RESUMEN

The anti-apoptotic molecule Aven was originally identified in a yeast two-hybrid screen for Bcl-x(L)-interacting proteins and has also been found to bind Apaf-1, thereby interfering with Apaf-1 self-association during apoptosome assembly. Aven is expressed in a wide variety of adult tissues and cell lines, and there is increasing evidence that its overexpression correlates with tumorigenesis, particularly in acute leukemias. The mechanism by which the anti-apoptotic activity of Aven is regulated remains poorly understood. Here we shed light on this issue by demonstrating that proteolytic removal of an inhibitory N-terminal Aven domain is necessary to activate the anti-apoptotic potential of the molecule. Furthermore, we identify Cathepsin D (CathD) as the protease responsible for Aven cleavage. On the basis of our results, we propose a model of Aven activation by which its N-terminal inhibitory domain is removed by CathD-mediated proteolysis, thereby unleashing its cytoprotective function.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Catepsina D/metabolismo , Proteínas de la Membrana/metabolismo , Proteolisis , Enfermedad Aguda , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Reguladoras de la Apoptosis/genética , Factor Apoptótico 1 Activador de Proteasas/genética , Factor Apoptótico 1 Activador de Proteasas/metabolismo , Catepsina D/genética , Línea Celular Tumoral , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Proteínas de la Membrana/genética , Estructura Terciaria de Proteína

RESUMEN

Increased generation of reactive oxygen species (ROS) leads to oxidative stress in diabetes. Catalase is a highly conserved heme-containing protein that reduces hydrogen peroxide to water and oxygen and is an important factor decreasing cellular injury owing to oxidative stress. Hyperglycemic conditions increase oxidative stress and angiotensinogen gene expression. Angiotensinogen conversion to angiotensin II leads to a furtherance in oxidative stress through increased generation of reactive oxygen species. In this study, we utilized mice transgenically overexpressing rat catalase in a kidney-specific manner to determine the impact on ROS, angiotensinogen and apoptotic gene expression in proximal tubule cells of diabetic animals. Proximal tubules isolated from wild-type and transgenic animals without or with streptozotocin-induced diabetes were incubated in low glucose media in the absence or presence of angiotensin II or in a high-glucose media. Our results show that the overexpression of catalase prevents the stimulation of ROS and angiotensinogen mRNA in tubules owing to elevated glucose or angiotensin II in vitro. Additionally, overexpression of catalase attenuated ROS generation, angiotensinogen and proapoptotic gene expression and apoptosis in the kidneys of diabetic mice in vivo. Our studies point to an important role of ROS in the pathophysiology of diabetic nephropathy.

Asunto(s)

Angiotensinógeno/genética , Catalasa/genética , Diabetes Mellitus Experimental/fisiopatología , Animales , Apoptosis , Caspasa 3/efectos de los fármacos , Caspasa 3/genética , Diabetes Mellitus Experimental/patología , Humanos , Insulina/uso terapéutico , Ratones , Ratones Transgénicos , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2/efectos de los fármacos , Proteína X Asociada a bcl-2/genética

RESUMEN

The present study investigated whether transforming growth factor-beta 1 (TGF-beta1) exerts an autocrine positive effect on angiotensinogen (ANG) gene expression in rat kidney proximal tubular cells, and delineates its underlying mechanism(s) of action. Rat immortalized renal proximal tubular cells (IRPTCs) and freshly isolated mouse renal proximal tubules were incubated in the absence or presence of active human TGF-beta1. IRPTCs were also stably transfected with rat TGF-beta1 or p53 tumor suppressor protein (p53) cDNA in sense (S) and antisense (AS) orientations. ANG mRNA and p53 protein expression were assessed by reverse transcription-polymerase chain reaction and Western blotting, respectively. Reactive oxygen species (ROS) generation was quantified by lucigenin assay. Active TGF-beta1 evoked ROS generation and stimulated ANG mRNA and p53 protein expression, whereas a superoxide scavenger and inhibitors of nicotinamide adenine dinucleotide oxidase and p38 mitogen-activated protein kinase (p38 MAPK) abolished the TGF-beta1 effect. Stable transfer of p53 cDNA (S) enhanced and p53 cDNA (AS) abolished the stimulatory effect of TGF-beta1 on ANG mRNA expression in IRPTCs. Our results demonstrate that TGF-beta1 stimulates ANG gene expression and its action is mediated, at least in part, via ROS generation, p38 MAPK activation, and p53 expression, suggesting that angiotensin II and TGF-beta1 may form a positive feedback loop to enhance their respective gene expression, leading to renal injury.

Asunto(s)

Angiotensinógeno/genética , Regulación de la Expresión Génica , Túbulos Renales Proximales/fisiología , Factor de Crecimiento Transformador beta/fisiología , Células Cultivadas , Humanos , Factor de Crecimiento Transformador beta1

RESUMEN

Angiotensinogen (ANG) is the sole substrate of the renin-angiotensin system (RAS). Clinical studies have shown that RAS activation may lead to hypertension, a major cardiovascular and renal risk factor. To delineate the underlying mechanisms of hypertension-induced nephropathy, we generated transgenic mice that overexpress rat ANG (rANG) in the kidney to establish whether intrarenal RAS activation alone can evoke hypertension and kidney damage and whether RAS blockade can reverse these effects. Transgenic mice overexpressing renal rANG were generated by employing the kidney-specific, androgen-regulated protein promoter linked to rANG cDNA. This promoter targets rANG cDNA to renal proximal tubules and responds to androgen stimulation. Transgenic mice displayed kidney-specific expression of rANG, significantly increased blood pressure (BP) and albuminuria in comparison to non-transgenic littermates. Administration of losartan (an angiotensin II (type 1)-receptor antagonist) or perindopril (an angiotensin-converting enzyme inhibitor) reversed these abnormalities in transgenic animals. Renal injury was evident on examination of the kidneys in transgenic mice, and attenuated by losartan and perindopril treatment. We conclude that the overproduction of ANG alone in the kidney induces an increase in systemic BP, proteinuria, and renal injury. RAS blockers prevent these abnormalities. These data support the role of the intrarenal RAS in the development of hypertension and renal injury.

Asunto(s)

Angiotensinógeno/análisis , Angiotensinógeno/genética , Presión Sanguínea/fisiología , Riñón/química , Proteinuria/fisiopatología , Sistema Renina-Angiotensina/fisiología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Western Blotting , ADN Complementario/análisis , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica , Hipertensión/complicaciones , Hipertensión/etiología , Hipertensión/genética , Hipertensión/fisiopatología , Inmunohistoquímica , Riñón/fisiopatología , Enfermedades Renales/etiología , Enfermedades Renales/fisiopatología , Losartán/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Perindopril/farmacología , Proteínas/análisis , Proteínas/genética , Ratas , Sistema Renina-Angiotensina/efectos de los fármacos

RESUMEN

Progression of diabetic nephropathy appears directly related to renal tubulointerstitial injury, but the involved genes are incompletely delineated. To identify such genes, DNA microarray analysis was performed with RNA from renal proximal tubules (RPTs) of streptozotocin-induced diabetic Wistar rats, spontaneously diabetic BioBreeding rats, and rat immortalized renal proximal tubular cells (IRPTCs) exposed to high glucose (25 mM) medium for 2 weeks. Osteopontin (OPN) mRNA expression was quantified by real time-quantitative polymerase chain reaction (RT-qPCR) or conventional reverse transcriptase-polymerase chain reaction (RT-PCR). OPN mRNA expression was upregulated (5-70-fold increase) in diabetic rat RPTs and in IRPTCs chronically exposed to high glucose compared to control RPTs and IRPTCs. High glucose, angiotensin II, phorbol 12-myristate 13-acetate and transforming growth factor-beta 1 (TGF-beta1) stimulated OPN mRNA expression in IRPTCs in a dose- and time-dependent manner. This effect was inhibited by tiron, taurine, diphenylene iodinium, losartan, perindopril, calphostin C, or LY 379196 but not PD123319. IRPTCs overexpressing dominant-negative protein kinase C-beta 1 (PKC-beta1) cDNA or antisense TGF-beta1 cDNA prevented the high glucose effect on OPN mRNA expression. We concluded that high glucose-mediated increases in OPN gene expression in diabetic rat RPTs and IRPTCs are mediated, at least in part, via reactive oxygen species generation, intrarenal rennin-angiotensin system activation, TGF-beta1 expression, and PKC-beta1 signaling.

Asunto(s)

Diabetes Mellitus Experimental/genética , Perfilación de la Expresión Génica , Glicoproteínas/análisis , Glicoproteínas/genética , Túbulos Renales Proximales/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Regulación hacia Arriba/genética , Angiotensina II/farmacología , Animales , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/fisiopatología , Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Masculino , Proteína Quinasa C/fisiología , Proteína Quinasa C beta , ARN Mensajero/análisis , Ratas , Ratas Endogámicas BB , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Sistema Renina-Angiotensina/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Acetato de Tetradecanoilforbol/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1

RESUMEN

BACKGROUND: High mobility group box 1 (HMGB1) is a non-histone chromosomal protein implicated in a variety of biologically important processes, including transcription, DNA repair, V(D)J recombination, differentiation, and development. Overexpression of HMGB1 inhibits apoptosis, arguing that the molecule may act as an antiapoptotic oncoprotein. Indeed, increased expression of HMGB1 has been reported for several different tumour types. In this study, we analysed human colon carcinoma for HMGB1 as well as for c-IAP2 expression levels. c-IAP2 is an antiapoptotic protein which may be upregulated as a consequence of nuclear factor kappaB (NFkappaB) activation via HMGB1. METHODS: A comparative genomic hybridisation (CGH) database comprising 1645 cases from different human tumour types was screened to detect cytogenetic changes at the HMGB1 locus. Immunohistochemical staining of human colon tissue microarrays and tumour biopsies, as well as western blot analysis of tumour lysates, were performed to detect elevated HMGB1 and c-IAP2 expression in colon carcinomas. The antiapoptotic potential of HMGB1 was analysed by measuring caspase activities, and luciferase reporter assays and quantitative polymerase chain reaction analysis were employed to confirm NFkappaB activation and c-IAP2 mRNA upregulation on HMGB1 overexpression. RESULTS: According to CGH analysis, the genomic locus containing the HMGB1 gene was overrepresented in one third (35/96) of colon cancers. Correspondingly, HMGB1 protein levels were significantly elevated in 90% of the 60 colon carcinomas tested compared with corresponding normal tissues evaluable from the same patients. HMGB1 increased NFkappaB activity and led to co-overexpression of the antiapoptotic NFkappaB target gene product c-IAP2 in vitro. Furthermore, increased HMGB1 levels correlated with enhanced amounts of c-IAP2 in colon tumours analysed by us. Finally, we demonstrated that HMGB1 overexpression suppressed caspase-9 and caspase-3 activity, suggesting that HMGB1 interferes with the apoptotic machinery at the level of apoptosomal caspase-9 activation. CONCLUSIONS: We identified in vitro a molecular pathway triggered by HMGB1 to inhibit apoptosis via c-IAP2 induction. Our data indicate a strong correlation between upregulation of the apoptosis repressing HMGB1 and c-IAP2 proteins in the pathogenesis of colon carcinoma.

Asunto(s)

Neoplasias del Colon/metabolismo , Proteína HMGB1/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas de Neoplasias/metabolismo , Apoptosis , Western Blotting , Inhibidores de Caspasas , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/metabolismo , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/genética , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transducción de Señal , Regulación hacia Arriba