RESUMO
Serum response factor (SRF) is a transcription factor essential for cell proliferation, differentiation, and migration and is required for primitive streak and mesoderm formation in the embryo. The canonical roles of SRF are mediated by a diverse set of context-dependent cofactors. Here, we show that SRF physically interacts with CTCF and cohesin subunits at topologically associating domain (TAD) boundaries and loop anchors. SRF promotes long-range chromatin loop formation and contributes to TAD insulation. In embryonic stem cells (ESCs), SRF associates with SOX2 and NANOG and contributes to the formation of three-dimensional (3D) pluripotency hubs. Our findings reveal additional roles of SRF in higher-order chromatin organization.
Assuntos
Cromatina , Proteína Homeobox Nanog , Fatores de Transcrição SOXB1 , Fator de Resposta Sérica , Fator de Resposta Sérica/metabolismo , Cromatina/metabolismo , Animais , Camundongos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Fator de Ligação a CCCTC/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Coesinas , Diferenciação Celular , Ligação Proteica , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologiaRESUMO
Esophageal fibrosis can develop due to caustic or radiation injuries. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are known to mitigate fibrosis in various organs. However, the potential effects of UC-MSCs on human esophageal fibrosis remain underexplored. This study investigated the anti-fibrogenic properties and mechanisms of UC-MSC-derived conditioned media (UC-MSC-CM) on human esophageal fibroblasts (HEFs). HEFs were treated with TGF-ß1 and then cultured with UC-MSC-CM, and the expression levels of extracellular matrix (ECM) components, RhoA, myocardin related transcription factor A (MRTF-A), serum response factor (SRF), Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ) were measured. UC-MSC-CM suppressed TGF-ß1-induced fibrogenic activation in HEFs, as evidenced by the downregulation of ECM. UC-MSC-CM diminished the expression of RhoA, MRTF-A, and SRF triggered by TGF-ß1. In TGF-ß1-stimulated HEFs, UC-MSC-CM decreased the nuclear localization of MRTF-A and YAP. Additionally, UC-MSC-CM diminished the TGF-ß1-induced nuclear expressions of YAP and TAZ, while concurrently enhancing the cytoplasmic presence of phosphorylated YAP. Furthermore, UC-MSC-CM reduced TGF-ß1-induced phosphorylation of Smad2. These findings suggest that UC-MSC-CM may inhibit TGF-ß1-induced fibrogenic activation in HEFs by targeting the Rho-mediated MRTF/SRF and YAP/TAZ pathways, as well as the Smad2 pathway. This indicates its potential as a stem cell therapy for esophageal fibrosis.
Assuntos
Esôfago , Fibroblastos , Fibrose , Células-Tronco Mesenquimais , Transativadores , Fatores de Transcrição , Fator de Crescimento Transformador beta1 , Proteína rhoA de Ligação ao GTP , Humanos , Células-Tronco Mesenquimais/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Esôfago/metabolismo , Esôfago/citologia , Fibroblastos/metabolismo , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Cordão Umbilical/citologia , Proteínas de Sinalização YAP/metabolismo , Fator de Resposta Sérica/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transdução de Sinais , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Proteína Smad2/metabolismoRESUMO
Paracrine IL-2 signalling drives the CD8 + T cell expansion and differentiation that allow protection against viral infections, but the underlying molecular events are incompletely understood. Here we show that the transcription factor SRF, a master regulator of cytoskeletal gene expression, is required for effective IL-2 signalling during L. monocytogenes infection. Acting cell-autonomously with its actin-regulated cofactors MRTF-A and MRTF-B, SRF is dispensible for initial TCR-mediated CD8+ T cell proliferation, but is required for sustained IL-2 dependent CD8+ effector T cell expansion, and persistence of memory cells. Following TCR activation, Mrtfab-null CD8+ T cells produce IL-2 normally, but homotypic clustering is impaired both in vitro and in vivo. Expression of cytoskeletal structural and regulatory genes, most notably actins, is defective in Mrtfab-null CD8+ T cells. Activation-induced cell clustering in vitro requires F-actin assembly, and Mrtfab-null cell clusters are small, contain less F-actin, and defective in IL-2 retention. Clustering of Mrtfab-null cells can be partially restored by exogenous actin expression. IL-2 mediated CD8+ T cell proliferation during infection thus depends on the control of cytoskeletal dynamics and actin gene expression by MRTF-SRF signalling.
Assuntos
Linfócitos T CD8-Positivos , Citoesqueleto , Interleucina-2 , Camundongos Endogâmicos C57BL , Fator de Resposta Sérica , Transativadores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/metabolismo , Interleucina-2/genética , Animais , Transativadores/metabolismo , Transativadores/genética , Citoesqueleto/metabolismo , Camundongos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Listeria monocytogenes/imunologia , Listeriose/imunologia , Listeriose/genética , Listeriose/microbiologia , Actinas/metabolismo , Regulação da Expressão Gênica , Transdução de Sinais , Camundongos Knockout , Proliferação de Células , Ativação LinfocitáriaRESUMO
Serum Response Factor (SRF) is a key regulatory transcription factor present in various cell types throughout the body, playing essential roles in cellular functions under physiological conditions. Mutations and abnormal expression of SRF have been linked to the development of various diseases and disorders. Recent evidence highlights that post-translational modifications (PTMs) are critical for regulating SRF function in different cell types and contribute to disease pathogenesis. Targeting SRF-related PTMs is emerging as a promising therapeutic approach for treating SRF-associated diseases. In this review, we summarize recent advances in understanding SRF PTMs and their underlying regulatory mechanisms. We also explore the implications of SRF-PTM in related cardiovascular and neurological diseases and their potential for therapeutic intervention. This information underscores the significance of SRF PTMs in both physiological and pathological contexts, enhancing our understanding of disease mechanisms and paving the way for the development of novel therapeutic strategies.
Assuntos
Doenças Cardiovasculares , Doenças do Sistema Nervoso , Processamento de Proteína Pós-Traducional , Fator de Resposta Sérica , Humanos , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/terapia , Doenças Cardiovasculares/genética , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/terapia , Doenças do Sistema Nervoso/tratamento farmacológico , Transdução de SinaisRESUMO
Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT.
Assuntos
Fibrose , Miofibroblastos , Proibitinas , Proteínas Repressoras , Animais , Proteínas Repressoras/metabolismo , Humanos , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fibrose/tratamento farmacológico , Antifibróticos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Movimento Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Células Cultivadas , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/antagonistas & inibidoresRESUMO
Rationale: Cardiomyocytes (CMs) undergo dramatic structural and functional changes in postnatal maturation; however, the regulatory mechanisms remain greatly unclear. Cypher/Z-band alternatively spliced PDZ-motif protein (ZASP) is an essential sarcomere component maintaining Z-disc stability. Deletion of mouse Cypher and mutation in human ZASP result in dilated cardiomyopathy (DCM). Whether Cypher/ZASP participates in CM maturation and thereby affects cardiac function has not been answered. Methods: Immunofluorescence, transmission electron microscopy, real-time quantitative PCR, and Western blot were utilized to identify the role of Cypher in CM maturation. Subsequently, RNA sequencing and bioinformatics analysis predicted serum response factor (SRF) as the key regulator. Rescue experiments were conducted using adenovirus or adeno-associated viruses encoding SRF, both in vitro and in vivo. The molecular mechanisms were elucidated through G-actin/F-actin fractionation, nuclear-cytoplasmic extraction, actin disassembly assays, and co-sedimentation assays. Results: Cypher deletion led to impaired sarcomere isoform switch and morphological abnormalities in mitochondria, transverse-tubules, and intercalated discs. RNA-sequencing analysis revealed significant dysregulation of crucial genes related to sarcomere assembly, mitochondrial metabolism, and electrophysiology in the absence of Cypher. Furthermore, SRF was predicted as key transcription factor mediating the transcriptional differences. Subsequent rescue experiments showed that SRF re-expression during the critical postnatal period effectively rectified CM maturation defects and notably improved cardiac function in Cypher-depleted mice. Mechanistically, Cypher deficiency resulted in the destabilization of F-actin and a notable increase in G-actin levels, thereby impeding the nuclear localisation of myocardin-related transcription factor A (MRTFA) and subsequently initiating SRF transcription. Conclusion: Cypher/ZASP plays a crucial role in CM maturation through actin-mediated MRTFA-SRF signalling. The linkage between CM maturation abnormalities and the late-onset of DCM is suggested, providing further insights into the pathogenesis of DCM and potential treatment strategies.
Assuntos
Actinas , Cardiomiopatia Dilatada , Miócitos Cardíacos , Fator de Resposta Sérica , Transdução de Sinais , Transativadores , Animais , Miócitos Cardíacos/metabolismo , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Camundongos , Actinas/metabolismo , Transativadores/metabolismo , Transativadores/genética , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Sarcômeros/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Camundongos KnockoutRESUMO
Serum response factor (SRF) controls gene transcription in vascular smooth muscle cells (VSMCs) and regulates VSMC phenotypic switch from a contractile to a synthetic state, which plays a key role in the pathogenesis of cardiovascular diseases (CVD). It is not known how post-translational SUMOylation regulates the SRF activity in CVD. Here we show that Senp1 deficiency in VSMCs increased SUMOylated SRF and the SRF-ELK complex, leading to augmented vascular remodeling and neointimal formation in mice. Mechanistically, SENP1 deficiency in VSMCs increases SRF SUMOylation at lysine 143, reducing SRF lysosomal localization concomitant with increased nuclear accumulation and switching a contractile phenotype-responsive SRF-myocardin complex to a synthetic phenotype-responsive SRF-ELK1 complex. SUMOylated SRF and phospho-ELK1 are increased in VSMCs from coronary arteries of CVD patients. Importantly, ELK inhibitor AZD6244 prevents the shift from SRF-myocardin to SRF-ELK complex, attenuating VSMC synthetic phenotypes and neointimal formation in Senp1-deficient mice. Therefore, targeting the SRF complex may have a therapeutic potential for the treatment of CVD.
Assuntos
Músculo Liso Vascular , Miócitos de Músculo Liso , Proteínas Nucleares , Fenótipo , Fator de Resposta Sérica , Sumoilação , Remodelação Vascular , Animais , Humanos , Masculino , Camundongos , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Neointima/patologia , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Transativadores/metabolismo , Transativadores/genéticaRESUMO
Dysregulated actin cytoskeleton gives rise to aberrant cell motility and metastatic spread of tumor cells. This study evaluates the effect of overexpression of wild-type versus functional mutants of MRTF-A on migration and invasion of breast cancer (BC) cells. Our studies indicate that SRF's interaction is critical for MRTF-A-induced promotion of both two-dimensional and three-dimensional cell migration, while the SAP-domain function is important selectively for three-dimensional cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction with MRTF. We demonstrate formin-family protein mDia2 as an important mediator of MRTF-stimulated actin polymerization at the leading edge and cell migration. Multiplexed quantitative immunohistochemistry and transcriptome analyses of clinical BC specimens further demonstrate a positive correlation between nuclear localization of MRTF with malignant traits of cancer cells and enrichment of MRTF-SRF gene signature in pair-matched distant metastases versus primary tumors. In conclusion, this study establishes a novel mechanism of MRTF-dependent regulation of cell migration and provides evidence for the association between MRTF activity and increased malignancy in human BC, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in BC.
Assuntos
Neoplasias da Mama , Movimento Celular , Forminas , Transativadores , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Forminas/metabolismo , Feminino , Transativadores/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Fator de Resposta Sérica/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genéticaRESUMO
BACKGROUND: Black women experience a disproportionate impact of uterine fibroids compared to White women, including earlier diagnosis, higher frequency, and more severe symptoms. The etiology underlying this racial disparity remains elusive. OBJECTIVE: The aim of this study was to evaluate the molecular differences in normal myometrium (fibroid-free uteri) and at-risk myometrium (fibroid-containing uteri) tissues in Black and White women. STUDY DESIGN: We conducted whole-genome RNA-seq on normal and at-risk myometrium tissues obtained from both self-identified Black and White women (not Hispanic or Latino) to determine global gene expression profiles and to conduct enriched pathway analyses (n=3 per group). We initially assessed the differences within the same type of tissue (normal or at-risk myometrium) between races. Subsequently, we analyzed the transcriptome of normal myometrium compared to at-risk myometrium in each race and determined the differences between them. We validated our findings through real-time PCR (sample size range=5-12), western blot (sample size range=5-6), and immunohistochemistry techniques (sample size range=9-16). RESULTS: The transcriptomic analysis revealed distinct profiles between Black and White women in normal and at-risk myometrium tissues. Interestingly, genes and pathways related to extracellular matrix and mechanosensing were more enriched in normal myometrium from Black than White women. Transcription factor enrichment analysis detected greater activity of the serum response transcription factor positional motif in normal myometrium from Black compared to White women. Furthermore, we observed increased expression levels of myocardin-related transcription factor-serum response factor and the serum response factor in the same comparison. In addition, we noted increased expression of both mRNA and protein levels of vinculin, a target gene of the serum response factor, in normal myometrium tissues from Black women as compared to White women. Importantly, the transcriptomic profile of normal to at-risk myometrium conversion differs between Black and White women. Specifically, we observed that extracellular matrix-related pathways are involved in the transition from normal to at-risk myometrium and that these processes are exacerbated in Black women. We found increased levels of Tenascin C, type I collagen alpha 1 chain, fibronectin, and phospho-p38 MAPK (Thr180/Tyr182, active) protein levels in at-risk over normal myometrium tissues from Black women, whereas such differences were not observed in samples from White women. CONCLUSION: These findings indicate that the racial disparities in uterine fibroids may be attributed to heightened production of extracellular matrix in the myometrium in Black women, even before the tumors appear. Future research is needed to understand early life determinants of the observed racial differences.
Assuntos
Negro ou Afro-Americano , Matriz Extracelular , Leiomioma , Miométrio , Neoplasias Uterinas , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Negro ou Afro-Americano/genética , Matriz Extracelular/metabolismo , Leiomioma/genética , Leiomioma/metabolismo , Leiomioma/etnologia , Miométrio/metabolismo , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Transcriptoma , Neoplasias Uterinas/genética , Neoplasias Uterinas/etnologia , Neoplasias Uterinas/metabolismo , Brancos/genéticaRESUMO
BACKGROUND: Fibrogenesis within ovarian endometrioma (endometrioma), mainly induced by transforming growth factor-ß (TGF-ß), is characterized by myofibroblast over-activation and excessive extracellular matrix (ECM) deposition, contributing to endometrioma-associated symptoms such as infertility by impairing ovarian reserve and oocyte quality. However, the precise molecular mechanisms that underpin the endometrioma- associated fibrosis progression induced by TGF-ß remain poorly understood. METHODS: The expression level of lysine acetyltransferase 14 (KAT14) was validated in endometrium biopsies from patients with endometrioma and healthy controls, and the transcription level of KAT14 was further confirmed by analyzing a published single-cell transcriptome (scRNA-seq) dataset of endometriosis. We used overexpression, knockout, and knockdown approaches in immortalized human endometrial stromal cells (HESCs) or human primary ectopic endometrial stromal cells (EcESCs) to determine the role of KAT14 in TGF-ß-induced fibrosis. Furthermore, an adeno-associated virus (AAV) carrying KAT14-shRNA was used in an endometriosis mice model to assess the role of KAT14 in vivo. RESULTS: KAT14 was upregulated in ectopic lesions from endometrioma patients and predominantly expressed in activated fibroblasts. In vitro studies showed that KAT14 overexpression significantly promoted a TGF-ß-induced profibrotic response in endometrial stromal cells, while KAT14 silencing showed adverse effects that could be rescued by KAT14 re-enhancement. In vivo, Kat14 knockdown ameliorated fibrosis in the ectopic lesions of the endometriosis mouse model. Mechanistically, we showed that KAT14 directly interacted with serum response factor (SRF) to promote the expression of α-smooth muscle actin (α-SMA) by increasing histone H4 acetylation at promoter regions; this is necessary for TGF-ß-induced ECM production and myofibroblast differentiation. In addition, the knockdown or pharmacological inhibition of SRF significantly attenuated KAT14-mediating profibrotic effects under TGF-ß treatment. Notably, the KAT14/SRF complex was abundant in endometrioma samples and positively correlated with α-SMA expression, further supporting the key role of KAT14/SRF complex in the progression of endometrioma-associated fibrogenesis. CONCLUSION: Our results shed light on KAT14 as a key effector of TGF-ß-induced ECM production and myofibroblast differentiation in EcESCs by promoting histone H4 acetylation via co-operating with SRF, representing a potential therapeutic target for endometrioma-associated fibrosis.
Assuntos
Endometriose , Fibrose , Fator de Resposta Sérica , Fator de Crescimento Transformador beta , Adulto , Animais , Feminino , Humanos , Camundongos , Endometriose/patologia , Endometriose/metabolismo , Endométrio/metabolismo , Endométrio/patologia , Histona Acetiltransferases/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fator de Resposta Sérica/metabolismo , Células Estromais/metabolismo , Células Estromais/patologia , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.
Assuntos
Transcrição Gênica , Animais , Transcrição Gênica/efeitos dos fármacos , RNA Polimerase II/metabolismo , RNA/metabolismo , RNA/genética , Camundongos , Humanos , Transativadores/metabolismo , Transativadores/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genéticaRESUMO
We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.
Assuntos
Núcleo Celular , Receptores ErbB , Receptor Tipo 1 de Angiotensina , Receptores ErbB/metabolismo , Humanos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Membrana Celular/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Ligação Proteica , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genéticaRESUMO
The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.
Assuntos
Glândulas Mamárias Animais , Proteínas Repressoras , Fator de Resposta Sérica , Células-Tronco , Fatores de Transcrição , Animais , Feminino , Camundongos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/citologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células-Tronco/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Humanos , Transativadores/metabolismo , Transativadores/genéticaRESUMO
The process of muscle growth directly affects the yield and quality of pork food products. Muscle fibers are created during the embryonic stage, grow following birth, and regenerate during adulthood; these are all considered to be phases of muscle development. A multilevel network of transcriptional, post-transcriptional, and pathway levels controls this process. An integrated toolbox of genetics and genomics as well as the use of genomics techniques has been used in the past to attempt to understand the molecular processes behind skeletal muscle growth and development in pigs under divergent selection processes. A class of endogenous noncoding RNAs have a major regulatory function in myogenesis. But the precise function of miRNA-423-5p in muscle development and the related molecular pathways remain largely unknown. Using target prediction software, initially, the potential target genes of miR-423-5p in the Guangxi Bama miniature pig line were identified using various selection criteria for skeletal muscle growth and development. The serum response factor (SRF) was found to be one of the potential target genes, and the two are negatively correlated, suggesting that there may be targeted interactions. In addition to being strongly expressed in swine skeletal muscle, miR-423-5p was also up-regulated during C2C12 cell development. Furthermore, real-time PCR analysis showed that the overexpression of miR-423-5p significantly reduced the expression of myogenin and the myogenic differentiation antigen (p < 0.05). Moreover, the results of the enzyme-linked immunosorbent assay (ELISA) demonstrated that the overexpression of miR-423-5p led to a significant reduction in SRF expression (p < 0.05). Furthermore, miR-423-5p down-regulated the luciferase activities of report vectors carrying the 3' UTR of porcine SRF, confirming that SRF is a target gene of miR-423-5p. Taken together, miR-423-5p's involvement in skeletal muscle differentiation may be through the regulation of SRF.
Assuntos
MicroRNAs , Desenvolvimento Muscular , Músculo Esquelético , Porco Miniatura , Animais , Camundongos , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Porco Miniatura/genética , Porco Miniatura/crescimento & desenvolvimentoRESUMO
The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.
Assuntos
Proliferação de Células , Contração Muscular , Miócitos de Músculo Liso , Proteínas Nucleares , Fenótipo , Fator de Resposta Sérica , Transativadores , Proteínas Elk-1 do Domínio ets , Proteína rhoA de Ligação ao GTP , Humanos , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/genética , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Proteína rhoA de Ligação ao GTP/metabolismo , Transativadores/metabolismo , Transativadores/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Células Cultivadas , Regulação da Expressão Gênica , Transdução de Sinais , Fator de Crescimento Epidérmico/metabolismoRESUMO
Serum response factor (SRF) is an essential transcription factor for brain development and function. Here, we explored how an SRF cofactor, the actin monomer-sensing myocardin-related transcription factor MRTF, is regulated in mouse cortical neurons. We found that MRTF-dependent SRF activity in vitro and in vivo was repressed by cyclase-associated protein CAP1. Inactivation of the actin-binding protein CAP1 reduced the amount of actin monomers in the cytoplasm, which promoted nuclear MRTF translocation and MRTF-SRF activation. This function was independent of cofilin1 and actin-depolymerizing factor, and CAP1 loss of function in cortical neurons was not compensated by endogenous CAP2. Transcriptomic and proteomic analyses of cerebral cortex lysates from wild-type and Cap1 knockout mice supported the role of CAP1 in repressing MRTF-SRF-dependent signaling in vivo. Bioinformatic analysis identified likely MRTF-SRF target genes, which aligned with the transcriptomic and proteomic results. Together with our previous studies that implicated CAP1 in axonal growth cone function as well as the morphology and plasticity of excitatory synapses, our findings establish CAP1 as a crucial actin regulator in the brain relevant for formation of neuronal networks.
Assuntos
Actinas , Córtex Cerebral , Proteínas dos Microfilamentos , Fator de Resposta Sérica , Transativadores , Fatores de Transcrição , Animais , Camundongos , Actinas/metabolismo , Actinas/genética , Proteínas de Transporte , Córtex Cerebral/metabolismo , Regulação da Expressão Gênica , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Neurônios/metabolismo , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genética , Transdução de Sinais , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.
Assuntos
Actinas , Fator de Resposta Sérica , Actinas/genética , Actinas/metabolismo , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Oligodendroglia/metabolismo , Bainha de Mielina/genética , Bainha de Mielina/metabolismo , Citoesqueleto/genética , Diferenciação Celular/genéticaRESUMO
RhoA-regulated gene transcription by serum response factor (SRF) and its transcriptional cofactor myocardin-related transcription factors (MRTFs) signaling pathway has emerged as a promising therapeutic target for pharmacological intervention in multiple diseases. Altered mitochondrial metabolism is one of the major hallmarks of cancer, therefore, this upregulation is a vulnerability that can be targeted with Rho/MRTF/SRF inhibitors. Recent advances identified a novel series of oxadiazole-thioether compounds that disrupt the SRF transcription, however, the direct molecular target of these compounds is unclear. Herein, we demonstrate the Rho/MRTF/SRF inhibition mechanism of CCG-203971 and CCG-232601 in normal cell lines of human lung fibroblasts and mouse myoblasts. Further studies investigated the role of these molecules in targeting mitochondrial function. We have shown that these molecules hyperacetylate histone H4K12 and H4K16 and regulate the genes involved in mitochondrial function and dynamics. These small molecule inhibitors regulate mitochondrial function as a compensatory mechanism by repressing oxidative phosphorylation and increasing glycolysis. Our data suggest that these CCG molecules are effective in inhibiting all the complexes of mitochondrial electron transport chains and further inducing oxidative stress. Therefore, our present findings highlight the therapeutic potential of CCG-203971 and CCG-232601, which may prove to be a promising approach to target aberrant bioenergetics.
Assuntos
Fator de Resposta Sérica , Fatores de Transcrição , Camundongos , Humanos , Animais , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Transdução de Sinais , Linhagem Celular , Mitocôndrias/metabolismoRESUMO
Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that ß1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of ß1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase ß1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.
Assuntos
Integrina beta1 , Metástase Neoplásica , Fator de Resposta Sérica , Proteínas Ativadoras de ras GTPase , Humanos , Integrina beta1/metabolismo , Integrina beta1/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Linhagem Celular Tumoral , Fator de Resposta Sérica/metabolismo , Masculino , Feminino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Animais , Transativadores/metabolismo , Adesão Celular , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Camundongos , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
INTRODUCTION: Serum response factor (SRF) is important in muscle development, tissue repair, and neuronal regulation. OBJECTIVES: This research aims to thoroughly examine the effects of SRF on spinal cord injury (SCI) and its ability to significantly impact the recovery and regeneration of neuronal axons. METHODS: The researchers created rat models of SCI and scratch injury to primary spinal cord neurons to observe the expression of relevant factors after neuronal injury. RESULTS: We found that the SRF, Ras, Raf, and cofilin levels increased after injury and gradually returned to normal levels. Afterward, researchers gave rats with SCI an SRF inhibitor (CCG1423) and studied the effects with nuclear magnetic resonance and transmission electron microscopy. The SRF inhibitor rodents had worse spinal cord recovery and axon regrowth than the control group. And the apoptosis of primary neurons after scratch injury was significantly higher in the SRF inhibitor group. Additionally, the researchers utilized lentiviral transfection to modify the SRF expression in neurons. SRF overexpression increased neuron migration while silencing SRF decreased it. Finally, Western blotting and RT-PCR were conducted to examine the expression changes of related factors upon altering SRF expression. The results revealed SRF overexpression increased Ras, Raf, and cofilin expression. Silencing SRF decreased Ras, Raf, and Cofilin expression. CONCLUSION: Based on our research, the SRF promotes axonal regeneration by activating the "Ras-Raf-Cofilin" signaling pathway.