RESUMEN
Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.
Asunto(s)
Aorta , Microscopía de Fuerza Atómica , Animales , Fenómenos Biomecánicos , Calibración , Módulo de Elasticidad , RatonesRESUMEN
Arterial stiffening is a hallmark of aging and risk factor for cardiovascular disease, yet its regulation is poorly understood. Here we use mouse modeling to show that matrix metalloproteinase-12 (MMP12), a potent elastase, is essential for acute and chronic arterial stiffening. MMP12 was induced in arterial smooth muscle cells (SMCs) after acute vascular injury. As determined by genome-wide analysis, the magnitude of its gene induction exceeded that of all other MMPs as well as those of the fibrillar collagens and lysyl oxidases, other common regulators of tissue stiffness. A preferential induction of SMC MMP12, without comparable effect on collagen abundance or structure, was also seen during chronic arterial stiffening with age. In both settings, deletion of MMP12 reduced elastin degradation and blocked arterial stiffening as assessed by atomic force microscopy and immunostaining for stiffness-regulated molecular markers. Isolated MMP12-null SMCs sense extracellular stiffness normally, indicating that MMP12 causes arterial stiffening by remodeling the SMC microenvironment rather than affecting the mechanoresponsiveness of the cells themselves. In human aortic samples, MMP12 levels strongly correlate with markers of SMC stiffness. We conclude that MMP12 causes arterial stiffening in mice and suggest that it functions similarly in humans.
Asunto(s)
Metaloproteinasa 12 de la Matriz/metabolismo , Rigidez Vascular , Enfermedad Aguda , Envejecimiento/patología , Animales , Enfermedad Crónica , Matriz Extracelular/metabolismo , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Miocitos del Músculo Liso/enzimologíaRESUMEN
Apolipoprotein E3 (apoE3) is thought to protect against atherosclerosis by enhancing reverse cholesterol transport. However, apoE3 also has cholesterol-independent effects that contribute to its anti-atherogenic properties. These include altering extracellular matrix protein synthesis and inhibiting vascular smooth muscle cell proliferation. Both of these cholesterol-independent effects result from an apoE3-mediated induction of cyclooxygenase-2 (Cox2). Nevertheless, how apoE3 regulates Cox2 remains unknown. Here, we show that apoE3 inhibits the activation of Rho, which reduces the formation of actin stress fibers and focal adhesions and results in cellular softening. Inhibition of Rho-Rho kinase signaling or direct cellular softening recapitulates the effect of apoE3 on Cox2 expression while a constitutively active Rho mutant overrides the apoE3 effect on both intracellular stiffness and Cox2. Thus, our results describe a previously unidentified mechanism by which an atheroprotective apolipoprotein uses Rho to control cellular mechanics and Cox2.
Asunto(s)
Apolipoproteína E3/metabolismo , Ciclooxigenasa 2/metabolismo , Actinas/metabolismo , Apolipoproteína E3/genética , Línea Celular , Guanosina Trifosfato/metabolismo , Humanos , Mecanotransducción Celular , Microscopía de Fuerza Atómica , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Paxillin/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transducción de Señal , Fibras de Estrés/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/metabolismoRESUMEN
In contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs) in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK)-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness- and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.
RESUMEN
Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.
Asunto(s)
Proteínas Portadoras/metabolismo , Ciclo Celular , Proteína Sustrato Asociada a CrK/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Transducción de Señal , Animales , Mecanotransducción Celular , RatonesRESUMEN
Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein (apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol.
Asunto(s)
Apolipoproteínas E/metabolismo , HDL-Colesterol/farmacología , Matriz Extracelular/metabolismo , Aminopropionitrilo/farmacología , Aminopropionitrilo/uso terapéutico , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Apolipoproteína E3/farmacología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Colágeno Tipo I/metabolismo , Ciclooxigenasa 2/metabolismo , Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Expresión Génica , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/metabolismo , Proteína-Lisina 6-Oxidasa/antagonistas & inhibidores , Proteína-Lisina 6-Oxidasa/genética , Proteína-Lisina 6-Oxidasa/metabolismo , Rigidez Vascular/efectos de los fármacosRESUMEN
Profilin-1 (Pfn1) is a ubiquitously expressed actin-monomer binding protein that has been linked to many cellular activities ranging from control of actin polymerization to gene transcription. Traditionally, Pfn1 has been considered to be an essential control element for actin polymerization and cell migration. Seemingly contrasting this view, a few recent studies have shown evidence of an inhibitory action of Pfn1 on motility of certain types of carcinoma cells. In this review, we summarize biochemistry and functional aspects of Pfn1 in normal cells and bring in newly emerged action of Pfn1 in cancer cells that may explain its context-specific role in cell migration.
Asunto(s)
Movimiento Celular , Regulación Neoplásica de la Expresión Génica , Profilinas/metabolismo , Actinas/química , Animales , Adhesión Celular , Línea Celular Tumoral , Extensiones de la Superficie Celular/metabolismo , Humanos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Polimerizacion , Profilinas/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de ProteínasRESUMEN
Profilin1, a ubiquitously expressed actin-binding protein, plays a critical role in cell migration through actin cytoskeletal regulation. Given the traditional view of profilin1 as a promigratory molecule, it is difficult to reconcile observations that profilin1 is down-regulated in various invasive adenocarcinomas and that reduced profilin1 expression actually confers increased motility to certain adenocarcinoma cells. In this study, we show that profilin1 negatively regulates lamellipodin targeting to the leading edge in MDA-MB-231 breast cancer cells and normal cells; profilin1 depletion increases lamellipodin concentration at the lamellipodial tip (where it binds Ena/VASP), and this mediates the hypermotility. We report that the molecular mechanism underlying profilin1's modulation of lamellipodin localization relates to phosphoinositide control. Specifically, we show that phosphoinositide binding of profilin1 inhibits the motility of MDA-MB-231 cells by negatively regulating PI(3,4)P(2) at the membrane and thereby limiting recruitment of lamellipodin [a PI(3,4)P(2)-binding protein] and Ena/VASP to the leading edge. In summary, this study uncovers a unique biological consequence of profilin1-phosphoinositide interaction, thus providing direct evidence of profilin1's regulation of cell migration independent of its actin-related activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Profilinas/metabolismo , Proteínas Portadoras/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Humanos , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Profilinas/genéticaRESUMEN
m-Calpain plays a critical role in cell migration enabling rear de-adhesion of adherent cells by cleaving structural components of the adhesion plaques. Growth factors and chemokines regulate keratinocyte, fibroblast, and endothelial cell migration by modulating m-calpain activity. Growth factor receptors activate m-calpain secondary to phosphorylation on serine 50 by ERK. Concurrently, activated m-calpain is localized to its inner membrane milieu by binding to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Opposing this, CXCR3 ligands inhibit cell migration by blocking m-calpain activity secondary to a PKA-mediated phosphorylation in the C2-like domain. The failure of m-calpain activation in the absence of PIP(2) points to a key regulatory role, although whether this PIP(2)-mediated membrane localization is regulatory for m-calpain activity or merely serves as a docking site for ERK phosphorylation is uncertain. Herein, we report the effects of two CXCR3 ligands, CXCL11/IP-9/I-TAC and CXCL10/IP-10, on the EGF- and VEGF-induced redistribution of m-calpain in human fibroblasts and endothelial cells. The two chemokines block the tail retraction and, thus, the migration within minutes, preventing and reverting growth factor-induced relocalization of m-calpain to the plasma membrane of the cells. PKA phosphorylation of m-calpain blocks the binding of the protease to PIP(2). Unexpectedly, we found that this was due to membrane anchorage itself and not merely serine 50 phosphorylation, as the farnesylation-induced anchorage of m-calpain triggers a strong activation of this protease, leading notably to an increased cell death. Moreover, the ERK and PKA phosphorylations have no effect on this membrane-anchored m-calpain. However, the presence of PIP(2) is still required for the activation of the anchored m-calpain. In conclusion, we describe a novel mechanism of m-calpain activation by interaction with the plasma membrane and PIP(2) specifically, this phosphoinositide acting as a cofactor for the enzyme. The phosphorylation of m-calpain by ERK and PKA by growth factors and chemokines, respectively, act in cells to regulate the enzyme only indirectly by controlling its redistribution.
Asunto(s)
Calpaína/metabolismo , Membrana Celular/metabolismo , Células Endoteliales/metabolismo , Fibroblastos/metabolismo , Fosfatos de Inositol/metabolismo , Animales , Calpaína/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular , Membrana Celular/genética , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Quimiocina CXCL11/genética , Quimiocina CXCL11/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/citología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/citología , Humanos , Fosfatos de Inositol/genética , Ratones , Fosforilación/fisiología , Estructura Terciaria de Proteína , Receptores CXCR3/genética , Receptores CXCR3/metabolismo , Receptores de Factores de Crecimiento/agonistas , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We previously showed that silencing profilin-1 (Pfn1) expression increases breast cancer cell motility, but the underlying mechanisms have not been explored. Herein, we demonstrate that loss of Pfn1 expression leads to slower but more stable lamellipodial protrusion thereby enhancing the net protrusion rate and the overall motility of MDA-MB-231 breast cancer cells. Interestingly, MDA-MB-231 cells showed dramatic enrichment of VASP at their leading edge when Pfn1 expression was downregulated and this observation was also reproducible in other cell types including human mammary epithelial cells and vascular endothelial cells. We further demonstrate that Pfn1 downregulation results in a hyper-motile phenotype of MDA-MB-231 cells in an Ena/VASP-dependent mechanism. Pfn1-depleted cells display a strong colocalization of VASP with lamellipodin (Lpd--a PI(3,4)P(2)-binding protein that has been previously implicated in lamellipodial targeting of Ena/VASP) at the leading edge. Finally, inhibition of PI3-kinase (important for generation of PI(3,4)P(2)) delocalizes VASP from the leading edge. This observation is consistent with a possible involvement of Lpd in enhanced membrane recruitment of VASP that results from loss of Pfn1 expression. Our findings for the first time highlight a possible mechanism of how reduced expression of a pro-migratory molecule like Pfn1 could actually promote motility of breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Profilinas/metabolismo , Actinas/metabolismo , Animales , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/genética , Línea Celular , Femenino , Silenciador del Gen , Humanos , Proteínas de Microfilamentos/genética , Fenotipo , Fosfoproteínas/genética , Profilinas/genética , Seudópodos/metabolismo , Seudópodos/ultraestructura , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Profilin-1 (Pfn1), a ubiquitously expressed actin-binding protein, has been regarded as a tumor-suppressor molecule for breast cancer. Since AKT signaling impacts cell survival and proliferation, in this study we investigated whether AKT activation in breast cancer cells is sensitive to perturbation of Pfn1 expression. We found that even a moderate overexpression of Pfn1 leads to a significant reduction in phosphorylation of AKT in MDA-MB-231 breast cancer cells. We further demonstrated that Pfn1 overexpression in MDA-MB-231 cells is associated with a significant reduction in the level of the phosphoinositide regulator of AKT, PIP(3), and impaired membrane translocation of AKT that is required for AKT activation, in response to EGF stimulation. Interestingly, Pfn1-overexpressing cells showed post-transcriptional upregulation of PTEN. Furthermore, when PTEN expression was silenced, AKT phosphorylation was rescued, suggesting PTEN upregulation is responsible for Pfn1-dependent attenuation of AKT activation in MDA-MB-231 cells. Pfn1 overexpression induced PTEN upregulation and reduced AKT activation were also reproducible features of BT474 breast cancer cells. These findings may provide mechanistic insights underlying at least some of the tumor-suppressive properties of Pfn1.
Asunto(s)
Neoplasias de la Mama/enzimología , Fosfohidrolasa PTEN/genética , Profilinas/genética , Profilinas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Regulación hacia Arriba/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Membrana Celular/enzimología , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.