Dataset Information

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos.

ABSTRACT: Chronic stimulation (24?h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP2) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP2. The resynthesis of PI requires the conversion of phosphatidic acid to CDP-diacylglycerol catalysed by CDP-diacylglycerol synthase (CDS) enzymes. To examine whether the resynthesis of PI is regulated by vasopressin stimulation, we focussed on the CDS enzymes. Three CDS enzymes are present in mammalian cells: CDS1 and CDS2 are integral membrane proteins localised at the endoplasmic reticulum and TAMM41 is a peripheral protein localised in the mitochondria. Vasopressin selectively stimulates an increase CDS1 mRNA that is dependent on protein kinase C, and can be inhibited by the AP-1 inhibitor, T-5224. Vasopressin also stimulates an increase in cFos protein which is inhibited by a protein kinase C inhibitor. We conclude that vasopressin stimulates CDS1 mRNA through phospholipase C, protein kinase C and cFos and provides a potential mechanism for maintenance of phosphatidylinositol levels during long-term phospholipase C signalling.

SUBMITTER: Blunsom NJ

PROVIDER: S-EPMC6495107 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos.

Blunsom Nicholas J NJ Gomez-Espinosa Evelyn E Ashlin Tim G TG Cockcroft Shamshad S

Biochimica et biophysica acta. Molecular and cell biology of lipids 20190309 7

Chronic stimulation (24 h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP<sub>2</sub>) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP<sub>2</sub>. The resynthesis of PI requires the conv ...[more]

PMID: 30862571

Similar Datasets

Project description:Cytidine diphosphate diacylglycerol (CDP-DAG) is a key intermediate in the synthesis of phosphatidylinositol (PI) and cardiolipin (CL). Both PI and CL have highly specialized roles in cells. PI can be phosphorylated and these phosphorylated derivatives play major roles in signal transduction, membrane traffic, and maintenance of the actin cytoskeletal network. CL is the signature lipid of mitochondria and has a plethora of functions including maintenance of cristae morphology, mitochondrial fission, and fusion and for electron transport chain super complex formation. Both lipids are synthesized in different organelles although they share the common intermediate, CDP-DAG. CDP-DAG is synthesized from phosphatidic acid (PA) and CTP by enzymes that display CDP-DAG synthase activities. Two families of enzymes, CDS and TAMM41, which bear no sequence or structural relationship, have now been identified. TAMM41 is a peripheral membrane protein localized in the inner mitochondrial membrane required for CL synthesis. CDS enzymes are ancient integral membrane proteins found in all three domains of life. In mammals, they provide CDP-DAG for PI synthesis and for phosphatidylglycerol (PG) and CL synthesis in prokaryotes. CDS enzymes are critical for maintaining phosphoinositide levels during phospholipase C (PLC) signaling. Hydrolysis of PI (4,5) bisphosphate by PLC requires the resynthesis of PI and CDS enzymes catalyze the rate-limiting step in the process. In mammals, the protein products of two CDS genes (CDS1 and CDS2) localize to the ER and it is suggested that CDS2 is the major CDS for this process. Expression of CDS enzymes are regulated by transcription factors and CDS enzymes may also contribute to CL synthesis in mitochondria. Studies of CDS enzymes in protozoa reveal spatial segregation of CDS enzymes from the rest of the machinery required for both PI and CL synthesis identifying a key gap in our understanding of how CDP-DAG can cross the different membrane compartments in protozoa and in mammals.

Project description:Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations: ATG: autophagy related; ATG7: autophagy related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OXPHOS: oxidative phosphorylation; PepA: pepstatin A; PLA: proximity ligation assay; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; RAB9A: RAB9A, member RAS oncogene family; ROS: reactive oxygen species; SIM: structured illumination microscopy; siRNA: short interfering RNA; SYNJ2BP: synaptojanin 2 binding protein; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.

Dataset Information

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos.

Publications

Sustained phospholipase C stimulation of H9c2 cardiomyoblasts by vasopressin induces an increase in CDP-diacylglycerol synthase 1 (CDS1) through protein kinase C and cFos.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets