High-throughput Exploration of the Network Dependent on AKT1 in Mouse Ovarian Granulosa Cells
2019; Elsevier BV; Volume: 18; Issue: 7 Linguagem: Inglês
10.1074/mcp.ra119.0014613
ISSN1535-9484
AutoresMaëva Elzaïat, Laetitia Herman, Bérangère Legois, Thibaut Léger, Anne‐Laure Todeschini, Reiner A. Veitia,
Tópico(s)Reproductive Biology and Fertility
ResumoThe PI3K/AKT signaling pathway is known to regulate a broad range of cellular processes, and it is often altered in several types of cancers. Recently, somatic AKT1 mutations leading to a strong activation of this kinase have been reported in juvenile granulosa cell tumors. However, the molecular role of AKT1 in the supporting cell lineage of the ovary is still poorly understood. To get insights into its function in such cells, we depleted Akt1 in murine primary granulosa cells and assessed the molecular consequences at both the transcript and protein levels. We were able to corroborate the involvement of AKT1 in the regulation of metabolism, apoptosis, cell cycle, or cytoskeleton dynamics in this ovarian cell type. Consistently, we showed in established granulosa cells that depletion of Akt1 provoked altered directional persistent migration and increased its velocity. This study also allowed us to put forward new direct and indirect targets of the kinase. Indeed, a series of proteins involved in intracellular transport and mitochondrial physiology were significantly affected by Akt1 depletion. Using in silico analyses, we also propose a set of kinases and transcription factors that can mediate the action of AKT1 on the deregulated transcripts and proteins. Taken altogether, our results provide a resource of direct and indirect AKT1 targets in granulosa cells and may help understand its roles in this ovarian cell type. The PI3K/AKT signaling pathway is known to regulate a broad range of cellular processes, and it is often altered in several types of cancers. Recently, somatic AKT1 mutations leading to a strong activation of this kinase have been reported in juvenile granulosa cell tumors. However, the molecular role of AKT1 in the supporting cell lineage of the ovary is still poorly understood. To get insights into its function in such cells, we depleted Akt1 in murine primary granulosa cells and assessed the molecular consequences at both the transcript and protein levels. We were able to corroborate the involvement of AKT1 in the regulation of metabolism, apoptosis, cell cycle, or cytoskeleton dynamics in this ovarian cell type. Consistently, we showed in established granulosa cells that depletion of Akt1 provoked altered directional persistent migration and increased its velocity. This study also allowed us to put forward new direct and indirect targets of the kinase. Indeed, a series of proteins involved in intracellular transport and mitochondrial physiology were significantly affected by Akt1 depletion. Using in silico analyses, we also propose a set of kinases and transcription factors that can mediate the action of AKT1 on the deregulated transcripts and proteins. Taken altogether, our results provide a resource of direct and indirect AKT1 targets in granulosa cells and may help understand its roles in this ovarian cell type. The AKT/PKB 1The abbreviations used are: AKTAKT serine/threonine protein kinase4E-BP1eukaryotic translation initiation factor 4E binding protein 1ASCC1activating signal cointegrator 1 complex subunit 1BRD8bromodomain containing 8CALM1calmodulin 1Capn1calpain 1CBX1chromobox 1Cdhr1cadherin-related family member 1CEBPBCCAAT enhancer binding protein betaCREB1cAMP-responsive element-binding protein 1Crip1cysteine-rich protein 1CTBP1C-terminal binding protein 1DDX1DEAD-box helicase 1DDX3XDEAD-box helicase 3 X-linkedDDX54DEAD-box helicase 54DEProtsproteins differentially expressedDHX36DEAH-box helicase 36DPhosphoproteins differentially phosphorylatedEcm1extracellular matrix protein 1EEF1Deukaryotic translation elongation factor 1 deltaEIF2S2eukaryotic translation initiation factor 2 subunit betaELP4elongator acetyltransferase complex subunit 4EMX2empty spiracles homeobox 2ENY2transcription and export complex 2 subunitEri3ERI1 exoribonuclease family member 3FOXP1forkhead box P1GATA4GATA-binding protein 4GOT2glutamic-oxaloacetic transaminase 2GTF2E2general transcription factor IIE subunit 2HMGB2high mobility group box 2HNRNPKheterogeneous nuclear ribonucleoprotein KHSF1heat shock transcription factor 1KDknock(ed)-downLog2FClog2fold changeMECP2methyl-CpG-binding protein 2MED23mediator complex subunit 23MED4mediator complex subunit 4MSmass spectrometrymTORC1mammalian target of rapamycin complex 1mTORC2mammalian target of rapamycin complex 2Mvdmevalonate diphosphate decarboxylaseNFE2L2nuclear factor, erythroid 2 like 2NFκBnuclear factor kappa B subunitOvgp1oviductal glycoprotein 1Pdlim7PDZ and LIM domain 7PELP1proline-, glutamate-, and leucine-rich protein 1pGCsmouse primary granulosa cellsPHDpleckstrin homology domainPHF6PHD finger protein 6PI3Kphosphoinositide3-kinasePIP2/PIP3phosphatidylinositol-di/trisphosphatePIRpirinPKBprotein kinase BPPIprotein-protein interactionsPpibpeptidylprolyl isomerase BPTMposttranslational modificationsPTMAprothymosin alphaRBPMSRNA-binding protein, MRNA-processing factorRNAiRNA interferenceRNA-seqRNA-sequencingRNF2ring finger protein 2RTN4reticulon 4S100a16S100 calcium-binding protein A16S6K1/2ribosomal protein S6 kinase B1/B2SFR1SWI5 dependent homologous recombination repair protein 1Smarca2SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2SRRM2serine/arginine repetitive matrix 2TCOF1treacle ribosome biogenesis factor 1TFAMtranscription factor A, mitochondrialTFAP2Ctranscription factor AP-2 gammaTFstranscription factorsTLE4transducing-like enhancer of split 4Tmem109transmembrane protein 109TSC1tuberous sclerosis 1 proteinTSC2tuberous sclerosis 2 proteinWT1Wilms tumor 1ZBED6zinc finger BED-type containing 6. 1The abbreviations used are: AKTAKT serine/threonine protein kinase4E-BP1eukaryotic translation initiation factor 4E binding protein 1ASCC1activating signal cointegrator 1 complex subunit 1BRD8bromodomain containing 8CALM1calmodulin 1Capn1calpain 1CBX1chromobox 1Cdhr1cadherin-related family member 1CEBPBCCAAT enhancer binding protein betaCREB1cAMP-responsive element-binding protein 1Crip1cysteine-rich protein 1CTBP1C-terminal binding protein 1DDX1DEAD-box helicase 1DDX3XDEAD-box helicase 3 X-linkedDDX54DEAD-box helicase 54DEProtsproteins differentially expressedDHX36DEAH-box helicase 36DPhosphoproteins differentially phosphorylatedEcm1extracellular matrix protein 1EEF1Deukaryotic translation elongation factor 1 deltaEIF2S2eukaryotic translation initiation factor 2 subunit betaELP4elongator acetyltransferase complex subunit 4EMX2empty spiracles homeobox 2ENY2transcription and export complex 2 subunitEri3ERI1 exoribonuclease family member 3FOXP1forkhead box P1GATA4GATA-binding protein 4GOT2glutamic-oxaloacetic transaminase 2GTF2E2general transcription factor IIE subunit 2HMGB2high mobility group box 2HNRNPKheterogeneous nuclear ribonucleoprotein KHSF1heat shock transcription factor 1KDknock(ed)-downLog2FClog2fold changeMECP2methyl-CpG-binding protein 2MED23mediator complex subunit 23MED4mediator complex subunit 4MSmass spectrometrymTORC1mammalian target of rapamycin complex 1mTORC2mammalian target of rapamycin complex 2Mvdmevalonate diphosphate decarboxylaseNFE2L2nuclear factor, erythroid 2 like 2NFκBnuclear factor kappa B subunitOvgp1oviductal glycoprotein 1Pdlim7PDZ and LIM domain 7PELP1proline-, glutamate-, and leucine-rich protein 1pGCsmouse primary granulosa cellsPHDpleckstrin homology domainPHF6PHD finger protein 6PI3Kphosphoinositide3-kinasePIP2/PIP3phosphatidylinositol-di/trisphosphatePIRpirinPKBprotein kinase BPPIprotein-protein interactionsPpibpeptidylprolyl isomerase BPTMposttranslational modificationsPTMAprothymosin alphaRBPMSRNA-binding protein, MRNA-processing factorRNAiRNA interferenceRNA-seqRNA-sequencingRNF2ring finger protein 2RTN4reticulon 4S100a16S100 calcium-binding protein A16S6K1/2ribosomal protein S6 kinase B1/B2SFR1SWI5 dependent homologous recombination repair protein 1Smarca2SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2SRRM2serine/arginine repetitive matrix 2TCOF1treacle ribosome biogenesis factor 1TFAMtranscription factor A, mitochondrialTFAP2Ctranscription factor AP-2 gammaTFstranscription factorsTLE4transducing-like enhancer of split 4Tmem109transmembrane protein 109TSC1tuberous sclerosis 1 proteinTSC2tuberous sclerosis 2 proteinWT1Wilms tumor 1ZBED6zinc finger BED-type containing 6. is the major downstream effector of the PI3K signaling pathway known to regulate a broad range of cellular functions such as: survival, proliferation, growth, metabolism, and migration (reviewed in (1Manning B.D. 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AKT serine/threonine protein kinase eukaryotic translation initiation factor 4E binding protein 1 activating signal cointegrator 1 complex subunit 1 bromodomain containing 8 calmodulin 1 calpain 1 chromobox 1 cadherin-related family member 1 CCAAT enhancer binding protein beta cAMP-responsive element-binding protein 1 cysteine-rich protein 1 C-terminal binding protein 1 DEAD-box helicase 1 DEAD-box helicase 3 X-linked DEAD-box helicase 54 proteins differentially expressed DEAH-box helicase 36 proteins differentially phosphorylated extracellular matrix protein 1 eukaryotic translation elongation factor 1 delta eukaryotic translation initiation factor 2 subunit beta elongator acetyltransferase complex subunit 4 empty spiracles homeobox 2 transcription and export complex 2 subunit ERI1 exoribonuclease family member 3 forkhead box P1 GATA-binding protein 4 glutamic-oxaloacetic transaminase 2 general transcription factor IIE subunit 2 high mobility group box 2 heterogeneous nuclear ribonucleoprotein K heat shock transcription factor 1 knock(ed)-down log2fold change methyl-CpG-binding protein 2 mediator complex subunit 23 mediator complex subunit 4 mass spectrometry mammalian target of rapamycin complex 1 mammalian target of rapamycin complex 2 mevalonate diphosphate decarboxylase nuclear factor, erythroid 2 like 2 nuclear factor kappa B subunit oviductal glycoprotein 1 PDZ and LIM domain 7 proline-, glutamate-, and leucine-rich protein 1 mouse primary granulosa cells pleckstrin homology domain PHD finger protein 6 phosphoinositide3-kinase phosphatidylinositol-di/trisphosphate pirin protein kinase B protein-protein interactions peptidylprolyl isomerase B posttranslational modifications prothymosin alpha RNA-binding protein, MRNA-processing factor RNA interference RNA-sequencing ring finger protein 2 reticulon 4 S100 calcium-binding protein A16 ribosomal protein S6 kinase B1/B2 SWI5 dependent homologous recombination repair protein 1 SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2 serine/arginine repetitive matrix 2 treacle ribosome biogenesis factor 1 transcription factor A, mitochondrial transcription factor AP-2 gamma transcription factors transducing-like enhancer of split 4 transmembrane protein 109 tuberous sclerosis 1 protein tuberous sclerosis 2 protein Wilms tumor 1 zinc finger BED-type containing 6. AKT serine/threonine protein kinase eukaryotic translation initiation factor 4E binding protein 1 activating signal cointegrator 1 complex subunit 1 bromodomain containing 8 calmodulin 1 calpain 1 chromobox 1 cadherin-related family member 1 CCAAT enhancer binding protein beta cAMP-responsive element-binding protein 1 cysteine-rich protein 1 C-terminal binding protein 1 DEAD-box helicase 1 DEAD-box helicase 3 X-linked DEAD-box helicase 54 proteins differentially expressed DEAH-box helicase 36 proteins differentially phosphorylated extracellular matrix protein 1 eukaryotic translation elongation factor 1 delta eukaryotic translation initiation factor 2 subunit beta elongator acetyltransferase complex subunit 4 empty spiracles homeobox 2 transcription and export complex 2 subunit ERI1 exoribonuclease family member 3 forkhead box P1 GATA-binding protein 4 glutamic-oxaloacetic transaminase 2 general transcription factor IIE subunit 2 high mobility group box 2 heterogeneous nuclear ribonucleoprotein K heat shock transcription factor 1 knock(ed)-down log2fold change methyl-CpG-binding protein 2 mediator complex subunit 23 mediator complex subunit 4 mass spectrometry mammalian target of rapamycin complex 1 mammalian target of rapamycin complex 2 mevalonate diphosphate decarboxylase nuclear factor, erythroid 2 like 2 nuclear factor kappa B subunit oviductal glycoprotein 1 PDZ and LIM domain 7 proline-, glutamate-, and leucine-rich protein 1 mouse primary granulosa cells pleckstrin homology domain PHD finger protein 6 phosphoinositide3-kinase phosphatidylinositol-di/trisphosphate pirin protein kinase B protein-protein interactions peptidylprolyl isomerase B posttranslational modifications prothymosin alpha RNA-binding protein, MRNA-processing factor RNA interference RNA-sequencing ring finger protein 2 reticulon 4 S100 calcium-binding protein A16 ribosomal protein S6 kinase B1/B2 SWI5 dependent homologous recombination repair protein 1 SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2 serine/arginine repetitive matrix 2 treacle ribosome biogenesis factor 1 transcription factor A, mitochondrial transcription factor AP-2 gamma transcription factors transducing-like enhancer of split 4 transmembrane protein 109 tuberous sclerosis 1 protein tuberous sclerosis 2 protein Wilms tumor 1 zinc finger BED-type containing 6. A number of AKT downstream target substrates have been described (1Manning B.D. Cantley L.C. AKT/PKB signaling: Navigating downstream.Cell. 2007; 129: 1261-1274Abstract Full Text Full Text PDF PubMed Scopus (4692) Google Scholar, 2Cecconi S. Mauro A. Cellini V. Patacchiola F. The role of Akt signalling in the mammalian ovary.Int. J. Dev. Biol. 2012; 56: 809-817Crossref PubMed Scopus (75) Google Scholar, 7Ersahin T. Tuncbag N. Cetin-Atalay R. The PI3K/AKT/mTOR interactive pathway.Mol. Biosyst. 2015; 11: 1946-1954Crossref PubMed Google Scholar). For example, AKT promotes cell survival via the phosphorylation of proapoptotic factors like BCL2 associated agonist of cell death (8Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. 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As a consequence of its central position in the physiology of the cell, AKT dysregulation is associated with several human diseases, including cancer. Indeed, many human cancers show elevated activity of AKT (reviewed in (26Altomare D.A. Testa J.R. Perturbations of the AKT signaling pathway in human cancer.Oncogene. 2005; 24: 7455-7464Crossref PubMed Scopus (1105) Google Scholar)). This hyperactivity can result from mutations in genes encoding upstream regulators of AKT, like PI3K (27Samuels Y. Wang Z. Bardelli A. Silliman N. Ptak J. Szabo S. Yan H. Gazdar A. Powell S.M. Riggins G.J. Willson J.K.V. Markowitz S. Kinzler K.W. Vogelstein B. Velculescu V.E. High frequency of mutations of the PIK3CA gene in human cancers.Science. 2004; 304: 554Crossref PubMed Scopus (2915) Google Scholar) or phosphatase and TENsin homolog (28Hyun T. Yam A. Pece S. Xie X. Zhang J. Miki T. Gutkind J.S. Li W. Loss of PTEN expression leading to high Akt activation in human multiple myelomas.Blood. 2000; 96: 3560-3568Crossref PubMed Google Scholar) and by rare mutations directly affecting AKT (29Stemke-Hale K. Gonzalez-Angulo A.M. Lluch A. Neve R.M. Kuo W.-L. Davies M. Carey M. Hu Z. Guan Y. Sahin A. Symmans W.F. Pusztai L. Nolden L.K. Horlings H. Berns K. Hung M.-C. van de Vijver M.J. Valero V. Gray J.W. Bernards R. Mills G.B. Hennessy B.T. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer.Cancer Res. 2008; 68: 6084-6091Crossref PubMed Scopus (808) Google Scholar, 30Kandoth C. McLellan M.D. Vandin F. Ye K. Niu B. Lu C. Xie M. Zhang Q. McMichael J.F. Wyczalkowski M.A. Leiserson M.D.M. Miller C.A. Welch J.S. Walter M.J. Wendl M.C. Ley T.J. Wilson R.K. Raphael B.J. Ding L. Mutational landscape and significance across 12 major cancer types.Nature. 2013; 502: 333-339Crossref PubMed Scopus (2931) Google Scholar). The most frequently reported AKT mutation, E17K, affects its PHD and results in an increased activity and a localization of to the plasma membrane (31Carpten J.D. Faber A.L. Horn C. Donoho G.P. Briggs S.L. Robbins C.M. Hostetter G. Boguslawski S. Moses T.Y. Savage S. Uhlik M. Lin A. Du J. Qian Y.-W. Zeckner D.J. Tucker-Kellogg G. Touchman J. Patel K. Mousses S. Bittner M. Schevitz R. Lai M.-H.T. Blanchard K.L. Thomas J.E. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer.Nature. 2007; 448: 439-444Crossref PubMed Scopus (999) Google Scholar). Point mutations of AKT are found spread all along the coding sequence (cBioPortal, http://www.cbioportal.org/). However, due to their low frequency, their status of drivers of tumorigenesis is not clear. Recently, we identified mutations affecting AKT1 as a hallmark of juvenile granulosa cell tumors. Indeed, we found that more than 60% of the tumor samples studied bore somatic in-frame tandem duplications affecting the PHD of AKT1 whereas the others carried potentially damaging point mutations at conserved residues (32Bessière L. Todeschini A.-L. Auguste A. Sarnacki S. Flatters D. Legois B. Sultan C. Kalfa N. Galmiche L. Veitia R.A. A hot-spot of in-frame duplications activates the oncoprotein AKT1 in juvenile granulosa cell tumors.EBioMedicine. 2015; 2: 421-431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Remarkably, the AKT1 variants bearing the tandem duplications were insensitive to serum deprivation, they were constitutively hyper-phosphorylated, and this was associated with hyperactivity and relocalization to the plasma membrane (32Bessière L. Todeschini A.-L. Auguste A. Sarnacki S. Flatters D. Legois B. Sultan C. Kalfa N. Galmiche L. Veitia R.A. A hot-spot of in-frame duplications activates the oncoprotein AKT1 in juvenile granulosa cell tumors.EBioMedicine. 2015; 2: 421-431Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Importantly, the mutational analysis of four juvenile granulosa cell tumor samples bearing AKT1 tandem duplications by RNA sequencing showed that these insertions were the sole detectable lesions common to the four tumors (33Auguste A. Bessière L. Todeschini A.-L. Caburet S. Sarnacki S. Prat J. D'angelo E. De La Grange P. Ariste O. Lemoine F. Legois B. Sultan C. Zider A. Galmiche L. Kalfa N. Veitia R.A. Molecular analyses of juvenile granulosa cell tumors bearing AKT1 mutations provide insights into tumor biology and therapeutic leads.Hum. Mol. Genet. 2015; 24: 6687-6698Crossref PubMed Scopus (36) Google Scholar), strongly suggesting that they constitute the driving event in these tumors. However, little is known about the role of AKT1 in granulosa cells at the molecular level. To date, only scarce data from studies performed in mouse knock-out models are available. Akt1−/− female mice show reduced fertility due to altered follicular development and abnormal oocyte growth (34Brown C. LaRocca J. Pietruska J. Ota M. Anderson L. Smith S.D. Weston P. Rasoulpour T. Hixon M.L. Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1.Biol. Reprod. 2010; 82: 246-256Crossref PubMed Scopus (83) Google Scholar). In the Akt1−/− ovaries, expression of cell-cycle positive regulators Cyclin D1 and Cyclin D3 are reduced as well as the expression of the anti-apoptotic factor BCL2 like 1 and of the survival factor KIT ligand (34Brown C. LaRocca J. Pietruska J. Ota M. Anderson L. Smith S.D. Weston P. Rasoulpour T. Hixon M.L. Subfertility caused by altered follicular development and oocyte growth in female mice lacking PKB alpha/Akt1.Biol. Reprod. 2010; 82: 246-256Crossref PubMed Scopus (83) Google Scholar). No molecular analyses have been performed, particularly, in healthy granulosa cells that could help understand the pathophysiological roles of AKT. In this study, we have performed high-throughput analyses of the proteins and transcripts deregulated after Akt1 depletion in mouse pGCs, which are the supporting cells of germ cell development and function (35Monniaux D. Driving folliculogenesis by the oocyte-somatic cell dialog: Lessons from genetic models.Theriogenology. 2016; 86: 41-53Crossref PubMed Scopus (74) Google Scholar). We found multiple cellular processes perturbed by the AKT1 KD, such as cytoskeleton regulation and focal adhesion. Consistently, we found that granulosa cells depleted for Akt1 displayed an altered directional persistent migration. Taken together, our transcriptomic and proteomic data can help understand the role of AKT1 in this critical ovarian cell type. Murine primary granulosa cells were collected from ovaries of 8-week-old female Swiss mice (Janvier Labs, Le Genest Saint Isle, France) and cultured as described in (36Georges A. L'Hôte D. Todeschini A.L. Auguste A. Legois B. Zider A. Veitia R.A. The transcription factor FOXL2 mobilizes estrogen signaling to maintain the identity of ovarian granulosa cells.eLife. 2014; 4: 3Google Scholar). The cell line AT29C derived from a mouse ovarian tumor was kindly provided by Dr. N. Di Clemente and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, Massachusetts) (37Dutertre M. Gouédard L. Xavier F. Long W.Q. di Clemente N. Picard J.Y. Rey R. Ovarian granulosa cell tumors express a functional membrane receptor for anti-Müllerian hormone in transgenic mice.Endocrinology. 2001; 142: 4040-4046Crossref PubMed Scopus (0) Google Scholar). Cells were transiently transfected in six-well plates with 30 pmoles of a pool of three different siRNAs against Akt1 (Dharmacon, Lafayette, Colorado; ON-TARGETplus siRNAs, pool KD1: J-040709-06-0005, J-040709-07-0005, J-040709-08-0005, KD2: J-040709-06-0005, J-040709-08-0005, J-040709-09-0005, KD3: J-040709-06-0005, J-040709-07-0005, J-040709-09-0005, and KD4: J-040709-07-0005, J-040709-08-0005, J-040709-09-0005) or 100 pmoles of a pool of two different siRNAs against Creb1 (Sigma-Aldrich, St. Louis, Missouri, SASI_Mm01_00191782 and SASI_Mm01_00191783), Nfkb2 (Sigma-Aldrich, SASI_Mm01_00151272 and SASI_Mm01_00151273), or Wt1 (Sigma-Aldrich, SASI_Mm01_00101396 and SASI_Mm01_00101398) or an equal amount of the nontargeting/scrambled s
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