RESUMEN
Osteoblast adherence to bone surfaces is important for remodeling bone tissue. This study demonstrates that deficiency of TG-interacting factor 1 (Tgif1) in osteoblasts results in altered cell morphology, reduced adherence to collagen type I-coated surfaces, and impaired migration capacity. Tgif1 is essential for osteoblasts to adapt a regular cell morphology and to efficiently adhere and migrate on collagen type I-rich matrices in vitro. Furthermore, Tgif1 acts as a transcriptional repressor of p21-activated kinase 3 (Pak3), an important regulator of focal adhesion formation and osteoblast spreading. Absence of Tgif1 leads to increased Pak3 expression, which impairs osteoblast spreading. Additionally, Tgif1 is implicated in osteoblast recruitment and activation of bone surfaces in the context of bone regeneration and in response to parathyroid hormone 1-34 (PTH 1-34) treatment in vivo in mice. These findings provide important novel insights in the regulation of the cytoskeletal architecture of osteoblasts.
Asunto(s)
Citoesqueleto , Proteínas de Homeodominio , Osteoblastos , Proteínas Represoras , Transducción de Señal , Quinasas p21 Activadas , Animales , Ratones , Adhesión Celular , Movimiento Celular , Citoesqueleto/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Osteoblastos/metabolismo , Quinasas p21 Activadas/metabolismo , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/deficiencia , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/deficienciaRESUMEN
Monoamine neurotransmitter disorders present predominantly with neurologic features, including dystonic or dyskinetic cerebral palsy and movement disorders. Genetic conditions that lead to secondary defects in the synthesis, catabolism, transport, and metabolism of biogenic amines can lead to neurotransmitter abnormalities, which can present with similar features. Eleven patients with secondary neurotransmitter abnormalities were enrolled between 2011 and 2015. All patients underwent research-based whole exome and/or whole genome sequencing (WES/WGS). A trial of treatment with levodopa/carbidopa and 5-hydroxytryptophan was initiated. In six families with abnormal neurotransmitter profiles and neurological phenotypes, variants in known disease-causing genes (KCNJ6, SCN2A, CSTB in 2 siblings, NRNX1, KIF1A and PAK3) were identified, while one patient had a variant of uncertain significance in a candidate gene (DLG4) that may explain her phenotype. In 3 patients, no compelling candidate genes were identified. A trial of neurotransmitter replacement therapy led to improvement in motor and behavioral symptoms in all but two patients. The patient with KCNJ6 variant did not respond to L-dopa therapy, but rather experienced increased dyskinetic movements even at low dose of medication. The patient's symptoms harboring the NRNX1 deletion remained unaltered. This study demonstrates the utility of genome-wide sequencing in further understanding the etiology and pathophysiology of neurometabolic conditions, and the potential of secondary neurotransmitter deficiencies to serve as novel therapeutic targets. As there was a largely favorable response to therapy in our case series, a careful trial of neurotransmitter replacement therapy should be considered in patients with cerebrospinal fluid (CSF) monoamines below reference range.
Asunto(s)
Aminas Biogénicas/metabolismo , Levodopa/genética , Neurotransmisores/líquido cefalorraquídeo , Quinasas p21 Activadas/deficiencia , Adolescente , Adulto , Carbidopa/metabolismo , Niño , Preescolar , Combinación de Medicamentos , Femenino , Humanos , Cinesinas/metabolismo , Levodopa/metabolismo , Levodopa/uso terapéutico , Masculino , Adulto Joven , Quinasas p21 Activadas/metabolismoRESUMEN
RATIONALE: Secreted and membrane-bound proteins, which account for 1/3 of all proteins, play critical roles in heart health and disease. The endoplasmic reticulum (ER) is the site for synthesis, folding, and quality control of these proteins. Loss of ER homeostasis and function underlies the pathogenesis of many forms of heart disease. OBJECTIVE: To investigate mechanisms responsible for regulating cardiac ER function, and to explore therapeutic potentials of strengthening ER function to treat heart disease. METHODS AND RESULTS: Screening a range of signaling molecules led to the discovery that Pak (p21-activated kinase)2 is a stress-responsive kinase localized in close proximity to the ER membrane in cardiomyocytes. We found that Pak2 cardiac deleted mice (Pak2-CKO) under tunicamycin stress or pressure overload manifested a defective ER response, cardiac dysfunction, and profound cell death. Small chemical chaperone tauroursodeoxycholic acid treatment of Pak2-CKO mice substantiated that Pak2 loss-induced cardiac damage is an ER-dependent pathology. Gene array analysis prompted a detailed mechanistic study, which revealed that Pak2 regulation of protective ER function was via the IRE (inositol-requiring enzyme)-1/XBP (X-box-binding protein)-1-dependent pathway. We further discovered that this regulation was conferred by Pak2 inhibition of PP2A (protein phosphatase 2A) activity. Moreover, IRE-1 activator, Quercetin, and adeno-associated virus serotype-9-delivered XBP-1s were able to relieve ER dysfunction in Pak2-CKO hearts. This provides functional evidence, which supports the mechanism underlying Pak2 regulation of IRE-1/XBP-1s signaling. Therapeutically, inducing Pak2 activation by genetic overexpression or adeno-associated virus serotype-9-based gene delivery was capable of strengthening ER function, improving cardiac performance, and diminishing apoptosis, thus protecting the heart from failure. CONCLUSIONS: Our findings uncover a new cardioprotective mechanism, which promotes a protective ER stress response via the modulation of Pak2. This novel therapeutic strategy may present as a promising option for treating cardiac disease and heart failure.
Asunto(s)
Estrés del Retículo Endoplásmico , Insuficiencia Cardíaca/enzimología , Miocitos Cardíacos/enzimología , Quinasas p21 Activadas/metabolismo , Animales , Apoptosis , Línea Celular , Modelos Animales de Enfermedad , Terapia Genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/terapia , Células Madre Pluripotentes Inducidas/enzimología , Macaca mulatta , Masculino , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Miocitos Cardíacos/patología , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Transducción de Señal , Proteína 1 de Unión a la X-Box/metabolismo , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
Epithelial-mesenchymal transition (EMT) facilitates cancer invasion and metastasis and thus accelerates cancer progression. p21-activated kinase 4 (PAK4) is a critical regulator of prostate cancer (PC) progression. Here, we report that PAK4 activation promotes PC progression through the EMT regulator Slug. We find that phosphorylated PAK4S474 (pPAK4) levels, an index of PAK4 activation, were tightly associated with Gleason score (p < 0.001), a clinical indicator of PC progression, but not with prostate serum antigen levels or tumor stage. Stable silencing of PAK4 in PC cells reduced their potential for EMT, cellular invasion, and metastasis in vivo. PAK4 bound and directly phosphorylated Slug at two previously unknown sites, S158 and S254, which resulted in its stabilization. The non-phosphorylatable form SlugS158A/S254A upregulated transcription of CDH1, which encodes E-cadherin, and thus suppressed EMT and invasion, to a greater extent than did wild-type Slug. The strong EMT inducer TGF-ß elevated pPAK4 and pSlugS158 levels; PAK4 knockdown or introduction of a dominant-negative form of PAK4 inhibited both TGF-ß-stimulated EMT and an increase in pSlugS158 levels. Finally, immunohistochemistry revealed a positive correlation between pPAK4 and pSlugS158 but an inverse correlation between pSlugS158 and E-cadherin. The results suggest that the PAK4-Slug axis represents a novel pathway that promotes PC progression.
Asunto(s)
Transición Epitelial-Mesenquimal , Neoplasias de la Próstata/patología , Factores de Transcripción de la Familia Snail/metabolismo , Quinasas p21 Activadas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Progresión de la Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Metástasis de la Neoplasia , Fosforilación , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Factores de Transcripción de la Familia Snail/química , Transcripción Genética , Factor de Crecimiento Transformador beta/metabolismo , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
Foxp3, a key transcription factor that drives lineage differentiation of regulatory T cells (Tregs), was thought to imprint a unique and irreversible genetic signature within Tregs. Recent evidence, however, suggests that loss or attenuation of Foxp3 expression can cause Tregs to de-differentiate into effector T cells capable of producing proinflammatory cytokines. Herein, we report that the signaling kinase, p21-activated kinase 2 (Pak2), is essential for maintaining Treg stability and suppressive function. Loss of Pak2, specifically in Tregs, resulted in reduced expression of multiple Treg functional molecules, including Foxp3, CD25, Nrp-1 and CTLA-4, coupled with a loss of Treg suppressive function in vitro and in vivo. Interestingly, Pak2-deficient Tregs gained expression of Th2-associated cytokines and the transcription factor, Gata3, becoming Th2-like cells, explaining their inability to regulate immune responses. Collectively, these findings suggest Pak2 as an important signaling molecule for guarding against aberrant immune responses through regulating the stability of Foxp3+ Tregs and maintaining a suppressive Treg phenotype.
Asunto(s)
Linfocitos T Reguladores/metabolismo , Quinasas p21 Activadas/genética , Animales , Autoanticuerpos/sangre , Linfocitos B/citología , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/metabolismo , Citocinas/sangre , Factores de Transcripción Forkhead/metabolismo , Factor de Transcripción GATA3/metabolismo , Inmunoglobulinas/sangre , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Células Th2/citología , Células Th2/inmunología , Células Th2/metabolismo , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/metabolismoRESUMEN
PAK1 inhibitors are known to markedly improve social and cognitive function in several animal models of brain disorders, including autism, but the underlying mechanisms remain elusive. We show here that disruption of PAK1 in mice suppresses inhibitory neurotransmission through an increase in tonic, but not phasic, secretion of endocannabinoids (eCB). Consistently, we found elevated levels of anandamide (AEA), but not 2-arachidonoylglycerol (2-AG) following PAK1 disruption. This increased tonic AEA signaling is mediated by reduced cyclooxygenase-2 (COX-2), and COX-2 inhibitors recapitulate the effect of PAK1 deletion on GABAergic transmission in a CB1 receptor-dependent manner. These results establish a novel signaling process whereby PAK1 upregulates COX-2, reduces AEA and restricts tonic eCB-mediated processes. Because PAK1 and eCB are both critically involved in many other organ systems in addition to the brain, our findings may provide a unified mechanism by which PAK1 regulates these systems and their dysfunctions including cancers, inflammations and allergies.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Endocannabinoides/metabolismo , Hipocampo/fisiología , Quinasas p21 Activadas/metabolismo , Animales , Ratones , Ratones Noqueados , Quinasas p21 Activadas/deficienciaRESUMEN
AIMS: Despite its known cardiovascular benefits, the intracellular signalling mechanisms underlying physiological cardiac growth remain poorly understood. Therefore, the purpose of this study was to investigate a novel role of p21-activated kinase-1 (Pak1) in the regulation of exercise-induced cardiac hypertrophy. METHODS AND RESULTS: Wild-type (WT) and Pak1 KO mice were subjected to 6 weeks of treadmill endurance exercise training (ex-training). Cardiac function was assessed via echocardiography, in situ haemodynamics, and the pCa-force relations in skinned fibre preparations at baseline and at the end of the training regimen. Post-translational modifications to the sarcomeric proteins and expression levels of calcium-regulating proteins were also assessed following ex-training. Heart weight/tibia length and echocardiography data revealed that there was marked hypertrophy following ex-training in the WT mice, which was not evident in the KO mice. Additionally, following ex-training, WT mice demonstrated an increase in cardiac contractility, myofilament calcium sensitivity, and phosphorylation of cardiac myosin-binding protein C, cardiac TnT, and tropomyosin compared with KO mice. With ex-training in WT mice, there were also increased protein levels of calcineurin and increased phosphorylation of phospholamban. CONCLUSIONS: Our data suggest that Pak1 is essential for adaptive physiological cardiac remodelling and support previous evidence that demonstrates Pak1 signalling is important for cardiac growth and survival.
Asunto(s)
Calcineurina/metabolismo , Cardiomegalia Inducida por el Ejercicio , Cardiomegalia/enzimología , Técnicas de Inactivación de Genes , Miocardio/enzimología , Esfuerzo Físico , Remodelación Ventricular , Quinasas p21 Activadas/deficiencia , Adaptación Fisiológica , Animales , Proteínas de Unión al Calcio/metabolismo , Cardiomegalia/genética , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Proteínas Portadoras/metabolismo , Tolerancia al Ejercicio , Genotipo , Hemodinámica , Ratones Noqueados , Contracción Miocárdica , Miocardio/patología , Miofibrillas/enzimología , Fenotipo , Fosforilación , Carrera , Transducción de Señal , Tropomiosina/metabolismo , Troponina T/metabolismo , Quinasas p21 Activadas/genéticaRESUMEN
Although significant effort has been devoted to understanding the thymic development of Foxp3(+) regulatory T cells (Tregs), the precise signaling pathways that govern their lineage commitment still remain enigmatic. Our findings show a novel role for the actin cytoskeletal remodeling protein, p21-activated kinase 2 (Pak2), in Treg development and homeostasis. The absence of Pak2 in T cells resulted in a marked reduction in both thymus- and peripherally derived Tregs, accompanied by the development of spontaneous colitis in Pak2-deficient mice. Additionally, Pak2 was required for the proper differentiation of in vitro-induced Tregs as well as maintenance of Tregs. Interestingly, Pak2 was necessary for generating the high-affinity TCR- and IL-2-mediated signals that are required by developing Tregs for their lineage commitment. These findings provide novel insight into how developing thymocytes translate lineage-specific high-affinity TCR signals to adopt the Treg fate, and they further posit Pak2 as an essential regulator for this process.
Asunto(s)
Tolerancia Periférica/genética , Tolerancia Periférica/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Quinasas p21 Activadas/genética , Animales , Diferenciación Celular , Colitis/genética , Colitis/inmunología , Colitis/metabolismo , Colitis/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Homeostasis , Inmunofenotipificación , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Ratones , Ratones Noqueados , Fenotipo , Transducción de Señal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Timo/inmunología , Timo/metabolismo , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/metabolismoRESUMEN
p21-Activated kinase 2 (Pak2), a serine/threonine kinase, has been previously shown to be essential for hematopoietic stem cell (HSC) engraftment. However, Pak2 modulation of long-term hematopoiesis and lineage commitment remain unreported. Using a conditional Pak2 knockout mouse model, we found that disruption of Pak2 in HSCs induced profound leukopenia and a mild macrocytic anemia. Although loss of Pak2 in HSCs leads to less efficient short- and long-term competitive hematopoiesis than wild-type cells, it does not affect HSC self-renewal per se. Pak2 disruption decreased the survival and proliferation of multicytokine stimulated immature progenitors. Loss of Pak2 skewed lineage differentiation toward granulocytopoiesis and monocytopoiesis in mice as evidenced by (a) a three- to sixfold increase in the percentage of peripheral blood granulocytes and a significant increase in the percentage of granulocyte-monocyte progenitors in mice transplanted with Pak2-disrupted bone marrow (BM); (b)Pak2-disrupted BM and c-kit(+) cells yielded higher numbers of more mature subsets of granulocyte-monocyte colonies and polymorphonuclear neutrophils, respectively, when cultured in the presence of granulocyte-macrophage colony-stimulating factor. Pak2 disruption resulted, respectively, in decreased and increased gene expression of transcription factors JunB and c-Myc, which may suggest underlying mechanisms by which Pak2 regulates granulocyte-monocyte lineage commitment. Furthermore, Pak2 disruption led to (a) higher percentage of CD4(+) CD8(+) double positive T cells and lower percentages of CD4(+) CD8(-) or CD4(-) CD8(+) single positive T cells in thymus and (b) decreased numbers of mature B cells and increased numbers of Pre-Pro B cells in BM, suggesting defects in lymphopoiesis.
Asunto(s)
Diferenciación Celular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Quinasas p21 Activadas/metabolismo , Anemia Macrocítica/patología , Animales , Apoptosis , Proliferación Celular , Supervivencia Celular , Eliminación de Gen , Regulación de la Expresión Génica , Hematopoyesis , Leucopenia/patología , Linfopoyesis , Ratones Noqueados , Células Mieloides/patología , Fenotipo , Factores de Transcripción/metabolismo , Quinasas p21 Activadas/deficienciaRESUMEN
BACKGROUND: Impaired sarcoplasmic reticular Ca(2+) uptake resulting from decreased sarcoplasmic reticulum Ca(2+)-ATPase type 2a (SERCA2a) expression or activity is a characteristic of heart failure with its associated ventricular arrhythmias. Recent attempts at gene therapy of these conditions explored strategies enhancing SERCA2a expression and the activity as novel approaches to heart failure management. We here explore the role of Pak1 in maintaining ventricular Ca(2+) homeostasis and electrophysiological stability under both normal physiological and acute and chronic ß-adrenergic stress conditions. METHODS AND RESULTS: Mice with a cardiomyocyte-specific Pak1 deletion (Pak1(cko)), but not controls (Pak1(f/f)), showed high incidences of ventricular arrhythmias and electrophysiological instability during either acute ß-adrenergic or chronic ß-adrenergic stress leading to hypertrophy, induced by isoproterenol. Isolated Pak1(cko) ventricular myocytes correspondingly showed aberrant cellular Ca(2+) homeostasis. Pak1(cko) hearts showed an associated impairment of SERCA2a function and downregulation of SERCA2a mRNA and protein expression. Further explorations of the mechanisms underlying the altered transcriptional regulation demonstrated that exposure to control Ad-shC2 virus infection increased SERCA2a protein and mRNA levels after phenylephrine stress in cultured neonatal rat cardiomyocytes. This was abolished by the Pak1-knockdown in Ad-shPak1-infected neonatal rat cardiomyocytes and increased by constitutive overexpression of active Pak1 (Ad-CAPak1). We then implicated activation of serum response factor, a transcriptional factor well known for its vital role in the regulation of cardiogenesis genes in the Pak1-dependent regulation of SERCA2a. CONCLUSIONS: These findings indicate that Pak1 is required to maintain ventricular Ca(2+) homeostasis and electrophysiological stability and implicate Pak1 as a novel regulator of cardiac SERCA2a through a transcriptional mechanism.
Asunto(s)
Calcio/metabolismo , Ventrículos Cardíacos/enzimología , Miocitos Cardíacos/enzimología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Taquicardia Ventricular/enzimología , Quinasas p21 Activadas/metabolismo , Agonistas Adrenérgicos beta , Animales , Estimulación Cardíaca Artificial , Cardiomegalia/enzimología , Células Cultivadas , Modelos Animales de Enfermedad , Electrocardiografía , Regulación Enzimológica de la Expresión Génica , Ventrículos Cardíacos/fisiopatología , Homeostasis , Isoproterenol , Masculino , Ratones Noqueados , Interferencia de ARN , ARN Mensajero/metabolismo , Ratas , Factores de Riesgo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , Taquicardia Ventricular/inducido químicamente , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatología , Factores de Tiempo , Transcripción Genética , Transfección , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
p21-activated protein kinase-1 (Pak1) plays a role in insulin secretion and glucagon-like peptide-1 (GLP-1) production. Pak1(-/-) mice were found to carry a defect in ip pyruvate tolerance test (IPPTT), leading us to speculate whether Pak1 represses hepatic gluconeogenesis. We show here that the defect in IPPTT became more severe in aged Pak1(-/-) mice. In primary hepatocytes, 2,2'-dihydroxy-1,1'-dinaphthyldisulfide, a potent inhibitor of group I Paks, reduced basal glucose production (GP), attenuated forskolin- or glucagon-stimulated GP, and attenuated the stimulation of forskolin on the expression of Pck1 and G6pc. In addition, the capacity of primary hepatocytes isolated from Pak1(-/-) mice in GP at the basal level is significantly lower than that of the control littermates. These in vitro observations imply that the direct effect of Paks in hepatocytes is the stimulation of gluconeogenesis and that the impairment in IPPTT in Pak1(-/-) mice is due to the lack of Pak1 elsewhere. Consecutive ip injection of forskolin for 2 weeks increased gut proglucagon expression, associated with improved IPPTT in aged Pak1(-/-) mice and wild-type controls. In addition, administration of the DPP-IV (dipeptidyl peptidase-4) inhibitor sitagliptin for 1 week reversed the defect in IPPTT in aged Pak1(-/-) mice, associated with increased plasma GLP-1 levels. Our observations indicate a potential role of Pak1 in the gut/pancreas/liver axis in controlling glucose disposal and affirmed the therapeutic application of GLP-1 and DPP-IV inhibitors in attenuating hepatic gluconeogenesis.
Asunto(s)
Glucosa/metabolismo , Hígado/metabolismo , Transducción de Señal , Quinasas p21 Activadas/genética , Animales , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Péptido 1 Similar al Glucagón/sangre , Péptido 1 Similar al Glucagón/metabolismo , Hepatocitos/metabolismo , Masculino , Ratones , Ratones Noqueados , Pirazinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Fosfato de Sitagliptina , Triazoles/farmacología , Quinasas p21 Activadas/deficienciaRESUMEN
P21-activated kinases (PAKs) are involved in the regulation of multiple processes including cell proliferation, adhesion and migration. However, the current knowledge about their function is mainly based on results obtained in adherent cell types. We investigated the effect of group I PAK inhibition using the compound IPA-3 in a variety of human leukemic cell lines (JURL-MK1, MOLM-7, K562, CML-T1, HL-60, Karpas-299, Jurkat, HEL) as well as in primary blood cells. IPA-3 induced cell death with EC50 ranging from 5 to more than 20 µM. Similar range was found for IPA-3-mediated dephosphorylation of a known PAK downstream effector, cofilin. The cell death was associated with caspase-3 activation, PARP cleavage and apoptotic DNA fragmentation. In parallel, 20 µM IPA-3 treatment induced rapid and marked decrease of the cell adhesivity to fibronectin. Per contra, partial reduction of PAK activity using lower dose IPA-3 or siRNA resulted in a slight increase in the cell adhesivity. The changes in the cell adhesivity were also studied using real-time microimpedance measurement and by interference reflection microscopy. Significant differences in the intracellular IPA-3 level among various cell lines were observed indicating that an active mechanism is involved in IPA-3 transport.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Sanguíneas/citología , Células Sanguíneas/efectos de los fármacos , Disulfuros/farmacología , Fibronectinas/metabolismo , Naftoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinasas p21 Activadas/antagonistas & inhibidores , Factores Despolimerizantes de la Actina/química , Factores Despolimerizantes de la Actina/metabolismo , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Disulfuros/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen , Hematopoyesis/efectos de los fármacos , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Leucemia/patología , Linfoma/patología , Naftoles/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , Serina/metabolismo , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
OBJECTIVES: PAK5 and PAK6 are protein kinases highly expressed in the brain. Previously, we observed that Pak6 knockout mice gained significantly more weight during development than Pak5 knockout mice as well as wild-type controls and double-knockout mice lacking both Pak5 and Pak6. In this study, we assessed the effects of exercise on food intake and weight gain of these mice as well as their sensitivity to the stimulant effects of amphetamine. METHODS: Mice of each genotype were placed in cages with free access to run wheel exercise or in cages without run wheels for a total of 74 days. Food and fluid intake as well as body weight of each mouse were measured on a weekly basis. Finally, mice were given a high dose of amphetamine and activity levels were observed immediately thereafter for 90 minutes. Brains and testes of mice were assayed for protein levels of the estrogen alpha and progesterone receptors. RESULTS: While run wheel mice consumed significantly more food, they weighed less than non-run wheel mice. In addition, although Pak6 knockout mice consumed the same amount of food as wild-type mice, they were significantly heavier regardless of run wheel condition. Pak5 knockout mice were found to be more active than other genotypes after amphetamine treatment. Finally, protein levels of the progesterone and estrogen alpha receptors were altered in brain and testes of the Pak6 knockout mice. DISCUSSION: Collectively, these data suggest that PAK6 play a role in weight gain unrelated to exercise and caloric intake and that Pak5 knockout mice are more sensitive to the stimulant effects of amphetamine.
Asunto(s)
Anfetamina/farmacología , Esfuerzo Físico/fisiología , Aumento de Peso/fisiología , Quinasas p21 Activadas/fisiología , Animales , Química Encefálica , Ingestión de Líquidos/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Energía , Receptor alfa de Estrógeno/análisis , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esfuerzo Físico/efectos de los fármacos , Receptores de Progesterona/análisis , Testículo/química , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
Defective DNA replication can result in genomic instability, cancer and developmental defects. To understand the roles of DNA damage response (DDR) genes on carcinogenesis in mutants defective for core DNA replication components, we utilized the Mcm4(Chaos3/Chaos3) ('Chaos3') mouse model that, by virtue of an amino-acid alteration in MCM4 that destabilizes the MCM2-7 DNA replicative helicase, has fewer dormant replication origins and an increased number of stalled replication forks. This leads to genomic instability and cancer in most Chaos3 mice. We found that animals doubly mutant for Chaos3 and components of the ataxia telangiectasia-mutated (ATM) double-strand break response pathway (Atm, p21/Cdkn1a and Chk2/Chek2) had decreased tumor latency and/or increased tumor susceptibility. Tumor latency and susceptibility differed between genetic backgrounds and genders, with females demonstrating an overall greater cancer susceptibility to Atm and p21 deficiency than males. Atm deficiency was semilethal in the Chaos3 background and impaired embryonic fibroblast proliferation, suggesting that ATM drug inhibitors might be useful against tumors with DNA replication defects. Hypomorphism for the 9-1-1 component Hus1 did not affect tumor latency or susceptibility in Chaos3 animals, and tumors in these mice did not exhibit impaired ATR pathway signaling. These and other data indicate that under conditions of systemic replication stress, the ATM pathway is particularly important both for cancer suppression and viability during development.
Asunto(s)
Carcinogénesis/genética , Daño del ADN , Replicación del ADN/genética , Modelos Biológicos , Transducción de Señal/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular , Proliferación Celular , Quinasa de Punto de Control 2/deficiencia , Susceptibilidad a Enfermedades/patología , Femenino , Masculino , Ratones , Componente 4 del Complejo de Mantenimiento de Minicromosoma/deficiencia , Quinasas p21 Activadas/deficienciaRESUMEN
Persistent DNA damage triggers cellular senescence, which may play an important role in the pathogenesis of cigarette smoke (CS)-induced lung diseases. Both p21(CDKN1A) (p21) and poly(ADP-ribose) polymerase-1 (PARP-1) are involved in DNA damage and repair. However, the role of p21-PARP-1 axis in regulating CS-induced lung DNA damage and cellular senescence remains unknown. We hypothesized that CS causes DNA damage and cellular senescence through a p21-PARP-1 axis. To test this hypothesis, we determined the levels of γH2AX (a marker for DNA double-strand breaks) as well as non-homologous end joining proteins (Ku70 and Ku80) in lungs of mice exposed to CS. We found that the level of γH2AX was increased, whereas the level of Ku70 was reduced in lungs of CS-exposed mice. Furthermore, p21 deletion reduced the level of γH2AX, but augmented the levels of Ku70, Ku80, and PAR in lungs by CS. Administration of PARP-1 inhibitor 3-aminobenzamide increased CS-induced DNA damage, but lowered the levels of Ku70 and Ku80, in lungs of p21 knockout mice. Moreover, 3-aminobenzamide increased senescence-associated ß-galactosidase activity, but decreased the expression of proliferating cell nuclear antigen in mouse lungs in response to CS. Interestingly, 3-aminobenzamide treatment had no effect on neutrophil influx into bronchoalveolar lavage fluid by CS. These results demonstrate that the p21-PARP-1 pathway is involved in CS-induced DNA damage and cellular senescence.
Asunto(s)
Senescencia Celular/genética , Pulmón/metabolismo , Poli(ADP-Ribosa) Polimerasas/genética , Transducción de Señal , Fumar/metabolismo , Quinasas p21 Activadas/genética , Animales , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Benzamidas/farmacología , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Autoantígeno Ku , Pulmón/patología , Ratones , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasa-1 , Poli(ADP-Ribosa) Polimerasas/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fumar/genética , Fumar/patología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Quinasas p21 Activadas/deficienciaRESUMEN
Altered neurogenesis in adult hippocampus is implicated in cognition impairment and depression. Inflammation is a potent inhibitor of neurogenesis. The cyclin-dependent kinase inhibitor p21(Cip1) (p21) restrains cell cycle progression and arrests the cell in the G1 phase. We recently showed that p21 is expressed in neuronal progenitors and regulates proliferation of these cells in the subgranular zone of the dentate gyrus of hippocampus where adult neurogenesis occurs. The current study suggests that p21 is induced in vivo in the hippocampus of WT mice in response to acute systemic inflammation caused by LPS injections, restrains neuronal progenitor proliferation and protects these cells from inflammation-induced apoptosis. In intact p21-/- hippocampus, neuronal progenitors proliferate more actively as assessed by BrdU incorporation, and give rise to increased number of DCX positive neuroblasts. However, when mice were treated with LPS, the number of neuroblasts decreased due to induced subgranular zone apoptosis. In vitro, differentiating Tuj-1 positive neuroblasts isolated from p21-/- hippocampus exhibited increased proliferation rate, measured by Ki-67 staining, as compared to WT cells (p<0.05). In WT neuronal progenitors treated with IL-6, the number of p21-positive cells was increased (p<0.05), and this led to Tuj-1(+) cell proliferation restraint, whereas the number of proliferating GFAP(+) astrocytes was increased ~ 2-fold. Thus, when p21 is intact, inflammation might divert neuronal progenitors towards astrogliogenesis by inducing p21. At the same time, when p21 is lacking, no effects of IL-6 on proliferation of Tuj-1(+) cells or GFAP(+) cells are detected in differentiating p21-/- neuronal progenitors. These results underscore the important role of p21 controlling hippocampal neuronal differentiation during inflammation.
Asunto(s)
Apoptosis/genética , Hipocampo/fisiopatología , Inflamación/patología , Células-Madre Neurales/fisiología , Neurogénesis/genética , Quinasas p21 Activadas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Proteína Doblecortina , Inflamación/inducido químicamente , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/efectos de los fármacos , Quinasas p21 Activadas/deficienciaRESUMEN
The p21-activated kinases are effector proteins for Rho-family GTPases. PAK4, PAK5, and PAK6 are the group II PAKs associated with neurite outgrowth, filopodia formation, and cell survival. Pak4 knockout mice are embryonic lethal, while Pak5, Pak6, and Pak5/Pak6 double knockout mice are viable and fertile. Our previous work found that the double knockout mice exhibit locomotor changes and learning and memory deficits. We also found some differences with Pak5 and Pak6 single knockout mice and the present work further explores the potential differences of the Pak5 knockout and Pak6 knockout mice in comparison with wild type mice. The Pak6 knockout mice were found to weigh significantly more than the other genotypes. The double knockout mice were found to be less active than the other genotypes. The Pak5 knockout mice and the double knockout mice performed worse on the rotorod test. All the knockout genotypes were found to be less aggressive in the resident intruder paradigm. The double knockout mice were, once again, found to perform worse in the active avoidance assay. These results indicate, that although some behavioral differences are seen in the Pak5 and Pak6 single knockout mice, the double knockout mice exhibit the greatest changes in locomotion and learning and memory.
Asunto(s)
Técnicas de Inactivación de Genes , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genética , Agresión , Animales , Ansiedad/enzimología , Ansiedad/genética , Conducta Animal , Peso Corporal/genética , Genotipo , Locomoción/genética , Masculino , Ratones , Retención en PsicologíaRESUMEN
There is an increasing evidence that the oncogenic kinase PAK1 is responsible not only for malignant transformation, but also for several other diseases such as inflammatory diseases (asthma and arthritis), infectious diseases including malaria, AIDS, and flu, as well as a series of neuronal diseases/disorders (neurofibromatosis, tuberous sclerosis, Alzheimer's diseases, Huntington's disease, epilepsy, depression, learning deficit, etc.) which often cause premature death. Interestingly, a few natural PAK1-blockers such as curcumin, caffeic acid (CA) and rosmarinic acid (RA) extend the lifespan of the nematode Caenorhabditis elegans or fruit flies. Here, to explore the possibility that C. elegans could provide us with a quick and inexpensive in vivo screening system for a series of more potent but safe (non-toxic) PAK1-blocking therapeutics, we examined the effects of PAK1-deficiency or down-regulation on a few selected functions of this worm, including reproduction, expression of HSP16.2 gene, and lifespan. In short, we found that PAK1 promotes reproduction, whereas it inactivates HSP16.2 gene and shortens lifespan, as do PI-3 kinase (AGE-1), TOR, and insulin-like signalling /ILS (Daf-2) in this worm. These findings not only support the "trade-off" theory on reproduction versus lifespan, but also suggest the possibility that the reduced reproduction (or HSP16.2 gene activation) of this worm could be used as the first indicator of extended lifespan for a quick in vivo screening for PAK1-blockers.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Choque Térmico/genética , Longevidad/genética , Quinasas p21 Activadas/antagonistas & inhibidores , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Ácidos Cafeicos/farmacología , Regulación hacia Abajo , Proteínas Fluorescentes Verdes/genética , Longevidad/efectos de los fármacos , Alcohol Feniletílico/análogos & derivados , Alcohol Feniletílico/farmacología , Fenilpropionatos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes de Fusión/genética , Reproducción/efectos de los fármacos , Reproducción/genética , Transducción de Señal/efectos de los fármacos , Quinasas p21 Activadas/deficiencia , Quinasas p21 Activadas/genéticaRESUMEN
The p21-activated kinase (PAK) serine/threonine kinases are important effectors of the small GTPases Rac and Cdc42, and play significant roles in controlling cell growth, motility, and transformation. Knockdown of PAK4 or PAK1 inhibited the proliferation of mutant KRAS or BRAF colon cancer cells in vitro. Dependence on PAK4 or PAK1 protein for colon cancer cell proliferation was independent of PAK4 or PAK1 protein expression levels. Mutant KRAS HCT116 colorectal cells were the most sensitive to PAK4 or PAK1 knockdown resulting in the potent inhibition of anchorage-dependent and -independent proliferation as well as the formation and proliferation of HCT116 colon cancer spheroids. This inhibition of proliferation did not correlate with inhibition of RAF/MEK/ERK or PI3K/AKT signaling. In HCT116 cells, knockdown of PAK4 or PAK1 caused changes to the actin cytoskeleton resulting in reduced basal spread and cell elongation and increased cell rounding. These cytoskeletal rearrangements seemed to be independent of LIMK/cofilin/paxillin phosphorylation. PAK4 or PAK1 knockdown initially induced growth arrest in HCT116 cells followed by cell death at later time points. Inhibition of the antiapoptotic proteins Bcl-2 and Bcl-X(L) with the pharmacologic inhibitor ABT-737 increased effector caspase activation and apoptosis, and reduced cell survival with PAK4 or PAK1 knockdown. These results support a role for the PAKs in the proliferation of mutant KRAS-driven colorectal carcinoma cells via pathways not involving RAF/MEK/ERK and PI3K/AKT signaling.
Asunto(s)
Neoplasias del Colon/genética , Genes ras , Proteínas Proto-Oncogénicas c-akt/genética , Quinasas p21 Activadas/deficiencia , Procesos de Crecimiento Celular/genética , Neoplasias del Colon/enzimología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HCT116 , Células HT29 , Humanos , Sistema de Señalización de MAP Quinasas , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Análisis de Supervivencia , Transfección , Quinasas p21 Activadas/genética , Quinasas p21 Activadas/metabolismoRESUMEN
The RAS genes are the most commonly mutated oncogenes in human cancer and present a particular therapeutic dilemma, as direct targeting of Ras proteins by small molecules has proved difficult. Signaling pathways downstream of Ras, in particular Raf/Mek/Erk and PI3K/Akt/mTOR, are dominated by lipid and protein kinases that provide attractive alternate targets in Ras-driven tumors. As p21-activated kinase 1 (Pak1) has been shown to regulate both these signaling pathways and is itself upregulated in many human cancers, we assessed the role of Pak1 in Ras-driven skin cancer. In human squamous cell carcinoma (SCC), we found a strong positive correlation between advanced stage and grade and PAK1 expression. Using a mouse model of Kras-driven SCC, we showed that deletion of the mouse Pak1 gene led to markedly decreased tumorigenesis and progression, accompanied by near total loss of Erk and Akt activity. Treatment of Kras(G12D) mice with either of two distinct small molecule Pak inhibitors (PF3758309 and FRAX597) caused tumor regression and loss of Erk and Akt activity. Tumor regression was also seen in mice treated with a specific Mek inhibitor, but not with an Akt inhibitor. These findings establish Pak1 as a new target in KRAS-driven tumors and suggest a mechanism of action through the Erk, but not the Akt, signaling pathway.