RESUMO
Objective: Ultraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKCα-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKCα nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation. Methods: We studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKCα nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation. Results: Our time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKCα in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKCα nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNFα, IFNγ, and IFNα in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency. Conclusions: ROCK regulated NETosis through modulation of PKCα nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB-induced skin inflammation by attenuation of NETosis and NET-associated cytokines.
Assuntos
Neutrófilos , Proteína Quinase C-alfa , Actinas/metabolismo , Actomiosina/metabolismo , Actomiosina/farmacologia , Animais , Inflamação/metabolismo , Camundongos , Neutrófilos/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Interphase phosphorylation of lamin-A,C depends dynamically on a cell's microenvironment, including the stiffness of extracellular matrix. However, phosphorylation dynamics is poorly understood for diseased forms such as progerin, a permanently farnesylated mutant of LMNA that accelerates aging of stiff and mechanically stressed tissues. Here, fine-excision alignment mass spectrometry (FEA-MS) is developed to quantify progerin and its phosphorylation levels in patient iPS cells differentiated to mesenchymal stem cells (MSCs). The stoichiometry of total A-type lamins (including progerin) versus B-type lamins measured for Progeria iPS-MSCs prove similar to that of normal MSCs, with total A-type lamins more abundant than B-type lamins. However, progerin behaves more like farnesylated B-type lamins in mechanically-induced segregation from nuclear blebs. Phosphorylation of progerin at multiple sites in iPS-MSCs cultured on rigid plastic is also lower than that of normal lamin-A and C. Reduction of nuclear tension upon i) cell rounding/detachment from plastic, ii) culture on soft gels, and iii) inhibition of actomyosin stress increases phosphorylation and degradation of lamin-C > lamin-A > progerin. Such mechano-sensitivity diminishes, however, with passage as progerin and DNA damage accumulate. Lastly, transcription-regulating retinoids exert equal effects on both diseased and normal A-type lamins, suggesting a differential mechano-responsiveness might best explain the stiff tissue defects in Progeria.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Lamina Tipo A/metabolismo , Mecanotransdução Celular , Células-Tronco Mesenquimais/metabolismo , Actomiosina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Lamina Tipo A/antagonistas & inibidores , Mecanotransdução Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica , Paxilina/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Quinases da Família src/metabolismo , Actomiosina/farmacologia , Anisotropia , Adesão Celular , Linhagem Celular Tumoral , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Matriz Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Microscopia de Fluorescência/métodos , RNA Interferente Pequeno/metabolismo , Complexo Shelterina , Transdução de Sinais , Vinculina/metabolismoRESUMO
In our previous general screening experiments, we found that NSU-242, a dibenzothiepine derivative (1-10 mg/kg), inhibited antigen-induced immediate asthmatic response in actively sensitized guinea pigs in a dose-dependent manner. The purpose of the present study was to assess the mechanism of the relaxing effect of NSU-242 on smooth muscle contractions in isolated smooth muscle tissues of the porcine trachea and rat aorta. NSU-242 administration resulted in a concentration-dependent inhibition of the tracheal-tissue contractions induced by carbachol and high K(+) and the aortic-tissue contractions induced by norepinephrine and high K(+). The IC(50) values of these inhibitions were 1-10 microM, and there was no selectivity for the type of stimulation. In tracheal tissue, the relaxations were accompanied by neither changes in cAMP nor changes in cGMP. Carbachol (1 microM) and high K(+) (59.2 mM) increased myosin light chain (MLC) phosphorylation in the trachea, and NSU-242 (3-30 microM) had no effect on the level of MLC phosphorylation. Furthermore, NSU-242 (300 microM) had no effect on contractions in membrane-permeabilized tracheal tissue. FITC-phalloidin staining of the actin fiber in cultured vascular smooth muscle cells (A7r5) indicated that NSU-242 (10-100 microM) altered the configuration of actin stress fiber in the cytosol. However, unlike cytochalasin D, NSU-242 did not inhibit actin polymerization as assessed by in vitro assay. These results suggest that NSU-242 inhibits smooth muscle contractions without any effect on the Ca(2+)-dependent MLC phosphorylation. NSU-242 may uncouple the force generated by the activated actomyosin interaction, possibly by modifying the actin assembly in smooth muscle cells without a direct effect on actin molecules.
Assuntos
Dibenzotiepinas/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Traqueia/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actomiosina/farmacologia , Animais , Broncoconstrição/efeitos dos fármacos , Carbacol/antagonistas & inibidores , Carbacol/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Cobaias , Técnicas In Vitro , Agonistas Muscarínicos/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Cadeias Leves de Miosina/metabolismo , Fosforilação , Cloreto de Potássio/antagonistas & inibidores , Cloreto de Potássio/farmacologia , SuínosRESUMO
Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Antineoplásicos/farmacologia , Axônios/fisiologia , Depsipeptídeos , Microtúbulos/metabolismo , Neurônios/metabolismo , Peptídeos Cíclicos/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Actomiosina/farmacologia , Animais , Axônios/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha/citologia , Embrião de Galinha/efeitos dos fármacos , Embrião de Galinha/metabolismo , Efrina-A2/farmacologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Técnicas Imunoenzimáticas , Peptídeos e Proteínas de Sinalização Intracelular , Microinjeções , Microscopia de Vídeo , Microtúbulos/efeitos dos fármacos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Neurônios/citologia , Neurônios/efeitos dos fármacos , Faloidina/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Retina/citologia , Retina/efeitos dos fármacos , Retina/metabolismo , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is <<1 microM at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a Kd below 0.1 microM. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for Kd's as low as 10 nM and we demonstrate that the Kd's can be estimated using only microgram quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.
Assuntos
Actinas/metabolismo , Miosinas/química , Miosinas/metabolismo , Actinas/efeitos dos fármacos , Actinas/isolamento & purificação , Actomiosina/metabolismo , Actomiosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Ligação Competitiva/fisiologia , Relação Dose-Resposta a Droga , Fluorometria , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/farmacologia , Miosinas/isolamento & purificação , Faloidina/farmacologia , Pirenos/farmacologia , CoelhosRESUMO
Nuclear histones bind to and precipitate the major contractile proteins, actin and myosin. The binding of histone to actin seems to reach saturation at 2:1 ratio, the interaction may serve some regulatory function(s) in intranuclear events. The binding of histone to myosin is not saturable, and, although it inhibits the actin-activated Mg2+-dependent myosin ATPase activity, does therefore not seem of physiological importance.
Assuntos
Proteínas Contráteis/análise , Histonas/análise , Miosinas/antagonistas & inibidores , Actinas/análise , Actinas/farmacologia , Actomiosina/farmacologia , Animais , Sítios de Ligação , Galinhas , Histonas/fisiologia , Miosinas/análise , Nefelometria e Turbidimetria , UltracentrifugaçãoAssuntos
Actomiosina/farmacologia , Músculo Liso Vascular/metabolismo , Vanádio/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Animais , Cálcio/fisiologia , Técnicas In Vitro , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , SuínosRESUMO
Reconstituted actomyosin (ATP phosphohydrolase, EC 3.6.1.3) (0.400 mg F-actin/mg myosin) in 10.0 muM ATP loses 96% of its specific ATPase activity when its reaction concentration is decreased from 42.0 mug/ml down to 0.700 mug/ml. The loss of specific activity at the very low enzyme concentrations is prevented by the addition of more F-actin to 17.6 mug/ml. It is concluded that at low actomyosin concentrations the complex dissociates into free myosin with a very low specific ATPase activity and free F-actin with no ATPase. The dissociation of the essential low molecular weight subunits of myosin from the heavy chains at very low actomyosin concentrations may be a contributing factor. Actomyosin has its maximum specific activity at pH 7.8-8.2. The Km for ATP is 9.4 muM, which is at least 20-fold greater than myosin's Km for ATP. The actin-activated ATPase of myosin follows hyperbolic kinetics with varying F-actin concentrations. The Km values for F-actin are 0.110 muM (4.95 mug/ml) at pH 7.4 and 0.241 muM (10.8 mug/ml) at pH 7.8. The actin-activated maximum turnover numbers for myosin are 9.3 s-1 at pH 7.4 and 11.6 s-1 at pH 7.8. The actomyosin ATPase is inhibited by KCl. This KCl inhibition is not competitive with respect to F-actin, and it is not a simple form of non-competitive inhibition.
Assuntos
Actinas/farmacologia , Adenosina Trifosfatases/metabolismo , Miosinas/metabolismo , Actomiosina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Ativação Enzimática , Concentração de Íons de Hidrogênio , Cinética , Cloreto de Potássio/farmacologia , CoelhosRESUMO
A nonlinear least squares analysis of Scatchard plots of Ca++ binding to troponin, native tropomyosin, and myosin B demonstrates that troponin does not possess two classes of independent binding sites. Since the data cannot be accounted for by assuming more than two classes of independent sites, we conclude that binding of one Ca decreases the affinity of troponin for binding the next. Negative interaction is increased in the presence of tropomyosin and further increased by actomyosin. The maximal Ca++ affinity of troponin at low fractional occupancy is not affected by tropomyosin but may be significantly reduced by actomyosin. Because of negative interaction, troponin's Ca++ affinity should vary during the course of contraction. This variation could profoundly affect translocations of Ca between troponin and reticulum and the time course of the active state.