Tetrandrine regulates NAADP-mediated calcium signalling through a novel LIMP-2-dependent, sphingosine-mediated mechanism
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ABSTRACT:
PROVIDER: PRJNA876411 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21412069_1.fastq.gz | Fastqsanger.gz | |||
| SRR21412069_2.fastq.gz | Fastqsanger.gz | |||
| SRR21412070_1.fastq.gz | Fastqsanger.gz | |||
| SRR21412070_2.fastq.gz | Fastqsanger.gz | |||
| SRR21412071_1.fastq.gz | Fastqsanger.gz |
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Project description:Tetrandrine (Tet) is a highly potent inhibitor of Ebola virus replication by blocking NAADP-dependent calcium release through endolysosomal two-pore channels (TPCs) and is a moderately potent anti-tumor agent. Using a clickable, photoaffinity probe of Tet, we identified lysosomal integral membrane protein-2 (LIMP-2) as the genuine molecular target of Tet and a novel regulator for NAADP-dependent calcium release. Tet binds to the ectodomain of LIMP-2 and inhibits its cholesterol and sphingosine transport function, resulting in the alteration of lipid profiles and metabolic pathways in cells and development of acute hypercholesterolemia and hepatosteatosis in mice. Cells treated with Tet or depleted for LIMP-2 similarly inhibits NAADP-dependent calcium release, which can be restored by eliminating lysosomal cholesterol and sphingosines. Importantly, addition of sphingosine dose-dependently triggers lysosomal calcium release through TPCs, and restores NAADP-dependent calcium release in Tet-treated or LIMP-2-depleted cells. At higher concentrations, Tet induced apoptosis associated with unfolded protein response activation in a LIMP-2-independent manner. We identified Tet as the first known inhibitor of LIMP-2, discerned its pharmacological principle in regulating calcium signalling and cell death, and discovered a novel LIMP-2-regulated, sphingosine-dependent lysosomal calcium signalling pathway. Our findings have profound implications in the understanding of lysosomal calcium signalling mechanisms and development of Tet and LIMP-2 inhibitors into clinical therapeutics.
2023-03-04 | GSE212648 | GEO
Project description:Tetrandrine (Tet) is a highly potent inhibitor of Ebola virus replication by blocking NAADP-dependent calcium release through endolysosomal two-pore channels (TPCs) and is a moderately potent anti-tumor agent. Using a clickable, photoaffinity probe of Tet, we identified lysosomal integral membrane protein-2 (LIMP-2) as the genuine molecular target of Tet and a novel regulator for NAADP-dependent calcium release. Tet binds to the ectodomain of LIMP-2 and inhibits its cholesterol and sphingosine transport function, resulting in the alteration of lipid profiles and metabolic pathways in cells and development of acute hypercholesterolemia and hepatosteatosis in mice. Cells treated with Tet or depleted for LIMP-2 similarly inhibits NAADP-dependent calcium release, which can be restored by eliminating lysosomal cholesterol and sphingosines. Importantly, addition of sphingosine dose-dependently triggers lysosomal calcium release through TPCs, and restores NAADP-dependent calcium release in Tet-treated or LIMP-2-depleted cells. At higher concentrations, Tet induced apoptosis associated with unfolded protein response activation in a LIMP-2-independent manner. We identified Tet as the first known inhibitor of LIMP-2, discerned its pharmacological principle in regulating calcium signalling and cell death, and discovered a novel LIMP-2-regulated, sphingosine-dependent lysosomal calcium signalling pathway. Our findings have profound implications in the understanding of lysosomal calcium signalling mechanisms and development of Tet and LIMP-2 inhibitors into clinical therapeutics.
2025-06-02 | PXD064455 | Pride
Project description:Identifying the signals that regulate the survival, lineage allocation and specification of pancreas progenitors will help elucidate the embryonic origins of pancreas dysfunction and provide important cues for the efficient conversion of pluripotent stem cells into fully functional β cells. Several transcription factors regulating the conversion of the early pancreatic progenitors into terminally differentiated cells have been identified but extracellular signals regulating pancreas development are less well understood. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata and genomics we have found that sphingosine-1-phosphate signalling through plays a key role in this process. S1p signalling stabilizes the Hippo pathway effector YAP to promote progenitor survival, acinar and endocrine specification. Endocrine cell specification relies on Gai subunits revealing an unexpected dependence of lineage specification on selected intracellular signalling components. Independently of YAP stabilization, S1p signalling attenuates Notch levels, thus regulating lineage allocation. These findings identify S1p signalling as a key pathway coordinating cell survival, lineage allocation and specification during pancreas development.
2017-01-24 | GSE76482 | GEO
Project description:Background: Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. Objective: To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. Methods: ASM cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and western blotting. Receptor signalling and function was determined by mRNA knockdown and intracellular calcium mobilisation experiments. Results: S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilisation and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. Conclusion: S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma. Airway smooth muscle cells from 3 healthy donors were cultured and stimulated for 4 h with sphingosine-1-phosphate (100 nM) or medium control. Total RNA was extracted and analysed using Affymetrix Human Exon 1.0 ST arrays.
2015-06-18 | E-GEOD-58657 | biostudies-arrayexpress
Project description:Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% presented with abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established CRISPR-Cas9 SGPL1 knock-out and a lentiviral induced SGPL1 overexpression in nTERT immortalised keratinocytes and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine and ceramide while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO and GSEA revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signalling genesets. SGPL1_KO upregulated differentiation markers while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum, sphingolipid accumulation in the spinous layer and a breakdown of E-cadherin junctions. We hypothesise that SPLIS associated ichthyosis is a multi-faceted disease caused by sphingolipid imbalance and excessive S1P signalling.
2023-03-06 | GSE207499 | GEO
Project description:Identifying the signals that regulate the survival, lineage allocation and specification of pancreas progenitors will help elucidate the embryonic origins of pancreas dysfunction and provide important cues for the efficient conversion of pluripotent stem cells into fully functional β cells. Several transcription factors regulating the conversion of the early pancreatic progenitors into terminally differentiated cells have been identified but extracellular signals regulating pancreas development are less well understood. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata and genomics we have found that sphingosine-1-phosphate signalling through plays a key role in this process. S1p signalling stabilizes the Hippo pathway effector YAP to promote progenitor survival, acinar and endocrine specification. Endocrine cell specification relies on Gai subunits revealing an unexpected dependence of lineage specification on selected intracellular signalling components. Independently of YAP stabilization, S1p signalling attenuates Notch levels, thus regulating lineage allocation. These findings identify S1p signalling as a key pathway coordinating cell survival, lineage allocation and specification during pancreas development.
2017-01-24 | GSE76678 | GEO
Project description:Pattern recognition receptors (PRRs) at the plasma membrane promote plant immunity through the detection of conserved microbe-associated molecular patterns (MAMPs). In plants, the PRR for bacterial flagellin (flg22) is encoded by the receptor kinase FLS2. One of the earliest MAMP responses is the rapid and transient increase of cytosolic calcium (Ca2+) ions, which is required for many of the well-described downstream responses, e.g. generation of reactive oxygen species (ROS) and the transcriptional activation of defence-associated genes. Despite its relevance, the molecular components regulating the Ca2+ burst remain largely unknown. Here, we show that the plasma membrane P2B-type Ca2+ ATPase ACA8 forms a dynamic complex with the PRR FLS2. ACA8 and its closest homologue ACA10 are required for immunity against virulent bacteria. Mutant aca8 aca10 plants are reduced in the flg22-induced Ca2+ burst, show reduced ROS production and exhibit altered transcriptional reprogramming. In particular, flg22-induced gene expression is elevated downstream of signalling mitogen-activated protein (MAP) kinases, but reduced downstream of the calcium-dependent protein (CDP) kinase cascade. These results demonstrate that the fine regulation of Ca2+ fluxes in the cytosol is critical for the coordination of the downstream MAMP responses and provide for the first time a link between the FLS2 receptor complex and signalling kinases via the secondary messenger Ca2+. ACA8 also interacted with the BRI1 and CLV1 receptor kinases, which correlated with the developmental phenotypes of aca8 aca10 mutants suggesting a broader role for Ca2+ ATPases in receptor-mediated signalling. We used Affymetrix Arabidopsis Tiling 1.0R Array to compare global transcript levels in 7 days-old sterile grown seedlings. Steady-state mRNA levels in total RNA samples of 7 days old sterile seedlings
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Project description:Background: Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. Objective: To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. Methods: ASM cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and western blotting. Receptor signalling and function was determined by mRNA knockdown and intracellular calcium mobilisation experiments. Results: S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilisation and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. Conclusion: S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma.
2015-06-18 | GSE58657 | GEO
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