RESUMO
Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a Padi6 missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of Padi6 mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from Padi6 mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.
Assuntos
Oócitos , Zigoto , Animais , Criança , Feminino , Humanos , Camundongos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Citoplasma/genética , Citoplasma/metabolismo , Metilação de DNA/genética , Desenvolvimento Embrionário/genética , Impressão Genômica/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Rearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance. METHODS: To gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student's paired t-test and statistical significance for the cell viability assay was a one-way ANOVA. RESULTS: GDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM. CONCLUSION: Outcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial , Tamoxifeno , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Retroalimentação , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Cinética , RNA Mensageiro , Transdução de Sinais , Tamoxifeno/farmacologia , Tamoxifeno/uso terapêutico , Fatores de Transcrição , HumanosRESUMO
BACKGROUND: Peptidylarginine deiminase enzymes (PADs) convert arginine residues to citrulline in a process called citrullination or deimination. Recently, two PADs, PAD2 and PAD4, have been linked to hormone signaling in vitro and the goal of this study was to test for links between PAD2/PAD4 and hormone signaling in vivo. METHODS: Preliminary analysis of Padi2 and Padi4 single knockout (SKO) mice did not find any overt reproductive defects and we predicted that this was likely due to genetic compensation. To test this hypothesis, we created a Padi2/Padi4 double knockout (DKO) mouse model and tested these mice along with wild-type FVB/NJ (WT) and both strains of SKO mice for a range of reproductive defects. RESULTS: Controlled breeding trials found that male DKO mice appeared to take longer to have their first litter than WT controls. This tendency was maintained when these mice were mated to either DKO or WT females. Additionally, unsexed 2-day old DKO pups and male DKO weanlings both weighed significantly less than their WT counterparts, took significantly longer than WT males to reach puberty, and had consistently lower serum testosterone levels. Furthermore, 90-day old adult DKO males had smaller testes than WT males with increased rates of germ cell apoptosis. CONCLUSIONS: The Padi2/Padi4 DKO mouse model provides a new tool for investigating PAD function and outcomes from our studies provide the first in vivo evidence linking PADs with hormone signaling.
Assuntos
Citrulina , Infertilidade , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Animais , Arginina , Modelos Animais de Doenças , Feminino , Gonadotropinas , Hidrolases/genética , Infertilidade/genética , Masculino , Camundongos , Camundongos Knockout , Proteína-Arginina Desiminase do Tipo 2/genética , Desiminases de Arginina em Proteínas/genética , TestosteronaRESUMO
Protein arginine deiminase (PAD) enzymes catalyze the conversion of protein-bound arginine into citrulline, an irreversible posttranslational modification with loss of a positive charge that can influence protein-protein interactions and protein structure. Protein arginine deiminase activity depends on high intracellular calcium concentrations occurring in dying cells. In this study, we demonstrate that protein citrullination is common during pyroptotic cell death in macrophages and that inhibition of PAD enzyme activity by Cl-amidine, a pan-PAD inhibitor, blocks NLRP3 inflammasome assembly and proinflammatory IL-1ß release in macrophages. Genetic deficiency of either PAD2 or PAD4 alone in murine macrophages does not impair IL-1ß release; however, pharmacological inhibition or small interfering RNA knockdown of PAD2 within PAD4-/- macrophages does. Our results suggest that PAD2 and 4 activity in macrophages is required for optimal inflammasome assembly and IL-1ß release, a finding of importance for autoimmune diseases and inflammation.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Animais , Citrulinação/fisiologia , Humanos , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose/fisiologiaRESUMO
BACKGROUND: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. RESULTS: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. CONCLUSION: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.
Assuntos
Doenças do Cão/patologia , Matriz Extracelular/patologia , Hemangiossarcoma/veterinária , Neoplasias Esplênicas/veterinária , Animais , Biomarcadores Tumorais , Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Cães , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Hemangiossarcoma/genética , Hemangiossarcoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Baço/metabolismo , Neoplasias Esplênicas/genética , Neoplasias Esplênicas/metabolismoRESUMO
Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the post-translational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor target genes. However, PAD2 lacks a canonical nuclear localization sequence, and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Binding of calcium to PAD2 weakens this cytoplasmic interaction, which generates a pool of calcium-bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into the nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus.
Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Transporte Ativo do Núcleo Celular , Sinalização do Cálcio , Células HEK293 , Humanos , Modelos Moleculares , Proteína-Arginina Desiminase do Tipo 2/análiseRESUMO
BACKGROUND: Mammary cancer is highly prevalent in dogs and cats and results in a poor prognosis due to critically lacking viable treatment options. Recent human and mouse studies have suggested that inhibiting peptidyl arginine deiminase enzymes (PAD) may be a novel breast cancer therapy. Based on the similarities between human breast cancer and mammary cancer in dogs and cats, we hypothesized that PAD inhibitors would also be an effective treatment for mammary cancer in these animals. METHODS: Canine and feline mammary cancer cell lines were treated with BB-Cl-Amidine (BB-CLA) and evaluated for viability and tumorigenicity. Endoplasmic reticulum stress was tested by western blot, immunofluorescence, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Canine and feline mammary cancer xenograft models were created using NOD scid gamma (NSG) mice, and were treated with BB-CLA for two weeks. RESULTS: We found that BB-CLA reduced viability and tumorigenicity of canine and feline mammary cancer cell lines in vitro. Additionally, we demonstrated that BB-CLA activates the endoplasmic reticulum stress pathway in these cells by downregulating 78 kDa Glucose-regulated Protein (GRP78), a potential target in breast cancer for molecular therapy, and upregulating the downstream target gene DNA Damage Inducible Transcript 3 (DDIT3). Finally, we established a mouse xenograft model of both canine and feline mammary cancer in which we preliminarily tested the effects of BB-CLA in vivo. CONCLUSION: We propose that our established mouse xenograft models will be useful for the study of mammary cancer in dogs and cats, and furthermore, that BB-CLA has potential as a novel therapeutic for mammary cancer in these species.
Assuntos
Amidinas/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Neoplasias Mamárias Animais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Amidinas/química , Animais , Gatos , Modelos Animais de Doenças , Cães , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We previously found that transgenic mice overexpressing MMTV-FLAG-hPAD2 (PAD2OE) developed spontaneous skin lesions, with a subset of these lesions progressing to invasive squamous cell carcinoma (SCC). The goal of this report was to better understand the potential mechanisms by which PAD2 overexpression promotes skin cancer. Here, PAD2OE mice were treated with the carcinogen, 9,10-dimethyl-1,2-benzanthracene and with O-tetradecanoylphorbol-13-acetate and then scored for papilloma formation. Additionally, tumor sections were evaluated for evidence of tumor cell invasion and inflammation. We found that the total number of papillomas was significantly increased in PAD2OE mice compared to controls. Histopathologic analysis of the lesions found that in PAD2OE skin tumors progressed to invasive SCC more frequently than controls. Additionally, we found that PAD2OE lesions were highly inflamed, with a dense inflammatory cell infiltrate and an associated increase in nuclear phospho-STAT3 (signal transducer and activator of transcription 3) in the transgenic tumors. These data suggest that overexpression of the hPAD2 transgene in the epidermis increases the malignant conversion rate of benign tumors by promoting an inflammatory microenvironment.
Assuntos
Inflamação/genética , Papiloma/genética , Desiminases de Arginina em Proteínas/genética , Neoplasias Cutâneas/genética , Regulação para Cima , 9,10-Dimetil-1,2-benzantraceno , Animais , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Carcinogênese/patologia , Carcinógenos , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Inflamação/complicações , Inflamação/patologia , Masculino , Camundongos , Camundongos Transgênicos , Papiloma/induzido quimicamente , Papiloma/complicações , Papiloma/patologia , Proteína-Arginina Desiminase do Tipo 2 , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/complicações , Neoplasias Cutâneas/patologia , Acetato de TetradecanoilforbolRESUMO
BACKGROUND: Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. METHODS: For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. RESULTS: Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. CONCLUSION: Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.
Assuntos
Carcinoma Intraductal não Infiltrante/patologia , Movimento Celular/fisiologia , Neoplasias Mamárias Experimentais/patologia , Desiminases de Arginina em Proteínas/metabolismo , Animais , Carcinoma Intraductal não Infiltrante/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Transgênicos , OrganoidesRESUMO
Transcription factor binding to DNA in vivo causes the recruitment of chromatin modifiers that can cause changes in chromatin structure, including the modification of histone tails. We previously showed that estrogen receptor (ER) target gene activation is facilitated by peptidylarginine deiminase 2 (PAD2)-catalyzed histone H3R26 deimination (H3R26Cit). Here we report that the genomic distributions of ER and H3R26Cit in breast cancer cells are strikingly coincident, linearly correlated, and observed as early as 2 minutes following estradiol treatment. The H3R26Cit profile is unlike that of previously described histone modifications and is characterized by sharp, narrow peaks. Paired-end MNase ChIP-seq indicates that the charge-neutral H3R26Cit modification facilitates ER binding to DNA by altering the fine structure of the nucleosome. Clinically, we find that PAD2 and H3R26Cit levels correlate with ER expression in breast tumors and that high PAD2 expression is associated with increased survival in ER+ breast cancer patients. These findings provide insight into how transcription factors gain access to nucleosomal DNA and implicate PAD2 as a novel therapeutic target for ER+ breast cancer.
Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Receptores de Estrogênio/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Montagem e Desmontagem da Cromatina , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Genômica , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Células MCF-7 , Prognóstico , Ligação Proteica , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em ProteínasRESUMO
Murine models are indispensible for the study of human breast cancer, but they have limitations: tumors arising spontaneously in humans must be induced in mice, and long-term follow up is limited by the short life span of rodents. In contrast, dogs and cats develop mammary tumors spontaneously and are relatively long-lived. This study examines the effects of the DNA methyltransferase (DNMT) inhibitor 5-Azacytidine (5-AzaC) on normal and tumoral mammary cell lines derived from dogs, cats and humans, as proof of concept that small companion animals are useful models of human breast cancer. Our findings show that treatment with 5-AzaC reduces in vitro tumorigenicity in all three species based on growth and invasion assays, mitochondrial activity and susceptibility to apoptosis. Interestingly, we found that the effects of 5-AzaC on gene expression varied not only between the different species but also between different tumoral cell lines within the same species, and confirmed the correlation between loss of methylation in a specific gene promotor region and increased expression of the associated gene using bisulfite sequencing. In addition, treatment with a high dose of 5-AzaC was toxic to tumoral, but not healthy, mammary cell lines from all species, indicating this drug has therapeutic potential. Importantly, we confirmed these results in primary malignant cells isolated from canine and feline adenocarcinomas. The similarities observed between the three species suggest dogs and cats can be useful models for the study of human breast cancer and the pre-clinical evaluation of novel therapeutics.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias Mamárias Animais/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/efeitos adversos , Azacitidina/efeitos adversos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Gatos , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/efeitos adversos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Estudo de Prova de Conceito , Especificidade da Espécie , Células Tumorais CultivadasRESUMO
Histone post-translational modifications (PTMs) alter the chromatin architecture, generating "open" and "closed" states, and these structural changes can modulate gene expression under specific cellular conditions. While methylation and acetylation are the best-characterized histone PTMs, citrullination by the protein arginine deiminases (PADs) represents another important player in this process. In addition to "fine tuning" chromatin structure at specific loci, histone citrullination can also promote rapid global chromatin decondensation during the formation of extracellular traps (ETs) in immune cells. Recent studies now show that PAD4-mediated citrullination of histone H1 at promoter elements can also promote localized chromatin decondensation in stem cells, thus regulating the pluripotent state. These observations suggest that PAD-mediated histone deimination profoundly affects chromatin structure, possibly above and beyond that of other PTMs. Additionally, these recent findings further enhance our understanding of PAD biology and the important contributions that these enzymes play in development, health, and disease.
Assuntos
Histonas/metabolismo , Hidrolases/fisiologia , Células-Tronco Pluripotentes Induzidas/enzimologia , Animais , Artrite Reumatoide/enzimologia , Reprogramação Celular , Montagem e Desmontagem da Cromatina , Citrulina/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Peptidylarginine deiminase 4 (PAD4) is a Ca(2+)-dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3ß (GSK3ß), both in vitro and in vivo. Among other functions, GSK3ß is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3ß protein levels, increased TGF-ß signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3ß is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3ß is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.
Assuntos
Citrulina/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Hidrolases/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Ionóforos de Cálcio , Imunofluorescência , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Células MCF-7 , Espectrometria de Massas , Microscopia de Interferência , Mutagênese Sítio-Dirigida , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não ParamétricasRESUMO
Ca(2+) oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca(2+) homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater(tm/tm) (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater(tm/tm) oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca(2+) oscillations was altered in Mater(tm/tm) oocytes after fertilization in vitro. Intriguingly, Ca(2+) oscillations in Mater(tm/tm) oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca(2+) oscillation defect in Mater(tm/tm) oocytes was likely caused by a reduced amount of Ca(2+) in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca(2+) homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.
Assuntos
Antígenos/metabolismo , Cálcio/metabolismo , Proteínas do Ovo/metabolismo , Retículo Endoplasmático/metabolismo , Homeostase/fisiologia , Metáfase/fisiologia , Microtúbulos/fisiologia , Animais , Western Blotting , Imunoprecipitação , Camundongos , Microscopia Confocal , Oócitos/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (â5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.
Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Tamanho da Partícula , Adsorção , Histidina/química , Cinética , Modelos Moleculares , Oligopeptídeos/química , Conformação ProteicaRESUMO
Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17ß-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation.
Assuntos
Arginina/metabolismo , Biocatálise , Citrulina/metabolismo , Receptor alfa de Estrogênio/metabolismo , Histonas/metabolismo , Hidrolases/metabolismo , Ativação Transcricional , Animais , Biocatálise/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatina/metabolismo , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/genética , Humanos , Camundongos , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas/genética , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Elementos de Resposta/genética , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline, and this activity has been linked to the repression of a limited number of target genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells. Results showed that PADI4 is enriched in gene promoter regions near transcription start sites (TSSs); and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found potential binding sites for Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. To better understand how PADI4 may facilitate gene transactivation, we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that, following Epidermal Growth Factor (EGF) stimulation, PADI4 catalytic activity facilitates Elk-1 phosphorylation, histone H4 acetylation, and c-Fos transcriptional activation. These results define a novel role for PADI4 as a transcription factor co-activator.
Assuntos
Neoplasias da Mama/genética , Genoma Humano , Hidrolases/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Elk-1 do Domínio ets/genética , Sítios de Ligação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Estudo de Associação Genômica Ampla , Humanos , Fosforilação , Regiões Promotoras Genéticas , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ativação Transcricional/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Matrix metalloproteinases (MMPs) are enzymes that regulate extracellular matrix composition and contribute to cell migration. Microarray studies in mouse placenta suggested that MMP-9 transcript abundance was dependent on distal-less 3 (Dlx3), a placental-specific transcriptional regulator; however, it was not clear if this was a direct or indirect effect. Here we investigate mechanism(s) for Dlx3-dependent MMP-9 gene transcription and gelatinase activity in placental trophoblasts. Initial studies confirmed that MMP-9 activity was reduced in placental explants from Dlx3(-/-) mice and that murine MMP-9 promoter activity was induced by Dlx3 overexpression. Two binding sites within a murine MMP-9 promoter fragment bound Dlx3, and mutations in both elements reduced basal MMP-9-luciferase reporter activity and abolished regulation by Dlx3. Chromatin immunoprecipitation studies in JEG3 cells confirmed Dlx3 binding to the endogenous human MMP-9 promoter at three distinct sites and knockdown of human Dlx3 resulted in reduced endogenous MMP-9 transcripts and secreted activity. These studies provide novel evidence that Dlx3 is involved directly in the transcriptional regulation of mouse and human MMP-9 gene expression in placental trophoblasts.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Trofoblastos/citologia , Animais , Western Blotting , Linhagem Celular Tumoral , Coriocarcinoma/metabolismo , Feminino , Gelatinases/genética , Gelatinases/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Masculino , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Knockout , Ligação Proteica , RNA Interferente Pequeno , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trofoblastos/enzimologiaRESUMO
Organelle positioning and movement in oocytes is largely mediated by microtubules (MTs) and their associated motor proteins. While yet to be studied in germ cells, cargo trafficking in somatic cells is also facilitated by specific recognition of acetylated MTs by motor proteins. We have previously shown that oocyte-restricted PADI6 is essential for formation of a novel oocyte-restricted fibrous structure, the cytoplasmic lattices (CPLs). Here, we show that α-tubulin appears to be associated with the PADI6/CPL complex. Next, we demonstrate that organelle positioning and redistribution is defective in PADI6-null oocytes and that alteration of MT polymerization or MT motor activity does not induce organelle redistribution in these oocytes. Finally, we report that levels of acetylated microtubules are dramatically suppressed in the cytoplasm of PADI6-null oocytes, suggesting that the observed organelle redistribution failure is due to defects in stable cytoplasmic MTs. These results demonstrate that the PADI6/CPL superstructure plays a key role in regulating MT-mediated organelle positioning and movement.
Assuntos
Citoplasma/ultraestrutura , Hidrolases/fisiologia , Microtúbulos/fisiologia , Oócitos/ultraestrutura , Organelas/fisiologia , Animais , Células Cultivadas , Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/ultraestrutura , Feminino , Hidrolases/análise , Camundongos , Microscopia Imunoeletrônica , Proteína-Arginina Desiminase do Tipo 6 , Desiminases de Arginina em Proteínas , Solubilidade , Fuso Acromático/fisiologia , Tubulina (Proteína)/análise , Tubulina (Proteína)/químicaRESUMO
BACKGROUND: The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. RESULTS: In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. CONCLUSIONS: Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation.