Mitochondrial TSPO Deficiency Triggers Retrograde Signaling in MA-10 Mouse Tumor Leydig Cells
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ABSTRACT:
PROVIDER: PRJNA690825 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13389430_1.fastq.gz | Fastqsanger.gz | |||
| SRR13389430_2.fastq.gz | Fastqsanger.gz | |||
| SRR13389431_1.fastq.gz | Fastqsanger.gz | |||
| SRR13389431_2.fastq.gz | Fastqsanger.gz | |||
| SRR13389432_1.fastq.gz | Fastqsanger.gz |
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Project description:The mitochondrial translocator protein (TSPO) has been shown to bind cholesterol with high affinity and is involved in mediating its availability for steroidogenesis. We recently reported that targeted Tspo gene deletion in MA-10 mouse tumor Leydig cells resulted in reduced cAMP-stimulated steroid formation and significant reduction in the mitochondrial membrane potential (ΔΨm) compared to control cells. We hypothesized that ΔΨm reduction in the absence of TSPO probably reflects the dysregulation and/or maintenance failure of some basic mitochondrial function(s). To explore the consequences of TSPO depletion via CRISPR-Cas9-mediated deletion (indel) mutation in MA-10 cells, we assessed the transcriptome changes in TSPO-mutant versus wild-type (Wt) cells using RNA-seq. Gene expression profiles were validated using real-time PCR. We report herein that there are significant changes in nuclear gene expression in Tspo mutant versus Wt cells. The identified transcriptome changes were mapped to several signaling pathways including the regulation of membrane potential, calcium signaling, extracellular matrix, and phagocytosis. This is a retrograde signaling pathway from the mitochondria to the nucleus and is probably the result of changes in expression of several transcription factors, including key members of the NF-κB pathway. In conclusion, TSPO regulates nuclear gene expression through intracellular signaling. This is the first evidence of a compensatory response to the loss of TSPO with transcriptome changes at the cellular level.
2021-01-09 | GSE164481 | GEO
Project description:The mitochondrial translocator protein (TSPO) has been shown to bind cholesterol with high affinity and is involved in mediating its availability for steroidogenesis. We recently reported that targeted Tspo gene deletion in MA-10 mouse tumor Leydig cells resulted in reduced cAMP-stimulated steroid formation and significant reduction in the mitochondrial membrane potential (??m) compared to control cells. We hypothesized that ??m reduction in the absence of TSPO probably reflects the dysregulation and/or maintenance failure of some basic mitochondrial function(s). To explore the consequences of TSPO depletion via CRISPR-Cas9-mediated deletion (indel) mutation in MA-10 cells, we assessed the transcriptome changes in TSPO-mutant versus wild-type (Wt) cells using RNA-seq. Gene expression profiles were validated using real-time PCR. We report herein that there are significant changes in nuclear gene expression in Tspo mutant versus Wt cells. The identified transcriptome changes were mapped to several signaling pathways including the regulation of membrane potential, calcium signaling, extracellular matrix, and phagocytosis. This is a retrograde signaling pathway from the mitochondria to the nucleus and is probably the result of changes in expression of several transcription factors, including key members of the NF-?B pathway. In conclusion, TSPO regulates nuclear gene expression through intracellular signaling. This is the first evidence of a compensatory response to the loss of TSPO with transcriptome changes at the cellular level.
| S-EPMC7795497 | biostudies-literature
Project description:Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) is a transcription factor recently identified in somatic cells of the male gonad. In other tissues, MEF2 is an essential regulator of organogenesis and cell differentiation. So far in the testis, MEF2 was found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MA-10 Leydig cells depleted in MEF2 and results were analyzed using the Partek and IPA softwares. Several genes were differentially expressed in MEF2-depleted Leydig cells and 15 were validated by qPCR. A large number of these genes are known to be involved in fertility, gonad morphology and steroidogenesis and include Pde8a, Por, Ahr, Bmal1, Cyp1a1, Cyp1b1, Map2k1, Tsc22d3, Nr0b2, Smad4, and Star, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2 binding sites within the first 2 kb upstream the transcription start site of the Por, Bmal1, and Nr0b2 promoters, which suggests a direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, siRNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1 and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction. MA-10 Leydig cells were treated with siRNA MEF2A/2D (siRNA MEF2) or scrambled siRNA as control (siRNA Ctrl) 48h before total RNA extraction.
2015-04-12 | E-GEOD-64128 | biostudies-arrayexpress
Project description:Testosterone production by Leydig cells is a tightly regulated process requiring synchronized expression of several steroidogenic genes by numerous transcription factors. Myocyte enhancer factor 2 (MEF2) is a transcription factor recently identified in somatic cells of the male gonad. In other tissues, MEF2 is an essential regulator of organogenesis and cell differentiation. So far in the testis, MEF2 was found to regulate Leydig cell steroidogenesis by controlling Nr4a1 and Star gene expression. To expand our understanding of the role of MEF2 in Leydig cells, we performed microarray analyses of MA-10 Leydig cells depleted in MEF2 and results were analyzed using the Partek and IPA softwares. Several genes were differentially expressed in MEF2-depleted Leydig cells and 15 were validated by qPCR. A large number of these genes are known to be involved in fertility, gonad morphology and steroidogenesis and include Pde8a, Por, Ahr, Bmal1, Cyp1a1, Cyp1b1, Map2k1, Tsc22d3, Nr0b2, Smad4, and Star, which were all downregulated in the absence of MEF2. In silico analyses revealed the presence of MEF2 binding sites within the first 2 kb upstream the transcription start site of the Por, Bmal1, and Nr0b2 promoters, which suggests a direct regulation by MEF2. Using transient transfections in MA-10 Leydig cells, siRNA knockdown, and a MEF2-Engrailed dominant negative, we found that MEF2 activates the Por, Bmal1 and Nr0b2 promoters and that this requires an intact MEF2 element. Our results identify novel target genes for MEF2 and define MEF2 as an important regulator of Leydig cell function and male reproduction.
2015-04-12 | GSE64128 | GEO
Project description:In males, Leydig cells are the main producers of testosterone and insulin-like 3, hormones which are both essential for sex differentiation and reproductive functions. Nuclear receptor chicken ovalbumin upstream promoter-transcription factors II (COUP-TFII) is expressed in the cells committed to give rise to the fully functional steroidogenic adult Leydig cells and has a major role in their function and differentiation. Up to date, only handful of COUP-TFII gene targets have been reported. A transcriptomic approach was used to identify additional genes affected by depletion of COUP-TFII in mouse MA-10 Leydig cell line.
2021-07-01 | GSE163283 | GEO
Project description:Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. MA-10 Leydig cells were treated with either DMSO (control), 10 uM forskolin or forskolin+Aicar (1 mM) for 1.5 h before total RNA extraction
2014-08-19 | E-GEOD-50118 | biostudies-arrayexpress
Project description:Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production. Steroid hormones regulate essential physiological processes and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by LH via its receptor leading to increased cAMP production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Leydig cell steroidogenesis then passively decreases following the rapid degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutive steroidogenic cell line R2C. Our data identify AMPK as an active repressor of steroid hormone biosynthesis in steroidogenic cells that is essential to preserve cellular energy and prevent excess steroid production.
2014-08-19 | GSE50118 | GEO
Project description:The purpose of this study was to identify differentially-expressed genes between WT MA-10 mouse tumor Leydig cells and MA-10 cells in which the steroidogenic acute regulatory protein (STAR) is knocked out.
2021-02-11 | GSE165392 | GEO
Project description:We hypothesized that the potential ability of the 18 kDa mitochondrial translocator protein (TSPO) to regulate gene expression may underlie its numerous reported functions, ranging from mitochondrial activity till mental disorders. Therefore, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 1 hr the TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. After 24 hrs primarily gene expression for enzymes and other proteins is changed. The associated gene products are known to affect various cellular functions: programmed cell death, cell cycle, viability, proliferation, migration, differentiation, development, tumorigenesis, and inflammatory / immune responses. Our microscopic studies showed intense TSPO mitochondrial labeling but no TSPO signal in the cell nuclei. Thus, it appears that the TSPO is part of the retrograde mitochondrial-nuclear signaling pathway for modulation of gene expression and thereby may exert its numerous effects on cell function, phenotype, and disease. U118MG cells (1.255 à 106) were seeded in Petri dishes (i.e. 28.5 à 103 cells / cm2) and allowed to proliferate for 3 days in full medium. Experiments for gene expression assayed with microarray typically consisted of three experimental groups and a vehicle control group (n = 3 for each group). Serum deprived medium was applied for 24 hrs, in the experimental groups ending with the inclusion a choice of various PK 11195 exposures i.e. either 1 hr, 3 hrs, or 24 hrs. Exposure to PK 11195 implies serum deprived medium with 1% alcohol (vehicle) and PK 11195 (25 µM final concentration) for the required time period. 25 µM of PK 11195 is the optimal concentration for these types of experiments with U118MG cells (Kugler et al., 2008). The cells were collected by trypsinization, washed by centrifugation in phosphate buffered saline (PBS) (400 à g, 5 min), and lysed [RLT lysis buffer provided with the RNeasy Mini Kit diluted with β-mercaptoethanol (1 : 100)], according to the manufacturer's instructions. Lysates were stored at -70oC.
2016-02-18 | E-GEOD-77998 | biostudies-arrayexpress
Project description:We hypothesized that the potential ability of the 18 kDa mitochondrial translocator protein (TSPO) to regulate gene expression may underlie its numerous reported functions, ranging from mitochondrial activity till mental disorders. Therefore, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 1 hr the TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. After 24 hrs primarily gene expression for enzymes and other proteins is changed. The associated gene products are known to affect various cellular functions: programmed cell death, cell cycle, viability, proliferation, migration, differentiation, development, tumorigenesis, and inflammatory / immune responses. Our microscopic studies showed intense TSPO mitochondrial labeling but no TSPO signal in the cell nuclei. Thus, it appears that the TSPO is part of the retrograde mitochondrial-nuclear signaling pathway for modulation of gene expression and thereby may exert its numerous effects on cell function, phenotype, and disease.
2016-02-18 | GSE77998 | GEO