RESUMEN
Previous studies have demonstrated that activation of calcineurin induces pathological cardiac hypertrophy (CH). In these studies, loss-of-function was mostly achieved by systemic administration of the calcineurin inhibitor cyclosporin A. The lack of conditional knockout models for calcineurin function has impeded progress toward defining the role of this protein during the onset and the development of CH in adults. Here, we exploited a mouse model of CH based on the infusion of a hypertensive dose of angiotensin II (AngII) to model the role of calcineurin in CH in adulthood. AngII-induced CH in adult mice was reduced by treatment with cyclosporin A, without affecting the associated increase in blood pressure, and also by induction of calcineurin deletion in adult mouse cardiomyocytes, indicating that cardiomyocyte calcineurin is required for AngII-induced CH. Surprisingly, cardiac-specific deletion of calcineurin, but not treatment of mice with cyclosporin A, significantly reduced AngII-induced cardiac fibrosis and apoptosis. Analysis of profibrotic genes revealed that AngII-induced expression of Tgfß family members and Lox was not inhibited by cyclosporin A but was markedly reduced by cardiac-specific calcineurin deletion. These results show that AngII induces a direct, calcineurin-dependent prohypertrophic effect in cardiomyocytes, as well as a systemic hypertensive effect that is independent of calcineurin activity.
Asunto(s)
Calcineurina/fisiología , Cardiomegalia/patología , Fibrosis/patología , Miocitos Cardíacos/patología , Angiotensina II/toxicidad , Animales , Cardiomegalia/inducido químicamente , Cardiomegalia/metabolismo , Progresión de la Enfermedad , Fibrosis/inducido químicamente , Fibrosis/metabolismo , Perfilación de la Expresión Génica , Ratones , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Transducción de Señal , Vasoconstrictores/toxicidadRESUMEN
OBJECTIVE: Atherosclerosis and restenosis are multifactorial diseases associated with abnormal vascular smooth muscle cell (VSMC) proliferation. Nuclear factor-Y (NF-Y) plays a major role in transcriptional activation of the CYCLIN B1 gene (CCNB1), a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. APPROACH AND RESULTS: We performed molecular and expression studies in cultured cells, animal models, and human tissues. We find upregulation of NF-Y and cyclin B1 expression in proliferative regions of murine atherosclerotic plaques and mechanically induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. NF-YA expression in neointimal lesions is detected in VSMCs, macrophages, and endothelial cells. Platelet-derived growth factor-BB, a main inductor of VSMC growth and neointima development, induces the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 and Akt activation in rat and human VSMCs. Moreover, adenovirus-mediated overexpression of a NF-YA-dominant negative mutant inhibits platelet-derived growth factor-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. CONCLUSIONS: Our results identify NF-Y as a key downstream effector of the platelet-derived growth factor-BB-dependent mitogenic pathway that is activated in experimental and human vasculoproliferative diseases. They also identify NF-Y inhibition as a novel and attractive strategy for the local treatment of neointimal formation induced by vessel denudation.
Asunto(s)
Factor de Unión a CCAAT/fisiología , Músculo Liso Vascular/citología , Neointima/etiología , Animales , Apolipoproteínas E/fisiología , Aterosclerosis/etiología , Becaplermina , Factor de Unión a CCAAT/antagonistas & inhibidores , Proliferación Celular , Células Cultivadas , Ciclina B1/genética , Células Endoteliales/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neointima/terapia , Proteínas Proto-Oncogénicas c-sis/farmacología , Ratas , Ratas WistarRESUMEN
Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca(2+)-calcineurin signaling.
Asunto(s)
Calcineurina/química , Calcineurina/metabolismo , Proteínas Musculares/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcineurina/genética , Línea Celular , Secuencia Conservada , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Datos de Secuencia Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Estructura Secundaria de Proteína , Alineación de Secuencia , Transducción de Señal , Especificidad por SustratoRESUMEN
The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.
Asunto(s)
Calcineurina/metabolismo , Inmunosupresores/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcineurina/química , Calcineurina/genética , Inhibidores de la Calcineurina , Clonación Molecular , Simulación por Computador , Secuencia Conservada , Genes Reporteros , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunofilinas/metabolismo , Inmunosupresores/farmacología , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Factores de Transcripción NFATC/metabolismo , Péptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Propiedades de Superficie , Proteína 1A de Unión a Tacrolimus/metabolismo , Transcripción Genética , TransfecciónRESUMEN
Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN). NFATs contain two CN binding motifs: PxIxIT and CnBP-B/CNBR2 (which we call LxVP). Here we carry out a detailed comparative analysis of the CN binding activity displayed by the PxIxIT and LxVP sites from different NFATs. Dose-response CN binding experiments with GST fusion proteins of NFATc1 and NFATc2 showed that NFATc1 binds CN in vitro more efficiently than does NFATc2. This difference in binding appears to be caused by the different CN binding potencies of the corresponding LxVP sites; thus while the LxVPc2 peptide fused to GST did not bind CN, GST-LxVPc1 bound it more efficiently than did GST-PxIxITc1 or GST-PxIxITc2. Furthermore, an NFATc2 chimera protein containing the LxVP motif from NFATc1 interacted with CN much more potently than did wild-type NFATc2. Free peptides spanning the LxVP motifs from NFATc1, c3 or c4 displaced CN from GST-NFATc1 and GST-NFATc2 more efficiently than any PxIxIT peptide. PxIxITc2 and LxVPc1 peptides were each able to cross-compete GST-LxVPc1-CN and GST-PxIxITc2-CN binding. In contrast with PxIxITc2, the LxVP peptide not only blocked CN-NFAT binding but also inhibited CN phosphatase activity in vitro. Furthermore, exogenous LxVPc1 blocked NFATc2 phosphorylation and nuclear translocation in vivo. These results suggest a model in which the different CN binding characteristics of the PxIxIT and LxVP sites enable different NFAT members to influence each others activities in cells where they are co-expressed.