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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.

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.

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.

Mitochondria regulate several physiological functions through mitochondrial Ca²⁺ dynamics. 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 demonstrate that mitochondrial Ca²⁺ uniporter (MCU) is crucial for melanogenesis while MCU rheostat, MCUb negatively control melanogenesis. Zebrafish, MCU^+/- and MCUb^-/- mice models show that MCU complex drives pigmentation in vivo. Mechanistically, MCU silencing activates transcription factor NFAT2 to induce expression of keratin (5, 7, and 8) filaments. Interestingly, keratin5 in turn augments mitochondrial Ca²⁺ uptake and potentiates melanogenesis by regulating melanosome biogenesis and maturation. Hence this signaling module acts as a negative feedback loop that fine-tunes both mitochondrial Ca²⁺ signaling and pigmentation. Notably, mitoxantrone, an FDA approved drug that inhibits MCU, reduces pigmentation thereby highlighting therapeutic potential of targeting mitochondrial Ca²⁺ uptake for clinical management of pigmentary disorders. Taken together, we reveal an MCU-NFAT2-Keratin5 driven signaling axis that acts as a critical determinant of mitochondrial Ca²⁺ uptake and pigmentation. Given the vital role of mitochondrial Ca²⁺ signaling and keratin filaments in cellular physiology, this feedback loop could be operational in a variety of other patho-physiological processes.

Pancreatic cancer (PC) is one of the most lethal forms of cancers with 5-year mean survival rate of less than 10%. Most of the PC associated deaths are due to metastasis to secondary sites. Calcium (Ca²⁺) signaling plays a critical role in regulating hallmarks of cancer progression including cell proliferation, migration and apoptotic resistance. Store operated Ca²⁺ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous pathway responsible for Ca²⁺ influx into non-excitable cells. 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 6 PC cell lines and found that Orai3 forms a functional SOCE 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 expression is inversely associated with the PC patients survival time, it appears to be a highly attractive therapeutic target.