Hypoxia-inducible factor 1α is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells

In melanocytes and melanoma cells α-melanocyte stimulating hormone (α-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell–specific manner and increases the expression of a functional hypoxia-inducible factor 1α (HIF1α) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF “silencing” abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the α-MSH/cAMP pathway, using MITF as a signal transducer and HIF1α as a target, might contribute to melanoma progression

Hypoxia Reduces Cell Attachment of SARS-CoV-2 Spike Protein by Modulating the Expression of ACE2, Neuropilin-1, Syndecan-1 and Cellular Heparan Sulfate

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1αdependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), the European Research Council (ERC) (grant numbers: ERC-2018-StG 804236-NEXTGEN-IO to A.P and ERC-2017-AdG 788143-RECGLYCANMR to J.J-B.), the Severo Ochoa Excellence Accreditation from MCIU (SEV-2016-0644) and the FERO Foundation. Personal fellowships: E.P. (Juan de la Cierva-Formación, FJC2018-035449-I), L.V. (Juan de la Cierva-Formación, FJCI-2017-34099), A.B. (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), A.G. (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), A.A (La Caixa Inphinit, LCF/BQ/DR20/11790022), B.J. (Basque Government, PRE_2019_1_0320), L.M. (Juan de la Cierva-Formación, FJC2019-039983-I), E.B. (MINECO, BFU2016-76872-R; Excellence Networks, SAF2017-90794-REDT) and A.P. (Ramón y Cajal, RYC2018-024183-I; Proyectos I+D+I, PID2019-107956RA-I00; and Ikerbasque Research Associate)

Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions

[Background] The adaptation to hypoxia is mainly controlled by the HIF transcription factors. Increased expression/activity of HIF-1α correlates with poor prognosis in cancer patients. PARP-1 inhibitors are used in the clinic to treat BRCAness breast/ovarian cancer and have been shown to regulate the hypoxic response; therefore, their use could be expanded.[Methods] In this work by integrating molecular/cell biology approaches, genome-wide ChIP-seq, and patient samples, we elucidate the extent to which PARP-1 exerts control over HIF-1-regulated genes.[Results] In human melanoma, PARP-1 and HIF-1α expression are strongly associated. In response to a hypoxic challenge poly(ADP-ribose) (PAR) is synthesized, HIF-1α is post-transcriptionally modified (PTM) and stabilized by PARylation at specific K/R residues located at its C-terminus. Using an unbiased ChIP-seq approach we demonstrate that PARP-1 dictates hypoxia-dependent HIF-recruitment to chromatin in a range of HIF-regulated genes while analysis of HIF-binding motifs (RCGTG) reveals a restriction on the recognition of hypoxia responsive elements in the absence of PARP-1. Consequently, the cells are poorly adapted to hypoxia, showing a reduced fitness during hypoxic induction.[Conclusions] These data characterize the fine-tuning regulation by PARP-1/PARylation of HIF activation and suggest that PARP inhibitors might have therapeutic potential against cancer types displaying HIF-1α over-activation.This work was supported by Junta de Andalucía, project of Excellence from Junta de Andalucía P10-CTS-0662, P12-CTS-383 to FJO, Spanish Ministry of Economy and Competitiveness SAF2012-40011-C02-01, SAF2015-70520- R, RTI2018-098968-B-I00, RTICC RD12/0036/0026 and CIBER Cáncer ISCIII CB16/12/00421 to FJO. EB1s lab is supported by the Basque Department of Industry, Tourism and Trade (Etortek) and the MINECO (CB16/12/00421) grants. Fundación Domingo Martínez (call 2019).Peer reviewe

Chronic hypoxia aggravates Alzheimer’s disease pathology by causing microglial dysfunction

Trabajo presentado en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celbrado en Granada del 9 al 12 de septiembre de 2014.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder and the most common form of dementia. In many cases AD patients present concomitant vascular pathology. Low oxygen levels are also frequently found in the brain of AD patients. The most accepted hypothesis to explain the correlation between hypoxia and AD is the deposition of amyloid ß (Aß) occurring in the microvasculature (amyloid angiopathy) and the affectation by the disease of the locus coeruleus, a brain region involved in the control of brain blood flow. However, few data has been collected to understand the relation between hypoxia and AD progression. We show here the accumulation of the hypoxic marker HIF1 α (Hypoxia-inducible-factor 1α), the major transcription factor for the adaptation to hypoxic conditions, in the brain of AD patients by western blot. We have also characterized the consequences of chronic exposition to hypoxia in the progression of the disease using a widely accepted AD mice model. AD mice were exposed to physiologic hypoxia (8.5% oxygen, 21 days) at initial and advances stages of the pathology. Brains from hypoxic animals showed no differences in the Aß content and number of plaques, but they showed a clear reduction in the total number of microglial cells that was even more evident around the Aß plaques. In vitro analyses suggest that hypoxia slows down proliferation and chemotaxis towards polymeric Aß in both cell line and primary microglial cultures. Interestingly, the brain cortex from the hypoxic animals showed a high increase in the number of dystrophic neurites surrounding the microglia- free Aß plaques. We observed also a decrease in the mRNA levels of two markers of interneurons, Somatostatin and Neuropeptide-Y, in the hippocampus of hypoxic mice. These data suggest that hypoxia accelerates the progression of AD pathology. The pathway underlying microglial affectation by hypoxia has an enormous potential in neurodegenerative disorders where microglia function is correlated with the progression of the disease.N

PI3K-regulated Glycine N-methyltransferase is required for the development of prostate cancer

[EN] Glycine N-Methyltransferase (GNMT) is a metabolic enzyme that integrates metabolism and epigenetic regulation. The product of GNMT, sarcosine, has been proposed as a prostate cancer biomarker. This enzyme is predominantly expressed in the liver, brain, pancreas, and prostate tissue, where it exhibits distinct regulation. Whereas genetic alterations in GNMT have been associated to prostate cancer risk, its causal contribution to the development of this disease is limited to cell line-based studies and correlative human analyses. Here we integrate human studies, genetic mouse modeling, and cellular systems to characterize the regulation and function of GNMT in prostate cancer. We report that this enzyme is repressed upon activation of the oncogenic Phosphoinositide-3-kinase (PI3K) pathway, which adds complexity to its reported dependency on androgen signaling. Importantly, we demonstrate that expression of GNMT is required for the onset of invasive prostate cancer in a genetic mouse model. Altogether, our results provide further support of the heavy oncogenic signal-dependent regulation of GNMT in prostate cancer.We are grateful to the Carracedo lab for valuable input, to Drs. Ana M. Aransay, James D. Sutherland and F. Elortza for technical advice, and Drs. Michelle Clasquin, Katie Sellers and Katya Marjon at Agios Pharmaceuticals for performing, processing and analyzing the metabolomics experiments. We thank the Basque Biobank for Research (BIOEF) for the support with prostate specimen acquisition and management. A.A-A. was funded by the Basque Government (predoctoral fellowship). V.T. is funded by Fundación Vasca de Innovación e Investigación Sanitarias, BIOEF (BIO15/CA/052), the AECC J.P. Bizkaia, the Basque Department of Health (2016111109) and the MICINN RTI2018-097267-B-I00. I.M. is supported by Fundación Cris Contra el Cáncer (PR_TPD_2020-19). The work of A. Carracedo is supported by the Basque Department of Industry, Tourism and Trade (Elkartek), the department of education (IKERTALDE IT1106-16) and health (RIS3), the BBVA foundation, the MICINN (SAF2016-79381-R; PID2019-108787RB-I00 (FEDER/EU); Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks RED2018-102769-T), the AECC (GCTRA18006CARR), Vencer el Cáncer Foundation, La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17/ and the European Research Council (Starting Grant 336343, PoC 754627, Consolidator Grant 819242). CIBERONC was co-funded with FEDER funds and funded by ISCIII. We are grateful for the support of Mondravember and Movembergara. A.E. was supported by MCIN/AEI/10.13039/501100011033 and the EU programme NextGenerationEU/PRTR (IJC2020-043583-I). The work of JM Mato was supported by NIH grant R01CA172086 and SAF2017-88041-R. EB is funded by the MICINN (BFU2016-76872-R (FEDER/EU), PID2019-108112RB-I00, and Excellence Networks SAF2017-90794-REDT)

Borrelia burgdorferi infection induces long-term memory-like responses in macrophages with tissue-wide consequences in the heart

Lyme carditis is an extracutaneous manifestation of Lyme disease characterized by episodes of atrioventricular block of varying degrees and additional, less reported cardiomyopathies. The molecular changes associated with the response to Borrelia burgdorferi over the course of infection are poorly understood. Here, we identify broad transcriptomic and proteomic changes in the heart during infection that reveal a profound down-regulation of mitochondrial components. We also describe the long-term functional modulation of macrophages exposed to live bacteria, characterized by an augmented glycolytic output, increased spirochetal binding and internalization, and reduced inflammatory responses. In vitro, glycolysis inhibition reduces the production of tumor necrosis factor (TNF) by memory macrophages, whereas in vivo, it produces the reversion of the memory phenotype, the recovery of tissue mitochondrial components, and decreased inflammation and spirochetal burdens. These results show that B. burgdorferi induces long-term, memory-like responses in macrophages with tissue-wide consequences that are amenable to be manipulated in vivo.Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (SAF2015-65327-R and RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB, AGL2017-86757-R to LA, SAF2017-87301-R to MLMC, SAF2015-64111-R to AP, SAF2015-73549-JIN to HR), Instituto de Salud Carlos III (PIE13/0004 to AP), the Basque Government Department of Health (2015111117 to LA), the Basque Foundation for Innovation and Health Research (BIOEF), through the EiTB Maratoia grant BIO15/CA/016/BS to MLMC, the regional Government of Andalusia co-funded by CEC and FEDER funds (Proyectos de Excelencia P12-CTS-2232) and Fundación Domingo Martínez (to AP). LA is supported by the Ramon y Cajal program (RYC-2013-13666). DB, MMR and TMM are recipients of MCIU FPI fellowships. ACG and AP are recipients of fellowships form the Basque Government. APC is a recipient of a fellowship from the University of the Basque Country. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia Province and the CIBERehd network. DB and JA are supported by a grant from the Jesús de Gangoiti Barrera Foundation

Systemic and Local Hypoxia Synergize Through HIF1 to Compromise the Mitochondrial Metabolism of Alzheimer's Disease Microglia

Microglial cells are key contributors to Alzheimer’s disease (AD), constituting the first cellular line against Aß plaques. Local hypoxia and hypoperfusion, which are typically present in peripheral inflammatory foci, are also common in the AD brain. We describe here that Aß deposits are hypoxic and hypoperfused and that Aß plaque-associated microglia (AßAM) are characterized by the expression of hypoxia-inducible factor 1 (HIF1)-regulated genes. Notably, AßAM simultaneously upregulate the expression of genes involved in anaerobic glycolysis and oxidative mitochondrial metabolism, show elongated mitochondria surrounded by rough endoplasmic reticulum, and blunt the HIF1-mediated exclusion of pyruvate from the mitochondria through the pyruvate dehydrogenase kinase 1 (PDK1). Overstabilization of HIF1 –by genetic (von Hippel-Lindau deficient microglia) or systemic hypoxia (an AD risk factor)– induces PDK1 in microglia and reduces microglial clustering in AD mouse models. The human AD brain exhibits increased HIF1 activity and a hypoxic brain area shows reduced microglial clustering. The loss of the microglial barrier associates with augmented Aß neuropathology both in the chronic hypoxia AD mouse model and the human AD brain. Thus, the synergy between local and systemic AD risk factors converges with genetic susceptibility to cause microglial dysfunction.Peer reviewe

The commensal bacterium Lactiplantibacillus plantarum imprints innate memory-like responses in mononuclear phagocytes

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.Supported by grants from the Spanish Ministry of Science, Innovation and Universities (MCIU) co-financed with FEDER funds (RTI2018-096494-B-100 to JA; BFU2016-76872-R to EB; AGL2017-86757-R to LA; SAF2015-73549-JIN to HR; SAF2016–77433-R and PID2019-110240RB-I00 to RPR). AP is supported by a Postdoctoral Fellowship from the Basque Government. DB and TMM are recipients of MCIU FPI fellowships. APC is a recipient of a fellowship from the University of the Basque Country. LA and RPR are supported by the Ramon y Cajal program from the Spanish Ministry of Economy and Competitiveness. We thank the MCIU for the Severo Ochoa Excellence accreditation (SEV-2016-0644), the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs) and the Innovation Technology Department of the Bizkaia Province. This work was further supported by grants from the Jesús de Gangoiti Barrera Foundation.Peer reviewe

Hypoxia compromises the mitochondrial metabolism of Alzheimer’s disease microglia via HIF1

Genetic Alzheimer’s disease (AD) risk factors associate with reduced defensive amyloid β plaque-associated microglia (AβAM), but the contribution of modifiable AD risk factors to microglial dysfunction is unknown. In AD mouse models, we observe concomitant activation of the hypoxia-inducible factor 1 (HIF1) pathway and transcription of mitochondrial-related genes in AβAM, and elongation of mitochondria, a cellular response to maintain aerobic respiration under low nutrient and oxygen conditions. Overactivation of HIF1 induces microglial quiescence in cellulo, with lower mitochondrial respiration and proliferation. In vivo, overstabilization of HIF1, either genetically or by exposure to systemic hypoxia, reduces AβAM clustering and proliferation and increases Aβ neuropathology. In the human AD hippocampus, upregulation of HIF1α and HIF1 target genes correlates with reduced Aβ plaque microglial coverage and an increase of Aβ plaque-associated neuropathology. Thus, hypoxia (a modifiable AD risk factor) hijacks microglial mitochondrial metabolism and converges with genetic susceptibility to cause AD microglial dysfunction.R.M.-D. was the recipient of a Sara Borrell fellowship from Instituto de Salud Carlos III (ISCIII) (CD09/0007). N.L.-U., C.O.-d.S.L., C.R.-M. and M.I.A.-V. were the recipients of FPU fellowships from Spanish Ministry of Education, Culture and Sport (FPU14/02115, AP2010‐1598, FPU16/02050 and FPU15/02898, respectively). A.H.-G. was the recipient of an FPI fellowship from the Spanish Ministry of Education, Culture and Sport (BES-2010-033886). This work was supported by grants from the Spanish MINEICO, ISCIII and FEDER (European Union) (SAF2012‐33816, SAF2015‐64111‐R, SAF2017-90794-REDT and PIE13/0004 to A.P.); by the Regional Government of Andalusia co-funded by CEC and FEDER funds (European Union) (‘Proyectos de Excelencia’; P12‐CTS‐2138 and P12‐CTS‐2232 to A.P.); by the ‘Ayuda de Biomedicina 2018’, Fundación Domingo Martínez (to A.P.) ; by the ISCIII of Spain, co-financed by FEDER funds (European Union) through grants PI18/01556 (to J.V.) and PI18/01557 (to A. Gutierrez); by Junta de Andalucía, co-financed by FEDER funds (grants UMA18-FEDERJA-211 (to A. Gutierrez) and US‐1262734 (to J.V.)); and by Spanish MINEICO (BFU2016-76872-R and BES-2011-047721 to E.B.).Peer reviewe

Resistir el déficit de oxígeno

Darwin wrote, "it is not the strongest of the species that survive, nor the most intelligent, but the one most responsive to change”. Oxygen is vital and its deficit can be dramatic. The organisms have evolved complex strategies to deal with the dangers of hypoxia. Adapt or die: it’s your choice.Ya lo dijo Darwin “no son las especies más fuertes las que sobre- viven, ni las más inteligentes, sino aquellas que se adaptan mejor al cambio”. El oxígeno es vital y su déficit puede resultar dramático. Los orga- nismos han ingeniado complejos dispositivos para resistir los peligros de la hipoxia. Adaptarse o morir: no queda otra