Corepressor C-terminal Binding Protein Research Articles

Juvenile hormone signal transducer hairy inhibits Krüppel homolog1 expression

Hairy and Krüppel homolog 1 (Kr-h1) are transcriptional repressors that act synergistically to mediate the gene-repressive action of juvenile hormone (JH). However, whether a regulatory relationship exists between Hairy and Kr-h1 remains unclear. In this study, an inhibitory effect of Hairy on Kr-h1 expression was found. Genetic studies in Drosophila have shown that the simultaneous overexpression of Hairy and Kr-h1 can rescue the defective phenotypes caused by the overexpression of a single factor. Reduced expression of Kr-h1 was observed in Hairy-overexpressing flies and cells, whereas the expression levels of Hairy were unaffected in cells with ectopic expression of Kr-h1. The inhibitory effect of Hairy on Kr-h1 expression was found to occur at the transcriptional level, as Hairy bound directly to the B-box within the Kr-h1 promoter via the bHLH motif and recruited the corepressors C-terminal binding protein (CtBP) and Groucho (Gro) through the PLSLV and WRPW motifs, respectively. Our findings revealed a regulatory relationship between two JH response factors, which advances our understanding of the molecular mechanism of JH signaling.

Glycolysis Promotes Angiotensin II-Induced Aortic Remodeling Through Regulating Endothelial-to-Mesenchymal Transition via the Corepressor C-Terminal Binding Protein 1.

Endothelial dysfunction plays a crucial role in aortic remodeling. Aerobic glycolysis and endothelial-to-mesenchymal transition (EndoMT) have, respectively, been suggested to contribute to endothelial dysfunction in many cardiovascular diseases. Here, we tested the hypothesis that glycolytic reprogramming is critical for EndoMT induction in aortic remodeling through an epigenetic mechanism mediated by a transcriptional corepressor CtBP1 (C-terminal binding protein 1), a sensor of glycolysis-derived NADH. EndoMT program, aortic remodeling, and endothelial expression of the glycolytic activator PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) were evaluated in Ang (angiotensin) II-infused mice. Mice with endothelial-specific Pfkfb3 deficiency or CtBP1 inactivation, immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assay were employed to elucidate whether and how PFKFB3/CtBP1 epigenetically controls EndoMT. The EndoMT program and increased endothelial PFKFB3 expression were induced in remodeled thoracic aortas. In TGF-β (transforming growth factor-β)-treated human endothelial cells, activated SMAD2/3 (SMAD Family Member 2/3) transcriptionally upregulated PFKFB3 expression. In turn, the TGF-β/SMAD signaling and EndoMT were compromised by silencing or inhibition of PFKFB3. Mechanistic studies revealed that PFKFB3-mediated glycolysis increased NADH content and activated the NADH-sensitive CtBP1. Through interaction with the transcription repressor E2F4 (E2F Transcription Factor 4), CtBP1 enhanced E2F4-mediated transcriptional repression of SMURF2 (SMAD ubiquitin regulatory factor 2), a negative regulator of TGF-β/SMAD2 signaling. Additionally, EC-specific Pfkfb3 deficiency or CtBP1 inactivation in mice led to attenuated Ang II-induced aortic remodeling. Our results demonstrate a glycolysis-mediated positive feedback loop of the TGF-β signaling to induce EndoMT and indicate that therapeutically targeting endothelial PFKFB3 or CtBP1 activity could provide a basis for treating EndoMT-linked aortic remodeling.

Aldolase B attenuates clear cell renal cell carcinoma progression by inhibiting CtBP2.

Aldolase B (ALDOB), a glycolytic enzyme, is uniformly depleted in clear cell renal cell carcinoma (ccRCC) tissues. We previously showed that ALDOB inhibited proliferation through a mechanism independent of its enzymatic activity in ccRCC, but the mechanism was not unequivocally identified. We showed that the corepressor C-terminal-binding protein 2 (CtBP2) is a novel ALDOB-interacting protein in ccRCC. The CtBP2-to-ALDOB expression ratio in clinical samples was correlated with the expression of CtBP2 target genes and was associated with shorter survival. ALDOB inhibited CtBP2-mediated repression of multiple cell cycle inhibitor, proapoptotic, and epithelial marker genes. Furthermore, ALDOB overexpression decreased the proliferation and migration of ccRCC cells in an ALDOB-CtBP2 interaction-dependent manner. Mechanistically, our findings showed that ALDOB recruited acireductone dioxygenase 1, which catalyzes the synthesis of an endogenous inhibitor of CtBP2, 4-methylthio 2-oxobutyric acid. ALDOB functions as a scaffold to bring acireductone dioxygenase and CtBP2 in close proximity to potentiate acireductone dioxygenase-mediated inhibition of CtBP2, and this scaffolding effect was independent of ALDOB enzymatic activity. Moreover, increased ALDOB expression inhibited tumor growth in a xenograft model and decreased lung metastasis in vivo. Our findings reveal that ALDOB is a negative regulator of CtBP2 and inhibits tumor growth and metastasis in ccRCC.

Angiotensin II Inhibits Adipogenic Differentiation and Promotes Mature Adipocyte Browning through the Corepressor CtBP1.

The mechanisms of angiotensin II (Ang II) on regulating adipogenic differentiation and function remain unknown. In this study, we focus on revealing the role of C-terminal-binding protein 1 (CtBP1) on Ang II-mediated adipogenic differentiation and mature adipocyte browning. Amounts of 3T3-L1 and CtBP1-KO 3T3-L1 were treated with Ang II for 24 h and then induced adipogenic differentiation, or cells were first induced differentiation and then treated with Ang II. The expressions of CtBP1 and adipogenic markers were checked by Western blot. Transcription of CtBP1 was assayed by Real-time RT-PCR. Lipid droplet formation and size were detected by Oil Red O. Mitochondrial content and reactive oxygenspecies (ROS) were detected by Mito-tracker and MitoSOX. Mitochondrial respiratory function was detected with the corresponding kits. Mitochondrial membrane potential (MMP) (∆Ψm) was assayed by JC-1. The results show that Ang II promoted CtBP1 transcription and expression via AT1 receptor during 3T3-L1 adipogenic differentiation. Ang II significantly inhibited lipid droplet formation and adipogenic markers expression in 3T3-L1 differentiation, which was blocked by CtBP1 knockout. In mature 3T3-L1, Ang II treatment increased uncoupling protein-1 (UCP-1) expression and the number of lipid droplets, and also reduced lipid droplet size and single cell lipid accumulation, which was reversed by CtBP1 knockout. In addition, Ang II treatment enhanced mitochondrial numbers, ATP production, oxygen consumption rate (OCR) and ROS generation, and reduced MMP (∆Ψm) via CtBP1 in mature 3T3-L1 adipocytes. In conclusion, this study demonstrates that CtBP1 plays a key role in the inhibitory effect of Ang II on adipogenesis. Moreover, Ang II regulates the function of mature adipocyte via CtBP1, including promoting adipocyte browning, mitochondrial respiration and ROS generation.

The transcriptional corepressor CTBP-1 acts with the SOX family transcription factor EGL-13 to maintain AIA interneuron cell identity in Caenorhabditis elegans.

Cell identity is characterized by a distinct combination of gene expression, cell morphology, and cellular function established as progenitor cells divide and differentiate. Following establishment, cell identities can be unstable and require active and continuous maintenance throughout the remaining life of a cell. Mechanisms underlying the maintenance of cell identities are incompletely understood. Here, we show that the gene ctbp-1, which encodes the transcriptional corepressor C-terminal binding protein-1 (CTBP-1), is essential for the maintenance of the identities of the two AIA interneurons in the nematode Caenorhabditis elegans. ctbp-1 is not required for the establishment of the AIA cell fate but rather functions cell-autonomously and can act in later larval stage and adult worms to maintain proper AIA gene expression, morphology and function. From a screen for suppressors of the ctbp-1 mutant phenotype, we identified the gene egl-13, which encodes a SOX family transcription factor. We found that egl-13 regulates AIA function and aspects of AIA gene expression, but not AIA morphology. We conclude that the CTBP-1 protein maintains AIA cell identity in part by utilizing EGL-13 to repress transcriptional activity in the AIAs. More generally, we propose that transcriptional corepressors like CTBP-1 might be critical factors in the maintenance of cell identities, harnessing the DNA-binding specificity of transcription factors like EGL-13 to selectively regulate gene expression in a cell-specific manner.

The transcriptional corepressor CtBP2 serves as a metabolite sensor orchestrating hepatic glucose and lipid homeostasis

Biological systems to sense and respond to metabolic perturbations are critical for the maintenance of cellular homeostasis. Here we describe a hepatic system in this context orchestrated by the transcriptional corepressor C-terminal binding protein 2 (CtBP2) that harbors metabolite-sensing capabilities. The repressor activity of CtBP2 is reciprocally regulated by NADH and acyl-CoAs. CtBP2 represses Forkhead box O1 (FoxO1)-mediated hepatic gluconeogenesis directly as well as Sterol Regulatory Element-Binding Protein 1 (SREBP1)-mediated lipogenesis indirectly. The activity of CtBP2 is markedly defective in obese liver reflecting the metabolic perturbations. Thus, liver-specific CtBP2 deletion promotes hepatic gluconeogenesis and accelerates the progression of steatohepatitis. Conversely, activation of CtBP2 ameliorates diabetes and hepatic steatosis in obesity. The structure-function relationships revealed in this study identify a critical structural domain called Rossmann fold, a metabolite-sensing pocket, that is susceptible to metabolic liabilities and potentially targetable for developing therapeutic approaches.

Limited Availability of General Co-Repressors Uncovered in an Overexpression Context during Wing Venation in Drosophila melanogaster.

Cell fate is determined by the coordinated activity of different pathways, including the conserved Notch pathway. Activation of Notch results in the transcription of Notch targets that are otherwise silenced by repressor complexes. In Drosophila, the repressor complex comprises the transcription factor Suppressor of Hairless (Su(H)) bound to the Notch antagonist Hairless (H) and the general co-repressors Groucho (Gro) and C-terminal binding protein (CtBP). The latter two are shared by different repressors from numerous pathways, raising the possibility that they are rate-limiting. We noted that the overexpression during wing development of H mutants HdNT and HLD compromised in Su(H)-binding induced ectopic veins. On the basis of the role of H as Notch antagonist, overexpression of Su(H)-binding defective H isoforms should be without consequence, implying different mechanisms but repression of Notch signaling activity. Perhaps excess H protein curbs general co-repressor availability. Supporting this model, nearly normal wings developed upon overexpression of H mutant isoforms that bound neither Su(H) nor co-repressor Gro and CtBP. Excessive H protein appeared to sequester general co-repressors, resulting in specific vein defects, indicating their limited availability during wing vein development. In conclusion, interpretation of overexpression phenotypes requires careful consideration of possible dominant negative effects from interception of limiting factors.

Transcriptional co-repressor CtBP2 orchestrates epithelial-mesenchymal transition through a novel transcriptional holocomplex with OCT1

The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-β (TGF-β) promotes the formation of the CtBP2/OCT1 complex.Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases.

NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

BackgroundAdenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear.MethodsThe dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding.ResultsPMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells.ConclusionsOur data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

Correction to: Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

HomeCirculationVol. 139, No. 2Correction to: Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1 Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1 Originally published7 Jan 2019https://doi.org/10.1161/CIR.0000000000000647Circulation. 2019;139:e1This article corrects the followingMetabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1In the article by Li et al, “Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1,” which published online August 25, 2016, and in print October 11, 2016 (Circulation. 2016;134:1105–1121. doi: 10.1161/CIRCULATIONAHA.116.023171), a correction is needed. The last name of one author, Radu Moldovan, was mistakenly misspelled and needs to be corrected.The author list should read as follows:“Min Li, Suzette Riddle, Hui Zhang, Angelo D’Alessandro, Amanda Flockton, Natalie J. Serkova, Kirk C. Hansen, Radu Moldovan, B. Alexandre McKeon, Maria Frid, Sushil Kumar, Hong Li, Hongbing Liu, Angela Caánovas, Juan F. Medrano, Milton G. Thomas, Dijana Iloska, Lydie Plecitá-Hlavatá, Petr Ježek, Soni Pullamsetti, Mehdi A. Fini, Karim C. El Kasmi, QingHong Zhang, and Kurt R. Stenmark”The correction has been made to the current online version of the article, which is available at https://www.ahajournals.org/doi/10.1161/CIRCULATIONAHA.116.023171.Footnoteshttps://www.ahajournals.org/journal/circ Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMetabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1Min Li, et al. Circulation. 2016;134:1105-1121 January 8, 2019Vol 139, Issue 2 Advertisement Article InformationMetrics © 2019 American Heart Association, Inc.https://doi.org/10.1161/CIR.0000000000000647PMID: 30615505 Originally publishedJanuary 7, 2019 PDF download Advertisement

MiR-34a Regulates Expression of the Stathmin-1 Oncoprotein and Prostate Cancer Progression.

In aggressive prostate cancers, the oncoprotein STMN1 (also known as stathmin 1 and oncoprotein 18) is often overexpressed. STMN1 is involved in various cellular processes, including cell proliferation, motility, and tumor metastasis. Here, it was found that the expression of STMN1 RNA and protein is elevated in metastatic prostate cancers. Knockdown of STMN1 resulted in reduced proliferation and invasion of cells and tumor growth and metastasis in vivo Furthermore, miR-34a downregulated STMN1 by directly binding to its 3'-UTR. Overexpression of miR-34a in prostate cancer cells reduced proliferation and colony formation, suggesting that it is a tumor suppressor. The transcriptional corepressor C-terminal binding protein 1 (CtBP1) negatively regulated expression of miR-34a. Furthermore, gene expression profiling of STMN1-modulated prostate cancer cells revealed molecular alterations, including elevated expression of growth differentiation factor 15 (GDF15), which is involved in cancer progression and potentially in STMN1-mediated oncogenesis. Thus, in prostate cancer, CtBP1-regulated miR-34a modulates STMN1 expression and is involved in cancer progression through the CtBP1\miR-34a\STMN1\GDF15 axis.Implications: The CtBP1\miR-34a\STMN1\GDF15 axis is a potential therapeutic target for treatment of aggressive prostate cancer. Mol Cancer Res; 16(7); 1125-37. ©2017 AACR.

CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs.

Regeneration following tissue damage often necessitates a mechanism for cellular re-programming, so that surviving cells can give rise to all cell types originally found in the damaged tissue. This process, if unchecked, can also generate cell types that are inappropriate for a given location. We conducted a screen for genes that negatively regulate the frequency of notum-to-wing transformations following genetic ablation and regeneration of the wing pouch, from which we identified mutations in the transcriptional co-repressor C-terminal Binding Protein (CtBP). When CtBP function is reduced, ablation of the pouch can activate the JNK/AP-1 and JAK/STAT pathways in the notum to destabilize cell fates. Ectopic expression of Wingless and Dilp8 precede the formation of the ectopic pouch, which is subsequently generated by recruitment of both anterior and posterior cells near the compartment boundary. Thus, CtBP stabilizes cell fates following damage by opposing the destabilizing effects of the JNK/AP-1 and JAK/STAT pathways.

Evaluation of metformin effects in the chronic phase of spontaneous seizures in pilocarpine model of temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a common form of drug-resistant epilepsy that sometimes responds to dietary manipulation such as the 'ketogenic diet'. Here we have investigated the effects of metformin in the rat pilocaroin model of TLE. Male rats were treated with intra peritoneal injection of pilocarpine hydrochloride, in dose of 360mg/kg to induce status epilepticus (SE). At 45day after induction of SE, metformin was injected intraperitoneally in dose of 250mg/kg/day for 5days. We show that metformin potently reduces the progression of seizures and blocks seizure-induced over-expression of brain-derived neurotropic factor (BDNF) and its receptor, Tropomyosin receptor kinase B (TrkB). We have shown that this reduced expression pattern is mediated by the transcriptional co-repressor CtBP (C-terminal binding protein). Moreover, metformin decreased mechanistic target of rapamycin (mTOR) activation through activation of AMP-activated protein kinase (AMPK) signaling pathway. Our findings have been shown that metformin has anticonvulsant and antiepileptic properties, and suggesting that antiglycolytic compounds such as metformin may represent a new class of drugs for treating epilepsy.

Abstract 475: Role of microRNA-34a in regulating oncoprotein STMN1

Abstract MicroRNAs, as negative regulators of gene expression, play a major role in cellular homeostasis. Downregulation of miRs is common in cancer which results in upregulation of multiple oncogenes in cancer. Here we show that stathmin (STMN1), which is over-expressed and oncogenic in wide variety of cancers, is regulated by microRNA-34 in prostate cancer. Earlier studies suggest that stathmin is involved in vital cellular processes such as cell proliferation, motility and metastasis. In the present study, we show elevated expression of stathmin in metastatic prostate cancer. Functionally, knockdown of stathmin in prostate cancer cells resulted in reduced cell proliferation and invasion in vitro, and tumor growth and metastasis in vivo. We also show that tumor suppressor microRNA miR-34a targets stathmin and mir-34 is regulated by transcriptional corepressor CtBP1 (C-Terminal Binding Protein 1). Further, Microarray analyses using stathmin modulated prostate cancer cell line RNA revealed multiple molecular alterations which may play a critical role in STMN1-mediated oncogenic activity. Thus, our study suggests that CtBP1-regulated miR-34a modulates stathmin expression in prostate cancer. This study therefore demonstrates functional role of CtBP1-miR-34a-Stathmin axis in prostate cancer progression. Citation Format: Balabhadrapatruni V.S.K. Chakravarthi, Rohit Mehra, Rui Wang, Darshan Shimoga Chandrashekar, Sai Akshaya Hodigere Balasubramanya, Irfan A. Asangani, Robert J. Lonigro, Arul M. Chinnaiyan, Sooryanarayana Varambally. Role of microRNA-34a in regulating oncoprotein STMN1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 475. doi:10.1158/1538-7445.AM2017-475

EVI1 Mediated Stimulation of 3T3-L1 Preadipocyte Differentiation Is CtBP Dependent

Myelodysplasia syndrome 1 (MDS1) and Ecotropic viral integration site 1 (EVI1) complex (MECOM) locus encode multiple isoforms of the EVI1 protein that are essential for normal vertebrate development and when inappropriately expressed play a significant role in malignancy and in particular leukaemias. However, the function of individual EVI1 isoforms is not fully understood. Recently, EVI1 or PRDM3, which is structurally closely related to the brown adipose tissue determining factor PRDM16, was shown to be required for differentiation of adipocytes. In this study, we show that 3T3-L1 preadipocytes sustain expression of all Evi1 isoforms examined, including Mds1-Evi1, Evi1FL, Evi1Δ324, Evi1FL + 9 and Evi1Δ105 throughout the adipogenesis differentiation programme. We also show that differentiation markers are enhanced by enforced expression of either Evi1, Evi1FL + 9 or Evi1Δ105 isoforms. Interestingly 3T3-L1 differentiation markers are also moderately enhanced by enforced expression of Evi1Δ324, which lacks part of the N-ter-minal zinc finger domain (ZF1), demonstrating a biological activity for this particular isoform. Enforced expression of an Evi1 mutant lacking C-terminal binding protein (CtBP) co-repressor protein binding activity fails to stimulate 3T3-L1 differentiation markers and may have dominant negative activity, causing partial inhibition of this developmental programme. These studies show that multiple EVI1 isoforms are expressed in adipocytes and can stimulate adipogenic markers in a manner that is partially independent of the ZF1 DNA binding domain but fully dependent upon interaction with co-repressor CtBP proteins.

Metabolic Reprogramming and Redox Signaling in Pulmonary Hypertension.

Pulmonary hypertension is a complex disease of the pulmonary vasculature, which in severe cases terminates in right heart failure. Complex remodeling of pulmonary arteries comprises the central issue of its pathology. This includes extensive proliferation, apoptotic resistance and inflammation. As such, the molecular and cellular features of pulmonary hypertension resemble hallmark characteristics of cancer cell behavior. The vascular remodeling derives from significant metabolic changes in resident cells, which we describe in detail. It affects not only cells of pulmonary artery wall, but also its immediate microenvironment involving cells of immune system (i.e., macrophages). Thus aberrant metabolism constitutes principle component of the cancer-like theory of pulmonary hypertension. The metabolic changes in pulmonary artery cells resemble the cancer associated Warburg effect, involving incomplete glucose oxidation through aerobic glycolysis with depressed mitochondrial catabolism enabling the fueling of anabolic reactions with amino acids, nucleotides and lipids to sustain proliferation. Macrophages also undergo overlapping but distinct metabolic reprogramming inducing specific activation or polarization states that enable their participation in the vascular remodeling process. Such metabolic synergy drives chronic inflammation further contributing to remodeling. Enhanced glycolytic flux together with suppressed mitochondrial bioenergetics promotes the accumulation of reducing equivalents, NAD(P)H. We discuss the enzymes and reactions involved. The reducing equivalents modulate the regulation of proteins using NAD(P)H as the transcriptional co-repressor C-terminal binding protein 1 cofactor and significantly impact redox status (through GSH, NAD(P)H oxidases, etc.), which together act to control the phenotype of the cells of pulmonary arteries. The altered mitochondrial metabolism changes its redox poise, which together with enhanced NAD(P)H oxidase activity and reduced enzymatic antioxidant activity promotes a pro-oxidative cellular status. Herein we discuss all described metabolic changes along with resultant alterations in redox status, which result in excessive proliferation, apoptotic resistance, and inflammation, further leading to pulmonary arterial wall remodeling and thus establishing pulmonary artery hypertension pathology.

Metabolic Reprogramming Regulates the Proliferative and Inflammatory Phenotype of Adventitial Fibroblasts in Pulmonary Hypertension Through the Transcriptional Corepressor C-Terminal Binding Protein-1.

Changes in metabolism have been suggested to contribute to the aberrant phenotype of vascular wall cells, including fibroblasts, in pulmonary hypertension (PH). Here, we test the hypothesis that metabolic reprogramming to aerobic glycolysis is a critical adaptation of fibroblasts in the hypertensive vessel wall that drives proliferative and proinflammatory activation through a mechanism involving increased activity of the NADH-sensitive transcriptional corepressor C-terminal binding protein 1 (CtBP1). RNA sequencing, quantitative polymerase chain reaction,13C-nuclear magnetic resonance, fluorescence-lifetime imaging, mass spectrometry-based metabolomics, and tracing experiments with U-13C-glucose were used to assess glycolytic reprogramming and to measure the NADH/NAD+ ratio in bovine and human adventitial fibroblasts and mouse lung tissues. Immunohistochemistry was used to assess CtBP1 expression in the whole-lung tissues. CtBP1 siRNA and the pharmacological inhibitor 4-methylthio-2-oxobutyric acid (MTOB) were used to abrogate CtBP1 activity in cells and hypoxic mice. We found that adventitial fibroblasts from calves with severe hypoxia-induced PH and humans with idiopathic pulmonary arterial hypertension (PH-Fibs) displayed aerobic glycolysis when cultured under normoxia, accompanied by increased free NADH and NADH/NAD+ ratios. Expression of the NADH sensor CtBP1 was increased in vivo and in vitro in fibroblasts within the pulmonary adventitia of humans with idiopathic pulmonary arterial hypertension and animals with PH and cultured PH-Fibs, respectively. Decreasing NADH pharmacologically with MTOB or genetically blocking CtBP1 with siRNA upregulated the cyclin-dependent genes (p15 and p21) and proapoptotic regulators (NOXA and PERP), attenuated proliferation, corrected the glycolytic reprogramming phenotype of PH-Fibs, and augmented transcription of the anti-inflammatory gene HMOX1. Chromatin immunoprecipitation analysis demonstrated that CtBP1 directly binds the HMOX1 promoter. Treatment of hypoxic mice with MTOB decreased glycolysis and expression of inflammatory genes, attenuated proliferation, and suppressed macrophage numbers and remodeling in the distal pulmonary vasculature. CtBP1 is a critical factor linking changes in cell metabolism to cell phenotype in hypoxic and other forms of PH and a therapeutic target.

The miR-644a/CTBP1/p53 axis suppresses drug resistance by simultaneous inhibition of cell survival and epithelial-mesenchymal transition in breast cancer.

Tumor cells develop drug resistance which leads to recurrence and distant metastasis. MicroRNAs are key regulators of tumor pathogenesis; however, little is known whether they can sensitize cells and block metastasis simultaneously. Here, we report miR-644a as a novel inhibitor of both cell survival and EMT whereby acting as pleiotropic therapy-sensitizer in breast cancer. We showed that both miR-644a expression and its gene signature are associated with tumor progression and distant metastasis-free survival. Mechanistically, miR-644a directly targets the transcriptional co-repressor C-Terminal Binding Protein 1 (CTBP1) whose knock-outs by the CRISPR-Cas9 system inhibit tumor growth, metastasis, and drug resistance, mimicking the phenotypes induced by miR-644a. Furthermore, downregulation of CTBP1 by miR-644a upregulates wild type- or mutant-p53 which acts as a ‘molecular switch’ between G1-arrest and apoptosis by inducing cyclin-dependent kinase inhibitor 1 (p21, CDKN1A, CIP1) or pro-apoptotic phorbol-12-myristate-13-acetate-induced protein 1 (Noxa, PMAIP1), respectively. Interestingly, an increase in mutant-p53 by either overexpression of miR-644a or downregulation of CTBP1 was enough to shift this balance in favor of apoptosis through upregulation of Noxa. Notably, p53-mutant patients, but not p53-wild type ones, with high CTBP1 have a shorter survival suggesting that CTBP1 could be a potential prognostic factor for breast cancer patients with p53 mutations. Overall, re-activation of the miR-644a/CTBP1/p53 axis may represent a new strategy for overcoming both therapy resistance and metastasis.

Quantitative proteomic analyses of mammary organoids reveals distinct signatures after exposure to environmental chemicals

Common environmental contaminants such as bisphenols and phthalates and persistent contaminants such as polychlorinated biphenyls are thought to influence tissue homeostasis and carcinogenesis by acting as disrupters of endocrine function. In this study we investigated the direct effects of exposure to bisphenol A (BPA), mono-n-butyl phthalate (Pht), and polychlorinated biphenyl 153 (PCB153) on the proteome of primary organotypic cultures of the mouse mammary gland. At low-nanomolar doses each of these agents induced distinct effects on the proteomes of these cultures. Although BPA treatment produced effects that were similar to those induced by estradiol, there were some notable differences, including a reduction in the abundance of retinoblastoma-associated protein and increases in the Rho GTPases Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle protein CDC42. Both Pht and PCB153 induced changes that were distinct from those induced by estrogen, including decreased levels of the transcriptional corepressor C-terminal binding protein 1. Interestingly, the three chemicals appeared to alter the abundance of distinct splice forms of many proteins as well as the abundance of several proteins that regulate RNA splicing. Our combined results indicate that the three classes of chemical have distinct effects on the proteome of normal mouse mammary cultures, some estrogen-like but most estrogen independent, that influence diverse biological processes including apoptosis, cell adhesion, and proliferation.

PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression.

Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway.