Catalina Ribas | Universidad Autónoma de Madrid (original) (raw)

Papers by Catalina Ribas

Different stable lines of CHO cells (previously characterised in [<a href="http:... more Different stable lines of CHO cells (previously characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B11" target="_blank">11</a>]) that stably overexpress wild-type or phosphorylation-deficient muscarinic M3 receptor (A) and wild-type or phosphorylation-deficient muscarinic M1 receptor (B) were utilised. All cell lines were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with acetylcholine (100µM) for the indicated times (A) or incubated for 15min with acetylcholine at various concentrations (B). ERK5 phosphorylation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#pone-0084174-g002" target="_blank">Figure 2</a>. Data (mean +/- SD of 2-3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions or over maximum activation (*p<0.05, **p<0.005; two-tailed T-test). </p

(A) CHO cells stably expressing wild-type muscarinic M3 receptor (CHO... more (A) CHO cells stably expressing wild-type muscarinic M3 receptor (CHO-M3 cells) were transfected with cDNAs encoding for HA-ERK5 and either pcDNA3, Gαq, β-arrestin1-Flag or β-arrestin2-GFP. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM) for the indicated times. ERK5 phosphorylation was assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#pone-0084174-g002" target="_blank">Figure 2</a>. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, **p<0.005, two-tailed T-test). Gαq, β-arrestin1-Flag and β-arrestin2-GFP, and α-tubulin expression levels were assessed with specific antibodies. (B) NIH-3T3-M1 cells were transfected with siRNA oligonucleotides targeting β-arrestin2 or non-targeting scrambled oligonucleotides (100nM) as detailed in the Materials and Methods section. After 72h of incubation, cells were serum-starved for 5h and challenged with carbachol (10µM) for the indicated times. Endogenous ERK5 phosphorylation was assessed in cell lysates with a phospho-ERK5 specific antibody. Total ERK5 appears as a double band corresponding to basal (lower) and hyper-phosphorylated (upper) kinase. A representative blot for 3 independent experiments with similar results is shown. (C) β-arrestin2 expression levels were also determined and quantified to estimate the overall efficiency of protein silencing. Data (mean +/- SEM of 3 independent experiments) were normalised using α-tubulin as loading control and expressed as the relative difference (%) to β-arrestin2 protein levels in scrambled siRNA-treated cells.</p

Antioxidants

All processes in human physiology relies on homeostatic mechanisms which require the activation o... more All processes in human physiology relies on homeostatic mechanisms which require the activation of specific control circuits to adapt the changes imposed by external stimuli. One of the critical modulators of homeostatic balance is autophagy, a catabolic process that is responsible of the destruction of long-lived proteins and organelles through a lysosome degradative pathway. Identification of the mechanism underlying autophagic flux is considered of great importance as both protective and detrimental functions are linked with deregulated autophagy. At the mechanistic and regulatory levels, autophagy is activated in response to diverse stress conditions (food deprivation, hyperthermia and hypoxia), even a novel perspective highlight the potential role of physical forces in autophagy modulation. To understand the crosstalk between all these controlling mechanisms could give us new clues about the specific contribution of autophagy in a wide range of diseases including vascular disor...

(A) CHO cells stably overexpressing wild-type muscarinic M3 receptor ... more (A) CHO cells stably overexpressing wild-type muscarinic M3 receptor or internalization-deficient M3 receptor (SASS motif mutant, characterised in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084174#B10" target="_blank">10</a>]) were transfected with HA-ERK5. Twenty-four hours after transfection, cells were serum-starved for 2h and stimulated with carbachol (10µM). HA-ERK5 was immunoprecipitated with an anti-HA agarose-conjugated antibody as detailed in the Materials and Methods section. ERK5 phosphorylation was assessed in the immunoprecipitate using a phosphospecific antibody. Data (mean +/- SEM of 3 independent experiments) were normalised using HA-ERK5 as loading control and expressed as fold-induction over basal conditions (*p<0.05, two-tailed T-test). (B) Quantification of cell surface receptor density was performed through [3H]-NMS binding at 4°C. The two cell types utilised in (A) were serum-starved for 2h and stimulated with carbachol (100µM) for 30 minutes. Data were normalised to unspecific binding (atropine treatment) and binding percentage was expressed as the mean +/- SEM of 3 independent experiments.</p

Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that... more Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of cardiovascular function. Enhanced Gαqmediated signaling plays an important role in cardiac hypertrophy and in the transition to heart failure. We have recently described that Gαq acts as an adaptor protein that facilitates PKCζmediated activation of ERK5 in epithelial cells. Since the ERK5 cascade is known to be involved in cardiac hypertrophy, we have investigated the potential relevance of this pathway in cardiovascular Gqdependent signaling using both cultured cardiac cell types and chronic administration of angiotensin II in mice. We find that PKCζ is required for the activation of the ERK5 pathway by Gqcoupled GPCR in neonatal and adult murine cardiomyocyte cultures and in cardiac fibroblasts. Stimulation of ERK5 by angiotensin II is blocked upon pharmacological inhibition or siRNAmediated silencing of PKCζ in primary cultures of cardiac cells and in neonatal ...

International Journal of Cancer, 2019

Cellular and Molecular Life Sciences, 2019

Accumulating evidence indicates that G protein-coupled receptor kinase 2 (GRK2) is a versatile pr... more Accumulating evidence indicates that G protein-coupled receptor kinase 2 (GRK2) is a versatile protein that acts as a signaling hub by modulating G protein-coupled receptor (GPCR) signaling and also via phosphorylation or scaffolding interactions with an extensive number of non-GPCR cellular partners. GRK2 multifunctionality arises from its multidomain structure and from complex mechanisms of regulation of its expression levels, activity, and localization within the cell, what allows the precise spatio-temporal shaping of GRK2 targets. A better understanding of the GRK2 interactome and its modulation mechanisms is helping to identify the GRK2-interacting proteins and its substrates involved in the participation of this kinase in different cellular processes and pathophysiological contexts.

Seminars in cancer biology, Feb 1, 2017

Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein k... more Increasing evidences point to G protein-coupled receptor kinases (GRKs), a subfamily of protein kinase A/G/C-like kinases, as relevant players in cancer progression, in a cell-type and tumor-specific way. Alterations in the expression and/or activity of particular GRKs have been identified in several types of tumors, and demonstrated to modulate the proliferation, survival or invasive properties of tumor cells by acting as integrating signaling nodes. GRKs are able to regulate the functionality of both G protein-coupled receptors (GPCR) and growth factor receptors and to directly control cytosolic, cytoskeletal or nuclear signaling components of pathways relevant for these processes. Furthermore, many chemokines as well as angiogenic and inflammatory factors present in the tumor microenvironment act through GPCR and other GRK-modulated signaling modules. Changes in the dosage of certain GRKs in the tumor stroma can alter tumor angiogenesis and the homing of immune cells, thus puttin...

Journal of Biological Chemistry, 2016

Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor ... more Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between G␣q and protein kinase C (PKC), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLC␤. We report herein that the activation-dependent G␣q/PKC complex involves the basic PB1-type II domain of PKC and a novel interaction module in G␣q different from the classical effector-binding site. Point mutations in this G␣q region completely abrogate ERK5 phosphorylation, indicating that G␣q/PKC association is required for the activation of the pathway. Indeed, PKC was demonstrated to directly bind ERK5 thus acting as a scaffold between G␣q and ERK5 upon GPCR activation. The inhibition of these protein complexes by G proteincoupled receptor kinase 2, a known G␣q modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that G␣q/ PKC complexes link G␣q to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in G␣q and the effector PKC is a key event in G␣q signaling.

Cellular and Molecular Life Sciences, 2019

VE-cadherin plays a central role in controlling endothelial barrier function, which is transientl... more VE-cadherin plays a central role in controlling endothelial barrier function, which is transiently disrupted by proinflammatory cytokines such as tumor necrosis factor (TNFα). Here we show that human endothelial cells compensate VE-cadherin degradation in response to TNFα by inducing VE-cadherin de novo synthesis. This compensation increases adherens junction turnover but maintains surface VE-cadherin levels constant. NF-κB inhibition strongly reduced VE-cadherin expression and provoked endothelial barrier collapse. Bacterial lipopolysaccharide and TNFα upregulated the transcription factor ETS1, in vivo and in vitro, in an NF-κB dependent manner. ETS1 gene silencing specifically reduced VE-cadherin protein expression in response to TNFα and exacerbated TNFα-induced barrier disruption. We propose that TNFα induces not only the expression of genes involved in increasing permeability to small molecules and immune cells, but also a homeostatic transcriptional program in which NF-κB-and ETS1-regulated VE-cadherin expression prevents the irreversible damage of endothelial barriers.

Basic Research in Cardiology, 2019

Inhibition of the Ca 2+-dependent proteases calpains attenuates post-infarction remodeling and he... more Inhibition of the Ca 2+-dependent proteases calpains attenuates post-infarction remodeling and heart failure. Recent data suggest that calpain activity is elevated in non-ischemic cardiomyopathies and that upregulation of the key cardiac G-protein-coupled receptor kinase 2 (GRK2) signaling hub promotes cardiac hypertrophy. However, the functional interactions between calpains and GRK2 in this context have not been explored. We hypothesized that calpain modulates GRK2 levels in myocardial hypertrophy of non-ischemic cause, and analyzed the mechanisms involved and the potential therapeutic benefit of inhibiting calpain activity in this situation. The oral calpain inhibitor SNJ-1945 was administered daily to male Sprague-Dawley rats or wild-type and hemizygous GRK2 mice treated with 5 mg/Kg/day isoproterenol intraperitoneally for 1 week. In isoproterenol-treated animals, calpains 1 and 2 were overexpressed in myocardium and correlated with increased calpain activity and ventricular hypertrophy. Oral co-administration of SNJ-1945 attenuated calpain activation and reduced heart hypertrophy as assessed using morphological and biochemical markers. Calpain activation induced by isoproterenol increased GRK2 protein levels, while genetic downregulation of GRK2 expression prevented isoproterenol-mediated hypertrophy independently of calpain inhibition. GRK2 upregulation was associated to calpain-dependent degradation of the GRK2 ubiquitin ligase MDM2 and to enhanced NF-κB-dependent GRK2 gene expression in correlation with calpainmediated IĸB proteolysis. These results demonstrate that calpain mediates isoproterenol-induced myocardial hypertrophy by modulating GRK2 protein content through mechanisms involving the control of GRK2 stability and expression. Sustained calpain inhibition attenuates isoproterenol-induced myocardial hypertrophy and could be an effective therapeutic strategy to limit ventricular remodeling of non-ischemic origin.

Current Medicinal Chemistry, 2019

Background: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by ... more Background: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease characterized by uncontrolled proliferation of precursor myeloid-lineage cells in the bone marrow. AML is also characterized by patients with poor long-term survival outcomes due to relapse. Many efforts have been made to understand the biological heterogeneity of AML and the challenges to develop new therapies are therefore enormous. G Protein-coupled Receptors (GPCRs) are a large attractive drug-targeted family of transmembrane proteins, and aberrant GPCR expression and GPCR-mediated signaling have been implicated in leukemogenesis of AML. This review aims to identify the molecular players of GPCR signaling, focusing on the hematopoietic system, which are involved in AML to help developing novel drug targets and therapeutic strategies. Methods: We undertook an exhaustive and structured search of bibliographic databases for research focusing on GPCR, GPCR signaling and expression in AML. Results and Con...

Current Medicinal Chemistry, 2019

Nature Communications

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gα... more The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular ho...

Cardiovascular Research, 2016

Transforming Growth Factors (TGF)-β regulate tissue fibrosis: TGF-β promotes fibrosis, whereas Bo... more Transforming Growth Factors (TGF)-β regulate tissue fibrosis: TGF-β promotes fibrosis, whereas Bone Morphogenetic Protein (BMP)-7 is antifibrotic. To demonstrate that: (i) Left ventricular (LV) remodelling after pressure overload is associated to disequilibrium in the signalling mediated by these cytokines; and (ii) BMP-7 exerts beneficial effects on LV remodelling and reverse remodelling. We studied patients with aortic stenosis (AS) and mice subjected to transverse aortic constriction (TAC) and TAC-release (de-TAC). LV morphology and function were assessed by echocardiography. LV biopsies were analysed by qPCR, immunoblotting and histology. Pressure overload reduced BMP-7 and pSmad1/5/8 and increased TGF-β and pSmad2/3 in AS-patients and TAC-mice. BMP-7 correlated inversely with collagen, fibronectin and β-MHC expressions, and with hypertrophy and diastolic dysfunction, and directly with the systolic function. Multiple linear regression disclosed BMP-7 and TGF-β as hypertrophy predictors, negative and positive, respectively. BMP-7 prevented TGF-β-elicited hypertrophic program in cardiomyocytes, and Col1A1 promoter activity in NIH-3T3 fibroblasts. The treatment of TAC-mice with rBMP-7 attenuated the development of structural damage and dysfunction, and halted ongoing remodelling. The reverse remodelling after pressure overload release was facilitated by rBMP-7, and hampered by disrupting BMP-7 function using a neutralizing antibody or genetic deletion. The disequilibrium between BMP-7 and TGF-β signals plays a relevant role in the LV remodelling response to hemodynamic stress in TAC-mice and AS-patients. Our observations may provide new important insights aimed at developing novel therapies designed to prevent, halt or reverse LV pathological remodelling in pressure overload cardiomyopathy.

PLoS ONE, 2012

Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associate... more Human papillomavirus (HPV) is the causative agent of human cervical cancer and has been associated with oropharyngeal squamous cell carcinoma development. Although prophylactic vaccines have been developed, there is a need to develop new targeted therapies for individuals affected with malignant infected lesions in these locations, which must be tested in appropriate models. Cutaneous beta HPV types appear to be involved in skin carcinogenesis. Virus oncogenicity is partly achieved by inactivation of retinoblastoma protein family members by the viral E7 gene. Here we show that the E7 protein of cutaneous beta HPV5 binds pRb and promotes its degradation. In addition, we described an in vivo model of HPVassociated disease in which artificial human skin prepared using primary keratinocytes engineered to express the E7 protein is engrafted onto nude mice. Expression of E7 in the transplants was stably maintained for up to 6 months, inducing the appearance of lesions that, in the case of HPV16 E7, histologically resembled human anogenital lesions caused by oncogenic HPVs. Moreover, it was confirmed through biomarker expression analysis via immunodetection and/or quantitative PCR from mRNA and miRNA that the 16E7-modified engrafted skin shares molecular features with human HPVassociated pretumoral and tumoral lesions. Finally, our findings indicate a decrease of the in vitro capacity of HPV5 E7 to reduce pRb levels in vivo, possibly explaining the phenotypical differences when compared with 16E7-grafts. Our model seems to be a valuable platform for basic research into HPV oncogenesis and preclinical testing of HPV-associated antitumor therapies. Citation: Buitrago-Pérez Á , Hachimi M, Dueñ as M, Lloveras B, Santos A, et al. (2012) A Humanized Mouse Model of HPV-Associated Pathology Driven by E7 Expression. PLoS ONE 7(7): e41743.

Journal of Biological Chemistry, 2012

We have recently described that G␣ q acts as an adaptor protein that facilitates PKC-mediated act... more We have recently described that G␣ q acts as an adaptor protein that facilitates PKC-mediated activation of ERK5. Results: Our results show that PKC is essential for Gq-dependent ERK5 activation in cardiomyocytes and cardiac fibroblasts. Conclusion: This novel signaling axis plays a key role in cardiac hypertrophy programs. Significance: The G␣ q /PKC/ERK5 pathway would be active in such pathological settings, providing new therapeutic targets.

Antioxidants

International Journal of Cancer, 2019

Cellular and Molecular Life Sciences, 2019

Seminars in cancer biology, Feb 1, 2017

Journal of Biological Chemistry, 2016

Cellular and Molecular Life Sciences, 2019

Basic Research in Cardiology, 2019

Current Medicinal Chemistry, 2019

Nature Communications

Cardiovascular Research, 2016

PLoS ONE, 2012

Journal of Biological Chemistry, 2012