Inorganic polyphosphate (polyP) is a linear polymer composed of up to a few hundred orthophosphates linked together by high-energy phosphoanhydride bonds, identical to those found in ATP. In mammalian mitochondria, polyP has been implicated in multiple processes, including energy metabolism, ion channels function, and the regulation of calcium signaling. However, the specific mechanisms of all these effects of polyP within the organelle remain poorly understood. The central goal of this study was to investigate how mitochondrial polyP participates in the regulation of the mammalian cellular energy metabolism. To accomplish this, we created HEK293 cells depleted of mitochondrial polyP, through the stable expression of the polyP hydrolyzing enzyme (scPPX). We found that these cells have significantly reduced rates of oxidative phosphorylation (OXPHOS), while their rates of glycolysis were elevated. Consistent with this, metabolomics assays confirmed increased levels of metabolites involved in glycolysis in these cells, compared with the wild-type samples. At the same time, key respiratory parameters of the isolated mitochondria were unchanged, suggesting that respiratory chain activity is not affected by the lack of mitochondrial polyP. However, we detected that mitochondria from cells that lack mitochondrial polyP are more fragmented when compared with those from wild-type cells. Based on these results, we propose that mitochondrial polyP plays an important role as a regulator of the metabolic switch between OXPHOS and glycolysis.

Enamel is the most calcified tissue in vertebrates. Enamel formation and mineralization is a two-step process that is mediated by ameloblast cells during their secretory and maturation stages. In these two stages, ameloblasts are characterized by different morphology and function, which is fundamental for proper mineral growth in the extracellular space. Ultrastructural studies have shown that the mitochondria in these cells localize to different subcellular regions in both stages. However, limited knowledge is available on the role/s of mitochondria in enamel formation. To address this issue, we analyzed mitochondrial biogenesis and respiration, as well as the redox status of rat primary enamel cells isolated from the secretory and maturation stages. We show that maturation stage cells have an increased expression of PGC1α, a marker of mitochondrial biogenesis, and of components of the electron transport chain. Oxygen consumption rate (OCR), a proxy for mitochondrial function, showed a significant increase in oxidative phosphorylation during the maturation stage, promoting ATP production. The GSH/GSSG ratio was lower in the maturation stage, indicative of increased oxidation. Because higher oxidative phosphorylation can lead to higher ROS production, we tested if ROS affected the expression of AmelX and Enam genes that are essential for enamel formation. The ameloblast cell line LS8 treated with H 2 O 2 to promote ROS elicited significant expression changes in AmelX and Enam. Our data highlight important metabolic and physiological differences across the two enamel stages, with higher ATP levels in the maturation stage indicative of a higher energy demand. Besides these metabolic shifts, it is likely that the enhanced ETC function results in ROS-mediated transcriptional changes.

Mibefradil is a tetralol derivative originally developed as an antagonist of T-type voltage-gated calcium (Ca2+) channels to treat hypertension when used at nanomolar dosage. More recently, its therapeutic application in hypertension has declined and has been instead repurposed as a treatment of cancer cell proliferation and solid tumor growth. Beyond its function as a Cav blocker, the micromolar concentration of mibefradil can stimulate a rise in [Ca2+]cyt although the mechanism is poorly known. The chanzyme TRPM7 (transient receptor potential melastanin 7), the release of intracellular Ca2+ pools, and Ca2+ influx by ORAI channels have been associated with the increase in [Ca2+]cyt triggered by mibefradil. This study aims to investigate the cellular targets and pathways associated with mibefradil’s effect on [Ca2+]cyt. To address these questions, we monitored changes in [Ca2+]cyt in the specialized mouse epithelial cells (LS8 and ALC) and the widely used HEK-293 cells by stimulating these cells with mibefradil (0.1 μM to 100 μM). We show that mibefradil elicits an increase in [Ca2+]cyt at concentrations above 10 μM (IC50 around 50 μM) and a fast Ca2+ increase capacity at 100 μM. We found that inhibiting IP3 receptors, depleting the ER-Ca2+ stores, or blocking phospholipase C (PLC), significantly decreased the capacity of mibefradil to elevate [Ca2+]cyt. Moreover, the transient application of 100 μM mibefradil triggered Ca2+ influx by store-operated Ca2+ entry (SOCE) mediated by the ORAI channels. Our findings reveal that IP3R and PLC are potential new targets of mibefradil offering novel insights into the effects of this drug.

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation‐stage ameloblasts focusing on their role in Ca2+ signaling. Quantification of the Ca2+ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca2+ pools by adenosine triphosphate in rhod2AM‐loaded cells showed similar mitochondrial Ca2+ (mCa2+) uptake. However, mCa2+ extrusion via Na+‐Li+‐Ca2+ exchanger was more prominent in maturation. To address if mCa2+ uptake via the mitochondrial Ca2+ uniporter (MCU) played a role in cytosolic Ca2+ (cCa2+) buffering, we stimulated Ca2+ influx via the store‐operated Ca2+ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented cCa2+ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher mCa2+ uptake in maturation ameloblasts. The uptake of Ca2+ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca2+ handling in maturation but only upon stimulation of Ca2+ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca2+ physiology, but also advance the concept that SOCE and mCa2+ uptake are complementary processes in biological mineralization.

Most cells use calcium (Ca2+) as a second messenger to convey signals that affect a multitude of biological processes. The ability of Ca2+ to bind to proteins to alter their charge and conformation is essential to achieve its signaling role. Cytosolic Ca2+ (cCa2+) concentration is maintained low at ~100 nM so that the impact of elevations in cCa2+ is readily sensed and transduced by cells. However, such elevations in cCa2+ must be transient to prevent detrimental effects. Cells have developed a variety of systems to rapidly clear the excess of cCa2+ including Ca2+ pumps, exchangers and sequestering Ca2+ within intracellular organelles. This Ca2+ signaling toolkit is evolutionarily adapted so that each cell, tissue, and organ can fulfill its biological function optimally. One of the most specialized cells in mammals are the enamel forming cells, the ameloblasts, which also handle large quantities of Ca2+. The end goal of ameloblasts is to synthesize, secrete and mineralize a unique proteinaceous matrix without the benefit of remodeling or repair mechanisms. Ca2+ uptake into ameloblasts is mainly regulated by the store operated Ca2+ entry (SOCE) before it is transported across the polarized ameloblasts to reach the insulated enamel space. Here we review the ameloblasts Ca2+ signaling toolkit and address how the common electronegative non-metal fluoride can alter its function, potentially addressing the biology of dental fluorosis.

Tobacco kills 6 million people annually and its global health costs are continuously rising. The main addictive component of every tobacco product is nicotine. Among the mechanisms by which nicotine, and its major metabolite, cotinine, contribute to heart disease is the renin‐angiotensin‐aldosterone system (RAAS) activation. This increases aldosterone production from the adrenals and circulating aldosterone levels. Aldosterone is a mineralocorticoid hormone with various direct harmful effects on the myocardium, including increased reactive oxygen species (ROS) generation, which contributes significantly to cardiac mitochondrial dysfunction and cardiac aging. Aldosterone is produced in the adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII), activating its type 1 receptor (AT 1 R). The AT 1 R is a G protein‐coupled receptor (GPCR) that leads to aldosterone biosynthesis and secretion, via signaling from both G q/11 proteins and the GPCR adapter protein βarrestin1, in AZG cells. Adrenal βarrestin1 is essential for AngII–dependent adrenal aldosterone production, which aggravates heart disease. Since adrenal βarrestin1 is essential for raising circulating aldosterone in the body and tobacco compounds are also known to elevate aldosterone levels in smokers, accelerating heart disease progression, our central hypothesis is that nicotine and cotinine increase aldosterone levels to induce cardiac injury by stimulating adrenal βarrestin1. In the present review, we provide an overview of the current literature of the physiology and pharmacology of adrenal aldosterone production regulation, of the effects of tobacco on this process and, finally, of the effects of tobacco and aldosterone on cardiac structure and function, with a particular focus on cardiac mitochondrial function. We conclude our literature account with a brief experimental outline, as well as with some therapeutic perspectives of our pharmacological hypothesis, that is that adrenal βarrestin1 is a novel molecular target for preventing tobacco–induced hyperaldosteronism, thereby also ameliorating tobacco–related heart disease development.

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Translational control and metabolic reprogramming are hallmarks of advanced cancers. Important genes involved in cancer progression express mRNAs that are selectively translated, including regulators of cancer cell metabolism. Cancer cells acquire an altered metabolism, switching from oxidative phosphorylation (OXPHOS) to a glycolytic phenotype (Warburg effect), to increase reliance on alternate metabolic pathways to support growth, proliferation, and metastasis. Triple-negative breast cancer (TNBC), one of the most aggressive and highly metastatic subtypes with the poorest outcome, is characterized by elevated glycolysis and low OXPHOS. Still, the exact mechanism for this metabolic switch is largely unknown. Using a TNBC cell model it has been shown an alternate mechanism of cap-dependent but mTORC1/eIF4Eindependent mRNA translation by the eIF4G homolog, DAP5, which directly binds the cap-binding protein eIF3d. Our research indicates that this alternate translation initiation is essential in regulating several mRNAs involved in breast cancer metabolism. DAVID analysis from genome-wide transcriptomic and translatomic studies, metabolomic assays including Glycolysis Assay [Extracellular Acidification], Oxidative Phosphorylation, and Mitotracker Assay, as well as RT-PCR and western blot analysis was performed from well-characterized triple-negative aggressive breast cancer cell lines,4T1 and MDA-231, Non-silencing (Nsi) control and silencing DAP5 (shDAP5) samples. Several mRNAs related to glucose metabolism decreased after silencing DAP5 and on the opposite, critical mRNAs associated with OXPHOS increased, independent of their steady-state mRNA abundance. Silencing DAP5 increased Oxidative Phosphorylation activity and decrease the glycolytic rate in TNBC breast cancer cells. Proteins correlated with aberrant glycolytic metabolism including the E-cadherin transcriptional repressors Snail (SNAI1) were reduced after silencing of DAP5. Our data indicates that the alternate mRNA translation initiation mechanism by DAP5 of specific mRNAs correlated to glycolysis and OXPHOS could play an essential role in the mechanism by which TNBC breast cancer cells orchestrate a metabolic switch required for malignant progression and metastasis. Citation Format: Hanna Rosenstock, Erna Mitaishvili, Columba de la Parra. The essential role of an alternate mRNA translation initiation in the regulation of breast cancer cell metabolism. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3704.