Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various ...age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.
Abstract
Cellular senescence is implicated in a great number of diseases including cancer. Although alterations in mitochondrial metabolism were reported as senescence drivers, the underlying ...mechanisms remain elusive. We report the mechanism altering mitochondrial function and OXPHOS in stress-induced senescent fibroblasts. We demonstrate that TRPC3 protein, acting as a controller of mitochondrial Ca
2+
load via negative regulation of IP
3
receptor-mediated Ca
2+
release, is down regulated in senescence regardless of the type of senescence inducer. This remodelling promotes cytosolic/mitochondrial Ca
2+
oscillations and elevates mitochondrial Ca
2+
load, mitochondrial oxygen consumption rate and oxidative phosphorylation. Re-expression of TRPC3 in senescent cells diminishes mitochondrial Ca
2+
load and promotes escape from OIS-induced senescence. Cellular senescence evoked by TRPC3 downregulation in stromal cells displays a proinflammatory and tumour-promoting secretome that encourages cancer epithelial cell proliferation and tumour growth in vivo. Altogether, our results unravel the mechanism contributing to pro-tumour behaviour of senescent cells.
Autophagy is a highly conserved process involved in lymphocyte development and differentiation. Herein, we demonstrated for the first time that triggering of TRPV1 by the specific agonist CPS induces ...autophagy in mouse thymocytes. TRPV1-dependent autophagy required Ca(2+)(i) and ROS generation, resulting in AMPK activation. CPS specifically increased Atg4C mRNA expression and induced oxidation of Atg4C protein by ROS generation. TRPV1-triggered autophagy was Atg6/Beclin-1-dependent, as demonstrated by the use of Beclin-1(+/-) transgenic mice, and involved ROS- and AMPK-mediated up-regulation of Beclin-1 expression. Autophagy is activated as a prosurvival process, as its inhibition triggered apoptosis of thymocytes: this effect was accompanied by down-regulation of Atg4C, Bcl-X(L), and Irgm1 mRNA expression, decreased Bcl-X(L) and Beclin-1 protein levels, and caspase-3 activation, suggesting the existence of a molecular interplay between autophagic and apoptotic programs. TRPV1 activation by CPS altered the expression of CD4 and CD8α antigens, inducing the development of DP(dull). Interestingly, we found that CPS induces autophagy of DP(dull) cells, and inhibition of CPS-induced autophagy by the 3-MA autophagic inhibitor induces apoptosis of DP(dull) cells, suggesting the presence of an interplay between autophagic survival and apoptotic cell death. Overall, our findings suggest that DP(dull) cells constitute a distinct thymocyte subpopulation involved in the homeostatic control of cellularity and in the responses to chemical stress signals during thymocyte maturation, via regulating autophagy and apoptosis in a TRPV1-dependent manner.
Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully ...understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.
The transient receptor potential vanilloid type-2 (TRPV2), belonging to the transient receptor potential channel family, is a specialized ion channel expressed in human and other mammalian immune ...cells. This channel has been found to be expressed in CD34(+) hematopoietic stem cells, where its cytosolic Ca(2) (+) activity is crucial for stem/progenitor cell cycle progression, growth, and differentiation. In innate immune cells, TRPV2 is expressed in granulocytes, macrophages, and monocytes where it stimulates fMet-Leu-Phe migration, zymosan-, immunoglobulin G-, and complement-mediated phagocytosis, and lipopolysaccharide-induced tumor necrosis factor-alpha and interleukin-6 production. In mast cells, activation of TRPV2 allows intracellular Ca(2) (+) ions flux, thus stimulating protein kinase A-dependent degranulation. In addition, TRPV2 is highly expressed in CD56(+) natural killer cells. TRPV2 orchestrates Ca(2) (+) signal in T cell activation, proliferation, and effector functions. Moreover, messenger RNA for TRPV2 are expressed in CD4(+) and CD8(+) T lymphocytes. Finally, TRPV2 is expressed in CD19(+) B lymphocytes where it regulates Ca(2) (+) release during B cell development and activation. Overall, the specific expression of TRPV2 in immune cells suggests a role in immune-mediated diseases and offers new potential targets for immunomodulation.
Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by an acidic microenvironment, which contributes to therapeutic failure. So far there is a lack of knowledge with respect to the role of the ...acidic microenvironment in the invasive process. This work aimed to study the phenotypic and genetic response of PDAC cells to acidic stress along the different stages of selection. To this end, we subjected the cells to short- and long-term acidic pressure and recovery to pH
7.4. This treatment aimed at mimicking PDAC edges and consequent cancer cell escape from the tumor. The impact of acidosis was assessed for cell morphology, proliferation, adhesion, migration, invasion, and epithelial-mesenchymal transition (EMT) via functional in vitro assays and RNA sequencing. Our results indicate that short acidic treatment limits growth, adhesion, invasion, and viability of PDAC cells. As the acid treatment progresses, it selects cancer cells with enhanced migration and invasion abilities induced by EMT, potentiating their metastatic potential when re-exposed to pH
7.4. The RNA-seq analysis of PANC-1 cells exposed to short-term acidosis and pH
-selected recovered to pH
7.4 revealed distinct transcriptome rewiring. We describe an enrichment of genes relevant to proliferation, migration, EMT, and invasion in acid-selected cells. Our work clearly demonstrates that upon acidosis stress, PDAC cells acquire more invasive cell phenotypes by promoting EMT and thus paving the way for more aggressive cell phenotypes.
There is evidence that calcium (Ca(2+)) increases the proliferation of human advanced prostate cancer (PCa) cells but the ion channels involved are not fully understood. Here, we investigated the ...correlation between alpha(1D)-adrenergic receptor (alpha(1D)-AR) and the transient receptor potential vanilloid type 1 (TRPV1) expression levels in human PCa tissues and evaluated the ability of alpha(1D)-AR to cross-talk with TRPV1 in PCa cell lines.
The expression of alpha1D-AR and TRPV1 was examined in human PCa tissues by quantitative RT-PCR and in PCa cell lines (DU145, PC3 and LNCaP) by cytofluorimetry. Moreover, alpha(1D)-AR and TRPV1 colocalization was investigated by confocal microscopy in PCa cell lines and by fluorescence microscopy in benign prostate hyperplasia (BPH) and PCa tissues. Cell proliferation was assessed by BrdU incorporation. Alpha(1D)-AR/TRPV1 knockdown was obtained using siRNA transfection. Signalling pathways were evaluated by measurement of extracellular acidification rate, Ca(2+) flux, IP3 production, western blot and MTT assay.
The levels of the alpha(1D)-AR and TRPV1 mRNAs are increased in PCa compared to BPH specimens and a high correlation between alpha(1D)-AR and TRPV1 expression levels was found. Moreover, alpha(1D)-AR and TRPV1 are co-expressed in prostate cancer cell lines and specimens. Noradrenaline (NA) induced an alpha(1D)-AR- and TRPV1-dependent protons release and Ca(2+) flux in PC3 cell lines; NA by triggering the activation of phospholipase C (PLC), protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways stimulated PC3 cell proliferation, that was completely inhibited by clopenphendioxan (WS433) and capsazepine (CPZ) combination or by alpha(1D)-AR/TRPV1 double knockdown.
We demonstrate a cross-talk between alpha1D-AR and TRPV1, that is involved in the control of PC3 cell proliferation. These data strongly support for a putative novel pharmacological approach in the treatment of PCa by targeting both alpha1D-AR and TRPV1 channels.
To cite this article: Farfariello V, Amantini C, Nabissi M, Morelli MB, Aperio C, Caprodossi S, Carlucci A, Bianchi AM, Santoni G. IL‐22 mRNA in peripheral blood mononuclear cells from allergic ...rhinitic and asthmatic pediatric patients. Pediatr Allergy Immunol 2011;: 419–423.
T helper (TH)‐17 lymphocytes are characterized by the expression of many regulatory cytokines, including IL‐17A and IL‐22, but at present no clinical data are available on the expression of these cytokines in peripheral blood mononuclear cells (PBMC) from allergic asthmatic and rhinitic (AR) children. The aim of this study was to investigate a possible relationship between IL‐22 and IL‐17A mRNAs and clinical parameters in seroatopic, AR, and asthmatic children. The study, conduced during the pollen season, included 18 healthy and 18 allergic (n = 9 asthmatic and n = 9 rhinitic) children. Serum total and specific IgE, eosinophil count, and skin prick test were performed; in addition, IL‐22 and IL‐17A mRNA levels were detected in PBMC from healthy and allergic subjects by quantitative real‐time PCR. Despite the absence of the mRNA for the IL‐17A cytokine, IL‐22 expression was found in PBMC from asthmatic patients, with increased IL‐22 mRNA levels in patients with chronic severe respect to those with moderate asthma. A positive correlation between IL‐22 mRNA and serum total IgE levels was found in asthmatic children. In addition, higher IL‐22 and IL‐17A mRNA levels were detected in both AR and asymptomatic seroatopic children, compared to healthy individuals, and a correlation between IL‐22 and IL17A mRNA and serum total IgE levels was demonstrated. Moreover, the mRNA level of retinoic acid‐related orphan receptor C, the TH17 transcription factor, was found to be increased in AR but not in asthmatic patients. This study provides the first evidence that IL‐22 mRNA might be expressed in chronic severe asthmatic and AR children. The expression of IL‐22 and IL‐17A mRNAs in asymptomatic monosensitized seroatopic children suggests a role of these cytokines in the early events involved in the development of these allergic diseases.
Oxidative stress is an imbalance between the production of free radicals and antioxidant defense mechanisms, potentially leading to tissue damage. Oxidative stress has a key role in the development ...of cerebrovascular and/or neurodegenerative diseases. This phenomenon is mainly mediated by an enhanced superoxide production by the vascular endothelium with its consequent dysfunction. Thioctic, also known as alpha-lipoic acid (1,2-dithiolane-3-pentanoic acid), is a naturally occurring antioxidant that neutralizes free radicals in the fatty and watery regions of cells. Both the reduced and oxidized forms of the compound possess antioxidant ability. Thioctic acid has two optical isomers designated as (+)- and (-)-thioctic acid. Naturally occurring thioctic acid is the (+)-thioctic acid form, but the synthetic compound largely used in the market for stability reasons is a mixture of (+)- and (-)-thioctic acid. The present study was designed to compare the antioxidant activity of the two enantiomers versus the racemic form of thioctic acid on hydrogen peroxide-induced apoptosis in a rat pheochromocytoma PC12 cell line. Cell viability was evaluated by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and free oxygen radical species (ROS) production was assessed by flow cytometry. Antioxidant activity of the two enantiomers and the racemic form of thioctic acid was also evaluated in spontaneously hypertensive rats (SHR) used as an in vivo model of increased oxidative stress. A 3-h exposure of PC12 cells to hydrogen peroxide (H(2)O(2)) significantly decreased cell viability and increased levels of intracellular ROS production. Pre-treatment with racemic thioctic acid or (+)-enantiomer significantly inhibited H(2)O(2)-induced decrease in cell viability from the concentration of 50 μmol/L and 20 μmol/L, respectively. Racemic thioctic acid and (+)-salt decreased levels of intracellular ROS, which were unaffected by (-)-thioctic acid. In the brain of SHR, the occurrence of astrogliosis and neuronal damage, with a decreased expression of neurofilament 200 kDa were observed. Treatment of SHR for 30 days with (+)-thioctic acid reduced the size of astrocytes and increased the neurofilament immunoreaction. The above findings could contribute to clarify the role played by thioctic acid in central nervous system injury related to oxidative stress. The more pronounced effect of (+)-thioctic acid observed in this study may have practical therapeutic implications worthy of being investigated in further preclinical and clinical studies.
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