RESUMEN
PURPOSE: Calpain-mediated C-terminal cleavage of alpha A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated alpha A-crystallins. METHODS: Recombinant wild-type (wt) alpha A-crystallin and C-terminal truncated alpha A(1-168)-, alpha A(1-163)-, and alpha A(1-162)-crystallins were expressed in Escherichia coli and purified to homogeneity. The wt and truncated alpha A-crystallins were labeled with (125)I, and proteolytic degradation was determined using both lens fiber lysate and reticulocyte lysate as sources of ubiquitinating and proteolytic enzymes. Far UV circular dichroism, tryptophan fluorescence intensity, and binding to the hydrophobic fluorescence probe Bis-ANS were used to characterize the wt and truncated alpha A-crystallins. Oligomer sizes of these crystallins were determined by multiangle light-scattering. RESULTS: Whereas wt alpha A-crystallin was degraded moderately in both lens fiber and reticulocyte lysates, alpha A(1-168)-crystallin was resistant to degradation. The susceptibility of alpha A(1-163)-crystallin to degradation was comparable to that of wt alpha A-crystallin. However, alpha A(1-162)-crystallin was much more susceptible than wt alpha A-crystallin to degradation in both lens fiber and reticulocyte lysates. The degradation of both wt and C-terminal truncated alpha A(1-162)-crystallins requires adenosine triphosphate (ATP) and was stimulated by addition of a ubiquitin-conjugating enzyme, Ubc4. The degradation was substantially inhibited by the proteasome inhibitor MG132 and a dominant negative mutant of ubiquitin, K6W-Ub, indicating that at least part of the proteolysis was mediated by the UPP. Spectroscopic analyses of wt and C-terminal truncated alpha A-crystallins revealed that C-terminal truncation of alpha A-crystallin resulted in only subtle changes in secondary structures. However, C-terminal truncations resulted in significant changes in surface hydrophobicity and thermal stability. Thus, these conformational changes may reveal or mask the signals for the ubiquitin-dependent degradation. CONCLUSIONS: The present data demonstrate that C-terminal cleavage of alpha A-crystallin not only alters its conformation and thermal stability, but also its susceptibility to degradation by the UPP. The rapid degradation of alpha A(1-162) by the UPP may prevent its accumulation in the lens.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/fisiología , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Ubiquitina/fisiología , Cadena A de alfa-Cristalina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Dicroismo Circular , Inhibidores de Cisteína Proteinasa/farmacología , Expresión Génica , Cristalino/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes , Cadena A de alfa-Cristalina/químicaRESUMEN
PURPOSE: This study aimed at elucidating the molecular mechanisms involved in the regulation of IL-8 production by several oxysterols in retinal pigment epithelium (RPE) cells. METHODS: A human cell line from RPE (ARPE-19) was used to test the role of cholesterol and several oxysterols (25-OH, 7-KC and 7ß-OH) in the expression and secretion of IL-8. Expression of IL-8 was assessed by real-time PCR, while IL-8 secretion was evaluated by ELISA. PI3K-, MEK1/2-, ERK1/2- and NF-κB-specific inhibitors were used to assess the specific role of the several players on the regulation of IL-8 production by oxysterols. A gene-reporter assay for AP-1 activity was also conducted to evaluate the putative role of this transcription factor on IL-8 expression induced by oxysterols. RESULTS: Here, we demonstrate that 25-OH specifically increases transcription and secretion of the cytokine IL-8 in ARPE-19 cells. Indeed, treatment of ARPE-19 with 25-OH, but not with 7-KC, 7ß-OH or cholesterol, induced the secretion of IL-8 from cells. 25-OH also induced the activation/phosphorylation of ERK1/2 through a mechanism dependent on MEK, ERK1/2 and PI3K kinase activity. Real-time PCR and ELISA experiments demonstrated that 25-OH increased transcription and secretion of IL-8 through a mechanism that is dependent on ERK1/2 and PI3K activity. Furthermore, 25-OH triggered the activation/phosphorylation of the AP-1 component c-Jun and, consistently, increased the transcriptional activity of AP-1. Additionally, we also found that 25-OH decreases the levels of IκB and increases the nuclear levels of NF-κB p65 subunit and that inhibition of NF-κB activity partially prevents the increased secretion of IL-8 induced by 25-OH. CONCLUSIONS: The results presented in this study suggest a role for 25-OH in inducing IL-8 production through pathways that are likely to involve AP-1 and NF-κB in ARPE-19 cells. Our data may also provide new molecular targets for the treatment of AMD.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hidroxicolesteroles/farmacología , Interleucina-8/genética , Interleucina-8/metabolismo , Epitelio Pigmentado de la Retina/efectos de los fármacos , Western Blotting , Línea Celular , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genes Reporteros , Humanos , Cetocolesteroles/farmacología , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Epitelio Pigmentado de la Retina/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. We have demonstrated previously that oxidative inactivation of the proteasome is a molecular link between oxidative stress and overexpression of interleukin (IL)-8. Here, we elucidated a novel signaling cascade that leads to up-regulation of IL-8 in response to proteasome inactivation. The sequence of events in this cascade includes proteasome inactivation, activation of mitogen-activated protein kinase kinase (MKK)3/MKK6, activation of p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor phosphorylation, phosphatidylinositol 3-kinase (PI3K) activation and increased IL-8 expression. Blocking any of these signaling pathways abolished the up-regulation of IL-8 induced by proteasome inhibition. Although Akt is also activated in response to proteasome inactivation, we found that the PI3K-dependent up-regulation of IL-8 is independent of 3-phosphoinositide-dependent protein kinase (PDK)1 and Akt. Inhibition of PDK1 and Akt with chemical inhibitors or expression of constitutive active Akt had little effects on IL-8 expression in response to proteasome inactivation. In contrast, inhibition of interleukin 2-inducible T cell kinase, a kinase downstream of PI3K, significantly reduced the expression and secretion of IL-8 in response to proteasome inactivation. Together, these data elucidate a novel signaling network that leads to increased IL-8 production in response to proteasome inactivation.
Asunto(s)
Células Epiteliales/metabolismo , Interleucina-8/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Epitelio Pigmentado de la Retina/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Activación Enzimática , Células Epiteliales/citología , Receptores ErbB/metabolismo , Humanos , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiologíaRESUMEN
NF-kappaB is a family of important transcription factors involved in many cellular functions, such as cell survival, proliferation, and stress responses. Many studies indicate that NF-kappaB is a stress-sensitive transcription factor and its activation is regulated by reactive oxygen species. In previous studies, we and others demonstrated that this transcription factor can be activated by transient oxidative stress. However, the effects of sustained oxidative stress on NF-kappaB activation are not clear. The objective of this study was to determine the effects of sustained oxidative stress on NF-kappaB activation and to elucidate the signaling events affected by sustained oxidative stress. Human lens epithelial cells (HLEC) that were subjected to 4 h of continuous influx of hydrogen peroxide were used to investigate the effects of sustained oxidative stress on NF-kappaB activation. The data showed that, unlike transient oxidative stress, sustained exposure of HLEC to physiologically relevant levels of H(2)O(2) (50-100 microM for 4 h) did not induce the degradation of I-kappaB and activation of NF-kappaB, but attenuated TNFalpha-induced degradation of I-kappaB and activation of NF-kappaB. Sustained exposure of HLEC to these levels of H(2)O(2) also inactivated proteasome activity by 50-80%. Consistent with the role of the proteasome in degradation of I-kappaB and activation of NF-kappaB, treatment of HLEC with proteasome inhibitors also attenuated TNFalpha-induced I-kappaB degradation and NF-kappaB activation. The data also indicate that activation of NF-kappaB is essential for the cells to recover from oxidative stress. Inhibiting NF-kappaB activation during recovery from transient oxidative stress significantly reduced the cell viability. Together, these data indicate that sustained oxidative stress may inactivate the proteasome and subsequently inhibit NF-kappaB activation by impeding the degradation of I-kappaB. The oxidative inactivation of the proteasome and subsequent impairment of NF-kappaB activation may contribute to the death of lens epithelial cells, a common feature associated with cataract.
Asunto(s)
Células Epiteliales/metabolismo , FN-kappa B/metabolismo , Estrés Oxidativo/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Catarata/etiología , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Cristalino/citología , FN-kappa B/genética , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/genética , Transducción de Señal , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
PURPOSE: Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study was to investigate the effect of oxidative stress on the UPP in cultured human retina pigment epithelial cells. METHODS: To mimic physiological oxidative stress, ARPE-19 cells were exposed to continuously generated H2O(2) or A2E-mediated photooxidation. Proteasome activity was monitored using fluorogenic peptides as substrates. The ubiquitin conjugation activity and activities of E1 and E2 were determined by the thiolester assays. Levels of ubiquitin and ubiquitin conjugates were determined by Western blotting. RESULTS: Exposure of ARPE-19 cells to 40 to 50 microM H2O(2) for 4 hours resulted in a 30% to 50% reduction in all three peptidase activities of the proteasome. Similarly, exposure of A2E-loaded ARPE-19 cells to blue light resulted in a 40% to 60% reduction in proteasome activity. Loading of A2E or exposure to blue light alone had little effect on proteasome activity. In contrast, exposure of ARPE-19 to low levels of H2O(2) (10 microM) stimulated ubiquitin conjugation activity. Loading of A2E, with or without exposure to blue light, upregulated the levels of ubiquitin-activating enzyme and increased conjugation activity. Exposure to H2O(2) or A2E-mediated photooxidation also resulted in a twofold to threefold increase in levels of endogenous ubiquitin conjugates. CONCLUSIONS: These data show that the proteasome in ARPE-19 is susceptible to oxidative inactivation, whereas activities of the ubiquitin-conjugating enzymes are more resistant to oxidative stress. Oxidative inactivation of the proteasome appears to be one of the mechanisms underlying stress-induced accumulation of ubiquitin conjugates in the cells.
Asunto(s)
Estrés Oxidativo/fisiología , Epitelio Pigmentado Ocular/efectos de los fármacos , Epitelio Pigmentado Ocular/efectos de la radiación , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Aldehídos/farmacología , Western Blotting , Células Cultivadas , Humanos , Peróxido de Hidrógeno/farmacología , Luz , Epitelio Pigmentado Ocular/metabolismo , Compuestos de Piridinio/farmacología , Retinoides/farmacología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Regulación hacia ArribaRESUMEN
Oxidative stress and inflammation are implicated in the pathogenesis of many age-related diseases. Stress-induced overproduction of inflammatory cytokines, such as interleukin-8 (IL-8), is one of the early events of inflammation. The objective of this study was to elucidate mechanistic links between oxidative stress and overproduction of IL-8 in retinal pigment epithelial (RPE) cells. We found that exposure of RPE cells to H(2)O(2), paraquat, or A2E-mediated photooxidation resulted in increased expression and secretion of IL-8. All of these oxidative stressors also inactivated the proteasome in RPE cells. In contrast, tert-butylhydroperoxide (TBH), a lipophilic oxidant that did not stimulate IL-8 production, also did not inactivate the proteasome. Moreover, prolonged treatment of RPE cells with proteasome-specific inhibitors recapitulated the stimulation of IL-8 production. These data suggest that oxidative inactivation of the proteasome is a potential mechanistic link between oxidative stress and up-regulation of the proinflammatory IL-8. The downstream signaling pathways that govern the production of IL-8 include NF-kappaB and p38 MAPK. Proteasome inhibition both attenuated the activation and delayed the turnoff of NF-kappaB, resulting in biphasic effects on the production of IL-8. Prolonged proteasome inhibition (>2 h) resulted in activation of p38 MAPK via activation of MKK3/6 and increased the production of IL-8. Chemically inhibiting the p38 MAPK blocked the proteasome inhibition-induced up-regulation of IL-8. Together, these data indicate that oxidative inactivation of the proteasome and the related activation of the p38 MAPK pathway provide a potential link between oxidative stress and overproduction of proinflammatory cytokines, such as IL-8.
Asunto(s)
Células Epiteliales/citología , Regulación de la Expresión Génica , Interleucina-8/metabolismo , Oxígeno/metabolismo , Epitelio Pigmentado Ocular/citología , Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular , Regulación Enzimológica de la Expresión Génica , Humanos , Peróxido de Hidrógeno/farmacología , Inflamación , Luz , Modelos Biológicos , Estrés Oxidativo , Oxígeno/química , Factores de TiempoRESUMEN
As in many other types of cells, retinal pigment epithelial (RPE) cells have an active ubiquitin-proteasome pathway (UPP). However, the function of the UPP in RPE remains to be elucidated. The objective of this study is to determine the role of the UPP in controlling the levels and activities of transcription factors hypoxia-inducible factor (HIF) and NF-kappaB. We inhibited the UPP with proteasome-specific inhibitors and determined the activation of HIF and NF-kappaB as well as the expression and secretion of pro-angiogenic factors. HIF-1alpha was not detectable in ARPE-19 cells under normal culture conditions. However, when proteasome activity was inhibited, HIF-1alpha accumulated in RPE in a time-dependent manner. Consistent with accumulation of HIF-1alpha in the cells, levels of mRNA for vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in RPE were up to 7-fold higher upon inhibition of the proteasome. Proteasome inhibition was also associated with a 2-fold increase in levels of mRNA for angiopoietin-1 (Ang-1). ARPE-19 cells secrete significant levels of VEGF under normal culture conditions. Inhibition of proteasome activity increased the secretion of VEGF by 2-fold. In contrast to the increase in HIF activity, NF-kappaB activation was reduced by proteasome inhibition. In addition, the expression and secretion of monocyte chemoattractant protein-1 (MCP-1) by RPE were substantially attenuated by the inhibition of proteasome activity. These data demonstrate that the UPP plays an important role in modulating the activities of HIF and NF-kappaB in the RPE. Consequences of an impairment of the UPP include accumulation of HIF-1alpha and diminished NF-kappaB activation, which lead to enhanced expression and secretion of pro-angiogenic factors and attenuated expression of MCP-1. Taken together, these data predict that the impairment of the UPP could lead to the development of AMD-related phenotypes.