Modulation of autophagic activity by extracellular pH.
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ABSTRACT:
SUBMITTER: Xu T
PROVIDER: S-EPMC3242796 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Purpose: Candida albicans avidly uses multiple carbon sources that are less preferred by related species. This project compared transcriptional profiles of cells growing in media containing these nutrients relative to glucose Methods: C. albicans strain SC5314 was incubated in minimal YNB media containing 2% glucose, casamino acids, glutamate or a-ketoglutarate for five hours. RNA was prepared and subjected to deep sequencing. Results: Substantial overlap was seen in the transcriptional changes in all alternative carbon sources relative to the glucose control. Source-specific changes reflected the unique metabolism of each nutrient. Conclusions: C. albicans is well-adapted to use a range of nutrients that can be found in the mammalian host and has a common transcriptional response to limiting carbon environments.
2016-11-01 | GSE87832 | GEO
Project description:The opportunistic fungal pathogen Candida albicans thrives within diverse niches in the mammalian host. Among the adaptations that underlie this fitness is an ability to utilize a wide array of nutrients, especially sources of carbon that are disfavored by many other fungi; this contributes to its ability to survive interactions with the phagocytes that serve as key barriers against disseminated infections. We have reported that C. albicans generates ammonia as a byproduct of amino acid catabolism to neutralize the acidic phagolysosome and promote hyphal morphogenesis in a manner dependent on the Stp2 transcription factor. Here, we report that this species rapidly neutralizes acidic environments when utilizing carboxylic acids like pyruvate, α-ketoglutarate (αKG), or lactate as the primary carbon source. Unlike in cells growing in amino acid-rich medium, this does not result in ammonia release, does not induce hyphal differentiation, and is genetically distinct. While transcript profiling revealed significant similarities in gene expression in cells grown on either carboxylic or amino acids, genetic screens for mutants that fail to neutralize αKG medium identified a nonoverlapping set of genes, including CWT1, encoding a transcription factor responsive to cell wall and nitrosative stresses. Strains lacking CWT1 exhibit retarded αKG-mediated neutralization in vitro, exist in a more acidic phagolysosome, and are more susceptible to macrophage killing, while double cwt1Δ stp2Δ mutants are more impaired than either single mutant. Together, our observations indicate that C. albicans has evolved multiple ways to modulate the pH of host-relevant environments to promote its fitness as a pathogen. The fungal pathogen Candida albicans is a ubiquitous and usually benign constituent of the human microbial ecosystem. In individuals with weakened immune systems, this organism can cause potentially life-threatening infections and is one of the most common causes of hospital-acquired infections. Understanding the interactions between C. albicans and immune phagocytic cells, such as macrophages and neutrophils, will define the mechanisms of pathogenesis in this species. One such adaptation is an ability to make use of nonstandard nutrients that we predict are plentiful in certain niches within the host, including within these phagocytic cells. We show here that the metabolism of certain organic acids enables C. albicans to neutralize acidic environments, such as those within macrophages. This phenomenon is distinct in several significant ways from previous reports of similar processes, indicating that C. albicans has evolved multiple mechanisms to combat the harmful acidity of phagocytic cells.
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Project description:TRPM7 channels participate in a variety of physiological/pathological processes. TRPM7 currents are modulated by protons but opposing effects of external pH (pHo) (potentiation vs inhibition) have been reported. TRPM7 has been less studied in human cardiomyocytes than in heart-derived non-cardiomyocyte cells. We used the whole-cell patch-clamp technique on isolated human atrial cardiomyocytes to investigate the impact of an acidic pHo on the TRPM7 current. With voltage-dependent and other ion channels inhibited, cardiomyocytes were challenged with external acidification in either the presence or the absence of extracellular divalent cations. TRPM7 outward and inward currents were increased by acidic pHo in extracellular medium containing Ca2+ and Mg2+, but suppressed by acidic pHo in the absence of extracellular Ca2+ and Mg2+. The potentiating effect in the presence of extracellular divalents occurred at pHo below 6 and was voltage-dependent. The inhibitory effect in the absence of extracellular divalents was already marked at pHo of 6 and was practically voltage-independent. TRPM7 current density was higher in cardiomyocytes from patients with history of coronary vascular disease and the difference compared to cardiomyocytes from patients without history of myocardial ischemia increased with acidic pHo. We demonstrate that proton-induced modification of TRPM7 currents depends on the presence of extracellular Ca2+ and Mg2+. Variability of the TRPM7 current density in human cardiomyocytes is related to the clinical history, being higher in atrial fibrillation and in ischemic cardiomyopathy.
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Project description:Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5-6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.
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Project description:Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3- transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.
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Project description:Acidic pH is an important feature of tumor microenvironment and a major determinant of tumor progression. We reported that cancer cells upregulate autophagy as a survival mechanism to acidic stress. Inhibition of autophagy by administration of chloroquine (CQ) in combination anticancer therapies is currently evaluated in clinical trials. We observed in 3 different human cancer cell lines cultured at acidic pH that autophagic flux is not blocked by CQ. This was consistent with a complete resistance to CQ toxicity in cells cultured in acidic conditions. Conversely, the autophagy-inhibiting activity of Lys-01, a novel CQ derivative, was still detectable at low pH. The lack of CQ activity was likely dependent on a dramatically reduced cellular uptake at acidic pH. Using cell lines stably adapted to chronic acidosis we could confirm that CQ lack of activity was merely caused by acidic pH. Moreover, unlike CQ, Lys-01 was able to kill low pH-adapted cell lines, although higher concentrations were required as compared with cells cultured at normal pH conditions. Notably, buffering medium pH in low pH-adapted cell lines reverted CQ resistance. In vivo analysis of tumors treated with CQ showed that accumulation of strong LC3 signals was observed only in normoxic areas but not in hypoxic/acidic regions. Our observations suggest that targeting autophagy in the tumor environment by CQ may be limited to well-perfused regions but not achieved in acidic regions, predicting possible limitations in efficacy of CQ in antitumor therapies.
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Project description:To investigate the impact of pH modulation on cell fate determination during mesendoderm differentiation using RNA-Seq data.
2024-08-08 | GSE211196 | GEO
Project description:The endothelium plays a central role in the regulation of vascular wall cellularity and tone by secreting an array of mediators of importance in intercellular communication. Nutrient deprivation of human endothelial cells (EC) evokes unconventional forms of secretion leading to the release of nanovesicles distinct from apoptotic bodies and bearing markers of multivesicular bodies (MVB). Nutrient deficiency is also a potent inducer of autophagy and vesicular transport pathways can be assisted by autophagy. Nutrient deficiency induced a significant and rapid increase in autophagic features, as imaged by electron microscopy and immunoblotting analysis of LC3-II/LC3-I ratios. Increased autophagic flux was confirmed by exposing serum-starved cells to bafilomycin A 1. Induction of autophagy was followed by indices of an apoptotic response, as assessed by microscopy and poly (ADP-ribose) polymerase cleavage in absence of cell membrane permeabilization indicative of necrosis. Pan-caspase inhibition with ZVAD-FMK did not prevent the development of autophagy but negatively impacted autophagic vacuole (AV) maturation. Adopting a multidimensional proteomics approach with validation by immunoblotting, we determined that nutrient-deprived EC released AV components (LC3I, LC3-II, ATG16L1 and LAMP2) whereas pan-caspase inhibition with ZVAD-FMK blocked AV release. Similarly, nutrient deprivation in aortic murine EC isolated from CASP3/caspase 3-deficient mice induced an autophagic response in absence of apoptosis and failed to prompt LC3 release. Collectively, the present results demonstrate the release of autophagic components by nutrient-deprived apoptotic human cells in absence of cell membrane permeabilization. These results also identify caspase-3 as a novel regulator of AV release.
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Project description:To investigate the genes that are modulated by acidic extracellular pH, we treated the mouse B16-BL6 cells with acidic medium and then analyzed them comprehensively by cDNA microarray.
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Project description:Epidemiological studies provide evidence that physical activity reduces the risk of cancer, particularly of breast cancer. However, little is known about the underlying molecular mechanisms as related to microRNAs. The goal of the herein presented study is to explore the involvement of miRNAs in beneficial effects exerted by physical activity in breast cancer prevention. Thirty subjects (mean age: 57.1 ± 14.7 years) underwent 45 minutes of treadmill walking under standardized conditions. The levels of extracellular miRNAs were evaluated in blood plasma before and after structured exercise by means of microarray analysis of 1,900 miRNAs identifying mostly modulated miRNAs. Structured exercise has been found to modulate the expression of 14 miRNAs involved in pathways relevant to cancer. The different expression of two miRNAs involved in breast cancer progression, i. e. up-regulation of miR-206 and down-regulation of anti-miR-30c, were the most striking effects induced by exercise. The biological effects of these miRNAs were investigated in MCF-7 human breast cancer cells. miR-206 transfection and anti-miR-30c silencing, inhibited cell growth and increased apoptosis of MCF-7 cells. Moreover, the combined use of the two miRNAs further enhanced apoptosis and induced growth arrest in the G1/S phase of cell cycle. Our results support that physical activity effectively change the expression of extracellular miRNAs. Specifically, miR-206 up-regulation and anti-miR-30c down-regulation act as suppressors in breast cancer cells. The evaluation of these miRNAs in blood can be used as non-invasive biomarkers for breast cancer prevention.
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