RESUMO
Expression levels of the lactate-H+ cotransporter MCT4 (also known as SLC16A3) and its chaperone CD147 (also known as basigin) are upregulated in breast cancers, correlating with decreased patient survival. Here, we test the hypothesis that MCT4 and CD147 favor breast cancer invasion through interdependent effects on extracellular matrix (ECM) degradation. MCT4 and CD147 expression and membrane localization were found to be strongly reciprocally interdependent in MDA-MB-231 breast cancer cells. Overexpression of MCT4 and/or CD147 increased, and their knockdown decreased, migration, invasion and the degradation of fluorescently labeled gelatin. Overexpression of both proteins led to increases in gelatin degradation and appearance of the matrix metalloproteinase (MMP)-generated collagen-I cleavage product reC1M, and these increases were greater than those observed upon overexpression of each protein alone, suggesting a concerted role in ECM degradation. MCT4 and CD147 colocalized with invadopodia markers at the plasma membrane. They also colocalized with MMP14 and the lysosomal marker LAMP1, as well as partially with the autophagosome marker LC3, in F-actin-decorated intracellular vesicles. We conclude that MCT4 and CD147 reciprocally regulate each other and interdependently support migration and invasiveness of MDA-MB-231 breast cancer cells. Mechanistically, this involves MCT4-CD147-dependent stimulation of ECM degradation and specifically of MMP-mediated collagen-I degradation. We suggest that the MCT4-CD147 complex is co-delivered to invadopodia with MMP14.
Assuntos
Basigina , Neoplasias da Mama , Matriz Extracelular , Proteína 1 de Membrana Associada ao Lisossomo , Metaloproteinase 14 da Matriz , Transportadores de Ácidos Monocarboxílicos , Invasividade Neoplásica , Podossomos , Feminino , Humanos , Basigina/metabolismo , Basigina/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Gelatina/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/genética , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 14 da Matriz/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/metabolismo , Proteínas Musculares/genética , Invasividade Neoplásica/genética , Podossomos/metabolismoRESUMO
Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.
Assuntos
Proteínas de Transporte , Cílios , Proteína 1 Homóloga a Discs-Large , Canais de Cátion TRPP , Animais , Cílios/metabolismo , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/genética , Camundongos , Proteína 1 Homóloga a Discs-Large/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Humanos , Transporte Proteico , Camundongos Knockout , Rim/metabolismo , Células Epiteliais/metabolismo , Ligação Proteica , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Anormalidades UrogenitaisRESUMO
The expansion of cancer cell mass in solid tumors generates a harsh environment characterized by dynamically varying levels of acidosis, hypoxia and nutrient deprivation. Because acidosis inhibits glycolytic metabolism and hypoxia inhibits oxidative phosphorylation, cancer cells that survive and grow in these environments must rewire their metabolism and develop a high degree of metabolic plasticity to meet their energetic and biosynthetic demands. Cancer cells frequently upregulate pathways enabling the uptake and utilization of lipids and other nutrients derived from dead or recruited stromal cells, and in particular lipid uptake is strongly enhanced in acidic microenvironments. The resulting lipid accumulation and increased reliance on ß-oxidation and mitochondrial metabolism increases susceptibility to oxidative stress, lipotoxicity and ferroptosis, in turn driving changes that may mitigate such risks. The spatially and temporally heterogeneous tumor microenvironment thus selects for invasive, metabolically flexible, and resilient cancer cells capable of exploiting their local conditions as well as of seeking out more favorable surroundings. This phenotype relies on the interplay between metabolism, acidosis and oncogenic mutations, driving metabolic signaling pathways such as peroxisome proliferator-activated receptors (PPARs). Understanding the particular vulnerabilities of such cells may uncover novel therapeutic liabilities of the most aggressive cancer cells.
RESUMO
Solid tumors comprise two major components: the cancer cells and the tumor stroma. The stroma is a mixture of cellular and acellular components including fibroblasts, mesenchymal and cancer stem cells, endothelial cells, immune cells, extracellular matrix, and tumor interstitial fluid. The insufficient tumor perfusion and the highly proliferative state and dysregulated metabolism of the cancer cells collectively create a physicochemical microenvironment characterized by altered nutrient concentrations and varying degrees of hypoxia and acidosis. Furthermore, both cancer and stromal cells secrete numerous growth factors, cytokines, and extracellular matrix proteins which further shape the tumor microenvironment (TME), favoring cancer progression.Transport proteins expressed by cancer and stromal cells localize at the interface between the cells and the TME and are in a reciprocal relationship with it, as both sensors and modulators of TME properties. It has been amply demonstrated how acid-base and nutrient transporters of cancer cells enable their growth, presumably by contributing both to the extracellular acidosis and the exchange of metabolic substrates and waste products between cells and TME. However, the TME also impacts other transport proteins important for cancer progression, such as multidrug resistance proteins. In this review, we summarize current knowledge of the cellular and acellular components of solid tumors and their interrelationship with key ion transport proteins. We focus in particular on acid-base transport proteins with known or proposed roles in cancer development, and we discuss their relevance for novel therapeutic strategies.
Assuntos
Neoplasias , Microambiente Tumoral , Proteínas de Transporte/uso terapêutico , Células Endoteliais , Humanos , Neoplasias/tratamento farmacológico , Processos NeoplásicosRESUMO
Acidic metabolic waste products accumulate in the tumor microenvironment because of high metabolic activity and insufficient perfusion. In tumors, the acidity of the interstitial space and the relatively well-maintained intracellular pH influence cancer and stromal cell function, their mutual interplay, and their interactions with the extracellular matrix. Tumor pH is spatially and temporally heterogeneous, and the fitness advantage of cancer cells adapted to extracellular acidity is likely particularly evident when they encounter less acidic tumor regions, for instance, during invasion. Through complex effects on genetic stability, epigenetics, cellular metabolism, proliferation, and survival, the compartmentalized pH microenvironment favors cancer development. Cellular selection exacerbates the malignant phenotype, which is further enhanced by acid-induced cell motility, extracellular matrix degradation, attenuated immune responses, and modified cellular and intercellular signaling. In this review, we discuss how the acidity of the tumor microenvironment influences each stage in cancer development, from dysplasia to full-blown metastatic disease.
Assuntos
Neoplasias/metabolismo , Microambiente Tumoral , Ácidos/metabolismo , Animais , Humanos , Concentração de Íons de Hidrogênio , Metástase Neoplásica , Neoplasias/patologia , Transdução de SinaisRESUMO
Aquaporin (AQP) water channels facilitate water transport across cellular membranes and are essential in regulation of body water balance. Moreover, several AQPs are overexpressed or ectopically expressed in breast cancer. Interestingly, several in vitro studies have suggested that AQPs can affect the response to conventional anticancer chemotherapies. Therefore, we took a systematic approach to test how AQP1, AQP3 and AQP5, which are often over-/ectopically expressed in breast cancer, affect total viability of 3-dimensional (3D) breast cancer cell spheroids when treated with the conventional anticancer chemotherapies Cisplatin, 5-Fluorouracil (5-FU) and Doxorubicin, a Combination of the three drugs as well as the Combination plus the Ras inhibitor Salirasib. Total viability of spheroids overexpressing AQP1 were decreased by all treatments except for 5-FU, which increased total viability by 20% compared to DMSO treated controls. All treatments reduced viability of spheroids overexpressing AQP3. In contrast, only Doxorubicin, Combination and Combination + Salirasib reduced total viability of spheroids overexpressing AQP5. Thus, this study supports a significant role of AQPs in the response to conventional chemotherapies. Evaluating the role of individual proteins that contribute to resistance to chemotherapies is essential in advancing personalized medicine in breast carcinomas.
Assuntos
Aquaporinas , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Aquaporinas/metabolismo , Fluoruracila/farmacologia , Doxorrubicina/farmacologia , Aquaporina 1/genética , Aquaporina 1/metabolismo , Aquaporina 5/metabolismo , Aquaporina 3/genética , Aquaporina 3/metabolismo , Aquaporina 4 , Aquaporina 2RESUMO
BACKGROUND: The lactate receptor GPR81 contributes to cancer development through unclear mechanisms. Here, we investigate the roles of GPR81 in three-dimensional (3D) and in vivo growth of breast cancer cells and study the molecular mechanisms involved. METHODS: GPR81 was stably knocked down (KD) in MCF-7 human breast cancer cells which were subjected to RNA-seq analysis, 3D growth, in situ- and immunofluorescence analyses, and cell viability- and motility assays, combined with KD of key GPR81-regulated genes. Key findings were additionally studied in other breast cancer cell lines and in mammary epithelial cells. RESULTS: GPR81 was upregulated in multiple human cancer types and further upregulated by extracellular lactate and 3D growth in breast cancer spheroids. GPR81 KD increased spheroid necrosis, reduced invasion and in vivo tumor growth, and altered expression of genes related to GO/KEGG terms extracellular matrix, cell adhesion, and Notch signaling. Single cell in situ analysis of MCF-7 cells revealed that several GPR81-regulated genes were upregulated in the same cell clusters. Notch signaling, particularly the Notch ligand Delta-like-4 (DLL4), was strikingly downregulated upon GPR81 KD, and DLL4 KD elicited spheroid necrosis and inhibited invasion in a manner similar to GPR81 KD. CONCLUSIONS: GPR81 supports breast cancer aggressiveness, and in MCF-7 cells, this occurs at least in part via DLL4. Our findings reveal a new GPR81-driven mechanism in breast cancer and substantiate GPR81 as a promising treatment target.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Ácido Láctico/metabolismo , Ligantes , Transdução de Sinais , Necrose , Receptor Notch1/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Post-translational modifications (PTMs) greatly expand the function and potential for regulation of protein activity, and O-glycosylation is among the most abundant and diverse PTMs. Initiation of O-GalNAc glycosylation is regulated by 20 distinct GalNAc-transferases (GalNAc-Ts), and deficiencies in individual GalNAc-Ts are associated with human disease, causing subtle but distinct phenotypes in model organisms. Here, we generate a set of isogenic keratinocyte cell lines lacking either of the three dominant and differentially expressed GalNAc-Ts. Through the ability of keratinocytes to form epithelia, we investigate the phenotypic consequences of the loss of individual GalNAc-Ts. Moreover, we probe the cellular responses through global transcriptomic, differential glycoproteomic, and differential phosphoproteomic analyses. We demonstrate that loss of individual GalNAc-T isoforms causes distinct epithelial phenotypes through their effect on specific biological pathways; GalNAc-T1 targets are associated with components of the endomembrane system, GalNAc-T2 targets with cell-ECM adhesion, and GalNAc-T3 targets with epithelial differentiation. Thus, GalNAc-T isoforms serve specific roles during human epithelial tissue formation.
Assuntos
N-Acetilgalactosaminiltransferases , Diferenciação Celular , Epitélio/metabolismo , Glicosilação , Humanos , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Polissacarídeos , Processamento de Proteína Pós-TraducionalRESUMO
In the brain, α-synuclein (aSN) partitions between free unbound cytosolic and membrane bound forms modulating both its physiological and pathological role and complicating its study due to structural heterogeneity. Here, we use an interdisciplinary, synergistic approach to characterize the properties of aSN:lipid mixtures, isolated aSN:lipid co-structures, and aSN in mammalian cells. Enabled by the isolation of the membrane-bound state, we show that within the previously described N-terminal membrane anchor, membrane interaction relies both on an N-terminal tail (NTT) head group layer insertion of 14 residues and a folded-upon-binding helix at the membrane surface. Both binding events must be present; if, for example, the NTT insertion is lost, the membrane affinity of aSN is severely compromised and formation of aSN:lipid co-structures hampered. In mammalian cells, compromised cooperativity results in lowered membrane association. Thus, avidity within the N-terminal anchor couples N-terminal insertion and helical surface binding, which is crucial for aSN membrane interaction and cellular localization, and may affect membrane fusion.
Assuntos
Membrana Celular/metabolismo , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Humanos , Mamíferos/metabolismo , Fusão de Membrana/fisiologiaRESUMO
Transport of ions and nutrients is a core mitochondrial function, without which there would be no mitochondrial metabolism and ATP production. Both ion homeostasis and mitochondrial phenotype undergo pervasive changes during cancer development, and both play key roles in driving the malignancy. However, the link between these events has been largely ignored. This review comprehensively summarizes and critically discusses the role of the reciprocal relationship between ion transport and mitochondria in crucial cellular functions, including metabolism, signaling, and cell fate decisions. We focus on Ca2+, H+, and K+, which play essential and highly interconnected roles in mitochondrial function and are profoundly dysregulated in cancer. We describe the transport and roles of these ions in normal mitochondria, summarize the changes occurring during cancer development, and discuss how they might impact tumorigenesis.
Assuntos
Transporte de Íons , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Cálcio/metabolismo , Movimento Celular , Proliferação de Células , Homeostase , Humanos , Canais Iônicos/metabolismo , Células-Tronco Neoplásicas/metabolismo , Potássio/metabolismo , Prótons , Microambiente TumoralRESUMO
KEY POINTS: Exogenous Na+ /H+ exchanger 1 (NHE1) expression stimulated the collective migration of epithelial cell sheets Stimulation with epidermal growth factor, a key morphogen, primarily increased migration of the front row of cells, whereas NHE1 increased that of submarginal cell rows, and the two stimuli were additive Accordingly, NHE1 localized not only to the leading edges of leader cells, but also in cryptic lamellipodia in submarginal cell rows NHE1 expression disrupted the morphology of epithelial cell sheets and three-dimensional cysts ABSTRACT: Collective cell migration plays essential roles in embryonic development, in normal epithelial repair processes, and in many diseases including cancer. The Na+ /H+ exchanger 1 (NHE1, SLC9A1) is an important regulator of motility in many cells and has been widely studied for its roles in cancer, although its possible role in collective migration of normal epithelial cells has remained unresolved. In the present study, we show that NHE1 expression in MDCK-II kidney epithelial cells accelerated collective cell migration. NHE1 localized to the leading edges of leader cells, as well as to cryptic lamellipodia in submarginal cell rows. Epidermal growth factor, a kidney morphogen, increased displacement of the front row of collectively migrating cells and reduced the number of migration fingers. NHE1 expression increased the number of migration fingers and increased displacement of submarginal cell rows, resulting in additive effects of NHE1 and epidermal growth factor. Finally, NHE1 expression resulted in disorganized development of MDCK-II cell cysts. Thus, NHE1 contributes to collective migration and epithelial morphogenesis, suggesting roles for the transporter in embryonic and early postnatal development.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/metabolismo , Pseudópodes/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Linhagem Celular , Cães , Desenvolvimento Embrionário/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Células Madin Darby de Rim CaninoRESUMO
As a result of elevated metabolic rates and net acid extrusion in the rapidly proliferating cancer cells, solid tumours are characterized by a highly acidic microenvironment, while cancer cell intracellular pH is normal or even alkaline. Two-dimensional (2D) cell monocultures, which have been used extensively in breast cancer research for decades, cannot precisely recapitulate the rich environment and complex processes occurring in tumours in vivo. The use of such models can consequently be misleading or non-predictive for clinical applications. Models mimicking the tumour microenvironment are particularly pivotal for studying tumour pH homeostasis, which is profoundly affected by the diffusion-limited conditions in the tumour. To advance the understanding of the mechanisms and consequences of dysregulated acid-base homeostasis in breast cancer, clinically relevant models that incorporate the unique microenvironment of these tumours are required. The development of three-dimensional (3D) cell cultures has provided new tools for basic research and pre-clinical approaches, allowing the culture of breast cancer cells under conditions that closely resemble tumour growth in a living organism. Here we provide an overview of the main 3D techniques relevant for breast cancer cell culture. We discuss the advantages and limitations of the classical 3D models as well as recent advances in 3D culture techniques, focusing on how these culture methods have been used to study acid-base transport in breast cancer. Finally, we outline future directions of 3D culture technology and their relevance for studies of acid-base transport.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Esferoides Celulares , Equilíbrio Ácido-Base , Animais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Microfluídica , Microambiente TumoralRESUMO
We present here the hypothesis that the unique microenvironmental pH landscape of acid-base transporting epithelia is an important factor in development of epithelial cancers, by rendering the epithelial and stromal cells pre-adapted to the heterogeneous extracellular pH (pHe ) in the tumor microenvironment. Cells residing in organs with net acid-base transporting epithelia such as the pancreatic ductal and gastric epithelia are exposed to very different, temporally highly variable pHe values apically and basolaterally. This translates into spatially and temporally non-uniform intracellular pH (pHi ) patterns. Disturbed pHe - and pHi -homeostasis contributes to essentially all hallmarks of cancer. Our hypothesis, that the physiological pHe microenvironment in acid-base secreting epithelia shapes cancers arising in these tissues, can be tested using novel imaging tools. The acidic tumor pHe in turn might be exploited therapeutically. Pancreatic cancers are used as our prime example, but we propose that this concept is also relevant for other cancers of acid-base transporting epithelia.
Assuntos
Carcinogênese , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Animais , Progressão da Doença , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/metabolismoRESUMO
Hippo pathway transcriptional coactivators TAZ and YAP and the TGF-ß1 (TGFß) effector Smad3 regulate a common set of genes, can physically interact, and exhibit multilevel cross-talk regulating cell fate-determining and fibrogenic pathways. However, a key aspect of this cross-talk, TGFß-mediated regulation of TAZ or YAP expression, remains uncharacterized. Here, we show that TGFß induces robust TAZ but not YAP protein expression in both mesenchymal and epithelial cells. TAZ levels, and to a lesser extent YAP levels, also increased during experimental kidney fibrosis. Pharmacological or genetic inhibition of Smad3 did not prevent the TGFß-induced TAZ up-regulation, indicating that this canonical pathway is dispensable. In contrast, inhibition of p38 MAPK, its downstream effector MK2 (e.g. by the clinically approved antifibrotic pirferidone), or Akt suppressed the TGFß-induced TAZ expression. Moreover, TGFß elevated TAZ mRNA in a p38-dependent manner. Myocardin-related transcription factor (MRTF) was a central mediator of this effect, as MRTF silencing/inhibition abolished the TGFß-induced TAZ expression. MRTF overexpression drove the TAZ promoter in a CC(A/T-rich)6GG (CArG) box-dependent manner and induced TAZ protein expression. TGFß did not act by promoting nuclear MRTF translocation; instead, it triggered p38- and MK2-mediated, Nox4-promoted MRTF phosphorylation and activation. Functionally, higher TAZ levels increased TAZ/TEAD-dependent transcription and primed cells for enhanced TAZ activity upon a second stimulus (i.e. sphingosine 1-phosphate) that induced nuclear TAZ translocation. In conclusion, our results uncover an important aspect of the cross-talk between TGFß and Hippo signaling, showing that TGFß induces TAZ via a Smad3-independent, p38- and MRTF-mediated and yet MRTF translocation-independent mechanism.
Assuntos
Proteína Smad3/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Células Cultivadas , Camundongos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
TGFß-induced expression of the NADPH oxidase Nox4 is essential for fibroblast-myofibroblast transition. Rho has been implicated in Nox4 regulation, but the underlying mechanisms are largely unknown. Myocardin-related transcription factor (MRTF), a Rho/actin polymerization-controlled coactivator of serum response factor, drives myofibroblast transition from various precursors. We have shown that TGFß is necessary but insufficient for epithelial-myofibroblast transition in intact epithelia; the other prerequisite is the uncoupling of intercellular contacts, which induces Rho-dependent nuclear translocation of MRTF. Because the Nox4 promoter harbors a serum response factor/MRTF cis-element (CC(A/T)6GG box), we asked if MRTF (and thus cytoskeleton organization) could regulate Nox4 expression. We show that Nox4 protein is robustly induced in kidney tubular cells exclusively by combined application of contact uncoupling and TGFß. Nox4 knockdown abrogates epithelial-myofibroblast transition-associated reactive oxygen species production. Laser capture microdissection reveals increased Nox4 expression in the tubular epithelium also during obstructive nephropathy. MRTF down-regulation/inhibition suppresses TGFß/contact disruption-provoked Nox4 protein and mRNA expression, Nox4 promoter activation, and reactive oxygen species production. Mutation of the CC(A/T)6GG box eliminates the synergistic activation of the Nox4 promoter. Jasplakinolide-induced actin polymerization synergizes with TGFß to facilitate MRTF-dependent Nox4 mRNA expression/promoter activation. Moreover, MRTF inhibition prevents Nox4 expression during TGFß-induced fibroblast-myofibroblast transition as well. Although necessary, MRTF is insufficient; Nox4 expression also requires TGFß-activated Smad3 and TAZ/YAP, two contact- and cytoskeleton-regulated Smad3-interacting coactivators. Down-regulation/inhibition of TAZ/YAP mitigates injury-induced epithelial Nox4 expression in vitro and in vivo. These findings uncover new MRTF- and TAZ/YAP-dependent mechanisms, which link cytoskeleton remodeling and redox state and impact epithelial plasticity and myofibroblast transition.
Assuntos
Citoesqueleto/metabolismo , Regulação Enzimológica da Expressão Gênica , NADPH Oxidases/genética , Fatores de Transcrição/metabolismo , Actinas/metabolismo , Animais , Epitélio/patologia , Fibrose , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Células LLC-PK1 , Masculino , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular , Miofibroblastos/metabolismo , Miofibroblastos/patologia , NADPH Oxidases/metabolismo , Oxirredução , Polimerização , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Suínos , Regulação para CimaRESUMO
Primary cilia are microtubule-based sensory organelles projecting from most quiescent mammalian cells, which disassemble in cells cultured in serum-deprived conditions upon re-addition of serum or growth factors. Platelet-derived growth factors (PDGF) are implicated in deciliation, but the specific receptor isoforms and mechanisms involved are unclear. We report that PDGFRß promotes deciliation in cultured cells and provide evidence implicating PLCγ and intracellular Ca(2+) release in this process. Activation of wild-type PDGFRα alone did not elicit deciliation. However, expression of constitutively active PDGFRα D842V mutant receptor, which potently activates PLCγ (also known as PLCG1), caused significant deciliation, and this phenotype was rescued by inhibiting PDGFRα D842V kinase activity or AURKA. We propose that PDGFRß and PDGFRα D842V promote deciliation through PLCγ-mediated Ca(2+) release from intracellular stores, causing activation of calmodulin and AURKA-triggered deciliation.
Assuntos
Aurora Quinase A/metabolismo , Cílios/metabolismo , Fosfolipase C gama/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Aurora Quinase A/genética , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Microscopia de Fluorescência , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genéticaRESUMO
BACKGROUND: Extracellular signal-regulated kinase 2 (ERK2) is an S/T kinase with more than 200 known substrates, and with critical roles in regulation of cell growth and differentiation and currently no membrane proteins have been linked to ERK2 scaffolding. METHODS AND RESULTS: Here, we identify the human Na(+)/H(+) exchanger 1 (hNHE1) as a membrane scaffold protein for ERK2 and show direct hNHE1-ERK1/2 interaction in cellular contexts. Using nuclear magnetic resonance (NMR) spectroscopy and immunofluorescence analysis we demonstrate that ERK2 scaffolding by hNHE1 occurs by one of three D-domains and by two non-canonical F-sites located in the disordered intracellular tail of hNHE1, mutation of which reduced cellular hNHE1-ERK1/2 co-localization, as well as reduced cellular ERK1/2 activation. Time-resolved NMR spectroscopy revealed that ERK2 phosphorylated the disordered tail of hNHE1 at six sites in vitro, in a distinct temporal order, with the phosphorylation rates at the individual sites being modulated by the docking sites in a distant dependent manner. CONCLUSIONS: This work characterizes a new type of scaffolding complex, which we term a "shuffle complex", between the disordered hNHE1-tail and ERK2, and provides a molecular mechanism for the important ERK2 scaffolding function of the membrane protein hNHE1, which regulates the phosphorylation of both hNHE1 and ERK2.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/química , Linhagem Celular , Ativação Enzimática , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Dobramento de Proteína , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/químicaRESUMO
The volume-regulated anion channel (VRAC), also known as the volume-sensitive outwardly rectifying (VSOR) anion channel or the volume-sensitive organic osmolyte/anion channel (VSOAC), is essential for cell volume regulation after swelling in most vertebrate cell types studied to date. In addition to its role in cell volume homeostasis, VRAC has been implicated in numerous other physiological and pathophysiological processes, including cancer, ischemic brain edema, cell motility, proliferation, angiogenesis, programmed cell death, and excitotoxic glutamate release. Although VRAC has been extensively biophysically, pharmacologically, and functionally characterized, its molecular identity was highly controversial until the recent identification of the leucine-rich repeats containing 8A (LRRC8A) protein as essential for the VRAC current in multiple cell types and a likely pore-forming subunit of VRAC. Members of this distantly pannexin-1-related protein family form heteromers, and in addition to LRRC8A, at least another LRRC8 family member is required for the formation of a functional VRAC. This review summarizes the biophysical and pharmacological properties of VRAC, highlights its main physiological functions and pathophysiological implications, and outlines the search for its molecular identity.
Assuntos
Ânions/metabolismo , Tamanho Celular , Canais Iônicos/metabolismo , Potenciais de Ação , Animais , Apoptose , Humanos , Transporte de Íons , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologiaRESUMO
Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.
Assuntos
Membrana Celular/metabolismo , Modelos Moleculares , Receptores da Prolactina/metabolismo , Receptores da Somatotropina/metabolismo , Linhagem Celular , Membrana Celular/química , Dicroísmo Circular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Dobramento de Proteína , Estrutura Terciária de Proteína , Receptores da Prolactina/química , Receptores da Prolactina/genética , Receptores da Somatotropina/química , Receptores da Somatotropina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Transdução de Sinais , Tirosina/metabolismo , Difração de Raios XRESUMO
In fibroblasts, platelet-derived growth factor receptor alpha (PDGFRα) is upregulated during growth arrest and compartmentalized to the primary cilium. PDGF-AA mediated activation of the dimerized ciliary receptor produces a phosphorylation cascade through the PI3K-AKT and MEK1/2-ERK1/2 pathways leading to the activation of the Na(+)/H(+) exchanger, NHE1, cytoplasmic alkalinization and actin nucleation at the lamellipodium that supports directional cell migration. We here show that AKT and MEK1/2-ERK1/2-p90(RSK) inhibition reduced PDGF-AA-induced cell migration by distinct mechanisms: AKT inhibition reduced NHE1 activity by blocking the translocation of NHE1 to the cell membrane. MEK1/2 inhibition did not affect NHE1 activity but influenced NHE1 localization, causing NHE1 to localize discontinuously in patches along the plasma membrane, rather than preferentially at the lamellipodium. We also provide direct evidence of NHE1 translocation through the cytoplasm to the leading edge. In conclusion, signals initiated at the primary cilium through the PDGFRαα cascade reorganize the cytoskeleton to regulate cell migration differentially through the AKT and the MEK1/2-ERK1/2-p90(RSK) pathways. The AKT pathway is necessary for initiation of NHE1 translocation, presumably in vesicles, to the leading edge and for its activation. In contrast, the MEK1/2-ERK1/2-p90(RSK) pathway controls the spatial organization of NHE1 translocation and incorporation, and therefore specifies the direction of the leading edge formation.