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.

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.