RESUMEN
Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the combination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-κB gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.
Asunto(s)
Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Humanos , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Resistencia a Antineoplásicos/genética , Secuenciación del Exoma , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
Angiogenesis and MYC expression associate with poor outcome in diffuse large B-cell lymphoma (DLBCL). MYC promotes neo-vasculature development but whether its deregulation in DLBCL contributes to angiogenesis is unclear. Examination of this relationship may uncover novel pathogenic regulatory circuitry as well as anti-angiogenic strategies in DLBCL. Here, we show that MYC expression positively correlates with vascular endothelial growth factor (VEGF) expression and angiogenesis in primary DLBCL biopsies, independently of dual expressor status or cell-of-origin classification. We found that MYC promotes VEGFA expression, a correlation that was validated in large datasets of mature B-cell tumours. Using DLBCL cell lines and patient-derived xenograft models, we identified the second messenger cyclic-AMP (cAMP) as a potent suppressor of MYC expression, VEGFA secretion and angiogenesis in DLBCL in normoxia. In hypoxia, cAMP switched targets and suppressed hypoxia-inducible factor 1α, a master regulator of VEGFA/angiogenesis in low oxygen environments. Lastly, we used the phosphodiesterase 4b (Pde4b) knockout mouse to demonstrate that the cAMP/PDE4 axis exercises additional anti-angiogenesis by directly targeting the lymphoma microenvironment. In conclusion, MYC could play a direct role in DLBCL angiogenesis, and modulation of cAMP levels, which can be achieved with clinical grade PDE4 inhibitors, has cell and non-cell autonomous anti-angiogenic activity in DLBCL.
Asunto(s)
AMP Cíclico , Subunidad alfa del Factor 1 Inducible por Hipoxia , Linfoma de Células B Grandes Difuso , Proteínas Proto-Oncogénicas c-myc , Adenosina Monofosfato , Animales , Línea Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Linfoma de Células B Grandes Difuso/patología , Ratones , Neovascularización Patológica/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mitochondria can function as signaling organelles, and part of this output leads to epigenetic remodeling. The full extent of this far-reaching interplay remains undefined. Here, we show that MYC transcriptionally activates IDH2 and increases alpha-ketoglutarate (αKG) levels. This regulatory step induces the activity of αKG-dependent DNA hydroxylases and RNA demethylases, thus reducing global DNA and RNA methylation. MYC, in a IDH2-dependent manner, also promotes the nuclear accumulation of TET1-TET2-TET3, FTO and ALKBH5. Notably, this subcellular movement correlated with the ability of MYC, in an IDH2-dependent manner, and, unexpectedly, of αKG to directly induce O-GlcNAcylation. Concordantly, modulation of the activity of OGT and OGA, enzymes that control the cycling of this non-canonical mono-glycosylation, largely recapitulated the effects of the MYC-IDH2-αKG axis on the subcellular movement of DNA and RNA demethylases. Together, we uncovered a hitherto unsuspected crosstalk between MYC, αKG and O-GlcNAcylation which could influence the epigenome and epitranscriptome homeostasis.
Asunto(s)
Metilación de ADN , ARN , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Mitocondrias/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
Cyclic-AMP (cAMP) exerts suppressive effects in the innate and adaptive immune system. The PD-1/PD-L1 immune checkpoint downregulates T-cell activity. Here, we examined if these two immunosuppressive nodes intersect. Using normal and malignant lymphocytes from humans, and the phosphodiesterase 4b (Pde4b) knockout mouse, we found that cAMP induces PD-L1 transcription and protein expression. Mechanistically, we discovered that the cAMP effectors PKA and CREB induce the transcription/secretion of IL-10, IL-8, and IL-6, which initiate an autocrine loop that activates the JAK/STAT pathway and ultimately increase PD-L1 expression in the cell surface. This signaling axis is disarmed at two specific nodes in subsets of diffuse large B-cell lymphoma, which may help explain the variable PD-L1 expression in these tumors. In vivo, we found that despite its immunosuppressive attributes, the PDE4 inhibitor roflumilast did not decrease the clinical activity of checkpoint inhibitors, an important clinical observation given the approved use of these agents in multiple diseases. In summary, we discovered that PD-L1 induction is a part of the repertoire of immunosuppressive actions mediated by cAMP, defined a cytokine-mediated autocrine loop that executes this action and, reassuringly, showed that PDE4 inhibition does not antagonize immune checkpoint blockade in an in vivo syngeneic lymphoma model.
Asunto(s)
Antígeno B7-H1/genética , AMP Cíclico/genética , Tolerancia Inmunológica/genética , Aminopiridinas/farmacología , Animales , Benzamidas/farmacología , Línea Celular Tumoral , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Ciclopropanos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Ratones , Ratones Noqueados , Inhibidores de Fosfodiesterasa 4/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
Several cancer cell types, including chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL) upregulate antiapoptotic Bcl-2 to cope with oncogenic stress. BH3 mimetics targeting Bcl-2's hydrophobic cleft have been developed, including venetoclax as a promising anticancer precision medicine for treating CLL patients. Recently, BDA-366 was identified as a small molecule BH4-domain antagonist that could kill lung cancer and multiple myeloma cells. BDA-366 was proposed to switch Bcl-2 from an antiapoptotic into a proapoptotic protein, thereby activating Bax and inducing apoptosis. Here, we scrutinized the therapeutic potential and mechanism of action of BDA-366 in CLL and DLBCL. Although BDA-366 displayed selective toxicity against both cell types, the BDA-366-induced cell death did not correlate with Bcl-2-protein levels and also occurred in the absence of Bcl-2. Moreover, although BDA-366 provoked Bax activation, it did neither directly activate Bax nor switch Bcl-2 into a Bax-activating protein in in vitro Bax/liposome assays. Instead, in primary CLL cells and DLBCL cell lines, BDA-366 inhibited the activity of the PI3K/AKT pathway, resulted in Bcl-2 dephosphorylation and reduced Mcl-1-protein levels without affecting the levels of Bcl-2 or Bcl-xL. Hence, our work challenges the current view that BDA-366 is a BH4-domain antagonist of Bcl-2 that turns Bcl-2 into a pro-apoptotic protein. Rather, our results indicate that other mechanisms beyond switching Bcl-2 conformation underlie BDA-366's cell-death properties that may implicate Mcl-1 downregulation and/or Bcl-2 dephosphorylation.
Asunto(s)
Antraquinonas/farmacología , Apoptosis , Etanolaminas/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Liposomas/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Fosforilación , Conformación Proteica , Dominios Proteicos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Transducción de Señal , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Mitochondrial D2HGDH and L2HGDH catalyze the oxidation of D-2-HG and L-2-HG, respectively, into αKG. This contributes to cellular homeostasis in part by modulating the activity of αKG-dependent dioxygenases. Signals that control the expression/activity of D2HGDH/L2HGDH are presumed to broadly influence physiology and pathology. Using cell and mouse models, we discovered that MYC directly induces D2HGDH and L2HGDH transcription. Furthermore, in a manner suggestive of D2HGDH, L2HGDH, and αKG dependency, MYC activates TET enzymes and RNA demethylases, and promotes their nuclear localization. Consistent with these observations, in primary B cell lymphomas MYC expression positively correlated with enhancer hypomethylation and overexpression of lymphomagenic genes. Together, these data provide additional evidence for the role of mitochondria metabolism in influencing the epigenome and epitranscriptome, and imply that in specific contexts wild-type TET enzymes could demethylate and activate oncogenic enhancers.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Epigenoma , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-myc/genética , Activación Transcripcional , Animales , Línea Celular , Femenino , Humanos , Masculino , Ratones Endogámicos C57BL , Transcriptoma , Células Tumorales CultivadasRESUMEN
The BCL-2 inhibitor venetoclax has only limited activity in DLBCL despite frequent BCL-2 overexpression. Since constitutive activation of the B cell receptor (BCR) pathway has been reported in both ABC and GCB DLBCL, we investigated whether targeting SYK or BTK will increase sensitivity of DLBCL cells to venetoclax. We report that pharmacological inhibition of SYK or BTK synergistically enhances venetoclax sensitivity in BCL-2-positive DLBCL cell lines with an activated BCR pathway in vitro and in a xenograft model in vivo, despite the only modest direct cytotoxic effect. We further show that these sensitizing effects are associated with inhibition of the downstream PI3K/AKT pathway and changes in the expression of MCL-1, BIM, and HRK. In addition, we show that BCR-dependent GCB DLBCL cells are characterized by deficiency of the phosphatase SHP1, a key negative regulator of the BCR pathway. Re-expression of SHP1 in GCB DBLCL cells reduces SYK, BLNK, and GSK3 phosphorylation and induces corresponding changes in MCL1, BIM, and HRK expression. Together, these findings suggest that SHP1 deficiency is responsible for the constitutive activation of the BCR pathway in GCB DLBCL and identify SHP1 and BCL-2 as potential predictive markers for response to treatment with a venetoclax/BCR inhibitor combination.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/metabolismo , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Sulfonamidas/farmacología , Quinasa Syk/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Linfoma de Células B Grandes Difuso/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Despite the well-known role of cystathionine γ-lyase (Cth) in cardiovascular pathophysiology, transcriptional regulation of Cth remains incompletely understood. Sequencing of the Cth promoter region in mouse models of genetic/essential hypertension (viz. Blood Pressure High [BPH], Blood Pressure Low [BPL] and Blood Pressure Normal [BPN] mice) identified several genetic variations. Transient transfections of BPH/BPL-Cth promoter-reporter plasmids into various cell types revealed higher promoter activity of BPL-Cth than that of BPH-Cth. Corroboratively, endogenous Cth mRNA levels in kidney and liver tissues were also elevated in BPL mice. Computational analysis of the polymorphic Cth promoter region predicted differential binding affinity of c-Rel, HOXA3 and IRF1 with BPL/BPH-Cth promoter domains. Over-expression of c-Rel/HOXA3/IRF1 modulated BPL/BPH-Cth promoter activities in a consistent manner. Gel shift assays using BPH/BPL-Cth-promoter oligonucleotides with/without binding sites for c-Rel/HOXA3/IRF1 displayed formation of specific complexes with c-Rel/HOXA3/IRF1; addition of antibodies to reaction mixtures resulted in supershifts/inhibition of Cth promoter-transcription factor complexes. Furthermore, chromatin immunoprecipitation (ChIP) assays proved differential binding of c-Rel, HOXA3 and IRF1 with the polymorphic promoter region of BPL/BPH-Cth. Tumor necrosis factor-α (TNF-α) reduced the activities of BPL/BPH-Cth promoters to different extents that were further declined by ectopic expression of IRF1; on the other hand, siRNA-mediated down-regulation of IRF1 rescued the TNF-α-mediated suppression of the BPL/BPH-Cth promoter activities. In corroboration, ChIP analysis revealed enhanced binding of IRF1 with BPH/BPL-Cth promoter following TNF-α treatment. BPL/BPH-Cth promoter activity was diminished upon exposure of hepatocytes and cardiomyoblasts to ischemia-like pathological condition due to reduced binding of c-Rel with BPL/BPH-Cth-promoter. Taken together, this study reveals the molecular basis for the differential expression of Cth in mouse models of essential hypertension under basal and pathophysiological conditions.
Asunto(s)
Cistationina gamma-Liasa/genética , Regulación de la Expresión Génica , Hipertensión/genética , Hipertensión/fisiopatología , Polimorfismo Genético , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Sitios de Unión , Presión Sanguínea , Mapeo Cromosómico , Biología Computacional/métodos , Modelos Animales de Enfermedad , Hipertensión Esencial , Genómica/métodos , Ratones , Motivos de Nucleótidos , Especificidad de Órganos/genética , Unión Proteica , Sitios de Carácter Cuantitativo , Ratas , Factores de Transcripción/metabolismo , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The Bcl-2 antagonist ABT-199 (venetoclax) has demonstrated promising clinical activity in patients with chronic lymphocytic leukemia (CLL). ABT-199 is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro but is much less effective against CLL cells that have received survival signals from the microenvironment. In particular, stimulation of CLL cells with CD40L results in substantial resistance mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-xL and Bfl-1. In this study, we investigated whether resistance to ABT-199 can be conferred by B-cell receptor (BCR) stimulation, which is another important survival signal from the leukemic microenvironment. We show that sustained BCR stimulation results in significant ABT-199 resistance, which correlates with induction of the antiapoptotic protein Mcl-1 and less consistently with downregulation of proapoptotic Bmf, Hrk, and BimEL A major role for Mcl-1 in conferring ABT-199 resistance is additionally supported by knockdown and enforced expression experiments with primary CLL cells. We further show that SYK, BTK, and phosphatidylinositol 3-kinase δ (PI3Kδ) inhibitors significantly downregulate Mcl-1, but with different efficacy. Complete Mcl-1 downregulation was consistently achieved only with SYK inhibitors R406 and GS-9973 (entospletinib), whereas the BTK inhibitor ibrutinib and the PI3Kδ inhibitor idelalisib in more than half of the cases had only a partial effect. The greater ability of SYK inhibitors to downregulate Mcl-1 correlated with their greater capacity to block BCR-mediated inactivation of GSK-3, a major negative regulator of Mcl-1. The finding that BCR signaling inhibitors differ in their ability to target Mcl-1 is relevant for the design of clinical trials combining these agents with ABT-199.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Sulfonamidas/uso terapéutico , Adenina/análogos & derivados , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Oxazinas/farmacología , Piperidinas , Purinas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Pirimidinas/farmacología , Quinazolinonas/farmacologíaRESUMEN
Monoamine oxidase A (MAOA) plays important roles in the pathogenesis of several neurological and cardiovascular disorders. The mechanism of transcriptional regulation of MAOA under basal and pathological conditions, however, remains incompletely understood. Here, we report systematic identification and characterization of cis elements and transcription factors that govern the expression of MAOA gene. Extensive computational analysis of MAOA promoter, followed by 5'-promoter deletion/reporter assays, revealed that the -71/-40 bp domain was sufficient for its basal transcription. Gel-shift and chromatin immunoprecipitation assays provided evidence of interactions of the transcription factors GATA-binding protein 2 (GATA2), Sp1 and TATA-binding protein (TBP) with this proximal promoter region. Consistently, over-expression of GATA2, Sp1 and TBP augmented MAOA promoter activity in a coordinated manner. In corroboration, siRNA-mediated down-regulation of GATA2/Sp1/TBP repressed the endogenous MAOA expression as well as transfected MAOA promoter activity. Tumor necrosis factor-α and forskolin activated MAOA transcription that was reversed by Sp1 siRNA; in support, tumor necrosis factor-α- and forskolin-induced activities were enhanced by ectopic over-expression of Sp1. On the other hand, MAOA transcription was diminished upon exposure of neuroblasts or cardiac myoblasts to ischemia-like conditions because of reduced binding of GATA2/Sp1/TBP with MAOA promoter. In conclusion, this study revealed previously unknown roles of GATA2, Sp1 and TBP in modulating MAOA expression under basal as well as pathophysiological conditions such as inflammation and ischemia, thus providing new insights into the molecular basis of aberrant MAOA expression in neuronal/cardiovascular disease states. Dysregulation of monoamine oxidase A (MAOA) have been implicated in several behavioral and neuronal disease states. Here, we identified three crucial transcription factors (GATA2, Sp1 and TBP) that regulate MAOA gene expression in a coordinated manner. Aberrant MAOA expression under pathophysiological conditions including inflammation and ischemia is mediated by altered binding of GATA2/Sp1/TBP with MAOA proximal promoter. Thus, these findings provide new insights into pathogenesis of several common diseases. GATA2, GATA-binding protein 2; Sp1, specificity protein 1; TBP, TATA-binding protein.
Asunto(s)
Factor de Transcripción GATA2/fisiología , Isquemia/metabolismo , Monoaminooxidasa/fisiología , Factor de Transcripción Sp1/fisiología , Proteína de Unión a TATA-Box/fisiología , Animales , Regulación de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Inflamación/genética , Inflamación/metabolismo , Isquemia/genética , Ratones , Datos de Secuencia MolecularRESUMEN
Renalase, a novel monoamine oxidase, is emerging as an important regulator of cardiovascular, metabolic, and renal diseases. However, the mechanism of transcriptional regulation of this enzyme remains largely unknown. We undertook a systematic analysis of the renalase gene to identify regulatory promoter elements and transcription factors. Computational analysis coupled with transfection of human renalase promoter/luciferase reporter plasmids (5'-promoter-deletion constructs) into various cell types (HEK-293, IMR32, and HepG2) identified two crucial promoter domains at base pairs -485 to -399 and -252 to -150. Electrophoretic mobility shift assays using renalase promoter oligonucleotides with and without potential binding sites for transcription factors Sp1, STAT3, and ZBP89 displayed formation of specific complexes with HEK-293 nuclear proteins. Consistently, overexpression of Sp1, STAT3, and ZBP89 augmented renalase promoter activity; additionally, siRNA-mediated downregulation of Sp1, STAT3, and ZBP89 reduced the level of endogenous renalase transcription as well as the transfected renalase promoter activity. In addition, chromatin immunoprecipitation assays showed in vivo interactions of these transcription factors with renalase promoter. Interestingly, renalase promoter activity was augmented by nicotine and catecholamines; while Sp1 and STAT3 synergistically activated the nicotine-induced effect, Sp1 appeared to enhance epinephrine-evoked renalase transcription. Moreover, renalase transcript levels in mouse models of human essential hypertension were concomitantly associated with endogenous STAT3 and ZBP89 levels, suggesting crucial roles for these transcription factors in regulating renalase gene expression in cardiovascular pathological conditions.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Monoaminooxidasa/genética , Factor de Transcripción STAT3/fisiología , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Secuencia de Consenso , Hipertensión Esencial , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Hipertensión/enzimología , Hipertensión/genética , Masculino , Ratones Endogámicos , Monoaminooxidasa/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Regiones Promotoras Genéticas , Transcripción Genética , Activación TranscripcionalRESUMEN
Although the transcript level of inducible heat shock protein 70.3 (Hsp70.3, also known as Hspa1a) is altered in various disease states, its transcriptional regulation remains incompletely understood. Here, we systematically analyzed the Hspa1a promoter to identify major cis elements and transcription factors that may govern the constitutive/inducible gene expression. Computational analyses coupled with extensive in vitro (promoter-reporter activity and electrophoretic mobility shift assays) and in vivo (chromatin immunoprecipitation assays) revealed interaction of several transcription factors with Hspa1a promoter motifs: HSF-1 (heat shock factor 1) at -114/-97 bp and -788/-777bp, NF-Y (nuclear transcription factor Y) at -73/-58 bp, NF-κB (nuclear factor kappa B) at -133/-124 bp, and CREB (cAMP response element binding protein) at -483/-476 bp. Consistently, siRNA (small interfering RNA)-mediated down-regulation of each of these transcription factors caused substantial reduction of endogenous Hspa1a expression. Heat-shock-induced activation of Hspa1a was coordinately regulated by HSF-1 and NF-Y/NF-κB. The Hspa1a expression was augmented by TNF-α (tumor necrosis factor-alpha) and forskolin in NF-κB and CREB-dependent manners, respectively. NF-κB and CREB also activated Hspa1a transcription in cardiac myoblasts upon exposure to ischemia-like conditions. Taken together, this study discovered previously unknown roles for NF-κB and CREB to regulate Hspa1a expression and a coordinated action by several transcription factors for Hspa1a transactivation under heat-shock/ischemia-like conditions and thereby provided new insights into the mechanism of Hspa1a regulation.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , FN-kappa B/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Fusión Artificial Génica , Inmunoprecipitación de Cromatina , Biología Computacional , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Factores de Transcripción del Choque TérmicoRESUMEN
Catestatin (CST), a chromogranin A (CHGA)-derived peptide, is a potent inhibitor of catecholamine release from adrenal chromaffin cells and postganglionic sympathetic axons. We re-sequenced the CST region of CHGA in an Indian population (n = 1010) and detected two amino acid substitution variants: G364S and G367V. Synthesized CST variant peptides (viz. CST-Ser-364 and CST-Val-367) were significantly less potent than the wild type peptide (CST-WT) to inhibit nicotine-stimulated catecholamine secretion from PC12 cells. Consistently, the rank-order of blockade of nicotinic acetylcholine receptor (nAChR)-stimulated inward current and intracellular Ca(2+) rise by these peptides in PC12 cells was: CST-WT > CST-Ser-364 > CST-Val-367. Structural analysis by CD spectroscopy coupled with molecular dynamics simulations revealed the following order of α-helical content: CST-WT > CST-Ser-364 > CST-Val-367; docking of CST peptides onto a major human nAChR subtype and molecular dynamics simulations also predicted the above rank order for their binding affinity with nAChR and the extent of occlusion of the receptor pore, providing a mechanistic basis for differential potencies. The G364S polymorphism was in strong linkage disequilibrium with several common CHGA genetic variations. Interestingly, the Ser-364 allele (detected in â¼15% subjects) was strongly associated with profound reduction (up to â¼2.1-fold) in plasma norepinephrine/epinephrine levels consistent with the diminished nAChR desensitization-blocking effect of CST-Ser-364 as compared with CST-WT. Additionally, the Ser-364 allele showed strong associations with elevated levels of plasma triglyceride and glucose levels. In conclusion, a common CHGA variant in an Indian population influences several biochemical parameters relevant to cardiovascular/metabolic disorders.
Asunto(s)
Alelos , Enfermedades Cardiovasculares , Cromogranina A , Enfermedades Metabólicas , Fragmentos de Péptidos , Sitios de Carácter Cuantitativo , Adulto , Animales , Glucemia/metabolismo , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/genética , Cromogranina A/química , Cromogranina A/genética , Cromogranina A/metabolismo , Cromogranina A/farmacología , Dicroismo Circular , Epinefrina/metabolismo , Femenino , Humanos , India , Masculino , Enfermedades Metabólicas/sangre , Enfermedades Metabólicas/genética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Norepinefrina/metabolismo , Células PC12 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Ratas , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Triglicéridos/sangreRESUMEN
Catestatin (CST), a chromogranin-A-derived peptide, is a potent endogenous inhibitor of the neuronal nicotinic acetylcholine receptor (nAChR). It exerts an anti-hypertensive effect by acting as a 'physiological brake' on transmitter release into the circulation. However, the mechanism of interaction of CST with nAChR is only partially understood. To unravel molecular interactions of the wild-type human CST (CST-WT) as well as its naturally occurring variants (CST-364S and CST-370L, which have GlyâSer and ProâLeu substitutions, respectively) with the human α3ß4 nAChR, we generated a homology-modeled human α3ß4 nAChR structure and solution structures of CST peptides. Docking and molecular dynamics simulations showed that ~90% of interacting residues were within 15 N-terminal residues of CST peptides. The rank order of binding affinity of these peptides with nAChR was: CST-370L>CST-WT>CST-364S; the extent of occlusion of the receptor pore by these peptides was also in the same order. In corroboration with computational predictions, circular dichroism analysis revealed significant differences in global structures of CST peptides (e.g. the order of α-helical content was: CST-370L>CST-WT>CST-364S). Consistently, CST peptides blocked various stages of nAChR signal transduction, such as nicotine- or acetylcholine-evoked inward current, rise in intracellular Ca(2+) and catecholamine secretion in or from neuron-differentiated PC12 cells, in the same rank order. Taken together, this study shows molecular interactions between human CST peptides and human α3ß4 nAChR, and demonstrates that alterations in the CST secondary structure lead to the gain of potency for CST-370L and loss of potency for CST-364S. These findings have implications for understanding the nicotinic cholinergic signaling in humans.
Asunto(s)
Antihipertensivos/metabolismo , Cromogranina A/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Receptores Nicotínicos/química , Transducción de Señal/efectos de los fármacos , Acetilcolina/farmacología , Sustitución de Aminoácidos , Animales , Antihipertensivos/síntesis química , Antihipertensivos/farmacología , Sitios de Unión , Calcio/metabolismo , Catecolaminas/metabolismo , Cromogranina A/síntesis química , Cromogranina A/farmacología , Dicroismo Circular , Humanos , Simulación de Dinámica Molecular , Nicotina/farmacología , Células PC12 , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/farmacología , Unión Proteica , Ratas , Receptores Nicotínicos/metabolismo , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
3-Hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase gene (Hmgcr) is a susceptibility gene for essential hypertension. Sequencing of the Hmgcr locus in genetically hypertensive BPH (blood pressure high), genetically hypotensive BPL (blood pressure low) and genetically normotensive BPN (blood pressure normal) mice yielded a number of single nucleotide polymorphisms (SNPs). BPH/BPL/BPN Hmgcr promoter-luciferase reporter constructs were generated and transfected into liver HepG2, ovarian CHO, kidney HEK-293 and neuronal N2A cells for functional characterization of the promoter SNPs. The BPH-Hmgcr promoter showed significantly less activity than the BPL-Hmgcr promoter under basal as well as nicotine/cholesterol-treated conditions. This finding was consistent with lower endogenous Hmgcr expression in liver and lower plasma cholesterol in BPH mice. Transfection experiments using 5'-promoter deletion constructs (strategically made to assess the functional significance of each promoter SNP) and computational analysis predicted lower binding affinities of transcription factors c-Fos, n-Myc and Max with the BPH-promoter as compared to the BPL-promoter. Corroboratively, the BPH promoter-luciferase reporter construct co-transfected with expression plasmids of these transcription factors displayed less pronounced augmentation of luciferase activity than the BPL construct, particularly at lower amounts of transcription factor plasmids. Electrophoretic mobility shift assays also showed diminished interactions of the BPH promoter with HepG2 nuclear proteins. Taken together, this study provides mechanistic basis for the differential Hmgcr expression in these mouse models of human essential hypertension and have implications for better understanding the role of this gene in regulation of blood pressure.
Asunto(s)
Hidroximetilglutaril-CoA Reductasas/genética , Hipertensión/genética , Polimorfismo Genético , Regiones Promotoras Genéticas/genética , Animales , Presión Sanguínea , Línea Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Polimorfismo de Nucleótido SimpleRESUMEN
Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by nanoparticles is an emerging area of research. We observed that TiO(2) nanoparticles of approximately 25 nm diameter caused significant enhancement of PCR efficiency for various types of templates (namely plasmid DNA, genomic DNA and complementary DNA). By a series of experiments, the optimal TiO(2) concentration was determined to be 0.4 nM, which resulted in up to a seven-fold increase in the amount of PCR product. As much as 50% reduction in overall reaction time (by reduction of the number of cycles and the time periods of cycles) was also achieved by utilizing TiO(2) nanoparticles without compromising the PCR yield. Investigations of the mechanism of such PCR enhancement by simulations using the 'Fluent K epsilon turbulent model' provided evidence of faster heat transfer in the presence of TiO(2) nanoparticles. Consistent with these findings, TiO(2) nanoparticles were observed to augment the denaturation of genomic DNA, indicating more efficient thermal conductivity through the reaction buffer. TiO(2) nanoparticle-assisted PCR may be useful for profound reduction of the overall PCR reaction period and for enhanced amplification of DNA amplicons from a variety of samples, including GC-rich templates that are often observed to yield unsatisfactory results.