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1.
Hum Mol Genet ; 30(15): 1413-1428, 2021 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-33987651

RESUMEN

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.


Asunto(s)
Canales de Cloruro/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/metabolismo , Nefrolitiasis/genética , Nefrolitiasis/metabolismo , Animales , Fenómenos Biológicos , Línea Celular , Canales de Cloruro/metabolismo , Enfermedad de Dent/genética , Endocitosis/fisiología , Estudios de Asociación Genética , Enfermedades Genéticas Ligadas al Cromosoma X/fisiopatología , Humanos , Hipercalciuria/genética , Túbulos Renales Proximales/metabolismo , Mutación , Nefrocalcinosis/genética , Nefrolitiasis/fisiopatología , Proteinuria/genética
2.
Brain ; 143(5): 1414-1430, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32282893

RESUMEN

Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.


Asunto(s)
Inflamasomas/inmunología , Interleucina-1beta/inmunología , Esclerosis Múltiple Crónica Progresiva/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Adulto , Animales , Biomarcadores/análisis , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Pronóstico
3.
Toxicol Appl Pharmacol ; 258(2): 275-87, 2012 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22155090

RESUMEN

Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells.


Asunto(s)
Apoptosis/efectos de los fármacos , Ciclosporina/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inmunosupresores/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Animales , Células Cultivadas , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Túbulos Renales Proximales/patología , Metaloproteinasas de la Matriz/metabolismo , Ratones , Proteína Quinasa C/metabolismo , Especies Reactivas de Oxígeno/metabolismo
4.
J Mol Cell Biol ; 12(7): 499-514, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32162654

RESUMEN

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial-mesenchymal transition (pEMT), proliferation, and further redifferentiation into specialized tubule epithelial cells (TECs). Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase (PPIase) activity of cyclophilin (Cyp) proteins, we hypothesized that cyclophilins could regulate TEC phenotype. Here we demonstrate that in cultured TECs, CypA silencing triggers loss of epithelial features and enhances transforming growth factor ß (TGFß)-induced EMT in association with upregulation of epithelial repressors Slug and Snail. This pro-epithelial action of CypA relies on its PPIase activity. By contrast, CypB emerges as an epithelial repressor, because CypB silencing promotes epithelial differentiation, prevents TGFß-induced EMT, and induces tubular structures in 3D cultures. In addition, in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction, inflammatory and pro-fibrotic events were attenuated. CypB silencing/knockout leads to Slug, but not Snail, downregulation. CypB support of Slug expression depends on its endoplasmic reticulum location, where it interacts with calreticulin, a calcium-buffering chaperone related to Slug expression. As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation, we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling. In conclusion, this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.


Asunto(s)
Ciclofilinas/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Túbulos Renales/citología , Animales , Basigina/metabolismo , Calcio/metabolismo , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células Epiteliales/efectos de los fármacos , Fibrosis , Silenciador del Gen/efectos de los fármacos , Humanos , Inflamación/patología , Ionomicina/farmacología , Ratones , Fenotipo , Transporte de Proteínas/efectos de los fármacos , Proteínas Smad/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Tapsigargina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Obstrucción Ureteral/patología
5.
PLoS One ; 15(1): e0227340, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31910234

RESUMEN

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit ß (shCK2ß cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2ß. However, in HK-2 cells the dependence on CK2ß was confirmed by rescue experiments (CK2ß re-expression in shCK2ß HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2ß downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2ß was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2ß HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3ß S9, was increased. Differently to what observed in response to CK2ß down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3ß pS9 mirrored those induced by CK2ß knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3ß pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2ß knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3ß pS9 was not reduced by CK2 blockade suggests that GSK3ß phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3ß pS9, and concomitantly decreased Snail1 levels (a GSK3ß target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2ß-downregulated cells, suggesting that reducing GSK3ß pS9 could be a strategy to control Snail1 levels in any situation where CK2ß is defective, as possibly occurring in cancer cells.


Asunto(s)
Quinasa de la Caseína II/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Proteína Oncogénica v-akt/genética , Factores de Transcripción de la Familia Snail/genética , Sistemas CRISPR-Cas/genética , Línea Celular , Cromonas/farmacología , Transición Epitelial-Mesenquimal/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Morfolinas/farmacología , Naftiridinas/farmacología , Fenazinas , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/efectos de los fármacos , Isoformas de Proteínas , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal/efectos de los fármacos
6.
J R Soc Interface ; 16(151): 20180709, 2019 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-30958186

RESUMEN

The scratch assay is an in vitro technique used to assess the contribution of molecular and cellular mechanisms to cell migration. The assay can also be used to evaluate therapeutic compounds before clinical use. Current quantification methods of scratch assays deal poorly with irregular cell-free areas and crooked leading edges which are features typically present in the experimental data. We introduce a new migration quantification method, called 'monolayer edge velocimetry', that permits analysis of low-quality experimental data and better statistical classification of migration rates than standard quantification methods. The new method relies on quantifying the horizontal component of the cell monolayer velocity across the leading edge. By performing a classification test on in silico data, we show that the method exhibits significantly lower statistical errors than standard methods. When applied to in vitro data, our method outperforms standard methods by detecting differences in the migration rates between different cell groups that the other methods could not detect. Application of this new method will enable quantification of migration rates from in vitro scratch assay data that cannot be analysed using existing methods.


Asunto(s)
Movimiento Celular , Proliferación Celular , Simulación por Computador , Modelos Biológicos , Línea Celular Tumoral , Humanos
7.
Oncotarget ; 9(5): 5736-5751, 2018 Jan 19.
Artículo en Inglés | MEDLINE | ID: mdl-29464030

RESUMEN

Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of renal cancer. STAT3 pathway is altered in these tumors and p-STAT3 Ser727 is an independent prognostic factor for ccRCC. Protein kinase CK2 is altered in different types of tumors and overexpression of CK2α is considered predictive of bad prognosis and metastatic risk. CK2 subunits analyses in ccRCC samples showed increased CK2α/α' nuclear content in all cases, but decreased cytosolic CK2ß (CK2ßcyt) levels in the more advanced tumors. Stable downregulation of CK2ß in renal proximal tubular (HK-2) and clear cell adenocarcinoma (786-O) cells triggered changes in E-cadherin, vimentin and Snail1 protein levels indicative of epithelial-to-mesenchymal transition (EMT), and increased HIF-α. Moreover, CK2ß was required in order to observe STAT3 Ser727 phosphorylation in HK-2 but not in 786-O cells. We also observed that CK2ß improved the prognostic value of p-STAT3 Ser727, as CK2ßcyt>41 (median value) discriminates patients free of disease for a period of 10 years upon surgery, from those with CK2ßcyt<41, when p-STAT3 Ser727levels are low. We conclude that CK2ß down-regulation might represent a mechanism to support EMT and angiogenesis and that CK2ßcyt levels are instrumental to refine prognosis of ccRCC patients with low p-STAT3 Ser727 levels.

8.
Biochem J ; 394(Pt 1): 227-36, 2006 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-16225457

RESUMEN

CK2 (protein kinase CK2) is known to phosphorylate eIF2 (eukaryotic translation initiation factor 2) in vitro; however, its implication in this process in living cells has remained to be confirmed. The combined use of chemical inhibitors (emodin and apigenin) of CK2 together with transfection experiments with the wild-type of the K68A kinase-dead mutant form of CK2alpha evidenced the direct involvement of this protein kinase in eIF2beta phosphorylation in cultured HeLa cells. Transfection of HeLa cells with human wild-type eIF2beta or its phosphorylation site mutants showed Ser2 as the main site for constitutive eIF2beta phosphorylation, whereas phosphorylation at Ser67 seems more restricted. In vitro phosphorylation of eIF2beta also pointed to Ser2 as a preferred site for CK2 phosphorylation. Overexpression of the eIF2beta S2/67A mutant slowed down the rate of protein synthesis stimulated by serum, although less markedly than the overexpression of the Delta2-138 N-terminal-truncated form of eIF2beta (eIF2beta-CT). Mutation at Ser2 and Ser67 did not affect eIF2beta integrating into the eIF2 trimer or being able to complex with eIF5 and CK2alpha. The eIF2beta-CT form was also incorporated into the eIF2 trimer but did not bind to eIF5. Overexpression of eIF2beta slightly decreased HeLa cell viability, an effect that was more evident when overexpressing the eIF2beta S2/67A mutant. Cell death was particularly marked when overexpressing the eIF2beta-CT form, being detectable at doses where eIF2beta and eIF2beta S2/67A were ineffective. These results suggest that Ser2 and Ser67 contribute to the important role of the N-terminal region of eIF2beta for its function in mammals.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/genética , Perfilación de la Expresión Génica , Células HeLa , Humanos , Mutación , Fosforilación , Unión Proteica , Biosíntesis de Proteínas , Estructura Terciaria de Proteína
9.
Am J Transl Res ; 9(9): 4173-4183, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28979691

RESUMEN

Immunosuppression based on calcineurin inhibitors (CNIs) has greatly improved organ transplantation, although subsequent nephrotoxicity significantly hinders treatment success. There are no currently available specific soluble biomarkers for CNI-induced nephrotoxicity and diagnosis relies on renal biopsy, which is costly, invasive and may cause complications. Accordingly, identification of non-invasive biomarkers distinguishing CNI-induced kidney tubular damage from that of other etiologies would greatly improve diagnosis and enable more precise dosage adjustment. For this purpose, HK-2 cells, widely used to model human proximal tubule, were treated with CNIs cyclosporine-A and FK506, or staurosporine as a calcineurin-independent toxic compound, and secretomes of each treatment were analyzed by proteomic means. Among the differentially secreted proteins identified, only fascin-1 was specifically released by both CNIs but not by staurosporine. To validate fascin-1 as a biomarker of CNI-induced tubular toxicity, fascin-1 levels were analyzed in serum and urine from kidney-transplanted patients under CNIs treatment presenting or not isometric vacuolization (IV), which nowadays represents the main histological hallmark of CNI-induced tubular damage. Patients with chronic kidney disease (CKD) and healthy volunteers were used as controls. Our results show that urinary fascin-1 was only significantly elevated in the subset of CNI-treated patients presenting IV. Moreover, fascin-1 anticipated the rise of sCr levels in serially collected urine samples from CNI-treated pulmonary-transplanted patients, where a decline in kidney function and serum creatinine (sCr) elevation was mainly attributed to CNIs treatment. In conclusion, our results point towards fascin-1 as a putative soluble biomarker of CNI-induced damage in the kidney tubular compartment.

10.
Biomed Res Int ; 2014: 750602, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24991565

RESUMEN

Serial surveillance renal allograft biopsies have shown that early subclinical inflammation constitutes a risk factor for the development of interstitial fibrosis. More recently, it has been observed that persistent inflammation is also associated with fibrosis progression and chronic humoral rejection, two histological conditions associated with poor allograft survival. Treatment of subclinical inflammation with steroid boluses prevents progression of fibrosis and preserves renal function in patients treated with a cyclosporine-based regimen. Subclinical inflammation has been reduced after the introduction of tacrolimus based regimens, and it has been shown that immunosuppressive schedules that are effective in preventing acute rejection and subclinical inflammation may prevent the progression of fibrosis and chronic humoral rejection. On the other hand, minimization protocols are associated with progression of fibrosis, and noncompliance with the immunosuppressive regime constitutes a major risk factor for chronic humoral rejection. Thus, adequate immunosuppressive treatment, avoiding minimization strategies and reinforcing educational actions to prevent noncompliance, is at present an effective approach to combat the progression of fibrosis.


Asunto(s)
Fibrosis/tratamiento farmacológico , Rechazo de Injerto/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Trasplante de Riñón/efectos adversos , Ciclosporina/uso terapéutico , Fibrosis/complicaciones , Fibrosis/patología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Supervivencia de Injerto/efectos de los fármacos , Humanos , Inmunosupresores/uso terapéutico , Inflamación/complicaciones , Inflamación/patología , Tacrolimus/uso terapéutico
11.
Cancer Res ; 74(5): 1416-28, 2014 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-24390735

RESUMEN

Renal cell carcinoma (RCC), the third most prevalent urological cancer, claims more than 100,000 lives/year worldwide. The clear cell variant (ccRCC) is the most common and aggressive subtype of this disease. While commonly asymptomatic, more than 30% of ccRCC are diagnosed when already metastatic, resulting in a 95% mortality rate. Notably, nearly one-third of organ-confined cancers treated by nephrectomy develop metastasis during follow-up care. At present, diagnostic and prognostic biomarkers to screen, diagnose, and monitor renal cancers are clearly needed. The gene encoding the cell surface molecule HAVCR1/KIM-1 is a suggested susceptibility gene for ccRCC and ectodomain shedding of this molecule may be a predictive biomarker of tumor progression. Microarray analysis of 769-P ccRCC-derived cells where HAVCR/KIM-1 levels have been upregulated or silenced revealed relevant HAVCR/KIM-1-related targets, some of which were further analyzed in a cohort of 98 ccRCC patients with 100 month follow-up. We found that HAVCR/KIM-1 activates the IL-6/STAT-3/HIF-1A axis in ccRCC-derived cell lines, which depends on HAVCR/KIM-1 shedding. Moreover, we found that pSTAT-3 S727 levels represented an independent prognostic factor for ccRCC patients. Our results suggest that HAVCR/KIM-1 upregulation in tumors might represent a novel mechanism to activate tumor growth and angiogenesis and that pSTAT-3 S727 is an independent prognostic factor for ccRCC.


Asunto(s)
Carcinoma de Células Renales/genética , Interleucina-6/genética , Neoplasias Renales/genética , Glicoproteínas de Membrana/genética , Receptores Virales/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/patología , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Interleucina-6/metabolismo , Neoplasias Renales/patología , Glicoproteínas de Membrana/metabolismo , Receptores Virales/metabolismo , Factor de Transcripción STAT3/metabolismo , Regulación hacia Arriba/genética
12.
Eur J Cancer ; 49(8): 2034-47, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23352434

RESUMEN

AIM OF THE STUDY: To correlate hepatitis A virus cellular receptor (HAVCR)/kidney injury molecule-1 (KIM-1) expression in clear cell renal cell carcinoma (ccRCC) tumours with patient outcome and study the consequences of HAVCR/KIM-1 ectodomain shedding. METHODS: HAVCR/KIM-1 expression in ccRCC, oncocytomes, papillary carcinomas and unaffected tissue counterparts was evaluated. Minimal change disease and pre-clamping normal and ccRCC tissue biopsies were included. Tissue microarrays from 98 ccRCC tumours were analysed. Tumour registry data and patient outcome were retrospectivelly collected. Deletions in HAVCR/KIM-1 ectodomain and lentiviral infection of 786-O cells with HAVCR/KIM-1 mutated constructs to determine their subcellular distribution and invasive capacity were performed. RESULTS: HAVCR/KIM-1 was expressed in ccRCC, papillary tumours and in tubule cells of adjacent and distal unaffected counterparts of ccRCC tumours. The latest was not related to ischemic or tumour-related paracrine effects since pre-clamping normal biopsies were positive for HAVCR/KIM-1 and unaffected counterparts of papillary tumours were negative. HAVCR/KIM-1 analyses in patients and the invasive capacity of HAVCR/KIM-1 shedding mutants in cell lines demonstrated that: (i) relative low HAVCR/KIM-1 membrane levels correlate with activated shedding in ccRCC patients and mutant cell lines; (ii) augmented shedding directly correlates with higher invasiveness and tumour malignancy. CONCLUDING STATEMENTS: Constitutive expression of HAVCR/KIM-1 in kidney might constitute a susceptibility trait for ccRCC tumour development. Enhanced HAVCR/KIM-1 ectodomain shedding promotes invasive phenotype in vitro and more aggressive tumours in vivo.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Virales/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Sitios de Unión/genética , Biomarcadores de Tumor/genética , Western Blotting , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad/genética , Células HEK293 , Receptor Celular 1 del Virus de la Hepatitis A , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Riñón/metabolismo , Riñón/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Glicoproteínas de Membrana/genética , Microscopía Fluorescente , Persona de Mediana Edad , Análisis Multivariante , Mutación , Pronóstico , Receptores Virales/genética , Estudios Retrospectivos
13.
FEBS Lett ; 585(2): 414-20, 2011 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-21187092

RESUMEN

In the present work we report the presence of protein kinase CK2 in lipid raft preparations from rat brain synaptosomes, obtained after detergent extraction and subsequent isolation of detergent-resistant membranes using sucrose gradient ultracentrifugation. Moreover, the phosphorylation of syntaxin-1 at Ser14, a specific CK2 target, has been detected in lipid rafts, as assessed by a phospho-specific antibody. Treatment with DMAT, a specific CK2 inhibitor, results in a decrease of syntaxin-1 Ser14 phosphorylation in lipid rafts, while the glutamate release from synaptosomes is enhanced. In conclusion, CK2 might control neurotransmitter release by acting on SNARE proteins attached to cholesterol-enriched microdomains.


Asunto(s)
Química Encefálica/fisiología , Quinasa de la Caseína II/antagonistas & inhibidores , Quinasa de la Caseína II/metabolismo , Microdominios de Membrana/metabolismo , Neurotransmisores/metabolismo , Sinaptosomas/química , Animales , Quinasa de la Caseína II/análisis , Microdominios de Membrana/química , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Proteínas SNARE/metabolismo , Sintaxina 1/metabolismo
14.
PLoS One ; 6(9): e25746, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21980535

RESUMEN

The use of cyclosporine A (CsA) is limited by its severe nephrotoxicity that includes reversible vasoconstrictor effects and proximal tubule cell injury, the latter associated whith chronic kidney disease progression. The mechanisms of CsA-induced tubular injury, mainly on the S3 segment, have not been completely elucidated. Kidney androgen-regulated protein (KAP) is exclusively expressed in kidney proximal tubule cells, interacts with the CsA-binding protein cyclophilin B and its expression diminishes in kidneys of CsA-treated mice. Since we reported that KAP protects against CsA toxicity in cultured proximal tubule cells, we hypothesized that low KAP levels found in kidneys of CsA-treated mice might correlate with proximal tubule cell injury. To test this hypothesis, we used KAP Tg mice developed in our laboratory and showed that these mice are more resistant to CsA-induced tubular injury than control littermates. Furthermore, we found that calpain, which was activated by CsA in cell cultures and kidney, is involved in KAP degradation and observed that phosphorylation of serine and threonine residues found in KAP PEST sequences by protein kinase CK2 enhances KAP degradation by calpain. Moreover, we also observed that CK2 inhibition protected against CsA-induced cytotoxicity. These findings point to a novel mechanism for CsA-induced kidney toxicity that might be useful in developing therapeutic strategies aimed at preventing tubular cell damage while maintaining the immunosuppressive effects of CsA.


Asunto(s)
Calpaína/metabolismo , Quinasa de la Caseína II/metabolismo , Ciclosporina/toxicidad , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/lesiones , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Línea Celular , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Túbulos Renales Proximales/enzimología , Túbulos Renales Proximales/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas/química , Proteínas/genética , Especificidad por Sustrato
15.
PLoS One ; 5(11): e13930, 2010 Nov 10.
Artículo en Inglés | MEDLINE | ID: mdl-21085665

RESUMEN

Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase ß1 subunit protein (Na/K-ß1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-ß1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.


Asunto(s)
Ciclofilinas/metabolismo , Túbulos Renales Proximales/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Western Blotting , Células COS , Línea Celular Transformada , Chlorocebus aethiops , Ciclofilinas/genética , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Túbulos Renales Proximales/citología , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , ATPasa Intercambiadora de Sodio-Potasio/genética , Técnicas del Sistema de Dos Híbridos
16.
Mol Cell Biochem ; 274(1-2): 53-61, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16335529

RESUMEN

The beta-subunit of eukaryotic translation initiation factor eIF2 is a substrate and a partner for protein kinase CK2. Surface plasmon resonance analysis shows that the truncated form corresponding to residues 138-333 of eIF2beta (eIF2beta-CT) interacts with CK2beta as efficiently as full length eIF2beta, whereas the form corresponding to residues 1-137, which contains the CK2 phosphorylation sites, (eIF2beta-NT) does not bind. The use of different mutants and truncated forms of CK2alpha allowed us to map the basic segment K74-K83 at the beginning of helix alphaC and residues R191R195K198 in the p + 1 loop as the main determinants for the binding to eIF2beta-CT of either the isolated CK2alpha subunit or the CK2 holoenzyme. The presence of eIF2beta-CT stimulated the activity of CK2alpha towards the RRRAADSDDDDD peptide substrate; effect that was not observed with the CK2a K74-77A whose ability to bind to eIF2beta-CT is severely impaired. Gel filtration analysis confirmed the ability of CK2alpha to form complexes with eIF2beta-CT, and the contribution of the basic cluster in CK2alpha (K74-K77) in this association.


Asunto(s)
Quinasa de la Caseína II/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Secuencias de Aminoácidos , Animales , Quinasa de la Caseína II/genética , Catálisis , Dominio Catalítico , Factor 2 Eucariótico de Iniciación/genética , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Mutación , Oligopéptidos/metabolismo , Fosforilación , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Especificidad por Sustrato
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