum (ER) Ca2+ stores. STIM1/STIM2 proteins sense the reduction in ER Ca2+ levels and activate Orai channels for restoring ER Ca2+ as well as for driving cellular functions. This signaling cascade is known as Store Operated Ca2+ Entry (SOCE). Although Orai1 is the ubiquitous SOCE channel protein, Orai2 and Orai3 mediate SOCE in certain specific tissues. Further, mammalian specific homolog Orai3 forms heteromultimeric channel with Orai1 for constituting Arachidonic acid regulated Ca2+ (ARC) channels or arachidonic acid metabolite Leukotriene C4 (LTC4) regulated Ca2+ (LRC) channels. Literature suggests that Orai3 regulates Breast, Prostate, Lung and Gastrointestinal cancers by either forming Store Operated Ca2+ (SOC) or ARC/LRC channels in the cancerous cells but not in healthy tissue. In this review, we would discuss the role of Orai3 in these cancers and would highlight the potential of therapeutic targeting of Orai3 for better management and treatment of cancer. Finally, we will deliberate on key outstanding questions in the field that demand critical attention and further studies.

Histone variant dictates fate biasing of neural crest cells to melanocyte lineage.

In the neural crest lineage, progressive fate restriction and stem cell assignment are crucial for both development and regeneration. Whereas fate commitment events have distinct transcriptional footprints, fate biasing is often transitory and metastable, and is thought to be moulded by epigenetic programmes. Therefore, the molecular basis of specification is difficult to define. In this study, we established a role for a histone variant, H2a.z.2, in specification of the melanocyte lineage from multipotent neural crest cells. H2a.z.2 silencing reduces the number of melanocyte precursors in developing zebrafish embryos and from mouse embryonic stem cells in vitro We demonstrate that this histone variant occupies nucleosomes in the promoter of the key melanocyte determinant mitf, and enhances its induction. CRISPR/Cas9-based targeted mutagenesis of this gene in zebrafish drastically reduces adult melanocytes, as well as their regeneration. Thereby, our study establishes the role of a histone variant upstream of the core gene regulatory network in the neural crest lineage. This epigenetic mark is a key determinant of cell fate and facilitates gene activation by external instructive signals, thereby establishing melanocyte fate identity.

Regulation of proto-oncogene Orai3 by miR18a/b and miR34a.

Store Operated Ca2+ Entry (SOCE) mediated by Orai channels is a ubiquitous Ca2+ influx pathway that regulates several cellular functions. We have earlier reported that Orai3, the mammalian specific Orai1 homolog, plays a critical role in breast cancer progression. More recently, Orai3 was demonstrated to regulate prostate and lung tumorigenesis. Although the tumorigenic potential of Orai3 is associated with increase in its expression, the molecular machinery regulating its expression remains largely unexplored. Here, by performing extensive bioinformatics analysis and functional studies, we identify and characterize micro-RNAs (miRNAs) that regulate Orai3 expression and function. We demonstrate that miR18a and miR18b positively regulate Orai3 whereas miR34a represses Orai3 expression and function. All these miRs exert their effect on Orai3 by virtue of their direct action on Orai3 3'UTR. These miRs provide novel opportunities for targeting Orai3 for better management of cancer. This study further opens up the possibility of targeting specific Orai homologs by different miRs in tissue and disease specific context.

STIM1 activation of adenylyl cyclase 6 connects Ca2+ and cAMP signaling during melanogenesis.

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions form functionally active microdomains that connect intracellular and extracellular environments. While the key role of these interfaces in maintenance of intracellular Ca2+ levels has been uncovered in recent years, the functional significance of ER-PM junctions in non-excitable cells has remained unclear. Here, we show that the ER calcium sensor protein STIM1 (stromal interaction molecule 1) interacts with the plasma membrane-localized adenylyl cyclase 6 (ADCY6) to govern melanogenesis. The physiological stimulus α-melanocyte-stimulating hormone (αMSH) depletes ER Ca2+ stores, thus recruiting STIM1 to ER-PM junctions, which in turn activates ADCY6. Using zebrafish as a model system, we further established STIM1's significance in regulating pigmentation in vivo STIM1 domain deletion studies reveal the importance of Ser/Pro-rich C-terminal region in this interaction. This mechanism of cAMP generation creates a positive feedback loop, controlling the output of the classical αMSH-cAMP-MITF axis in melanocytes. Our study thus delineates a signaling module that couples two fundamental secondary messengers to drive pigmentation. Given the central role of calcium and cAMP signaling pathways, this module may be operative during various other physiological processes and pathological conditions.

Role of SOCE architects STIM and Orai proteins in Cell Death.

Calcium (Ca2+) signaling plays a critical role in regulating plethora of cellular functions including cell survival, proliferation and migration. The perturbations in cellular Ca2+ homeostasis can lead to cell death either by activating autophagic pathways or through induction of apoptosis. Endoplasmic reticulum (ER) is the major storehouse of Ca2+ within cells and a number of physiological agonists mediate ER Ca2+ release by activating IP3 receptors (IP3R). This decrease in ER Ca2+ levels is sensed by STIM, which physically interacts and activates plasma membrane Ca2+ selective Orai channels. Emerging literature implicates a key role for STIM1, STIM2, Orai1 and Orai3 in regulating both cell survival and death pathways. In this review, we will retrospect the work highlighting the role of STIM and Orai homologs in regulating cell death signaling. We will further discuss the rationales that could explain the dual role of STIM and Orai proteins in regulating cell fate decisions.

Cardiovascular and Hemostatic Disorders: Role of STIM and Orai Proteins in Vascular Disorders.

Store-operated Ca2+ entry (SOCE) mediated by STIM and Orai proteins is a highly regulated and ubiquitous signaling pathway that plays an important role in various cellular and physiological functions. Endoplasmic reticulum (ER) serves as the major site for intracellular Ca2+ storage. Stromal Interaction Molecule 1/2 (STIM1/2) sense decrease in ER Ca2+ levels and transmits the message to plasma membrane Ca2+ channels constituted by Orai family members (Orai1/2/3) resulting in Ca2+ influx into the cells. This increase in cytosolic Ca2+ in turn activates a variety of signaling cascades to regulate a plethora of cellular functions. Evidence from the literature suggests that SOCE dysregulation is associated with several pathophysiologies, including vascular disorders. Interestingly, recent studies have suggested that STIM proteins may also regulate vascular functions independent of their contribution to SOCE. In this updated book chapter, we will focus on the physiological role of STIM and Orai proteins in the vasculature (endothelial cells and vascular smooth muscle cells). We will further retrospect the literature implicating a critical role for these proteins in vascular disease.

Metal based imaging probes of DO3A-Act-Met for LAT1 mediated methionine specific tumors: synthesis and preclinical evaluation.

PURPOSE: Tumor cells are known to have an elevated requirement for methionine due to increased protein synthesis and trans-methylation reactions. A methionine based macrocyclic tumor imaging system, DO3A-Act-Met, has been designed to provide a novel platform for tumor imaging via modalities, PET/MRI using metal ions, (68)Ga and (157)Gd. METHODS: Synthesis of DO3A-Act-Met was confirmed through NMR and mass spectrometric techniques. Cytotoxicity of complexes was evaluated using MTT assay whereas receptor binding and trans-stimulation studies were performed on EAT and U-87 MG cell lines. Tumor targeting was assessed through imaging and biodistribution experiments on U-87 MG xenograft model. RESULTS: DO3A-Act-Met was synthesized and radiolabeled with (68)Ga in high radiochemical purity (85-92%). The receptor binding assay on EAT cells predicted high binding affinity with Kd of 0.78 nM. Efflux of (35)S-L-methionine trans-stimulated by extracellular DO3A-Act-Met on U-87MG cells suggested an L-system transport. MR studies revealed a longitudinal relaxivity of 4.35 mM(-1) s(-1) for Gd-DO3A-Act-Met and a 25% signal enhancement at tumor site. The biodistribution studies in U-87MG xenografts validated tumor specificity. CONCLUSION: DO3A-Act-Met, a methionine conjugated probe is a promising agent for targeted molecular imaging, exhibiting high specificity towards tumor owing to its essential role in proliferation of cancer cells mediated through LAT1.

Multidrug resistance: an emerging crisis.

The resistance among various microbial species (infectious agents) to different antimicrobial drugs has emerged as a cause of public health threat all over the world at a terrifying rate. Due to the pacing advent of new resistance mechanisms and decrease in efficiency of treating common infectious diseases, it results in failure of microbial response to standard treatment, leading to prolonged illness, higher expenditures for health care, and an immense risk of death. Almost all the capable infecting agents (e.g., bacteria, fungi, virus, and parasite) have employed high levels of multidrug resistance (MDR) with enhanced morbidity and mortality; thus, they are referred to as "super bugs." Although the development of MDR is a natural phenomenon, the inappropriate use of antimicrobial drugs, inadequate sanitary conditions, inappropriate food-handling, and poor infection prevention and control practices contribute to emergence of and encourage the further spread of MDR. Considering the significance of MDR, this paper, emphasizes the problems associated with MDR and the need to understand its significance and mechanisms to combat microbial infections.

Design and synthesis of calcium responsive magnetic resonance imaging agent: Its relaxation and luminescence studies.

Calcium concentration modulation both inside and outside cell is of considerable interest for nervous system function in normal and pathological conditions. MRI has potential for very high spatial resolution at molecular/cellular level. Design, synthesis and evaluation of Gd-DO3A-AME-NPHE, a calcium responsive MRI contrast agent is presented. The probe is comprised of a Gd(3+)-DO3A core coupled to iminoacetate coordinating groups for calcium induced relaxivity switching. In the absence of Ca(2+) ions, inner sphere water binding to the Gd-DO3A-AME-NPHE is restricted with longitudinal relaxivity, r1 = 4.37 mM(-1) s(-1) at 4.7 T. However, addition of Ca(2+) triggers a marked enhancement in r1 = 6.99 mM(-1) s(-1) at 4.7 T (60% increase). The construct is highly selective for Ca(2+) over competitive metal ions at extracellular concentration. The r1 is modulated by changes in the hydration number (0.2 to 1.05), which was confirmed by luminescence emission lifetimes of the analogous Eu(3+) complex. T1 phantom images establish the capability of complex of visualizing changes in [Ca(2+)] by MRI.

Facile synthesis of non-ionic dimeric molecular resonance imaging contrast agent: its relaxation and luminescence studies.

A bis-polyazamacrocycle, 10'-bis(acetamido)ethane-bis[1,4,7-tri(carboxymethane)-1,4,7,10-tetraazacyclododecane] (DO3A-AME-DO3A) was synthesized for application in magnetic resonance imaging. The efficacy of DO3A-AME-DO3A as non ionic magnetic contrast agent was tested by performing relaxometric studies on its gadolinium complex. The longitudinal relaxivity, r(1) and transverse relaxivity, r(2) values were found to be 5.84 mM(-1)s(-1) and 6.82 mM(-1)s(-1), per Gd(III) at pH 7.0, 37 °C. The luminescence properties of europium complex of DO3A-AME-DO3A were investigated in aqueous medium. The lifetime of Eu(2)-DO3A-AME-DO3A in water was found to be 0.786 ms. Emission and luminescence lifetime measurements on the europium complex of DO3A-AME-DO3A gives a hydration number of q = 1.9. The reaction enthalpy and entropy were found to be, ΔH(0) = -(6.2 ± 2) kJ mol(-1), ΔS(0) = - (1.8 ± 0.4) kJ mol(-1)K(-1), and K(Eu)(298) = (1.8 ± 0.1).

Preclinical evaluation of DO3P-AME-DO3P: a polyazamacrocyclic methylene phosphonate for diagnosis and therapy of skeletal metastases.

A phosphonate derivative 10'-bis(acetamido)-ethane-bis[1,4,7-tri(methylene phosphonic acid)-1,4,7,10-tetraazacyclododecane] (DO3P-AME-DO3P), was synthesized with 90% yield in high purity. It was labeled with (99m)Tc in 97.5% efficiency and specific activity of 112-250 MBq/µmol. The binding affinity of (99m)Tc-DO3P-AME-DO3P towards bone minerals was tested in vitro by using hydroxy apatite as a bone model with absorption of 93% during the first hour of the experiment. Receptor binding assay on human bone cell line SAOS-2 demonstrated K(d) value of 1.07 nM. Cell binding studies of DO3P-AME-DO3P on osteoblasts and osteoclasts cells performed in vitro displayed preferential affinity of the compound towards osteoclast (167.95 ± 3.56% dose/mg protein). The serum stability of (99m)Tc complex was found to be 96.8% after 24 h. Blood kinetics of (99m)Tc-DO3P-AME-DO3P performed on normal rabbits showed fast clearance with t(1/2)(F) = 15 min ± 0.014 min and t(1/2)(S) = 4 h 3 min ± 0.09 min. Biodistribution studies carried out in normal BALB/c mice showed bone-to-blood ratio of 20 and bone-to-muscle ratio of 33. The bone tissue demonstrated highest concentration of bound radioactivity with 10.73% ID/g at 1 h post injection. The protonation and stability constants were determined by pH-potentiometry titrations. The stability constants of DO3P-AME-DO3P with Lu(III), Sm(III), and Ho(III) were 19.7, 21.8, and 20.2 determined by "out of cell" method. The excellent bone seeking properties of DO3P-AME-DO3P make it a candidate of choice for SPECT imaging and preferential uptake of the compound in osteoclasts in comparison to osteoblasts; BMM and BMC can be used to understand the pathway of pathogenesis of osteoporosis and skeletal metastases.