RESUMO
Membrane polyphosphoinositides (PPIs) are lipid-signaling molecules that undergo metabolic turnover and influence a diverse range of cellular functions. PPIs regulate the activity and/or spatial localization of a number of actin-binding proteins (ABPs) through direct interactions; however, it is much less clear whether ABPs could also be an integral part in regulating PPI signaling. In this study, we show that ABP profilin1 (Pfn1) is an important molecular determinant of the cellular content of PI(4,5)P2 (the most abundant PPI in cells). In growth factor (EGF) stimulation setting, Pfn1 depletion does not impact PI(4,5)P2 hydrolysis but enhances plasma membrane (PM) enrichment of PPIs that are produced downstream of activated PI3-kinase, including PI(3,4,5)P3 and PI(3,4)P2, the latter consistent with increased PM recruitment of SH2-containing inositol 5' phosphatase (SHIP2) (a key enzyme for PI(3,4)P2 biosynthesis). Although Pfn1 binds to PPIs in vitro, our data suggest that Pfn1's affinity to PPIs and PM presence in actual cells, if at all, is negligible, suggesting that Pfn1 is unlikely to directly compete with SHIP2 for binding to PM PPIs. Additionally, we provide evidence for Pfn1's interaction with SHIP2 in cells and modulation of this interaction upon EGF stimulation, raising an alternative possibility of Pfn1 binding as a potential restrictive mechanism for PM recruitment of SHIP2. In conclusion, our findings challenge the dogma of Pfn1's binding to PM by PPI interaction, uncover a previously unrecognized role of Pfn1 in PI(4,5)P2 homeostasis and provide a new mechanistic avenue of how an ABP could potentially impact PI3K signaling byproducts in cells through lipid phosphatase control.
Assuntos
Fosfatidilinositóis , Profilinas , Fator de Crescimento Epidérmico/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosfatidilinositóis/metabolismo , Humanos , Células HEK293 , Profilinas/metabolismoRESUMO
Overexpression of actin-binding protein profilin-1 (Pfn1) correlates with advanced disease features and adverse clinical outcome of patients with clear cell renal carcinoma, the most prevalent form of renal cancer. We previously reported that Pfn1 is predominantly overexpressed in tumor-associated vascular endothelial cells in human clear cell renal carcinoma. In this study, we combined in vivo strategies involving endothelial cell-specific depletion and overexpression of Pfn1 to demonstrate a role of vascular endothelial Pfn1 in promoting tumorigenicity and enabling progressive growth and metastasis of renal carcinoma cells in a syngeneic orthotopic mouse model of kidney cancer. We established an important role of endothelial Pfn1 in tumor angiogenesis and further identified endothelial Pfn1-dependent regulation of several pro- (VEGF, SERPINE1, CCL2) and anti-angiogenic factors (platelet factor 4) in vivo. Endothelial Pfn1 overexpression increases tumor infiltration by macrophages and concomitantly diminishes tumor infiltration by T cells including CD8+ T cells in vivo, correlating with the pattern of endothelial Pfn1-dependent changes in tumor abundance of several prominent immunomodulatory cytokines. These data were also corroborated by multiplexed quantitative immunohistochemistry and immune deconvolution analyses of RNA-seq data of clinical samples. Guided by Upstream Regulator Analysis of tumor transcriptome data, we further established endothelial Pfn1-induced Hif1α elevation and suppression of STAT1 activation. In conclusion, this study demonstrates for the first time a direct causal relationship between vascular endothelial Pfn1 dysregulation, immunosuppressive tumor microenvironment, and disease progression with mechanistic insights in kidney cancer. Our study also provides a conceptual basis for targeting Pfn1 for therapeutic benefit in kidney cancer.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Profilinas , Microambiente Tumoral , Animais , Humanos , Camundongos , Carcinoma de Células Renais/genética , Células Endoteliais/metabolismo , Neoplasias Renais/genética , Profilinas/genética , Profilinas/metabolismo , Progressão da DoençaRESUMO
Dynamic remodeling of the actin cytoskeleton is an essential feature for virtually all actin-dependent cellular processes, including cell migration, cell cycle progression, chromatin remodeling and gene expression, and even the DNA damage response. An altered actin cytoskeleton is a structural hallmark associated with numerous pathologies ranging from cardiovascular diseases to immune disorders, neurological diseases and cancer. The actin cytoskeleton in cells is regulated through the orchestrated actions of a myriad of actin-binding proteins. In this Review, we provide a brief overview of the structure and functions of the actin-monomer-binding protein profilin-1 (Pfn1) and then discuss how dysregulated expression of Pfn1 contributes to diseases associated with the cardiovascular system.
Assuntos
Doenças Cardiovasculares , Profilinas , Citoesqueleto de Actina/genética , Actinas/genética , Doenças Cardiovasculares/genética , Humanos , Proteínas dos Microfilamentos , Profilinas/genéticaRESUMO
Mitochondria perform diverse functions in the cell and their roles during processes such as cell survival, differentiation, and migration are increasingly being appreciated. Mitochondrial and actin cytoskeletal networks not only interact with each other, but this multifaceted interaction shapes their functional dynamics. The interrelation between mitochondria and the actin cytoskeleton extends far beyond the requirement of mitochondrial ATP generation to power actin dynamics, and impinges upon several major aspects of cellular physiology. Being situated at the hub of cell signaling pathways, mitochondrial function can alter the activity of actin regulatory proteins and therefore modulate the processes downstream of actin dynamics such as cellular migration. As we will discuss, this regulation is highly nuanced and operates at multiple levels allowing mitochondria to occupy a strategic position in the regulation of migration, as well as pathological events that rely on aberrant cell motility such as cancer metastasis. In this review, we summarize the crosstalk that exists between mitochondria and actin regulatory proteins, and further emphasize on how this interaction holds importance in cell migration in normal as well as dysregulated scenarios as in cancer.
Assuntos
Actinas , Neoplasias , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimento Celular , Humanos , Mitocôndrias/metabolismo , Neoplasias/metabolismoRESUMO
Angiogenesis-mediated neovascularization in the eye is usually associated with visual complications. Pathological angiogenesis is particularly prominent in the retina in the settings of proliferative diabetic retinopathy, in which it can lead to permanent loss of vision. In this study, by bioinformatics analyses, we provide evidence for elevated expression of actin-binding protein PFN1 (profilin1) in the retinal vascular endothelial cells (VECs) of individuals with proliferative diabetic retinopathy, findings further supported by gene expression analyses for PFN1 in experimentally induced abnormal retinal neovascularization in an oxygen-induced retinopathy murine model. We observed that in a conditional knockout mouse model, postnatal deletion of the Pfn1 gene in VECs leads to defects in tip cell activity (marked by impaired filopodial protrusions) and reduced vascular sprouting, resulting in hypovascularization during developmental angiogenesis in the retina. Consistent with these findings, an investigative small molecule compound targeting the PFN1-actin interaction reduced random motility, proliferation, and cord morphogenesis of retinal VECs in vitro and experimentally induced abnormal retinal neovascularization in vivo In summary, these findings provide the first direct in vivo evidence that PFN1 is required for formation of actin-based protrusive structures and developmental angiogenesis in the retina. The proof of concept of susceptibility of abnormal angiogenesis to small molecule intervention of PFN1-actin interaction reported here lays a conceptual foundation for targeting PFN1 as a possible strategy in angiogenesis-dependent retinal diseases.
Assuntos
Movimento Celular , Proliferação de Células , Células Endoteliais/metabolismo , Profilinas/metabolismo , Neovascularização Retiniana/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células Endoteliais/patologia , Humanos , Camundongos , Camundongos Knockout , Oxigênio/metabolismo , Profilinas/genética , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologia , Neovascularização Retiniana/terapiaRESUMO
Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC.
Assuntos
Carcinoma de Células Renais/patologia , Neoplasias Renais/patologia , Profilinas/metabolismo , Actinas/antagonistas & inibidores , Actinas/metabolismo , Animais , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Cofilina 1/genética , Cofilina 1/metabolismo , Bases de Dados Genéticas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Neoplasias Renais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Profilinas/antagonistas & inibidores , Profilinas/genética , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Microambiente Tumoral , Regulação para CimaRESUMO
Aberrant angiogenesis lies at the heart of a wide range of ocular pathologies such as proliferative diabetic retinopathy, wet age-related macular degeneration and retinopathy of prematurity. This study explores the anti-angiogenic activity of a novel small molecule investigative compound capable of inhibiting profilin1-actin interaction recently identified by our group. We demonstrate that our compound is capable of inhibiting migration, proliferation and angiogenic activity of microvascular endothelial cells in vitro as well as choroidal neovascularization (CNV) ex vivo. In mouse model of laser-injury induced CNV, intravitreal administration of this compound diminishes sub-retinal neovascularization. Finally, our preliminary structure-activity relationship study (SAR) demonstrates that this small molecule compound is amenable to improvement in biological activity through structural modifications.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Neovascularização Retiniana/tratamento farmacológico , Actinas/antagonistas & inibidores , Animais , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Humanos , Injeções Intravítreas , Camundongos , Camundongos Endogâmicos C57BL , Profilinas/antagonistas & inibidores , Neovascularização Retiniana/metabolismo , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Degeneração Macular Exsudativa/tratamento farmacológico , Degeneração Macular Exsudativa/metabolismoRESUMO
Dynamic regulation of the actin cytoskeleton is an essential feature of cell motility. Action of Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP), a family of conserved actin-elongating proteins, is an important aspect of regulation of the actin cytoskeletal architecture at the leading edge that controls membrane protrusion and cell motility. In this study, we performed mutagenesis experiments in overexpression and knockdown-rescue settings to provide, for the first time, direct evidence of the role of the actin-binding protein profilin1 (Pfn1) in VASP-mediated regulation of cell motility. We found that VASP's interaction with Pfn1 is promoted by cell-substrate adhesion and requires down-regulation of PKA activity. Our experimental data further suggest that PKA-mediated Ser137 phosphorylation of Pfn1 potentially negatively regulates the Pfn1-VASP interaction. Finally, Pfn1's ability to be phosphorylated on Ser137 was partly responsible for the anti-migratory action elicited by exposing cells to a cAMP/PKA agonist. On the basis of these findings, we propose a mechanism of adhesion-protrusion coupling in cell motility that involves dynamic regulation of Pfn1 by PKA activity.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Movimento Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Profilinas/metabolismo , Células HEK293 , Humanos , Fosforilação , Profilinas/química , Ligação Proteica , Serina/metabolismoRESUMO
Actin-based cell migration is a fundamental cellular activity that plays a crucial role in a wide range of physiological and pathological processes. An essential feature of the remodeling of actin cytoskeleton during cell motility is the de novo synthesis of factors involved in the regulation of the actin cytoskeleton and cell adhesion in response to growth-factor signaling, and this aspect of cell migration is critically regulated by serum-response factor (SRF)-mediated gene transcription. Myocardin-related transcription factors (MRTFs) are key coactivators of SRF that link actin dynamics to SRF-mediated gene transcription. In this Review, we provide a comprehensive overview of the role of MRTF in both normal and cancer cell migration by discussing its canonical SRF-dependent as well as its recently emerged SRF-independent functions, exerted through its SAP domain, in the context of cell migration. We conclude by highlighting outstanding questions for future research in this field.
Assuntos
Movimento Celular , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Animais , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Neoplasias/patologia , Domínios Proteicos , Transdução de Sinais , Transativadores/químicaRESUMO
Profilin 1 (Pfn1) is an important regulator of the actin cytoskeleton and plays a vital role in many actin-based cellular processes. Therefore, identification of a small-molecule intervention strategy targeted against the Pfn1-actin interaction could have broad utility in cytoskeletal research and further our understanding of the role of Pfn1 in actin-mediated biological processes. Based on an already resolved Pfn1-actin complex crystal structure, we performed structure-based virtual screening of small-molecule libraries to seek inhibitors of the Pfn1-actin interaction. We identified compounds that match the pharmacophore of the key actin residues of Pfn1-actin interaction and therefore have the potential to act as competitive inhibitors of this interaction. Subsequent biochemical assays identified two candidate compounds with nearly identical structures that can mitigate the effect of Pfn1 on actin polymerization in vitro As a further proof-of-concept test for cellular effects of these compounds, we performed proximity ligation assays in endothelial cells (ECs) to demonstrate compound-induced inhibition of Pfn1-actin interaction. Consistent with the important role of Pfn1 in regulating actin polymerization and various fundamental actin-based cellular activities (migration and proliferation), treatment of these compounds reduced the overall level of cellular filamentous (F) actin, slowed EC migration and proliferation, and inhibited the angiogenic ability of ECs both in vitro and ex vivo In summary, this study provides the first proof of principle of small-molecule-mediated interference with the Pfn1-actin interaction. Our findings may have potential general utility for perturbing actin-mediated cellular activities and biological processes.
Assuntos
Actinas/metabolismo , Profilinas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/antagonistas & inibidores , Actinas/genética , Animais , Aorta Torácica/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Polimerização/efeitos dos fármacos , Profilinas/antagonistas & inibidores , Profilinas/química , Profilinas/genética , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Megakaryoblastic leukemia (MKL)/serum-response factor (SRF)-mediated gene transcription is a highly conserved mechanism that connects dynamic reorganization of the actin cytoskeleton to regulation of expression of a wide range of genes, including SRF itself and many important structural and regulatory components of the actin cytoskeleton. In this study, we examined the possible role of MKL/SRF in the context of regulation of profilin (Pfn), a major controller of actin dynamics and actin cytoskeletal remodeling in cells. We demonstrated that despite being located on different genomic loci, two major isoforms of Pfn (Pfn1 and Pfn2) are co-regulated by a common mechanism involving the action of MKL that is independent of its SRF-related activity. We found that MKL co-regulates the expression of Pfn isoforms indirectly by modulating signal transducer and activator of transcription 1 (STAT1) and utilizing its SAP-domain function. Unexpectedly, our studies revealed that cellular externalization, rather than transcription of Pfn1, is affected by the perturbations of MKL. We further demonstrated that MKL can influence cell migration by modulating Pfn1 expression, indicating a functional connection between MKL and Pfn1 in actin-dependent cellular processes. Finally, we provide initial evidence supporting the ability of Pfn to influence MKL and SRF expression. Collectively, these findings suggest that Pfn may play a role in a possible feedback loop of the actin/MKL/SRF signaling circuit.
Assuntos
Regulação da Expressão Gênica , Profilinas/metabolismo , Fator de Transcrição STAT1/metabolismo , Transativadores/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Células HEK293 , Humanos , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Profilinas/genética , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Fator de Resposta Sérica/antagonistas & inibidores , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/genéticaRESUMO
BACKGROUND: Adhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes. METHODS: Quantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1's association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition. RESULTS: Lower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1. CONCLUSION: Overall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC.
Assuntos
Neoplasias da Mama/patologia , Técnicas de Cultura de Células/métodos , Profilinas/deficiência , Proteína Smad3/genética , Regulação para Cima , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Gradação de Tumores , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Análise Serial de Tecidos , Microambiente TumoralRESUMO
De novo synthesis of cytoskeleton-regulatory proteins triggered by the megakaryoblastic leukemia (MKL)/serum response factor (SRF) transcriptional system in response to pro-angiogenic growth factors lies at the heart of endothelial cell (EC) migration (a critical element of angiogenesis) and neovascularization. This study explores whether pharmacological intervention of MKL/SRF signaling axis by CCG-1423 is able to suppress angiogenesis. Our studies show that CCG-1423 inhibits migration and cord morphogenesis of EC in vitro and sprouting angiogenesis ex vivo and in vivo, suggesting CCG-1423 could be a novel anti-angiogenic agent. Kymography analyses of membrane dynamics of EC revealed that CCG-1423 treatment causes a major defect in membrane protrusion. CCG-1423 treatment led to attenuated expression of several actin-binding proteins that are important for driving membrane protrusion including ArpC2, VASP, and profilin1 (Pfn1) with the most drastic effect seen on the expression of Pfn1. Finally, depletion of Pfn1 alone is also sufficient for a dramatic decrease in sprouting angiogenesis of EC in vitro and ex vivo, further suggesting that Pfn1 depletion may be one of the mechanisms of the anti-angiogenic action of CCG-1423.
Assuntos
Anilidas/farmacologia , Benzamidas/farmacologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Resposta Sérica/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Actinas/metabolismo , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Linhagem Celular , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Células Endoteliais/efeitos dos fármacos , Humanos , Camundongos Knockout , Profilinas/metabolismo , Peixe-Zebra/embriologiaRESUMO
Isolation of a monomeric SiO2 compound 3 as a stable donor-acceptor complex with two different ligands -a σ-donating ligand (pyridine, dimethylaminopyridine, N-heterocyclic carbene) and a donor-acceptor ligand (iminophosphorane)-is presented. The SiO2 complexâ 3 is soluble in ordinary organic solvents and is stable at room temperature in solution and in the solid state. Of particular interest, 3 remains reactive and can be used as a stable and soluble unimolecular SiO2 reagent.
RESUMO
A donor-stabilized 1,3-disila-2,4-diazacyclobutadiene presents an exceptionally short nonbonded Siâ â â Si distance (2.23â Å), which is as short as that of Si=Si bonds (2.15-2.23â Å). Theoretical investigations indicate that there is no bond between the two silicon atoms, and that the unusual geometry can be related to a significant coulomb repulsion between the two ring nitrogen atoms. This chemical pressure phenomenon could provide an alternative and superior way of squeezing out van der Waals space in highly strained structures, as compared to the classical physical methods.
RESUMO
Actin cytoskeleton plays an important role in various aspects of atherosclerosis, a key driver of ischemic heart disease. Actin-binding protein Profilin1 (Pfn1) is overexpressed in atherosclerotic plaques in human disease, and Pfn1, when partially depleted globally in all cell types, confers atheroprotection in vivo. This study investigates the impact of endothelial cell (EC)-specific partial loss of Pfn1 expression in atherosclerosis development. We utilized mice engineered for conditional heterozygous knockout of the Pfn1 gene in ECs, with atherosclerosis induced by depletion of hepatic LDL receptor by gene delivery of PCSK9 combined with high-cholesterol diet. Our studies show that partial depletion of EC Pfn1 has certain beneficial effects marked by dampening of select pro-atherogenic cytokines (CXCL10 and IL7) with concomitant reduction in cytotoxic T cell abundance but is not sufficient to reduce hyperlipidemia and confer atheroprotection in vivo. In light of these findings, we conclude that atheroprotective phenotype conferred by global Pfn1 haplo-insufficiency requires contributions of additional cell types that are relevant for atherosclerosis progression.
RESUMO
Actin cytoskeleton plays an important role in various aspects of atherosclerosis, a key driver of ischemic heart disease. Actin-binding protein Profilin1 (Pfn1) is overexpressed in atherosclerotic plaques in human disease, and Pfn1, when partially depleted globally in all cell types, confers atheroprotection in vivo . This study investigates the impact of endothelial cell (EC)-specific partial loss of Pfn1 expression in atherosclerosis development. We utilized mice engineered for conditional heterozygous knockout of the Pfn1 gene in ECs, with atherosclerosis induced by depletion of hepatic LDL receptor by gene delivery of PCSK9 combined with high-cholesterol diet. Our studies show that partial depletion of EC Pfn1 has certain beneficial effects marked by dampening of select pro-atherogenic cytokines (CXCL10 and IL7) with concomitant reduction in cytotoxic T cell abundance but is not sufficient to reduce hyperlipidemia and confer atheroprotection in vivo . In light of these findings, we conclude that atheroprotective phenotype conferred by global Pfn1 haplo-insufficiency requires contributions of additional cell types that are relevant for atherosclerosis progression.
RESUMO
Bone is a frequent site for breast cancer metastasis. Conditioning of the local tumor microenvironment (TME) through crosstalk between tumor cells and bone resident cells in the metastatic niche is a major driving force for bone colonization of breast cancer cells. The vast majority of breast cancer-associated metastasis is osteolytic in nature, and RANKL-induced differentiation of bone marrow-derived macrophages to osteoclasts (OCLs) is a key requirement for osteolytic metastatic growth of cancer cells. In this study, we demonstrate that breast cancer cell-secreted factors stimulate RANKL-induced OCL differentiation of BMDMs requiring the function of Myocardin-related transcription factor (MRTF) in tumor cells. This is partly attributed to the critical role of MRTF in maintaining the basal cellular expression of connective tissue growth factor (CTGF), a pro-osteoclastogenic matricellular factor known to promote bone metastasis in human breast cancer. Supporting these in vitro findings, bioinformatics analyses of multiple human breast cancer transcriptome datasets reveal a strong positive correlation between CTGF expression and MRTF gene signature further establishing the relevance of our findings in a human disease context. By Luminex analyses, we show that MRTF depletion in breast cancer cells has a broad impact on OCL-regulatory cell-secreted factors that extends beyond CTGF. These findings, taken together with demonstration of MRTF-dependence for bone colonization breast cancer cells in vivo, suggest that MRTF inhibition could be an effective strategy to diminish OCL formation and skeletal involvement in breast cancer. In summary, this study highlights a novel tumor-extrinsic function of MRTF relevant to breast cancer metastasis.
RESUMO
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 vs 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 2D and 3D cell migration, while the SAP-domain function is important selectively for 3D cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction of 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 vs 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 breast cancer, justifying future development of a specific small molecule inhibitor of the MRTF-SRF transcriptional complex as a potential therapeutic agent in breast cancer. SIGNIFICANCE: Actin cytoskeletal dysregulation gives rise to metastatic dissemination of cancer cells. This study mechanistically investigates the impact of specific functional disruption of MRTF (a transcriptional co-factor of SRF) on breast cancer cell migration.This study establishes a novel mechanism linking mDia2 to MRTF-dependent regulation of cell migration and provides clinical evidence for the association between MRTF activity and increased malignancy in human breast cancer.Findings from these studies justify future exploration of specific small molecule inhibitor of the MRTF-SRF transcriptional complex as a potential therapeutic agent in breast cancer.
RESUMO
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 vs 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 2D and 3D cell migration, while the SAP-domain function is important selectively for 3D cell migration. Increased MRTF-A activity is associated with more effective membrane protrusion, a phenotype that is attributed predominantly to SRF's interaction of 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 vs 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 breast cancer, justifying future development of specific small molecule inhibitors of the MRTF-SRF transcriptional complex as potential therapeutic agents in breast cancer.