Cyclin B1 deficiency in renal tubular epithelial cells promotes kidney ageing and fibrosis in mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE224039 | GEO | 2024/01/01
REPOSITORIES: GEO
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Project description:Renal tubular epithelial cells (TECs) are critical mediators of renal fibrogenesis. Telomere dysfunction has been associated with renal injury and fibrosis. However, the role of telomere dysfunction specifically in TECs in the onset and progression of renal fibrosis remains poorly understood. To investigate the impact of telomere dysfunction on renal injury and fibrosis, we generated mice depleted for the shelterin component TRF1 specifically in TECs. Genetic ablation of Trf1 caused decline in renal function, tubular injury and tubulointerstitial fibrosis eight weeks after TRF1 depletion, concomitant with excessive accumulation of extracellular matrix (ECM), cell cycle arrest at G2/M phase, and telomeric damage. Trf1Δ/Δ mice activated regenerative repair mechanism, increasing the risk of of chronic kidney disease (CKD) progression following acute kidney injury (AKI), supporting proliferation-mediated telomere shortening in tubular cells. Mechanistically, Trf1 deletion upregulated Ras–Raf–Mek–Erk, PI3k/Akt/mTOR and p38 pathways. At humane endpoint, Trf1Δ/Δ mice displayed elevated urinary albumin-to-creatinine ratio (uACR), associated with augmented interstitial fibrosis and tubular atrophy eventually leading to CKD. In folic acid (FA)-induced nephropathy, depletion of Trf1 in TECs mitigated the fibrogenic phenotype of CKD. Collectively, our study underlies the important role of TECs in the development and progression of renal fibrosis and CKD associated to dysfunctional telomeres.
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Project description:Proximal tubular epithelial cells (TECs) demand high energy and rely on mitochondrial oxidative phosphorylation as a main energy source. However, this is disturbed in renal fibrosis. Acetylation is an important posttranslational modification for mitochondrial metabolism. The mitochondrial protein NAD-dependent deacetylase sirtuin 3 (SIRT3) regulates mitochondrial metabolic function. Therefore, we aimed to identify the changes in the acetylome in tubules from fibrotic kidneys and determine their association with mitochondria. We found that decreased SIRT3 expression was accompanied by increased acetylation in mitochondria that have separated from TECs during the early phase of renal fibrosis. SIRT3 knockout mice were susceptible to hyper-acetylated mitochondrial proteins and to severe renal fibrosis. The activation of SIRT3 by honokiol ameliorated acetylation and prevented renal fibrosis. Analysis of the acetylome in separated tubules using LC-MS/MS showed that most kidney proteins were hyper-acetylated after unilateral ureteral obstruction. The increased acetylated proteins with 26.76% were mitochondrial proteins which were mapped to a broad range of mitochondrial pathways including fatty acid β-oxidation, the TCA cycle and oxidative phosphorylation. Pyruvate dehydrogenase E1α (PDHE1α), which is the primary link between glycolysis and the TCA cycle, was hyper-acetylated at lysine 385 in TECs after TGF-β1 stimulation, and was regulated by SIRT3. Our findings showed that mitochondrial proteins involved in regulating energy metabolism were acetylated and targeted by SIRT3 in TECs. The deacetylation of PDHE1α by SIRT3 at lysine 385 plays a key role in metabolic reprogramming associated with renal fibrosis.
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Project description:The APC complex was purified from insect cells and an in vitro phosphorylation purified WT cyclin B1 or pocket mutant cyclin B1 was performed. Phosphorylation sites were detected by mass spectrometry (in vitro data). WT cyclin B1 and mutant cyclin B1 was transfected in cells and the APC complex immunoprecipitated with an anti-APC3 antibody and binders identified by MS
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Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors. We measured gene expression changes in U2OS cells after treatment with 0.2% hypoxia for 24 hours. The data were used in the exploration of hyopxia induced alterations in the G2 checkpoint.
2016-01-04 | E-GEOD-70530 | biostudies-arrayexpress
Project description:Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, in addition to DNA repair, genome stability is also controlled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can alter the DNA damage cell cycle checkpoints. Here, we show that hypoxia (24h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, flow cytometric bar-coding analysis of individual cells showed that the levels of several G2 checkpoint regulators were reduced in G2 phase cells after hypoxic exposure, in particular cyclin B1. These effects were accompanied by decreased Cyclin dependent kinase (CDK) activity in G2 phase cells after hypoxia. Furthermore, cells pre-exposed to hypoxia showed a longer G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (~0.03 % O2 20h and 0.2% O2 72h). These results demonstrate that the DNA damage G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.
2016-01-04 | GSE70530 | GEO
Project description:Cellular proliferation is highly regulated to ensure proper tissue homeostasis and to prevent diseases such as cancer. In actively proliferating cells, entry into S phase of the cell cycle and initiation of DNA replication is promoted by inactivation of the E3 ubiquitin ligase APC/C (anaphase promoting complex/cyclosome), though the critical S phase-promoting substrates have not been fully elucidated. The APC/C is also active in cells that have exited the cell cycle and entered an arrested state, but whether APC/C activity is essential to maintain arrest is unclear. We found that APC/C inactivation is sufficient to bypass cellular arrest induced by the CDK4/6 inhibitor Palbociclib. To identify potential APC/C substrates responsible for driving the escape from cell cycle arrest, we arrested cells using Palbociclib and then induced EMI1, inactivating the APC/C. Samples were collected at 0, 8, 16 and 20h after arrest and analyzed using proteomics. We found that inactivation of the APC/C promotes cell cycle re-entry by inducing RB phosphorylation and E2F-dependent gene transcription. Stabilization of both cyclin A and cyclin B contributes to arrest bypass, but only cyclin A accumulation is absolutely required. Direct expression of APC/C-resistant cyclin A, but not cyclin B, in arrested cells induces S phase entry analogous to APC/C inactivation. Cells bypassing arrest initiate DNA replication with severely reduced origin licensing, at least in part due to premature geminin accumulation. As a result, cells exhibit reduced rates of DNA synthesis, which leads to the accumulation of replication stress and long-term proliferation defects. Our findings suggest that CDK4/6 inhibition in cancers with reduced APC/C activity may be ineffective at promoting cytostatic cell cycle arrest but may nonetheless lead to elevated levels of replication stress that ultimately leads to a more durable cell cycle withdrawal.
2024-07-18 | PXD046783 | Pride
Project description:Cyclin B1 deficiency in renal tubular epithelial cells promotes kidney ageing and fibrosis in mice
| PRJNA929467 | ENA
Project description:The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is under the control of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The Checkpoint prevents the APC/C from recognizing Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live cell imaging, in vitro reconstitution, biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We further show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an ‘arginine-anchor’ in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the nucleosome binding motif on Cyclin B1 reduces its interaction with APC/C and delays its degradation, and cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes constitute a platform to promote the efficient interaction between Cyclin B1 and APC/C and ensure the timely degradation of Cyclin B1 and genomic stability.
2025-05-07 | PXD052677 | Pride
Project description:The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is under the control of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The Checkpoint prevents the APC/C from recognizing Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live cell imaging, in vitro reconstitution, biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We further show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an ‘arginine-anchor’ in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the nucleosome binding motif on Cyclin B1 reduces its interaction with APC/C and delays its degradation, and cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes constitute a platform to promote the efficient interaction between Cyclin B1 and APC/C and ensure the timely degradation of Cyclin B1 and genomic stability.
2025-05-06 | PXD046458 | Pride