H(2)O(2)-induced Ca(2+) overload in NRVM involves ERK1/2 MAP kinases: role for an NHE-1-dependent pathway

Generation of reactive oxygen species (ROS) and intracellular Ca(2+) overload are key mechanisms involved in ischemia-reperfusion (I/R)-induced myocardial injury. The relationship between I/R injury and Ca(2+) overload has not been fully characterized. The increase in Na(+)/H(+) exchanger (NHE-1) activity observed during I/R injury is an attractive candidate to link increased ROS production with Ca(2+) overload. We have shown that low doses of H(2)O(2) increase NHE-1 activity in an extracellular signal-regulated kinase (ERK)-dependent manner. In this study, we examined the effect of low doses of H(2)O(2) on intracellular Ca(2+) in fura 2-loaded, spontaneously contracting neonatal rat ventricular myocytes. H(2)O(2) induced a time- and concentration-dependent increase in diastolic intracellular Ca(2+) concentration that was blocked by inhibition of ERK1/2 activation with 5 microM U-0126 (88%) or inhibition of NHE-1 with 5 microM HOE-642 (50%). Increased NHE activity was associated with phosphorylation of the NHE-1 carboxyl tail that was blocked by U-0126. These results suggest that H(2)O(2) induced Ca(2+) overload is partially mediated by NHE-1 activation secondary to phosphorylation of NHE-1 by the ERK1/2 MAP kinase pathway.     

Role of calcium in pancreatic islet cell death by IFN-gamma/TNF-alpha

We studied the intracellular events associated with pancreatic beta cell apoptosis by IFN-gamma/TNF-alpha synergism. IFN-gamma/TNF-alpha treatment of MIN6N8 insulinoma cells increased the amplitude of high voltage-activated Ca(2+) currents, while treatment with IFN-gamma or TNF-alpha alone did not. Cytosolic Ca(2+) concentration ([Ca(2+)](c)) was also increased by IFN-gamma/TNF-alpha treatment. Blockade of L-type Ca(2+) channel by nifedipine abrogated death of insulinoma cells by IFN-gamma/TNF-alpha. Diazoxide that attenuates voltage-activated Ca(2+) currents inhibited MIN6N8 cell death by IFN-gamma/TNF-alpha, while glibenclamide that accentuates voltage-activated Ca(2+) currents augmented insulinoma cell death. A protein kinase C inhibitor attenuated MIN6N8 cell death and the increase in [Ca(2+)](c) by IFN-gamma/TNF-alpha. Following the increase in [Ca(2+)](c), calpain was activated, and calpain inhibitors decreased insulinoma cell death by IFN-gamma/TNF-alpha. As a downstream of calpain, calcineurin was activated and the inhibition of calcineurin activation by FK506 diminished insulinoma cell death by IFN-gamma/TNF-alpha. BAD phosphorylation was decreased by IFN-gamma/TNF-alpha because of the increased calcineurin activity, which was reversed by FK506. IFN-gamma/TNF-alpha induced cytochrome c translocation from mitochondria to cytoplasm and activation of caspase-9. Effector caspases such as caspase-3 or -7 were also activated by IFN-gamma/TNF-alpha treatment. These results indicate that IFN-gamma/TNF-alpha synergism induces pancreatic beta cell apoptosis by Ca(2+) channel activation followed by downstream intracellular events such as mitochondrial events and caspase activation and also suggest the therapeutic potential of Ca(2+) modulation in type 1 diabetes.     

The cellular resistance against oxidative stress (H2O2) is independent of neutral trehalase (Ntc1p) activity in Candida albicans

The protective role of trehalose against oxidative stress caused by hydrogen peroxide in Candida albicans has been investigated in the homozygous mutant ntc1Delta/ntc1Delta, disrupted in the NTC1 gene, which encodes the neutral (cytosolic) trehalase (Ntc1p). After a severe oxidative exposure (50 mM H(2)O(2)), both parental (CAI-4) and ntc1Delta/ntc1Delta exponential-phase cells stored large amounts of intracellular trehalose. In turn, the degree of cell survival was roughly equivalent in both strains, although slightly higher in ntc1Delta/ntc1Delta cultures. The mechanism of 'adaptive tolerance' was functional in the two strains. Thus, a gently oxidative pretreatment (5 mM H(2)O(2)) increased the recovery of cellular viability when it was followed by a severe challenge (50 mM H(2)O(2)); this phenomenon was accompanied by a significant elevation of the endogenous trehalose content. Oxidative stress also induced specific activation of the antioxidant enzymes catalase and glutathione reductase upon gentle oxidative treatment (5 mM H(2)O(2)), whereas superoxide dismutase activity was only activated upon prolonged exposure. Taken together, these results strongly suggest that in C. albicans neutral trehalase activity does not play an essential role in the protective response against oxidative stress. They also suggest that a diminished Ntc1p activity might favour the growth of C. albicans cells subjected to a strong oxidative exposure.     

Adaptation to environmental pH: integrating the Rim101 and calcineurin signal transduction pathways

The ability to appropriately respond to environmental conditions is critical for the survival of simple microbes and for development of complex multicellular organisms. Sensing and responding to a given environmental condition requires the integration of numerous signals through one or more signal transduction pathways. This leads to changes in gene expression, and potentially post-translational modifications, that favour growth in the given environment. In the fungus Candida albicans, an important opportunistic pathogen, environmental pH has profound effects on morphology and proper adaptation to extracellular pH is critical for pathogenesis. Here, we demonstrate that the Rim101/PacC pH-sensing pathway acts in parallel to Crz1, via calcineurin, to adapt to alkaline pH. We also show that the Rim101 pathway acts in parallel to Crz2, independent of calcineurin, to adapt to high lithium concentrations and to repress filamentation at acidic pH. Our studies also revealed a novel requirement for Crz1, Crz2 and calcineurin for growth at acidic pH. From these studies, we propose that the Crz1 homologue Crz2 is calcineurin-independent, but like Crz1, acts in parallel to promote specific Rim101-dependent processes. These results establish and begin to dissect the complex interactions between important signal transduction pathways in C. albicans, which are critical for virulence.     

Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections.     

Alkaline stress triggers an immediate calcium fluctuation in Candida albicans mediated by Rim101p and Crz1p transcription factors

In the human fungal pathogen Candida albicans, environmental pH has profound effects on morphogenesis and response to extracellular pH is clearly relevant to the pathogenicity of this fungus. Yeast cells have evolved a complex network of mechanisms in response to the environmental pH and they often require the integration of the Rim101 and calcineurin/Crz1 signaling pathways. Ca(2+) burst is a common cellular response when cells are exposed to environmental stresses; therefore, in this study, we asked whether it follows the same case under alkaline stress and whether this calcium change is regulated by Rim101p and Crz1p. We confirmed the calcium influx was activated by KOH stimuli using a flow cytometry-based method, but it was obviously abolished in cells lacking MID1 or CCH1. We also found that alkaline pH-induced activation of the PHO89 promoter was blocked without the same gene; moreover, the response was Crz1p- and Rim101p-dependent. Finally, we investigated the regulation role of Rim101p and Crz1p in calcium influx through MID1, CCH1 and YVC1 genes, which were all downregulated in rim101Δ/Δ and crz1Δ/Δ mutants. The important role of calcium influx in the alkaline stress response and its regulation suggested a potential integration effect of Rim101 and Crz1 signaling pathways in C. albicans.     

Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H(2)O(2)-activated calcium signaling pathway in mouse hippocampal neurons

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H(2)S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O(2), 5% CO(2) and 93% N(2) at 37°C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H(2)S) for 30min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca(2+)](i)), and pretreatment with NaHS prevented the increase in [Ca(2+)](i). Additionally, polyethylene glycol (PEG)-catalase (a H(2)O(2) scavenger) but not PEG-SOD (an O(2)(-) scavenger) conferred an inhibitory effect similar to H(2)S on the hypoxia-induced increase in [Ca(2+)](i). Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP(3)) receptor blocker xestospongin C. Taken together, these findings suggest that H(2)S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H(2)O(2))-activated Ca(2+) signaling pathway in mouse hippocampal neurons.     

Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

Hsp90 Is Involved in Apoptosis of Candida albicans by Regulating the Calcineurin-Caspase Apoptotic Pathway

Candida albicans is the most common human fungal pathogen. Recent evidence has revealed the occurrence of apoptosis in C. albicans that is inducible by environmental stresses such as hydrogen peroxide, acetic acid, and amphotericin B. Apoptosis is regulated by the calcineurin-caspase pathway in C. albicans, and calcineurin is under the control of Hsp90 in echinocandin resistance. However, the role of Hsp90 in apoptosis of C. albicans remains unclear. In this study, we investigated the role of Hsp90 in apoptosis of C. albicans by using an Hsp90-compromised strain tetO-HSP90/hsp90 and found that upon apoptotic stimuli, including hydrogen peroxide, acetic acid or amphotericin B treatment, less apoptosis occurred, less ROS was produced, and more cells survived in the Hsp90-compromised strain compared with the Hsp90/Hsp90 wild-type strain. In addition, Hsp90-compromised cells were defective in up-regulating caspase-encoding gene CaMCA1 expression and activating caspase activity upon the apoptotic stimuli. Investigations on the relationship between Hsp90 and calcineurin revealed that activation of calcineurin could up-regulate apoptosis but could not further down-regulate apoptosis in Hsp90-compromised cells, indicating that calcineurin was downstream of Hsp90. Hsp90 inhibitor geldanamycin (GdA) could further decrease the apoptosis in calcineurin-pathway-defect strains, indicating that compromising Hsp90 function had a stronger effect than compromising calcineurin function on apoptosis. Collectively, this study demonstrated that compromised Hsp90 reduced apoptosis in C. albicans, partially through downregulating the calcineurin-caspase pathway.