RESUMEN
High CD44 expression is associated with enhanced malignant potential in esophageal squamous cell carcinoma (ESCC), among the deadliest of all human carcinomas. Although alterations in autophagy and CD44 expression are associated with poor patient outcomes in various cancer types, the relationship between autophagy and cells with high CD44 expression remains incompletely understood. In transformed oesophageal keratinocytes, CD44Low-CD24High (CD44L) cells give rise to CD44High-CD24-/Low (CD44H) cells via epithelial-mesenchymal transition (EMT) in response to transforming growth factor (TGF)-ß. We couple patient samples and xenotransplantation studies with this tractable in vitro system of CD44L to CD44H cell conversion to investigate the functional role of autophagy in generation of cells with high CD44 expression. We report that high expression of the autophagy marker cleaved LC3 expression correlates with poor clinical outcome in ESCC. In ESCC xenograft tumours, pharmacological autophagy inhibition with chloroquine derivatives depletes cells with high CD44 expression while promoting oxidative stress. Autophagic flux impairment during EMT-mediated CD44L to CD44H cell conversion in vitro induces mitochondrial dysfunction, oxidative stress and cell death. During CD44H cell generation, transformed keratinocytes display evidence of mitophagy, including mitochondrial fragmentation, decreased mitochondrial content and mitochondrial translocation of Parkin, essential in mitophagy. RNA interference-mediated Parkin depletion attenuates CD44H cell generation. These data suggest that autophagy facilitates EMT-mediated CD44H generation via modulation of redox homeostasis and Parkin-dependent mitochondrial clearance. This is the first report to implicate mitophagy in regulation of tumour cells with high CD44 expression, representing a potential novel therapeutic avenue in cancers where EMT and CD44H cells have been implicated, including ESCC.
Asunto(s)
Autofagia/fisiología , Receptores de Hialuranos/metabolismo , Mitocondrias/fisiología , Estrés Oxidativo/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago , Humanos , Queratinocitos/metabolismo , Queratinocitos/fisiología , Mitocondrias/metabolismo , Oxidación-Reducción , Interferencia de ARN/fisiología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Retrograde signaling is a mechanism by which mitochondrial dysfunction is communicated to the nucleus for inducing a metabolic shift essential for cell survival. Previously, we showed that partial mitochondrial DNA (mtDNA) depletion in different cell types induced mitochondrial retrograde signaling pathway (MtRS) involving Ca+2-sensitive Calcineurin (Cn) activation as an immediate upstream event of stress response. In multiple cell types, this stress signaling was shown to induce tumorigenic phenotypes in immortalized cells. In this study we show that MtRS also induces p53 expression, which was abrogated by Ca2+ chelators and short hairpin RNA-mediated knockdown of CnAß mRNA. Mitochondrial dysfunction induced by mitochondrial ionophore, carbonyl cyanide m-chlorophenyl hydrazone and other respiratory inhibitors, which perturb the transmembrane potential, were equally efficient in inducing the expression of p53 and downregulation of MDM2. Stress-induced p53 physically interacted with hypoxia-inducible factor-1α (HIF-1α) and attenuated the latter's binding to promoter DNA motifs. In addition, p53 promoted ubiquitination and degradation of HIF-1α in partial mtDNA-depleted cells. The mtDNA depleted cells, with inhibited HIF-1α, showed upregulation of glycolytic pathway genes, glucose transporter 1-4 (Glut1-4), phosphoglycerate kinase 1 and Glucokinase but not of prolyl hydroxylase isoforms. For the first time we show that p53 is induced as part of MtRS and it renders HIF-1α inactive by physical interaction. In this respect, our results show that MtRS induces tumor growth independent of the HIF-1α pathway.
Asunto(s)
Calcineurina/metabolismo , Calcio/metabolismo , ADN Mitocondrial/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Mitocondrias/genética , Proteína p53 Supresora de Tumor/metabolismo , Células A549 , Animales , Secuencia de Bases , Células COS , Línea Celular Tumoral , Células HCT116 , Humanos , Ratones , Unión Proteica , Ratas , Eliminación de Secuencia , Transducción de Señal , UbiquitinaciónRESUMEN
Defects in mitochondrial oxidative phosphorylation complexes, altered bioenergetics and metabolic shift are often seen in cancers. Here we show a role for the dysfunction of the electron transport chain component cytochrome c oxidase (CcO) in cancer progression. We show that genetic silencing of the CcO complex by shRNA expression and loss of CcO activity in multiple cell types from the mouse and human sources resulted in metabolic shift to glycolysis, loss of anchorage-dependent growth and acquired invasive phenotypes. Disruption of the CcO complex caused loss of transmembrane potential and induction of Ca2+/Calcineurin-mediated retrograde signaling. Propagation of this signaling includes activation of PI3-kinase, IGF1R and Akt, Ca2(+)-sensitive transcription factors and also TGFß1, MMP16 and periostin, which are involved in oncogenic progression. Whole-genome expression analysis showed the upregulation of genes involved in cell signaling, extracellular matrix interactions, cell morphogenesis, cell motility and migration. The transcription profiles reveal extensive similarity to retrograde signaling initiated by partial mitochondrial DNA depletion, although distinct differences are observed in signaling induced by CcO dysfunction. The possible CcO dysfunction as a biomarker for cancer progression was supported by data showing that esophageal tumors from human patients show reduced CcO subunits IVi1 and Vb in regions that were previously shown to be the hypoxic core of the tumors. Our results show that mitochondrial electron transport chain defect initiates a retrograde signaling. These results suggest that a defect in the CcO complex can potentially induce tumor progression.
Asunto(s)
Complejo IV de Transporte de Electrones/metabolismo , Animales , Línea Celular , Complejo IV de Transporte de Electrones/genética , Silenciador del Gen , Ratones , Estrés Oxidativo , Transducción de SeñalRESUMEN
Epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44(low)-CD24(high) (CD44L) epithelial phenotype to a CD44(high)-CD24(-/low) (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT, although excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT, as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference and flow cytometric approaches, we find that transforming growth factor (TGF)-ß stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-ß-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. These data provide novel mechanistic insights into the dynamic expression of SOD2 during EMT. In addition, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.
Asunto(s)
Transición Epitelial-Mesenquimal , Superóxido Dismutasa/fisiología , Línea Celular , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Receptores de Hialuranos , Mitocondrias/enzimología , FN-kappa B/metabolismo , Proteínas Represoras/metabolismo , Caja Homeótica 2 de Unión a E-Box con Dedos de ZincRESUMEN
Metastatic breast tumors undergo epithelial-to-mesenchymal transition (EMT), which renders them resistant to therapies targeted to the primary cancers. The mechanistic link between mtDNA (mitochondrial DNA) reduction, often seen in breast cancer patients, and EMT is unknown. We demonstrate that reducing mtDNA content in human mammary epithelial cells (hMECs) activates Calcineurin (Cn)-dependent mitochondrial retrograde signaling pathway, which induces EMT-like reprogramming to fibroblastic morphology, loss of cell polarity, contact inhibition and acquired migratory and invasive phenotype. Notably, mtDNA reduction generates breast cancer stem cells. In addition to retrograde signaling markers, there is an induction of mesenchymal genes but loss of epithelial markers in these cells. The changes are reversed by either restoring the mtDNA content or knockdown of CnAα mRNA, indicating the causal role of retrograde signaling in EMT. Our results point to a new therapeutic strategy for metastatic breast cancers targeted to the mitochondrial retrograde signaling pathway for abrogating EMT and attenuating cancer stem cells, which evade conventional therapies. We report a novel regulatory mechanism by which low mtDNA content generates EMT and cancer stem cells in hMECs.
Asunto(s)
Neoplasias de la Mama/genética , ADN Mitocondrial/genética , Transición Epitelial-Mesenquimal/genética , Células Madre Neoplásicas/metabolismo , Transducción de Señal/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Calcineurina/genética , Calcineurina/metabolismo , Línea Celular , Movimiento Celular/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Femenino , Dosificación de Gen , Expresión Génica , Humanos , Immunoblotting , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Células MCF-7 , Ratones SCID , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Células Madre Neoplásicas/patología , Consumo de Oxígeno/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
In this study, we show that partial mitochondrial DNA (mtDNA) depletion (mitochondrial stress) induces resistance to staurosporine (STP)-mediated apoptosis in C2C12 myoblasts. MtDNA-depleted cells show a 3-4-fold increased proapoptotic proteins (Bax, BAD and Bid), markedly increased antiapoptotic Bcl-2, and reduced processing of p21 Bid to active tBid. The protein levels and also the ability to undergo STP-mediated apoptosis were restored in reverted cells containing near-normal mtDNA levels and restored mitochondrial transmembrane potential. Inhibition of apoptosis closely correlated with sequestration of Bax, Bid and BAD in the mitochondrial inner membrane, increased Bcl-2 and Bcl-X(L), and inability to process p21 Bid. These factors, together with the reduced activation of caspases 3, 9 and 8 are possible causes of mitochondrial stress-induced resistance to apoptosis. Our results suggest that a highly proliferative and invasive behavior of mtDNA-depleted C2C12 cells is related to their resistance to apoptosis.
Asunto(s)
Apoptosis , ADN Mitocondrial/fisiología , Células Musculares/citología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Biomarcadores/metabolismo , Proteínas Portadoras/biosíntesis , Caspasas/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Activación Enzimática , Humanos , Membranas Intracelulares/metabolismo , Potenciales de la Membrana , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Transducción de Señal , Estaurosporina/farmacología , Proteína X Asociada a bcl-2 , Proteína Letal Asociada a bclRESUMEN
We have investigated the sites of N-terminally truncated cytochrome P4501A1 targeted to mitochondria (P450MT2) which interact with adrenodoxin (Adx), cytochrome P450 reductase (CPR) and bacterial flavodoxin (Fln). The binding site was mapped by a combination of in vitro mutagenesis, in vivo screening with a mammalian two-hybrid system, spectral analysis, reconstitution of enzyme activity and homology-based structural modeling. Our results show that part of an aqueous accessible helix (putative helix G, residues 264-279) interacts with all three electron donor proteins. Mutational studies revealed that Lys267 and Lys271 are crucial for binding to Adx, while Lys268 and Arg275 are important for binding to CPR and FLN: Additive effects of different electron donor proteins on enzyme activity and models on protein docking show that Adx and CPR bind in a non-overlapping manner to the same helical domain in P450MT2 at different angular orientations, while CPR and Fln compete for the same binding site. We demonstrate that evolutionarily divergent electron donor proteins interact with the same domain but subtly different contact points of P450MT2.
Asunto(s)
Adrenodoxina/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Evolución Molecular , Flavodoxina/metabolismo , Mitocondrias Hepáticas/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Sitios de Unión , Bovinos , Mapeo Cromosómico , Reactivos de Enlaces Cruzados , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Mamíferos , Modelos Moleculares , Mutagénesis , NADPH-Ferrihemoproteína Reductasa/genética , Estructura Secundaria de Proteína , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
Hepatic mitochondria contain an inducible cytochrome P450, referred to as P450 MT5, which cross-reacts with antibodies to microsomal cytochrome P450 2E1. In the present study, we purified, partially sequenced, and determined enzymatic properties of the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1 was purified from pyrazole-induced rat livers using a combination of hydrophobic and ion-exchange chromatography. Mass spectrometry analysis of tryptic fragments of the purified protein further ascertained its identity. N-terminal sequencing of the purified protein showed that its N terminus is identical to that of the microsomal cytochrome P450 2E1. In reconstitution experiments, the mitochondrial cytochrome P450 2E1 displayed the same catalytic activity as the microsomal counterpart, although the activity of the mitochondrial enzyme was supported exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry analysis of tryptic fragments and also immunoblot analysis of proteins with anti-serine phosphate antibody demonstrated that the mitochondrial cytochrome P450 2E1 is phosphorylated at a higher level compared with the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be responsible for its observed preference for adrenodoxin and adrenodoxin reductase electron transfer proteins.
Asunto(s)
Adrenodoxina/metabolismo , Citocromo P-450 CYP2E1/química , Transporte de Electrón , Ferredoxina-NADP Reductasa/metabolismo , Animales , Citocromo P-450 CYP2E1/metabolismo , Masculino , Espectrometría de Masas , Microsomas Hepáticos/metabolismo , Mitocondrias Hepáticas/enzimología , Fragmentos de Péptidos/química , Conformación Proteica , Pirazoles/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Recently we showed that partial depletion of mitochondrial DNA (genetic stress) or treatment with mitochondrial-specific inhibitors (metabolic stress) induced a stress signaling that was associated with increased cytoplasmic-free Ca(2+) [Ca(2+)](c). In the present study we show that the mitochondria-to-nucleus stress signaling induces invasive phenotypes in otherwise non-invasive C2C12 myoblasts and human pulmonary carcinoma A549 cells. Tumor-specific markers cathepsin L and transforming growth factor beta (TGFbeta) are overexpressed in cells subjected to mitochondrial genetic as well as metabolic stress. C2C12 myoblasts subjected to stress showed 4- to 6-fold higher invasion through reconstituted Matrigel membrane as well as rat tracheal xenotransplants in Scid mice. Activation of Ca(2+)-dependent protein kinase C (PKC) under both genetic and metabolic stress conditions was associated with increased cathepsin L gene expression, which contributes to increased invasive property of cells. Reverted cells with approximately 70% of control cell mtDNA exhibited marker mRNA contents, cell morphology and invasive property closer to control cells. These results provide insights into a new pathway by which mitochondrial DNA and membrane damage can contribute to tumor progression and metastasis.
Asunto(s)
Núcleo Celular/metabolismo , Transformación Celular Neoplásica , Endopeptidasas , Mitocondrias/metabolismo , Invasividad Neoplásica , Animales , Carcinoma/patología , Catepsina L , Catepsinas/genética , Cisteína Endopeptidasas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Membranas Intracelulares/metabolismo , Neoplasias Pulmonares/patología , Potenciales de la Membrana , Ratones , Ratones SCID , Músculos/patología , Transducción de SeñalRESUMEN
We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a "weak" Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN , Proteínas Proto-Oncogénicas/genética , Receptores de Glucocorticoides/genética , Proteínas Represoras , Esteroide Hidroxilasas/genética , Transactivadores/genética , Factores de Transcripción , Activación Transcripcional , Células 3T3 , Animales , Colestanotriol 26-Monooxigenasa , Sistema Enzimático del Citocromo P-450/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Proto-Oncogénica c-ets-2 , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Receptores de Glucocorticoides/metabolismo , Transducción de Señal/genética , Esteroide Hidroxilasas/metabolismo , Transactivadores/metabolismoRESUMEN
The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to beta-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH(2) termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.
Asunto(s)
Encéfalo/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Mitocondrias/efectos de los fármacos , beta-naftoflavona/farmacología , Animales , Encéfalo/enzimología , Catálisis , Masculino , Mitocondrias/enzimología , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Ratas , Ratas Sprague-DawleyRESUMEN
We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab(34)) and anti-IP(3)R (Ab(40)) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab(40), anti-RyR-1, anti-RyR-2 (Ab(129)), and anti-RyR-3 (Ab(180)). Only anti-RyR-1 and Ab(40) showed bands corresponding, respectively, to full-length RyR-1 ( approximately 500 kDa) and IP(3)R-1 (approximately 250 kDa). Band intensity was reduced by just approximately 20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca(2+) concentration ([Ca(2+)](np)) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca(2+) via Ca(2+)-ATPase activation (1 mM ATP and approximately 100 nM Ca(2+)). Adequate Ca(2+) loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca(2+)-loaded nuclei to IP(3) or cADP ribose resulted in a rapid and sustained [Ca(2+)](np) elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca(2+) influx in osteoblasts through nuclear membrane-resident IP(3)Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP(3)-Ca(2+) pathway.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Núcleo Celular/metabolismo , Osteoblastos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Células 3T3 , Animales , Inmunohistoquímica , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Ratones , Membrana Nuclear/metabolismo , Isoformas de Proteínas/metabolismoRESUMEN
We have isolated a cDNA encoding full length microsomal glutathione S-transferase (MGST) from mouse liver. The cDNA was isolated by RT-PCR using primers designed from published cDNA sequence of rat MGST with the addition of 5' Nde-1 and 3' HindIII sites, and cloned into bacterial expression vector pSP19T7LT. Deduced amino acid sequence (155 amino acids, calculated mol.mass 17512 Dalton) confirmed the identity of microsomal GST from mouse liver which has sequence homology with that of rat and human liver MGST1. Recombinant GST cDNA (Genbank accession # 159050) was expressed in BL21(DE3) in the presence of 1 mM IPTG at 30 degrees C. The expressed GST protein was found to be localised in the bacterial membrane as determined by measuring catalytic activity using CDNB and cumene hydroperoxide substrates, SDS-PAGE and Western blot analysis. We have demonstrated the cloning and expression of full length cDNA for MGST from mouse liver and have characterised the functionally active product as MGST protein. These results should facilitate studies on the role of MGST in the regulation of chemical carcinogenesis and in the prevention of oxidative stress caused by endogenous and exogenous chemicals.
Asunto(s)
Glutatión Transferasa/genética , Microsomas Hepáticos/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Escherichia coli , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Datos de Secuencia Molecular , Ratas , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
Nucleoplasmic calcium ions (Ca2+) influence nuclear functions as critical as gene transcription, apoptosis, DNA repair, topoisomerase activation and polymerase unfolding. Although both inositol trisphosphate receptors and ryanodine receptors, types of Ca2+ channel, are present in the nuclear membrane, their role in the homeostasis of nuclear Ca2+ remains unclear. Here we report the existence in the inner nuclear membrane of a functionally active CD38/ADP-ribosyl cyclase that has its catalytic site within the nucleoplasm. We propose that the enzyme catalyses the intranuclear cyclization of nicotinamide adenine dinucleotide to cyclic adenosine diphosphate ribose. The latter activates ryanodine receptors of the inner nuclear membrane to trigger nucleoplasmic Ca2+ release.
Asunto(s)
Antígenos CD , Antígenos de Diferenciación/metabolismo , Calcio/metabolismo , Núcleo Celular/metabolismo , NAD+ Nucleosidasa/metabolismo , Membrana Nuclear/metabolismo , Células 3T3 , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/farmacología , Animales , Fraccionamiento Celular/métodos , ADP-Ribosa Cíclica , Genes Reporteros/genética , Immunoblotting , Inositol 1,4,5-Trifosfato/farmacología , Glicoproteínas de Membrana , Ratones , Microscopía Confocal , Complejos Multienzimáticos , NAD/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue-specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by approximately 50% reduced, mitochondrial genome-encoded COX I and II mRNAs and nuclear genome-encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50-80% reduction in mitochondrial transcription and translation rates in heme-depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme-depleted tissues contained a 30-50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme-depleted tissues. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.
Asunto(s)
Cobalto/farmacología , Complejo IV de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/farmacología , Hemo/antagonistas & inhibidores , Heptanoatos/farmacología , Mitocondrias/enzimología , Complejos Multienzimáticos/metabolismo , Adenosina Trifosfato/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/embriología , Catálisis , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Complejo IV de Transporte de Electrones/genética , Inducción Enzimática , Hemo/biosíntesis , Hemo Oxigenasa (Desciclizante)/biosíntesis , Hemo Oxigenasa (Desciclizante)/genética , Hemoproteínas/biosíntesis , Hierro/metabolismo , Riñón/efectos de los fármacos , Riñón/enzimología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Complejos Multienzimáticos/genética , Especificidad de Órganos , Porfobilinógeno Sintasa/antagonistas & inhibidores , ARN Mensajero/biosíntesisRESUMEN
In vitro and in vivo studies have shown that the sterol 27-hydroxylase (CYP27) gene is transcriptionally repressed by hydrophobic bile acids. The molecular mechanism(s) of repression of CYP27 by bile acids is unknown. To identify the bile acid responsive element (BARE) and transcription factor(s) that mediate the repression of CYP27 by bile acids, constructs of the CYP27 5'-flanking DNA were linked to either the CAT or luciferase reporter gene and transiently transfected into primary rat hepatocytes. Taurocholate (TCA), taurodeoxycholate (TDCA) and taurochenodeoxycholate (TCDCA) significantly reduced CAT activities of the -840/+23, -329/+23, and -195/+23 mCAT constructs. A -76/+23 construct showed no regulation by bile acids. When a DNA fragment (-110/-86) from this region was cloned in front of an SV 40 promoter it showed down-regulation by TDCA. 'Super'-electrophoretic mobility shift assays (EMSA) indicated that both HNF1alpha and C/EBP bind to the -110 to -86 bp DNA fragment. Recombinant rat HNF1alpha and C/EBPalpha competitively bound to this DNA fragment. 'Super'-EMSA showed that TDCA addition to hepatocytes in culture decreased HNF1alpha, but not C/EBP, binding to the -110/-86 bp DNA fragment. A four base pair substitution mutation (-103 to -99) in this sequence eliminated TCA and TDCA regulation of the (-840/+23) construct. The substitution mutation also eliminated (>95%) HNF1alpha, but not C/EBP, binding to this DNA fragment. We conclude that bile acids repress CYP27 transcription through a putative BARE located between -110 and -86 bp of the CYP27 promoter. The data suggest that bile acids repress CYP27 transcriptional activity by decreasing HNF1alpha binding to the CYP27 promoter.
Asunto(s)
Ácidos y Sales Biliares/fisiología , Sistema Enzimático del Citocromo P-450/genética , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo , Hígado/enzimología , Proteínas Nucleares/fisiología , Esteroide Hidroxilasas/genética , Factores de Transcripción/fisiología , Animales , Proteínas Potenciadoras de Unión a CCAAT , Células Cultivadas , Colestanotriol 26-Monooxigenasa , Mapeo Cromosómico , Clonación Molecular , Sistema Enzimático del Citocromo P-450/biosíntesis , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Masculino , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Análisis de Secuencia de ADN , Esteroide Hidroxilasas/biosíntesis , Transcripción Genética , TransfecciónRESUMEN
We have investigated mechanisms of mitochondrial targeting of the phenobarbital-inducible hepatic mitochondrial P450MT4, which cross-reacts with antibody to microsomal P4502B1. Results show that P4502B1 and P450MT4 have identical primary sequence but different levels of phosphorylation and secondary structure. We demonstrate that P4502B1 contains a chimeric mitochondrial and endoplasmic reticulum (ER) targeting signal at its N-terminus. Inducers of cAMP and protein kinase A-mediated phosphorylation of P4502B1 at Ser128 activate the signal for mitochondrial targeting and modulate its mitochondrial or ER destination. S128A mutation inhibits in vitro mitochondrial transport and also in vivo mitochondrial targeting in COS cells. A fragment of P4502B1 containing the N-terminal signal and the phosphorylation site could drive the transport of dihydrofolate reductase (DHFR) into mitochondria. Ser128 phosphorylation reduced the affinity of 2B1 protein for binding to SRP, but increased the affinity of the 2B1-DHFR fusion protein for binding to yeast mitochondrial translocase proteins, TOM40 and TIM44, and matrix Hsp70. We describe a novel regulatory mechanism by which cAMP modulates the targeting of a protein to two distinct organelles.
Asunto(s)
Citocromo P-450 CYP2B1/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Transporte Biológico Activo , Células COS , AMP Cíclico/metabolismo , Citocromo P-450 CYP2B1/química , Citocromo P-450 CYP2B1/genética , Técnicas In Vitro , Mitocondrias Hepáticas/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Fenobarbital/farmacología , Fosforilación , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Recent studies from our laboratory showed that the beta-naphthoflavone-inducible cytochrome P4501A1 is targeted to both the endoplasmic reticulum (ER) and mitochondria. In the present study, we have further investigated the ability of the N-terminal signal sequence (residues 1-44) of P4501A1 to target heterologous proteins, dihydrofolate reductase, and the mature portion of the rat P450c27 to the two subcellular compartments. In vitro transport and in vivo expression experiments show that N-terminally fused 1-44 signal sequence of P4501A1 targets heterologous proteins to both the ER and mitochondria, whereas the 33-44 sequence strictly functions as a mitochondrial targeting signal. Site-specific mutations show that positively charged residues at the 34th and 39th positions are critical for mitochondrial targeting. Cholesterol 27-hydroxylase activity of the ER-associated 1-44/1A1-CYP27 fusion protein can be reconstituted with cytochrome P450 reductase, but the mitochondrial associated fusion protein is functional with adrenodoxin + adrenodoxin reductase. Consistent with these differences, the fusion protein in the two organelle compartments exhibited distinctly different membrane topology. The results on the chimeric nature of the N-terminal signal of P4501A1 coupled with interaction with different electron transport proteins suggest a co-evolutionary nature of some of the xenobiotic inducible microsomal and mitochondrial P450s.
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Citocromo P-450 CYP1A1/metabolismo , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Señales de Clasificación de Proteína/metabolismo , Animales , Transporte Biológico , Células COS , Colestanotriol 26-Monooxigenasa , Citocromo P-450 CYP1A1/química , Sistema Enzimático del Citocromo P-450/metabolismo , Inmunohistoquímica , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Esteroide Hidroxilasas/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The presence of xenobiotic-inducible CYP1A1, 2B1/2, and 3A1/2 in rat lung mitochondria was investigated using mitochondrial preparations of defined purity. The mitochondrial P450 content in uninduced lung was 1.5-fold higher compared to microsomes. Administration of BNF induced the P450 contents by twofold in both mitochondrial and microsomal membrane fractions. BNF treatment induced EROD activity to about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction. The microsomal induction was observed at 4 days of BNF treatment, while the mitochondrial induction required 10 days of treatment. Consistent with the activity profile, Western blot analysis showed the presence of CYP1A1 antibody reactive protein only in lung mitochondria from BNF-treated rats. BNF administration also caused a 50 to 80% reduction in the CYP2B1/2-associated PROD and BROD activities and CYP3A1/2-associated ERND activity in both mitochondria and microsomes. There was also a parallel reduction in the antibody reactive CYP2B1/2 and 3A1/2 proteins in both of these membrane fractions. Administration of DEX for 4 days induced mitochondrial and microsomal ERND activity by 1. 7- and 2.5-fold, respectively. Mitochondrial EROD activity was inhibited by antibodies to P450MT2, as well as Adx, but not by antibody against P450 reductase, indicating the mitochondrial localization of CYP1A1. Protease protection and alkaline extraction experiments indicated that CYP1A1 associated with lung mitochondria is localized inside the inner membrane and exists as a membrane extrinsic protein. In summary, this is probably the first report of inducible P450s in rat lung mitochondria, and our results suggest a possible functional role for these mitochondrial enzymes in xenobiotic metabolism.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Pulmón/enzimología , Mitocondrias/enzimología , Xenobióticos/farmacología , Animales , Northern Blotting , Catálisis , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP2B1/biosíntesis , Citocromo P-450 CYP3A , Inducción Enzimática/efectos de los fármacos , Técnicas In Vitro , Isoenzimas/biosíntesis , Masculino , Proteínas de la Membrana/metabolismo , Microsomas/efectos de los fármacos , Microsomas/enzimología , Oxigenasas de Función Mixta/biosíntesis , Ratas , Ratas Sprague-Dawley , Esteroide Hidroxilasas/biosíntesis , Fracciones Subcelulares/enzimologíaRESUMEN
We have investigated the mechanism of mitochondrial-nuclear crosstalk during cellular stress in mouse C2C12 myocytes. For this purpose, we used cells with reduced mitochondrial DNA (mtDNA) contents by ethidium bromide treatment or myocytes treated with known mitochondrial metabolic inhibitors, including carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin, valinomycin and azide. Both genetic and metabolic stresses similarly affected mitochondrial membrane potential (Deltapsim) and electron transport-coupled ATP synthesis, which was also accompanied by an elevated steady-state cytosolic Ca2+ level ([Ca2+]i). The mitochondrial stress resulted in: (i) an enhanced expression of the sarcoplasmic reticular ryanodine receptor-1 (RyR-1), hence potentiating the Ca2+ release in response to its modulator, caffeine; (ii) enhanced levels of Ca2+-responsive factors calineurin, calcineurin-dependent NFATc (cytosolic counterpart of activated T-cell-specific nuclear factor) and c-Jun N-terminal kinase (JNK)-dependent ATF2 (activated transcription factor 2); (iii) reduced levels of transcription factor, NF-kappaB; and (iv) enhanced transcription of cytochrome oxidase Vb (COX Vb) subunit gene. These cellular changes, including the steady-state [Ca2+]i were normalized in genetically reverted cells which contain near-normal mtDNA levels. We propose that the mitochondria-to-nucleus stress signaling occurs through cytosolic [Ca2+]i changes, which are likely to be due to reduced ATP and Ca2+ efflux. Our results indicate that the mitochondrial stress signal affects a variety of cellular processes, in addition to mitochondrial membrane biogenesis.