RESUMO
In cardiac wound healing following myocardial infarction (MI), relatively inactive resident cardiac fibroblasts phenoconvert to hypersynthetic/secretory myofibroblasts that produce large quantities of extracellular matrix (ECM) and fibrillar collagen proteins. Our laboratory and others have identified TGFß1 as being a persistent stimulus in the chronic and inappropriate wound healing phase that is marked by hypertrophic scarring and eventual stiffening of the entire myocardium, ultimately leading to the pathogenesis of heart failure following MI. Ski is a potent negative regulator of TGFß/Smad signaling with known antifibrotic effects. Conversely, Scleraxis is a potent profibrotic basic helix-loop-helix transcription factor that stimulates fibrillar collagen expression. We hypothesize that TGFß1 induces Scleraxis expression by a novel Smad-independent pathway. Our data support the hypothesis that Scleraxis expression is induced by TGFß1 through a Smad-independent pathway in the cardiac myofibroblast. Specifically, we demonstrate that TGFß1 stimulates p42/44 (Erk1/2) kinases, which leads to increased Scleraxis expression. Inhibition of MEK1/2 using U0126 led to a sequential temporal reduction of phospho-p42/44 and subsequent Scleraxis expression. We also found that adenoviral Ski expression in primary myofibroblasts caused a significant repression of endogenous Scleraxis expression at both the mRNA and protein levels. Thus we have identified a novel TGFß1-driven, Smad-independent, signaling cascade that may play an important role in regulating the fibrotic response in activated cardiac myofibroblasts following cardiac injury.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Miócitos Cardíacos/metabolismo , Proteínas Smad/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Células 3T3 , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Butadienos/farmacologia , Células COS , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Chlorocebus aethiops , Fibrose/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Remodeling of the extracellular matrix is beneficial during the acute wound healing stage following tissue injury. In the short term, resident fibroblasts and myofibroblasts regulate the matrix remodeling process through production of matricellular protein components that provide structural support to the damaged tissue. This process is largely governed by the transforming growth factor-ß1 (TGF-ß1) pathway, a critical mediator of the remodeling process. In the long term, chronic activation of the TGF-ß1 pathway promotes excessive synthesis and deposition of matrix proteins, including fibrillar collagens, which ultimately leads to organ failure. SnoN (and its alternatively-spliced isoforms SnoN2, SnoA, and SnoI) is one of four members of a family of negative regulators of TGF-ß1 signaling that includes Ski and functional Smad-suppressing elements on chromosomes 15 and 18. SnoN has been shown to be structurally and functionally similar to Ski and has been demonstrated to directly interact with Ski to abrogate gene expression. Despite this, little progress has been made in delineating a specific role for SnoN in the regulation of myofibroblast phenotype and function. This review outlines the current body of knowledge of what we refer to as the "Ski-Sno superfamily," with a focus on the structural and functional importance of SnoN in mediating the fibrotic response by myofibroblasts following tissue injury.
Assuntos
Pulmão/metabolismo , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Smad/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Colágeno/genética , Colágeno/metabolismo , Fibrose/metabolismo , Humanos , Pulmão/patologia , Miocárdio/patologia , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
µ-Calpain is a Ca(2+)-activated protease abundant in mammalian tissues. Here, we examined the effects of µ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to µ-calpain and their Na(+)-dependent (I(1)) and Ca(2+)-dependent (I(2)) regulatory phenotypes were assessed. For these exchangers, I(1) inactivation is evident as a Na(+)(i)-dependent decay of peak outward currents whereas I(2) regulation manifests as outward current activation by micromolar Ca(2+)(i) concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca(2+)(i) levels alleviate I(1) inactivation. Our results show that (i) µ-calpain selectively ablates Ca(2+)-dependent (I(2)) regulation leading to a constitutive activation of exchange current, (ii) µ-calpain has much smaller effects on Na(+)-dependent (I(1)) regulation, produced by a slight destabilization of the I(1) state, and (iii) Ca(2+)-dependent regulation (I(2)) and Ca(2+)-mediated alleviation of I(1) appear to be functionally distinct mechanisms, the latter of which is left largely intact after µ-calpain treatment. The ability of µ-calpain to selectively and constitutively activate Na(+)-Ca(2+) exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.
Assuntos
Processamento Alternativo/fisiologia , Encéfalo/metabolismo , Calpaína/metabolismo , Rim/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Calpaína/genética , Cães , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador de Sódio e Cálcio/genética , Xenopus laevisRESUMO
Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.
Assuntos
Antiporters/química , Antiporters/metabolismo , Cálcio/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Sódio/metabolismo , Animais , Antiporters/genética , Sítios de Ligação , Cristalografia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Domínios e Motivos de Interação entre Proteínas/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Células Receptoras Sensoriais/fisiologiaRESUMO
Members of the Na+/Ca2+ exchanger (NCX) family are important regulators of cytosolic Ca2+ in myriad tissues and are highly conserved across a wide range of species. Three distinct NCX genes and numerous splice variants exist in mammals, many of which have been characterized in a variety of heterologous expression systems. Recently, however, we discovered a fourth NCX gene (NCX4), which is found exclusively in teleost, amphibian, and reptilian genomes. Zebrafish (Danio rerio) NCX4a encodes for a protein of 939 amino acids and shows a high degree of identity with known NCXs. Although knockdown of NCX4a activity in zebrafish embryos has been shown to alter left-right patterning, it has not been demonstrated that NCX4a functions as a NCX. In this study, we 1) demonstrated, for the first time, that this gene encodes for a novel NCX; 2) characterized the tissue distribution of zebrafish NCX4a; and 3) evaluated its kinetic and transport properties. While ubiquitously expressed, the highest levels of NCX4a expression occurred in the brain and eyes. NCX4a exhibits modest levels of Na+-dependent inactivation and requires much higher levels of regulatory Ca2+ to activate outward exchange currents. NCX4a also exhibited extremely fast recovery from Na+-dependent inactivation of outward currents, faster than any previously characterized wild-type exchanger. While this result suggests that the Na+-dependent inactive state of NCX4a is far less stable than in other NCX family members, this exchanger was still strongly inhibited by 2 microM exchanger inhibitory peptide. We demonstrated that a new putative member of the NCX gene family, NCX4a, encodes for a NCX with unique functional properties. These data will be useful in understanding the role that NCX4a plays in embryological development as well as in the adult, where it is expressed ubiquitously.
Assuntos
Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Encéfalo/enzimologia , Olho/enzimologia , Cinética , Potenciais da Membrana , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Alinhamento de Sequência , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
The activity of the cardiac Na(+)/Ca(2+) exchanger (NCX1.1) undergoes continuous modulation during the contraction-relaxation cycle because of the accompanying changes in the electrochemical gradients for Na(+) and Ca(2+). In addition, NCX1.1 activity is also modulated via secondary, ionic regulatory mechanisms mediated by Na(+) and Ca(2+). In an effort to evaluate how ionic regulation influences exchange activity under pulsatile conditions, we studied the behavior of the cloned NCX1.1 during frequency-controlled changes in intracellular Na(+) and Ca(+) (Na(i)(+) and Ca(i)(2+)). Na(+)/Ca(2+) exchange activity was measured by the giant excised patch-clamp technique with conditions chosen to maximize the extent of Na(+)- and Ca(2+)-dependent ionic regulation so that the effects of variables such as pulse frequency and duration could be optimally discerned. We demonstrate that increasing the frequency or duration of solution pulses leads to a progressive decline in pure outward, but not pure inward, Na(+)/Ca(2+) exchange current. However, when the exchanger is permitted to alternate between inward and outward transport modes, both current modes exhibit substantial levels of inactivation. Changes in regulatory Ca(2+), or exposure of patches to limited proteolysis by alpha-chymotrypsin, reveal that this "coupling" is due to Na(+)-dependent inactivation originating from the outward current mode. Under physiological ionic conditions, however, evidence for modulation of exchange currents by Na(i)(+)-dependent inactivation was not apparent. The current approach provides a novel means for assessment of Na(+)/Ca(2+) exchange ionic regulation that may ultimately prove useful in understanding its role under physiological and pathophysiological conditions.
Assuntos
Coração/fisiologia , Modelos Biológicos , Trocador de Sódio e Cálcio/fisiologia , Animais , Cálcio/metabolismo , Quimotripsina/farmacologia , Clonagem Molecular , Citoplasma/metabolismo , Feminino , Potenciais da Membrana/fisiologia , Oócitos/citologia , Oócitos/fisiologia , Técnicas de Patch-Clamp , Sódio/metabolismo , Trocador de Sódio e Cálcio/genética , Xenopus laevisRESUMO
SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline) has recently been described as a potent and selective inhibitor of Na(+)-Ca(2+) exchange in cardiac, neuronal, and renal preparations. The inhibitory effects of SEA0400 were investigated on the cloned cardiac Na(+)-Ca(2+) exchanger, NCX1.1, expressed in Xenopus laevis oocytes to gain insight into its inhibitory mechanism. Na(+)-Ca(2+) exchange currents were measured using the giant excised patch technique using conditions to evaluate both inward and outward currents. SEA0400 inhibited outward Na(+)-Ca(2+) exchange currents with high affinity (IC(50) = 78 +/- 15 and 23 +/- 4 nM for peak and steady-state currents, respectively). Considerably less inhibitory potency (i.e., micromolar) was observed for inward currents. The inhibitory profile was reexamined after proteolytic treatment of excised patches with alpha-chymotrypsin, a procedure that eliminates ionic regulatory mechanisms. After this treatment, an IC(50) value of 1.2 +/- 0.6 microM was estimated for outward currents, whereas inward currents became almost insensitive to SEA0400. The inhibitory effects of SEA0400 on outward exchange currents were evident at both high and low concentrations of regulatory Ca(2+), although distinct features were noted. SEA0400 accelerated the inactivation rate of outward currents. Based on paired pulse experiments, SEA0400 altered the recovery of exchangers from the Na(+)(i)-dependent inactive state, particularly at higher regulatory Ca(2+)(i) concentrations. Finally, the inhibitory potency of SEA0400 was strongly dependent on the intracellular Na(+) concentration. Our data confirm that SEA0400 is the most potent inhibitor of the cardiac Na(+)-Ca(2+) exchanger described to date and provide a reasonable explanation for its apparent transport mode selectivity.
Assuntos
Compostos de Anilina/farmacologia , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Compostos de Anilina/uso terapêutico , Animais , Camundongos , Éteres Fenílicos/uso terapêutico , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Transfecção , Xenopus laevisRESUMO
SEA0400 (SEA) blocks cardiac and neuronal Na+-Ca2+ exchange with the highest affinity of any known inhibitor, yet very little is known about its molecular mechanism of action. Previous data from our lab suggested that SEA stabilizes or modulates the transition of NCX1.1 exchangers into a Na+i-dependent (I1) inactive state. To test this hypothesis, we examined the effects of SEA on mutant exchangers with altered ionic regulatory properties. With mutants where Na+i-dependent inactivation is absent, the effects of SEA were greatly reduced. Conversely, with mutants displaying accelerated Na+i-dependent inactivation, block of NCX1.1 by SEA was either enhanced or unchanged, depending upon the phenotype of the particular mutation. With mutant exchangers where Ca2+i-dependent (I2) inactivation was suppressed, block of exchange currents by SEA was similar to that observed for wild-type NCX1.1. These data strongly support the involvement of I1 inactivation in the inhibitory mechanism of NCX1.1 by SEA, whereas I2 inactivation does not seem to serve an important role. The involvement of processes regulated by intracellular Na+ in the inhibitory mechanism of SEA may prove to be particularly important when considering the potential cardioprotective effects of this agent.
Assuntos
Compostos de Anilina/farmacologia , Oócitos/efeitos dos fármacos , Éteres Fenílicos/farmacologia , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Eletrofisiologia , Oócitos/metabolismo , Oócitos/fisiologia , Sódio/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidores , Xenopus laevisRESUMO
The electrophysiological effects of the benzothiazepine 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-one (CGP-37157) (CGP) were investigated on the canine (NCX1.1) and Drosophila (CALX1.1) plasmalemmal Na+-Ca2+ exchangers. These exchangers were selected for study because they show opposite responses to cytoplasmic regulatory Ca2+, thereby allowing us to examine the role of this regulatory mechanism in the inhibitory effects of CGP. CGP blocked Na+-Ca2+ exchange current mediated by both transporters with moderate potency (IC50 values = approximately 3-17 microM) compared with other recently reported blockers of Na+-Ca2+ exchange [e.g., 2-[4-[2,5-difluorophenyl) methoxy]phenoxy]phenoxy]-5-ethoxyaniline (KB-R7943) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (SEA0400)]. Experiments using alpha-chymotrypsin to remove autoregulation of Na+-Ca2+ exchange showed that block by CGP was reduced, suggesting that part of the effects of this drug may require intact ionic regulatory mechanisms. For NCX1.1, the inhibition produced by CGP was greater for outward Na+-Ca2+ exchange currents compared with inward currents. When CALX1.1 was examined, the extent of inhibition was similar for both inward and outward exchange currents. Although the extent and potency of CGP-mediated inhibition of Na+-Ca2+ exchange are less than those observed with SEA0400 and KB-R7943, our data demonstrate that CGP constitutes a novel class of plasmalemmal Na+-Ca2+ exchange inhibitors. Moreover, the widespread use of CGP as a selective mitochondrial Na+-Ca2+ exchange inhibitor should be reconsidered in light of these additional inhibitory effects.