Katrin J Svensson

All Publications

• AlphaFold2 enables accurate deorphanization of ligands to single-pass receptors. Cell systems Banhos Danneskiold-Samsoe, N., Kavi, D., Jude, K. M., Nissen, S. B., Wat, L. W., Coassolo, L., Zhao, M., Santana-Oikawa, G. A., Broido, B. B., Garcia, K. C., Svensson, K. J. 2024

  Abstract

  Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold2 (AF2) as a screening approach to identify extracellular ligands to single-pass transmembrane receptors. Key to the approach is the optimization of AF2 input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. The predictions were performed on ligand-receptor pairs not used for AF2 training. We demonstrate high discriminatory power and a success rate of close to 90% for known ligand-receptor pairs and 50% for a diverse set of experimentally validated interactions. Further, we show that screen accuracy does not correlate linearly with prediction of ligand-receptor interaction. These results demonstrate a proof of concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

  View details for DOI 10.1016/j.cels.2024.10.004

  View details for PubMedID 39541981

• Size matters: the biochemical logic of ligand type in endocrine crosstalk. Life metabolism Lone, J. B., Long, J. Z., Svensson, K. J. 2024; 3 (1)

  Abstract

  The endocrine system is a fundamental type of long-range cell-cell communication that is important for maintaining metabolism, physiology, and other aspects of organismal homeostasis. Endocrine signaling is mediated by diverse blood-borne ligands, also called hormones, including metabolites, lipids, steroids, peptides, and proteins. The size and structure of these hormones are fine-tuned to make them bioactive, responsive, and adaptable to meet the demands of changing environments. Why has nature selected such diverse ligand types to mediate communication in the endocrine system? What is the chemical, signaling, or physiologic logic of these ligands? What fundamental principles from our knowledge of endocrine communication can be applied as we continue as a field to uncover additional new circulating molecules that are claimed to mediate long-range cell and tissue crosstalk? This review provides a framework based on the biochemical logic behind this crosstalk with respect to their chemistry, temporal regulation in physiology, specificity, signaling actions, and evolutionary development.

  View details for DOI 10.1093/lifemeta/load048

  View details for PubMedID 38425548

  View details for PubMedCentralID PMC10904031

• A class of secreted mammalian peptides with potential to expand cell-cell communication. Nature communications Wiggenhorn, A. L., Abuzaid, H. Z., Coassolo, L., Li, V. L., Tanzo, J. T., Wei, W., Lyu, X., Svensson, K. J., Long, J. Z. 2023; 14 (1): 8125

  Abstract

  Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

  View details for DOI 10.1038/s41467-023-43857-0

  View details for PubMedID 38065934

  View details for PubMedCentralID 5946320

• Hallmarks of the metabolic secretome. Trends in endocrinology and metabolism: TEM Reghupaty, S. C., Dall, N. R., Svensson, K. J. 2023

  Abstract

  The identification of novel secreted factors is advancing at an unprecedented pace. However, there is a critical need to consolidate and integrate this knowledge to provide a framework of their diverse mechanisms, functional significance, and inter-relationships. Complicating this effort are challenges related to nonstandardized methods, discrepancies in sample handling, and inconsistencies in the annotation of unknown molecules. This Review aims to synthesize the rapidly expanding field of the metabolic secretome, encompassing the five major types of secreted factors: proteins, peptides, metabolites, lipids, and extracellular vesicles. By systematically defining the functions and detection of the components within the metabolic secretome, this Review provides a primer into the advances of the field, and how integration of the techniques discussed can provide a deeper understanding of the mechanisms underlying metabolic homeostasis and its disorders.

  View details for DOI 10.1016/j.tem.2023.09.006

  View details for PubMedID 37845120

• Protocol for invivo measurement of basal and insulin-stimulated glucose uptake in mouse tissues. STAR protocols Zhao, M., Wat, L. W., Svensson, K. J. 2023; 4 (2): 102179

  Abstract

  Here, we present an invivo protocol for measuring basal and insulin-stimulated glucose uptake in tissues from mice. We describe steps for administering 2-deoxy-D-[1,2-3H]glucose in the presence or absence of insulin via intraperitoneal injections. We then detail tissue collection, tissue processing to measure 3H counts on a scintillation counter, and data interpretation. This protocol can be applied to other glucoregulatory hormones, genetic mouse models, and other species. For complete details on the use and execution of this protocol, please refer to Jiang etal. (2021).1.

  View details for DOI 10.1016/j.xpro.2023.102179

  View details for PubMedID 36933224

• Mapping transcriptional heterogeneity and metabolic networks in fatty livers at single-cell resolution. iScience Coassolo, L., Liu, T., Jung, Y., Taylor, N. P., Zhao, M., Charville, G. W., Nissen, S. B., Yki-Jarvinen, H., Altman, R. B., Svensson, K. J. 2023; 26 (1): 105802

  Abstract

  Non-alcoholic fatty liver disease is a heterogeneous disease with unclear underlying molecular mechanisms. Here, we perform single-cell RNA sequencing of hepatocytes and hepatic non-parenchymal cells to map the lipid signatures in mice with non-alcoholic fatty liver disease (NAFLD). We uncover previously unidentified clusters of hepatocytes characterized by either high or low srebp1 expression. Surprisingly, the canonical lipid synthesis driver Srebp1 is not predictive of hepatic lipid accumulation, suggestive of other drivers of lipid metabolism. By combining transcriptional data at single-cell resolution with computational network analyses, we find that NAFLD is associated with high constitutive androstane receptor (CAR) expression. Mechanistically, CAR interacts with four functional modules: cholesterol homeostasis, bile acid metabolism, fatty acid metabolism, and estrogen response. Nuclear expression of CAR positively correlates with steatohepatitis in human livers. These findings demonstrate significant cellular differences in lipid signatures and identify functional networks linked to hepatic steatosis in mice and humans.

  View details for DOI 10.1016/j.isci.2022.105802

  View details for PubMedID 36636354

  View details for PubMedCentralID PMC9830221

• Rapid and accurate deorphanization of ligand-receptor pairs using AlphaFold BioRxiv Banhos Danneskiold-Samsøe, N., Kavi, D., Jude, K., Nissen, S., Wat, L., Coassolo, L., Zhao, M., Santana-Oikawa, G., Broido, B., Garcia, K., Svensson, K. J. 2023

  View details for DOI 10.1101/2023.03.16.531341

• New players of the adipose secretome: Therapeutic opportunities and challenges. Current opinion in pharmacology Coassolo, L., Dannieskiold-Samsøe, N. B., Zhao, M., Allen, H., Svensson, K. J. 2022; 67: 102302

  Abstract

  Adipose tissue is a functional endocrine organ comprised of adipocytes and other cell types that are known to secrete a multiplicity of adipose-derived factors, including lipids and proteins. It is well established that adipose tissue and its secretome can impact systemic energy homeostasis. The endocrine and paracrine effects of adipose-derived factors have been widely studied over the last several decades. Owing to technological advances in genomics and proteomics, several additional adipose-derived protein factors have recently been identified. By learning from previous efforts, the next challenge will be to leverage these discoveries for the prevention or treatment of metabolic disorders. Here, we discuss recently discovered adipose-derived proteins secreted from white or brown adipose tissue and the opportunities and challenges of translating these biological findings into disease therapeutics.

  View details for DOI 10.1016/j.coph.2022.102302

  View details for PubMedID 36195010

• Phosphoproteomic mapping reveals distinct signaling actions and activation of muscle protein synthesis by Isthmin-1 eLife Zhao, M., Banhos Danneskiold-Samsøe, N., Ulicna, L., Nguyen, Q., Voilquin, L., Lee, D. E., White, J. P., Jiang, Z., Cuthbert, N., Paramasivam, S., Bielczyk-Maczynska, E., van Rechem, C., Svensson, K. J. 2022

  View details for DOI 10.7554/eLife.80014

• Isthmin-1 is an adipokine that promotes glucose uptake and improves glucose tolerance and hepatic steatosis. Cell metabolism Jiang, Z., Zhao, M., Voilquin, L., Jung, Y., Aikio, M. A., Sahai, T., Dou, F. Y., Roche, A. M., Carcamo-Orive, I., Knowles, J. W., Wabitsch, M., Appel, E. A., Maikawa, C. L., Camporez, J. P., Shulman, G. I., Tsai, L., Rosen, E. D., Gardner, C. D., Spiegelman, B. M., Svensson, K. J. 2021

  Abstract

  With the increasing prevalence of type 2 diabetes and fatty liver disease, there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Here, we identify isthmin-1 (Ism1) as an adipokine and one that has a dual role in increasing adipose glucose uptake while suppressing hepatic lipid synthesis. Ism1 ablation results in impaired glucose tolerance, reduced adipose glucose uptake, and reduced insulin sensitivity, demonstrating an endogenous function for Ism1 in glucose regulation. Mechanistically, Ism1 activates a PI3K-AKT signaling pathway independently of the insulin and insulin-like growth factor receptors. Notably, while the glucoregulatory function is shared with insulin, Ism1 counteracts lipid accumulation in the liver by switching hepatocytes from a lipogenic to a protein synthesis state. Furthermore, therapeutic dosing of recombinant Ism1 improves diabetes in diet-induced obese mice and ameliorates hepatic steatosis in a diet-induced fatty liver mouse model. These findings uncover an unexpected, bioactive protein hormone that might have simultaneous therapeutic potential for diabetes and fatty liver disease.

  View details for DOI 10.1016/j.cmet.2021.07.010

  View details for PubMedID 34348115

• Isolation, culture, and functional analysis of hepatocytes from mice with fatty liver disease. STAR protocols Jung, Y., Zhao, M., Svensson, K. J. 2020; 1 (3): 100222

  Abstract

  We present a protocol for isolating hepatocytes from mice with established non-alcoholic fatty liver disease. This protocol consists of liver perfusion using a peristaltic pump, followed by a modified 25% and 90% Percoll gradient centrifugation protocol to capture lipid-laden hepatocytes that are usually lost using traditional isolation protocols. This protocol enables simultaneous isolation of normal and lipid-filled hepatocytes. Lipid-filled hepatocytes can be used in cell culture systems to study drug metabolism, hepatotoxicity, or glucose and lipid metabolism. For complete details on the use and execution of this protocol, please refer to Sharabi etal. (2017) and Kegel etal. (2016).

  View details for DOI 10.1016/j.xpro.2020.100222

  View details for PubMedID 33377114

• A β-hydroxybutyrate shunt pathway generates anti-obesity ketone metabolites. Cell Moya-Garzon, M. D., Wang, M., Li, V. L., Lyu, X., Wei, W., Tung, A. S., Raun, S. H., Zhao, M., Coassolo, L., Islam, H., Oliveira, B., Dai, Y., Spaas, J., Delgado-Gonzalez, A., Donoso, K., Alvarez-Buylla, A., Franco-Montalban, F., Letian, A., Ward, C. P., Liu, L., Svensson, K. J., Goldberg, E. L., Gardner, C. D., Little, J. P., Banik, S. M., Xu, Y., Long, J. Z. 2024

  Abstract

  β-Hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve the interconversion of BHB and primary energy intermediates. Here, we identify a previously undescribed BHB secondary metabolic pathway via CNDP2-dependent enzymatic conjugation of BHB and free amino acids. This BHB shunt pathway generates a family of anti-obesity ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. The most abundant BHB-amino acid, BHB-Phe, is a ketosis-inducible congener of Lac-Phe that activates hypothalamic and brainstem neurons and suppresses feeding. Conversely, CNDP2-KO mice exhibit increased food intake and body weight following exogenous ketone ester supplementation or a ketogenic diet. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, enzymatic amino acid BHB-ylation defines a ketone shunt pathway and bioactive ketone metabolites linked to energy balance.

  View details for DOI 10.1016/j.cell.2024.10.032

  View details for PubMedID 39536746

• GLUT1 overexpression in CAR-T cells induces metabolic reprogramming and enhances potency. Nature communications Guerrero, J. A., Klysz, D. D., Chen, Y., Malipatlolla, M., Lone, J., Fowler, C., Stuani, L., May, A., Bashti, M., Xu, P., Huang, J., Michael, B., Contrepois, K., Dhingra, S., Fisher, C., Svensson, K. J., Davis, K. L., Kasowski, M., Feldman, S. A., Sotillo, E., Mackall, C. L. 2024; 15 (1): 8658

  Abstract

  The intensive nutrient requirements needed to sustain T cell activation and proliferation, combined with competition for nutrients within the tumor microenvironment, raise the prospect that glucose availability may limit CAR-T cell function. Here, we seek to test the hypothesis that stable overexpression (OE) of the glucose transporter GLUT1 in primary human CAR-T cells would improve their function and antitumor potency. We observe that GLUT1OE in CAR-T cells increases glucose consumption, glycolysis, glycolytic reserve, and oxidative phosphorylation, and these effects are associated with decreased T cell exhaustion and increased Th17 differentiation. GLUT1OE also induces broad metabolic reprogramming associated with increased glutathione-mediated resistance to reactive oxygen species, and increased inosine accumulation. When challenged with tumors, GLUT1OE CAR-T cells secrete more proinflammatory cytokines and show enhanced cytotoxicity in vitro, and demonstrate superior tumor control and persistence in mouse models. Our collective findings support a paradigm wherein glucose availability is rate limiting for effector CAR-T cell function and demonstrate that enhancing glucose availability via GLUT1OE could augment antitumor immune function.

  View details for DOI 10.1038/s41467-024-52666-y

  View details for PubMedID 39370422

  View details for PubMedCentralID PMC11456602

• A secondary β-hydroxybutyrate metabolic pathway linked to energy balance. bioRxiv : the preprint server for biology Moya-Garzon, M. D., Wang, M., Li, V. L., Lyu, X., Wei, W., Tung, A. S., Raun, S. H., Zhao, M., Coassolo, L., Islam, H., Oliveira, B., Dai, Y., Spaas, J., Delgado-Gonzalez, A., Donoso, K., Alvarez-Buylla, A., Franco-Montalban, F., Letian, A., Ward, C., Liu, L., Svensson, K. J., Goldberg, E. L., Gardner, C. D., Little, J. P., Banik, S. M., Xu, Y., Long, J. Z. 2024

  Abstract

  β-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.

  View details for DOI 10.1101/2024.09.09.612087

  View details for PubMedID 39314488

  View details for PubMedCentralID PMC11418978

• Novel secreted regulators of glucose and lipid metabolism in the development of metabolic diseases. Diabetologia Wat, L. W., Svensson, K. J. 2024

  Abstract

  The tight regulation of glucose and lipid metabolism is crucial for maintaining metabolic health. Dysregulation of these processes can lead to the development of metabolic diseases. Secreted factors, or hormones, play an essential role in the regulation of glucose and lipid metabolism, thus also playing an important role in the development of metabolic diseases such as type 2 diabetes and obesity. Given the important roles of secreted factors, there has been significant interest in identifying new secreted factors and new functions for existing secreted factors that control glucose and lipid metabolism. In this review, we evaluate novel secreted factors or novel functions of existing factors that regulate glucose and lipid metabolism discovered in the last decade, including secreted isoform of endoplasmic reticulum membrane complex subunit 10, vimentin, cartilage intermediate layer protein 2, isthmin-1, lipocalin-2, neuregulin-1 and neuregulin-4. We discuss their discovery, tissues of origin, mechanisms of action and sex differences, emphasising their potential to regulate metabolic processes central to diabetes. Additionally, we discuss the translational barriers, particularly the absence of identified receptors, that hamper their functional characterisation and further therapeutic development. Ultimately, the identification of new secreted factors may give insights into previously unidentified pathways of disease progression and mechanisms of glucose and lipid homeostasis.

  View details for DOI 10.1007/s00125-024-06253-x

  View details for PubMedID 39180580

  View details for PubMedCentralID 8232639

• PTER is a N-acetyltaurine hydrolase that regulates feeding and obesity. Nature Wei, W., Lyu, X., Markhard, A. L., Fu, S., Mardjuki, R. E., Cavanagh, P. E., Zeng, X., Rajniak, J., Lu, N., Xiao, S., Zhao, M., Moya-Garzon, M. D., Truong, S. D., Chou, J. C., Wat, L. W., Chidambaranathan-Reghupaty, S., Coassolo, L., Xu, D., Shen, F., Huang, W., Ramirez, C. B., Jang, C., Li, L., Svensson, K. J., Fischbach, M. A., Long, J. Z. 2024

  Abstract

  Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in the biosynthesis of taurine from cysteine and in the downstream metabolism of secondary taurine metabolites4,5. One taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by stimuli that alter taurine or acetate flux, including endurance exercise7, dietary taurine supplementation8 and alcohol consumption6,9. So far, the identities of the enzymes involved in N-acetyltaurine metabolism, and the potential functions of N-acetyltaurine itself, have remained unknown. Here we show that the body mass index associated orphan enzyme phosphotriesterase-related (PTER)10 is a physiological N-acetyltaurine hydrolase. In vitro, PTER catalyses the hydrolysis of N-acetyltaurine to taurine and acetate. In mice, PTER is expressed in the kidney, liver and brainstem. Genetic ablation of Pter in mice results in complete loss of tissue N-acetyltaurine hydrolysis activity and a systemic increase in N-acetyltaurine levels. After stimuli that increase taurine levels, Pter knockout mice exhibit reduced food intake, resistance to diet-induced obesity and improved glucose homeostasis. Administration of N-acetyltaurine to obese wild-type mice also reduces food intake and body weight in a GFRAL-dependent manner. These data place PTER into a central enzymatic node of secondary taurine metabolism and uncover a role for PTER and N-acetyltaurine in body weight control and energy balance.

  View details for DOI 10.1038/s41586-024-07801-6

  View details for PubMedID 39112712

  View details for PubMedCentralID 3501277

• Loss of RREB1 reduces adipogenesis and improves insulin sensitivity in mouse and human adipocytes. bioRxiv : the preprint server for biology Yu, G. Z., Krentz, N. A., Bentley, L., Zhao, M., Paphiti, K., Sun, H., Honecker, J., Nygård, M., Dashti, H., Bai, Y., Reid, M., Thaman, S., Wabitsch, M., Rajesh, V., Yang, J., Mattis, K. K., Abaitua, F., Casero, R., Hauner, H., Knowles, J. W., Wu, J. Y., Mandrup, S., Claussnitzer, M., Svensson, K. J., Cox, R. D., Gloyn, A. L. 2024

  Abstract

  There are multiple independent genetic signals at the Ras-responsive element binding protein 1 (RREB1) locus associated with type 2 diabetes risk, fasting glucose, ectopic fat, height, and bone mineral density. We have previously shown that loss of RREB1 in pancreatic beta cells reduces insulin content and impairs islet cell development and function. However, RREB1 is a widely expressed transcription factor and the metabolic impact of RREB1 loss in vivo remains unknown. Here, we show that male and female global heterozygous knockout (Rreb1 +/-) mice have reduced body length, weight, and fat mass on high-fat diet. Rreb1+/- mice have sex- and diet-specific decreases in adipose tissue and adipocyte size; male mice on high-fat diet had larger gonadal adipocytes, while males on standard chow and females on high-fat diet had smaller, more insulin sensitive subcutaneous adipocytes. Mouse and human precursor cells lacking RREB1 have decreased adipogenic gene expression and activated transcription of genes associated with osteoblast differentiation, which was associated with Rreb1 +/- mice having increased bone mineral density in vivo. Finally, human carriers of RREB1 T2D protective alleles have smaller adipocytes, consistent with RREB1 loss-of-function reducing diabetes risk.

  View details for DOI 10.1101/2024.07.30.605923

  View details for PubMedID 39131393

  View details for PubMedCentralID PMC11312556

• A PTER-dependent pathway of taurine metabolism linked to energy balance. bioRxiv : the preprint server for biology Wei, W., Lyu, X., Markhard, A. L., Fu, S., Mardjuki, R. E., Cavanagh, P. E., Zeng, X., Rajniak, J., Lu, N., Xiao, S., Zhao, M., Moya-Garzon, M. D., Truong, S. D., Chou, J. C., Wat, L. W., Chidambaranathan-Reghupaty, S., Coassolo, L., Xu, D., Shen, F., Huang, W., Ramirez, C. B., Jang, C., Svensson, K. J., Fischbach, M. A., Long, J. Z. 2024

  Abstract

  Taurine is a conditionally essential micronutrient and one of the most abundant amino acids in humans1-3. In endogenous taurine metabolism, dedicated enzymes are involved in biosynthesis of taurine from cysteine as well as the downstream derivatization of taurine into secondary taurine metabolites4,5. One such taurine metabolite is N-acetyltaurine6. Levels of N-acetyltaurine are dynamically regulated by diverse physiologic perturbations that alter taurine and/or acetate flux, including endurance exercise7, nutritional taurine supplementation8, and alcohol consumption6,9. While taurine N-acetyltransferase activity has been previously detected in mammalian cells6,7, the molecular identity of this enzyme, and the physiologic relevance of N-acetyltaurine, have remained unknown. Here we show that the orphan body mass index-associated enzyme PTER (phosphotriesterase-related)10 is the principal mammalian taurine N-acetyltransferase/hydrolase. In vitro, recombinant PTER catalyzes bidirectional taurine N-acetylation with free acetate as well as the reverse N-acetyltaurine hydrolysis reaction. Genetic ablation of PTER in mice results in complete loss of tissue taurine N-acetyltransferase/hydrolysis activities and systemic elevation of N-acetyltaurine levels. Upon stimuli that increase taurine levels, PTER-KO mice exhibit lower body weight, reduced adiposity, and improved glucose homeostasis. These phenotypes are recapitulated by administration of N-acetyltaurine to wild-type mice. Lastly, the anorexigenic and anti-obesity effects of N-acetyltaurine require functional GFRAL receptors. Together, these data uncover enzymatic control of a previously enigmatic pathway of secondary taurine metabolism linked to energy balance.

  View details for DOI 10.1101/2024.03.21.586194

  View details for PubMedID 38562797

  View details for PubMedCentralID PMC10983888

• Mitochondrial uncoupler and retinoic acid synergistically induce differentiation and inhibit proliferation in neuroblastoma. bioRxiv : the preprint server for biology Jiang, H., Tiche, S. J., He, C. J., Jedoui, M., Forgo, B., Zhao, M., He, B., Li, Y., Li, A. M., Truong, A. T., Ho, J., Simmermaker, C., Yang, Y., Zhou, M., Hu, Z., Cuthbertson, D. J., Svensson, K. J., Hazard, F. K., Shimada, H., Chiu, B., Ye, J. 2024

  Abstract

  Neuroblastoma is a leading cause of death in childhood cancer cases. Unlike adult malignancies, which typically develop from aged cells through accumulated damage and mutagenesis, neuroblastoma originates from neural crest cells with disrupted differentiation. This distinct feature provides novel therapeutic opportunities beyond conventional cytotoxic methods. Previously, we reported that the mitochondrial uncoupler NEN (niclosamide ethanolamine) activated mitochondria respiration to reprogram the epigenome, promoting neuronal differentiation. In the current study, we further combine NEN with retinoic acid (RA) to promote neural differentiation both in vitro and in vivo. The treatment increased the expression of RA signaling and neuron differentiation-related genes, resulting in a global shift in the transcriptome towards a more favorable prognosis. Overall, these results suggest that the combination of a mitochondrial uncoupler and the differentiation agent RA is a promising therapeutic strategy for neuroblastoma.

  View details for DOI 10.1101/2024.01.22.576741

  View details for PubMedID 38328117

• Roles of Activin A and Gpnmb in metabolic dysfunction-associated steatotic liver disease (MASLD). Diabetes Liu, H., Yerevanian, A., Westerhoff, M., Hastings, M. H., Guerra, J. R., Zhao, M., Svensson, K. J., Cai, B., Soukas, A. A., Rosenzweig, A. 2023

  Abstract

  Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as nonalcoholic fatty liver disease (NAFLD)) and metabolic dysfunction-associated steatohepatitis (MASH, formerly known as nonalcoholic steatohepatitis (NASH)) are leading chronic liver diseases, driving cirrhosis, hepatocellular carcinoma, and mortality. MASLD/MASH is associated with increased senescence proteins, including Activin A, and senolytics have been proposed as a therapeutic approach. To test the role of Activin A, we induced hepatic expression of Activin A in a murine MASLD/MASH model. Surprisingly, overexpression of hepatic Activin A dramatically mitigated MASLD, reducing liver steatosis and inflammation as well as systemic fat accumulation, while improving insulin sensitivity. Further studies identified a dramatic decrease in the lipid-associated macrophages (LAM) marker glycoprotein NMB (Gpnmb) by Activin A and Gpnmb knockdown in the same model produced similar benefits and transcriptional changes to Activin A expression. These studies reveal a surprising protective role for Activin A in MASLD and the potential for SASP proteins to have context-specific beneficial effects. Moreover, they implicate both Activin A and Gpnmb as potential therapeutic targets for this condition.

  View details for DOI 10.2337/db23-0357

  View details for PubMedID 37934943

• A single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes. American journal of physiology. Cell physiology Bielczyk-Maczynska, E., Sharma, D., Blencowe, M., Saliba Gustafsson, P., Gloudemans, M. J., Yang, X., Carcamo-Orive, I., Wabitsch, M., Svensson, K. J., Park, C. Y., Quertermous, T., Knowles, J. W., Li, J. 2023

  Abstract

  CROP-Seq combines gene silencing using CRISPR interference with single-cell RNA sequencing. Here, we applied CROP-Seq to study adipogenesis and adipocyte biology. Human preadipocyte SGBS cell line expressing KRAB-dCas9 was transduced with a sgRNA library. Following selection, individual cells were captured using microfluidics at different timepoints during adipogenesis. Bioinformatic analysis of transcriptomic data was used to determine the knock-down effects, the dysregulated pathways, and to predict cellular phenotypes. Single-cell transcriptomes recapitulated adipogenesis states. For all targets, over 400 differentially expressed genes were identified at least at one timepoint. As a validation of our approach, the knock-down of PPARG and CEBPB (which encode key proadipogenic transcription factors) resulted in the inhibition of adipogenesis. Gene set enrichment analysis generated hypotheses regarding the molecular function of novel genes. MAFF knock-down led to downregulation of transcriptional response to proinflammatory cytokine TNF-α in preadipocytes and to decreased CXCL-16 and IL-6 secretion. TIPARP knock-down resulted in increased expression of adipogenesis markers. In summary, this powerful, hypothesis-free tool can identify novel regulators of adipogenesis, preadipocyte and adipocyte function associated with metabolic disease.

  View details for DOI 10.1152/ajpcell.00148.2023

  View details for PubMedID 37486064

• Extracellular Vesicles in Systemic Juvenile Idiopathic Arthritis. Journal of leukocyte biology Maller, J., Morgan, T., Morita, M., McCarthy, F., Jung, Y., Svensson, K. J., Elias, J. E., Macaubas, C., Mellins, E. 2023

  Abstract

  Systemic juvenile idiopathic arthritis (sJIA) is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles (EVs), contributes to sJIA pathogenesis and that the number and cellular sources of EVs would differ between inactive and active states of sJIA and healthy controls.We evaluated plasma from healthy pediatric controls and sJIA patients with active systemic flare or inactive disease. We isolated EVs by size-exclusion chromatography and determined total EV abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific EV subpopulations were measured by nanoscale flow cytometry. Isolated EVs were validated using a variety of ways, including Nanotracking and Cryo-EM. EV protein content was analyzed in pooled samples using mass spectrometry.Total EV concentration did not significantly differ between controls and sJIA patients. EVs with diameters <200 nm were the most abundant, including the majority of cell-specific EV subpopulations. sJIA patients had significantly higher levels of EVs from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active sJIA relative to inactive disease and controls. Protein analysis of isolated EVs from active patients showed a pro-inflammatory profile, uniquely expressing heat shock protein 47 (HP47), a stress-inducible protein.Our findings indicate that multiple cell types contribute to altered EV profiles in sJIA. The EV differences between sJIA disease states and healthy controls implicate EV-mediated cellular crosstalk as a potential driver of sJIA disease activity.

  View details for DOI 10.1093/jleuko/qiad059

  View details for PubMedID 37201912

• Organism-wide, cell-type-specific secretome mapping of exercise training in mice. Cell metabolism Wei, W., Riley, N. M., Lyu, X., Shen, X., Guo, J., Raun, S. H., Zhao, M., Moya-Garzon, M. D., Basu, H., Sheng-Hwa Tung, A., Li, V. L., Huang, W., Wiggenhorn, A. L., Svensson, K. J., Snyder, M. P., Bertozzi, C. R., Long, J. Z. 2023

  Abstract

  There is a significant interest in identifying blood-borne factors that mediate tissue crosstalk and function as molecular effectors of physical activity. Although past studies have focused on an individual molecule or cell type, the organism-wide secretome response to physical activity has not been evaluated. Here, we use a cell-type-specific proteomic approach to generate a 21-cell-type, 10-tissue map of exercise training-regulated secretomes in mice. Our dataset identifies >200 exercise training-regulated cell-type-secreted protein pairs, the majority of which have not been previously reported. Pdgfra-cre-labeled secretomes were the most responsive to exercise training. Finally, we show anti-obesity, anti-diabetic, and exercise performance-enhancing activities for proteoforms of intracellular carboxylesterases whose secretion from the liver is induced by exercise training.

  View details for DOI 10.1016/j.cmet.2023.04.011

  View details for PubMedID 37141889

• Rapid and accurate deorphanization of ligand-receptor pairs using AlphaFold. bioRxiv : the preprint server for biology Danneskiold-Samsøe, N. B., Kavi, D., Jude, K. M., Nissen, S. B., Wat, L. W., Coassolo, L., Zhao, M., Santana-Oikawa, G. A., Broido, B. B., Garcia, K. C., Svensson, K. J. 2023

  Abstract

  Secreted proteins are extracellular ligands that play key roles in paracrine and endocrine signaling, classically by binding cell surface receptors. Experimental assays to identify new extracellular ligand-receptor interactions are challenging, which has hampered the rate of novel ligand discovery. Here, using AlphaFold-multimer, we developed and applied an approach for extracellular ligand-binding prediction to a structural library of 1,108 single-pass transmembrane receptors. We demonstrate high discriminatory power and a success rate of close to 90 % for known ligand-receptor pairs where no a priori structural information is required. Importantly, the prediction was performed on de novo ligand-receptor pairs not used for AlphaFold training and validated against experimental structures. These results demonstrate proof-of-concept of a rapid and accurate computational resource to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.

  View details for DOI 10.1101/2023.03.16.531341

  View details for PubMedID 36993313

  View details for PubMedCentralID PMC10055078

• A class of secreted mammalian peptides with potential to expand cell-cell communication BioRxiv Wiggenhorn, A. L. 2023

  View details for DOI 10.1101/2023.06.02.543503

• A human TRPV1 genetic variant within the channel gating domain regulates pain sensitivity in rodents. The Journal of clinical investigation He, S., Zambelli, V. O., Sinharoy, P., Brabenec, L., Bian, Y., Rwere, F., Hell, R. C., Stein Neto, B., Hung, B., Yu, X., Zhao, M., Luo, Z., Wu, C., Xu, L., Svensson, K. J., McAllister, S. L., Stary, C. M., Wagner, N. M., Zhang, Y., Gross, E. R. 2022

  Abstract

  Pain signals are relayed to the brain via a nociceptive system, and in rare situations, this nociceptive system contains genetic variants that can limit pain response. Here we questioned whether a human transient receptor potential vanilloid 1 (TRPV1) missense variant causes a resistance to noxious stimuli and further if we can target this region by a cell-permeable peptide as a pain therapeutic. Initially using a computational approach, we identified a human K710N TRPV1 missense variant in an otherwise highly conserved region of mammalian TRPV1. After generating a TRPV1K710N knock-in mouse using CRISPR/Cas9, we discovered the K710N variant reduced capsaicin-induced calcium influx in dorsal root ganglion neurons. The TRPV1K710N rodents also had less acute behavioral response to chemical noxious stimuli and less hypersensitivity to nerve injury-induced pain, while leaving the response to noxious heat intact. Furthermore, blocking this K710 region in wild-type rodents by a cell-penetrating peptide limited acute behavioral responses to noxious stimuli and rescued pain hypersensitivity induced by nerve injury back to baseline. These findings identify K710 TRPV1 as a discrete site crucial for the control of nociception and provides new insights into how to leverage rare genetic variants in humans to uncover fresh strategies for developing pain therapeutics.

  View details for DOI 10.1172/JCI163735

  View details for PubMedID 36472910

• Isthmin-1 As A Modulator Of Metabolic Health ENDO Zhao, M., Banhos Danneskiold-Samsøe, N., Voilquin, L., Jiang, Z., Svensson, K. J. 2022

  View details for DOI 10.1210/jendso/bvac150.1216.

• Organism-wide secretome mapping uncovers pathways of tissue crosstalk in exercise BioRxiv Wei, W., et al 2022

  View details for DOI 10.1101/2022.11.21.517385

• CROP-Seq: a single-cell CRISPRi platform for characterizing candidate genes relevant to metabolic disorders in human adipocytes BioRxiv Bielczyk-Maczynska, E., Sharma, D., Blencowe, M., Gustafsson, P. S., Gloudemans, M. J., Xia, X., Carcamo-Orive, I., Wabitsch, M., Svensson, K. J., Park, C. Y., Quertermous, T., Knowles, J. W., Li, J. 2022

  View details for DOI 10.1101/2022.06.27.497796

• Single-cell analysis of non-alcoholic fatty livers identifies a role for the constitutive androstane receptor BioRxiv Coassolo, L., Liu, T., Jung, Y., Taylor, N. P., Zhao, M., Charville, G. W., Nissen, S. B., Yki-Jarvinen, H., Altman, R. B., Svensson, K. J. 2022

  View details for DOI 10.1101/2022.08.15.504026

• G protein-coupled receptor 151 regulates glucose metabolism and hepatic gluconeogenesis Nature Communications Bielczyk-Maczynska, E., Zhao, M., Zushin, P. H., Schnurr, T. M., Kim, H., Li, J., Sangwung, P., Nallagatla, P., Park, C., Cornn, C., Stahl, A., Svensson, K. J., Knowles, J. W. 2022

  View details for DOI 10.1038/s41467-022-35069-9

• Mitochondria-Rich Extracellular Vesicles Rescue Patient-Specific Cardiomyocytes From Doxorubicin Injury: Insights Into the SENECA Trial. JACC. CardioOncology O'Brien, C. G., Ozen, M. O., Ikeda, G., Vaskova, E., Jung, J. H., Bayardo, N., Santoso, M. R., Shi, L., Wahlquist, C., Jiang, Z., Jung, Y., Zeng, Y., Egan, E., Sinclair, R., Gee, A., Witteles, R., Mercola, M., Svensson, K. J., Demirci, U., Yang, P. C. 2021; 3 (3): 428-440

  Abstract

  Anthracycline-induced cardiomyopathy (AIC) is a significant source of morbidity and mortality in cancer survivors. The role of mesenchymal stem cells (MSCs) in treating AIC was evaluated in the SENECA trial, a Phase 1 National Heart, Lung, and Blood Institute-sponsored study, but the mechanisms underpinning efficacy in human tissue need clarification.The purpose of this study was to perform an in vitro clinical trial evaluating the efficacy and putative mechanisms of SENECA trial-specific MSCs in treating doxorubicin (DOX) injury, using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iCMs) generated from SENECA patients.Patient-specific iCMs were injured with 1 μmol/L DOX for 24 hours, treated with extracellular vesicles (EVs) from MSCs by either coculture or direct incubation and then assessed for viability and markers of improved cellular physiology. MSC-derived EVs were separated into large extracellular vesicles (L-EVs) (>200 nm) and small EVs (<220nm) using a novel filtration system.iCMs cocultured with MSCs in a transwell system demonstrated improved iCM viability and attenuated apoptosis. L-EVs but not small EVs recapitulated this therapeutic effect. L-EVs were found to be enriched in mitochondria, which were shown to be taken up by iCMs. iCMs treated with L-EVs demonstrated improved contractility, reactive oxygen species production, ATP production, and mitochondrial biogenesis. Inhibiting L-EV mitochondrial function with 1-methyl-4-phenylpyridinium attenuated efficacy.L-EV-mediated mitochondrial transfer mitigates DOX injury in patient-specific iCMs. Although SENECA was not designed to test MSC efficacy, consistent tendencies toward a positive effect were observed across endpoints. Our results suggest a mechanism by which MSCs may improve cardiovascular performance in AIC independent of regeneration, which could inform future trial design evaluating the therapeutic potential of MSCs.

  View details for DOI 10.1016/j.jaccao.2021.05.006

  View details for PubMedID 34604804

  View details for PubMedCentralID PMC8463733

• Mesenchymal Stem Cells Rescue Patient-Specific Cardiomyocyte Viability and Function Following Doxorubicin Injury via Microvesicle Mediated Mitochondrial Transfer to Recapitulate Human Clinical Trial Results OBrien, C. G., Ozen, M. O., Vaskova, E., Jung, J., Santoso, M., Shi, L., Wahlquist, C. A., Jiang, Z., Jung, Y., Ikeda, G., Zeng, Y., Sinclair, R., Gee, A., Witteles, R. M., Mercola, M., Svensson, K., Demirci, U., Yang, P. C. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for Web of Science ID 000607190402282

• Regulation of Energy Metabolism by Receptor Tyrosine Kinase Ligands. Frontiers in physiology Zhao, M. n., Jung, Y. n., Jiang, Z. n., Svensson, K. J. 2020; 11: 354

  Abstract

  Metabolic diseases, such as diabetes, obesity, and fatty liver disease, have now reached epidemic proportions. Receptor tyrosine kinases (RTKs) are a family of cell surface receptors responding to growth factors, hormones, and cytokines to mediate a diverse set of fundamental cellular and metabolic signaling pathways. These ligands signal by endocrine, paracrine, or autocrine means in peripheral organs and in the central nervous system to control cellular and tissue-specific metabolic processes. Interestingly, the expression of many RTKs and their ligands are controlled by changes in metabolic demand, for example, during starvation, feeding, or obesity. In addition, studies of RTKs and their ligands in regulating energy homeostasis have revealed unexpected diversity in the mechanisms of action and their specific metabolic functions. Our current understanding of the molecular, biochemical and genetic control of energy homeostasis by the endocrine RTK ligands insulin, FGF21 and FGF19 are now relatively well understood. In addition to these classical endocrine signals, non-endocrine ligands can govern local energy regulation, and the intriguing crosstalk between the RTK family and the TGFβ receptor family demonstrates a signaling network that diversifies metabolic process between tissues. Thus, there is a need to increase our molecular and mechanistic understanding of signal diversification of RTK actions in metabolic disease. Here we review the known and emerging molecular mechanisms of RTK signaling that regulate systemic glucose and lipid metabolism, as well as highlighting unexpected roles of non-classical RTK ligands that crosstalk with other receptor pathways.

  View details for DOI 10.3389/fphys.2020.00354

  View details for PubMedID 32372975

  View details for PubMedCentralID PMC7186430

• A CRISPR-based genome-wide screen for adipogenesis reveals new insights into mitotic expansion and lipogenesis bioRxiv Hilgendorf, K. I., Johnson, C. T., Han, K., Rabiee, A., Demeter, J., Cheng, R., Zhu, Y., Jiang, Z., Svensson, K. J., Bassik, M. C., Jackson, P. K. 2020
• Discovery of Hydrolysis-resistant Isoindoline N-Acyl Amino Acid Analogs that Stimulate Mitochondrial Respiration. Journal of medicinal chemistry Lin, H. n., Long, J. Z., Roche, A. M., Svensson, K. J., Dou, F. n., Chang, M. R., Strutzenberg, T. n., Ruiz, C. n., Cameron, M. D., Novick, S. J., Berdan, C. M., Louie, S. n., Nomura, D. K., Spiegelman, B. M., Griffin, P. R., Kamenecka, T. M. 2018

  Abstract

  N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. We found that administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure indicating that this pathway might be useful for treating obesity and associated disorders. We report the full account of the synthesis and mitochondrial uncoupling bioactivity of lipidated N-acyl amino acids and their unnatural analogs. Unsaturated fatty acid chains of medium length and neutral amino acid head groups are required for optimal uncoupling activity on mammalian cells. A class of unnatural N-acyl amino acid analogs, characterized by isoindoline-1-carboxylate head groups (37), were resistant to enzymatic degradation by PM20D1 and maintained uncoupling bioactivity in cells and in mice.

  View details for PubMedID 29533650

• Ablation of PM20D1 reveals N-acyl amino acid control of metabolism and nociception. Proceedings of the National Academy of Sciences of the United States of America Long, J. Z., Roche, A. M., Berdan, C. A., Louie, S. M., Roberts, A. J., Svensson, K. J., Dou, F. Y., Bateman, L. A., Mina, A. I., Deng, Z. n., Jedrychowski, M. P., Lin, H. n., Kamenecka, T. M., Asara, J. M., Griffin, P. R., Banks, A. S., Nomura, D. K., Spiegelman, B. M. 2018

  Abstract

  N-acyl amino acids (NAAs) are a structurally diverse class of bioactive signaling lipids whose endogenous functions have largely remained uncharacterized. To clarify the physiologic roles of NAAs, we generated mice deficient in the circulating enzyme peptidase M20 domain-containing 1 (PM20D1). Global PM20D1-KO mice have dramatically reduced NAA hydrolase/synthase activities in tissues and blood with concomitant bidirectional dysregulation of endogenous NAAs. Compared with control animals, PM20D1-KO mice exhibit a variety of metabolic and pain phenotypes, including insulin resistance, altered body temperature in cold, and antinociceptive behaviors. Guided by these phenotypes, we identify N-oleoyl-glutamine (C18:1-Gln) as a key PM20D1-regulated NAA. In addition to its mitochondrial uncoupling bioactivity, C18:1-Gln also antagonizes certain members of the transient receptor potential (TRP) calcium channels including TRPV1. Direct administration of C18:1-Gln to mice is sufficient to recapitulate a subset of phenotypes observed in PM20D1-KO animals. These data demonstrate that PM20D1 is a dominant enzymatic regulator of NAA levels in vivo and elucidate physiologic functions for NAA signaling in metabolism and nociception.

  View details for DOI 10.1073/pnas.1803389115

  View details for PubMedID 29967167

• Metastasis Stimulation by Hypoxia and Acidosis-Induced Extracellular Lipid Uptake Is Mediated by Proteoglycan-Dependent Endocytosis CANCER RESEARCH Menard, J. A., Christianson, H. C., Kucharzewska, P., Bourseau-Guilmain, E., Svensson, K. J., Lindqvist, E., Chandran, V. I., Kjellen, L., Welinder, C., Bengzon, J., Johansson, M. C., Belting, M. 2016; 76 (16): 4828-4840

  Abstract

  Hypoxia and acidosis are inherent stress factors of the tumor microenvironment and have been linked to increased tumor aggressiveness and treatment resistance. Molecules involved in the adaptive mechanisms that drive stress-induced disease progression constitute interesting candidates of therapeutic intervention. Here, we provide evidence of a novel role of heparan sulfate proteoglycans (HSPG) in the adaptive response of tumor cells to hypoxia and acidosis through increased internalization of lipoproteins, resulting in a lipid-storing phenotype and enhanced tumor-forming capacity. Patient glioblastoma tumors and cells under hypoxic and acidic stress acquired a lipid droplet (LD)-loaded phenotype, and showed an increased recruitment of all major lipoproteins, HDL, LDL, and VLDL. Stress-induced LD accumulation was associated with increased spheroid-forming capacity during reoxygenation in vitro and lung metastatic potential in vivo On a mechanistic level, we found no apparent effect of hypoxia on HSPGs, whereas lipoprotein receptors (VLDLR and SR-B1) were transiently upregulated by hypoxia. Importantly, however, using pharmacologic and genetic approaches, we show that stress-mediated lipoprotein uptake is highly dependent on intact HSPG expression. The functional relevance of HSPG in the context of tumor cell stress was evidenced by HSPG-dependent lipoprotein cell signaling activation through the ERK/MAPK pathway and by reversal of the LD-loaded phenotype by targeting of HSPGs. We conclude that HSPGs may have an important role in the adaptive response to major stress factors of the tumor microenvironment, with functional consequences on tumor cell signaling and metastatic potential. Cancer Res; 76(16); 4828-40. ©2016 AACR.

  View details for DOI 10.1158/0008-5472.CAN-15-2831

  View details for Web of Science ID 000382297700024

  View details for PubMedID 27199348

• The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria CELL Long, J. Z., Svensson, K. J., Bateman, L. A., Lin, H., Kamenecka, T., Lokurkar, I. A., Lou, J., Rao, R. R., Chang, M. R., Jedrychowski, M. P., Paulo, J. A., Gygi, S. P., Griffin, P. R., Nomura, D. K., Spiegelman, B. M. 2016; 166 (2): 424-435

  Abstract

  Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders.

  View details for DOI 10.1016/j.cell.2016.05.071

  View details for Web of Science ID 000380255400019

  View details for PubMedID 27374330

  View details for PubMedCentralID PMC4947008

• A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function CELL METABOLISM Svensson, K. J., Long, J. Z., Jedrychowski, M. P., Cohen, P., Lo, J. C., Serag, S., Kir, S., Shinoda, K., Tartaglia, J. A., Rao, R. R., Chedotal, A., Kajimura, S., Gygi, S. P., Spiegelman, B. M. 2016; 23 (3): 454-466

  Abstract

  Activation of brown and beige fat can reduce obesity and improve glucose homeostasis through nonshivering thermogenesis. Whether brown or beige fat also secretes paracrine or endocrine factors to promote and amplify adaptive thermogenesis is not fully explored. Here we identify Slit2, a 180 kDa member of the Slit extracellular protein family, as a PRDM16-regulated secreted factor from beige fat cells. In isolated cells and in mice, full-length Slit2 is cleaved to generate several smaller fragments, and we identify an active thermogenic moiety as the C-terminal fragment. This Slit2-C fragment of 50 kDa promotes adipose thermogenesis, augments energy expenditure, and improves glucose homeostasis in vivo. Mechanistically, Slit2 induces a robust activation of PKA signaling, which is required for its prothermogenic activity. Our findings establish a previously unknown peripheral role for Slit2 as a beige fat secreted factor that has therapeutic potential for the treatment of obesity and related metabolic disorders.

  View details for DOI 10.1016/j.cmet.2016.01.008

  View details for Web of Science ID 000373614100013

  View details for PubMedID 26876562

  View details for PubMedCentralID PMC4785066

• Exosome and microvesicle mediated phene transfer in mammalian cells SEMINARS IN CANCER BIOLOGY Christianson, H. C., Svensson, K. J., Belting, M. 2014; 28: 31-38

  Abstract

  Extracellular vesicles (EVs), e.g. exosomes and microvesicles, emerge as new signaling organelles in the exchange of information between cells at the paracrine and systemic level. It is clear that these virus like particles carry complex biological information that can elicit a pleiotropic response in recipient cells with potential relevance in physiology as well as in cancer and other pathological conditions. Numerous studies convincingly show that the molecular composition of EVs closely reflects their cell or tissue of origin. Thus, the signaling status of donor cells, more specifically their endosomal compartments, may largely determine the biological output in recipient cells, a process that we then may conceptualize as vesicle mediated phene transfer. Whereas more conventional modes of cell-cell communication mostly depend on extracellular ligand concentration and cell-surface receptor availability, the magnitude of the EV signaling response relies on the capture and uptake by target cells, allowing release of the EV content. Numerous reports point at the intriguing possibility that, among thousands of mRNAs, miRNAs, and proteins, single EV constituents effectuate the biological response, e.g. stimulation of angiogenesis and cancer cell metastasis, in recipient cells; however, we find it conceivable that strategies targeted at general mechanisms of EV function should provide more rational avenues for therapeutic intervention directed at the EV system. Such strategies include manipulation of EV formation in the endolysosomal system, EV stability in the extracellular milieu, and EV entry into target cells. Here, we provide important insights into potential mechanisms of EV transport in mammalian cells and how these may be targeted.

  View details for DOI 10.1016/j.semcancer.2014.04.007

  View details for Web of Science ID 000343019900005

  View details for PubMedID 24769057

• Meteorin-like Is a Hormone that Regulates Immune-Adipose Interactions to Increase Beige Fat Thermogenesis CELL Rao, R. R., Long, J. Z., White, J. P., Svensson, K. J., Lou, J., Lokurkar, I., Jedrychowski, M. P., Ruas, J. L., Wrann, C. D., Lo, J. C., Camera, D. M., Lachey, J., Gygi, S., Seehra, J., Hawley, J. A., Spiegelman, B. M. 2014; 157 (6): 1279-1291

  Abstract

  Exercise training benefits many organ systems and offers protection against metabolic disorders such as obesity and diabetes. Using the recently identified isoform of PGC1-α (PGC1-α4) as a discovery tool, we report the identification of meteorin-like (Metrnl), a circulating factor that is induced in muscle after exercise and in adipose tissue upon cold exposure. Increasing circulating levels of Metrnl stimulates energy expenditure and improves glucose tolerance and the expression of genes associated with beige fat thermogenesis and anti-inflammatory cytokines. Metrnl stimulates an eosinophil-dependent increase in IL-4 expression and promotes alternative activation of adipose tissue macrophages, which are required for the increased expression of the thermogenic and anti-inflammatory gene programs in fat. Importantly, blocking Metrnl actions in vivo significantly attenuates chronic cold-exposure-induced alternative macrophage activation and thermogenic gene responses. Thus, Metrnl links host-adaptive responses to the regulation of energy homeostasis and tissue inflammation and has therapeutic potential for metabolic and inflammatory diseases.

  View details for DOI 10.1016/j.cell.2014.03.065

  View details for Web of Science ID 000340881400007

  View details for PubMedID 24906147

  View details for PubMedCentralID PMC4131287

• A Smooth Muscle-Like Origin for Beige Adipocytes CELL METABOLISM Long, J. Z., Svensson, K. J., Tsai, L., Zeng, X., Roh, H. C., Kong, X., Rao, R. R., Lou, J., Lokurkar, I., Baur, W., Castellot, J. J., Rosen, E. D., Spiegelman, B. M. 2014; 19 (5): 810-820

  Abstract

  Thermogenic UCP1-positive cells, which include brown and beige adipocytes, transform chemical energy into heat and increase whole-body energy expenditure. Using a ribosomal profiling approach, we present a comprehensive molecular description of brown and beige gene expression from multiple fat depots in vivo. This UCP1-TRAP data set demonstrates striking similarities and important differences between these cell types, including a smooth muscle-like signature expressed by beige, but not classical brown, adipocytes. In vivo fate mapping using either a constitutive or an inducible Myh11-driven Cre demonstrates that at least a subset of beige cells arise from a smooth muscle-like origin. Finally, ectopic expression of PRDM16 converts bona fide vascular smooth muscle cells into Ucp1-positive adipocytes in vitro. These results establish a portrait of brown and beige adipocyte gene expression in vivo and identify a smooth muscle-like origin for beige cells.

  View details for DOI 10.1016/j.cmet.2014.03.025

  View details for Web of Science ID 000335561200010

  View details for PubMedID 24709624

  View details for PubMedCentralID PMC4052772

• Ablation of PRDM16 and Beige Adipose Causes Metabolic Dysfunction and a Subcutaneous to Visceral Fat Switch CELL Cohen, P., Levy, J. D., Zhang, Y., Frontini, A., Kolodin, D. P., Svensson, K. J., Lo, J. C., Zeng, X., Ye, L., Khandekar, M. J., Wu, J., Gunawardana, S. C., Banks, A. S., Camporez, J. P., Jurczak, M. J., Kajimura, S., Piston, D. W., Mathis, D., Cinti, S., Shulman, G. I., Seale, P., Spiegelman, B. M. 2014; 156 (1-2): 304-316

  Abstract

  A clear relationship exists between visceral obesity and type 2 diabetes, whereas subcutaneous obesity is comparatively benign. Here, we show that adipocyte-specific deletion of the coregulatory protein PRDM16 caused minimal effects on classical brown fat but markedly inhibited beige adipocyte function in subcutaneous fat following cold exposure or β3-agonist treatment. These animals developed obesity on a high-fat diet, with severe insulin resistance and hepatic steatosis. They also showed altered fat distribution with markedly increased subcutaneous adiposity. Subcutaneous adipose tissue in mutant mice acquired many key properties of visceral fat, including decreased thermogenic and increased inflammatory gene expression and increased macrophage accumulation. Transplantation of subcutaneous fat into mice with diet-induced obesity showed a loss of metabolic benefit when tissues were derived from PRDM16 mutant animals. These findings indicate that PRDM16 and beige adipocytes are required for the "browning" of white fat and the healthful effects of subcutaneous adipose tissue.

  View details for DOI 10.1016/j.cell.2013.12.021

  View details for Web of Science ID 000329912200029

  View details for PubMedID 24439384

  View details for PubMedCentralID PMC3922400

• Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Christianson, H. C., Svensson, K. J., van Kuppevelt, T. H., Li, J., Belting, M. 2013; 110 (43): 17380-17385

  Abstract

  Extracellular vesicle (EV)-mediated intercellular transfer of signaling proteins and nucleic acids has recently been implicated in the development of cancer and other pathological conditions; however, the mechanism of EV uptake and how this may be targeted remain as important questions. Here, we provide evidence that heparan sulfate (HS) proteoglycans (PGs; HSPGs) function as internalizing receptors of cancer cell-derived EVs with exosome-like characteristics. Internalized exosomes colocalized with cell-surface HSPGs of the syndecan and glypican type, and exosome uptake was specifically inhibited by free HS chains, whereas closely related chondroitin sulfate had no effect. By using several cell mutants, we provide genetic evidence of a receptor function of HSPG in exosome uptake, which was dependent on intact HS, specifically on the 2-O and N-sulfation groups. Further, enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous PG biosynthesis by xyloside significantly attenuated exosome uptake. We provide biochemical evidence that HSPGs are sorted to and associate with exosomes; however, exosome-associated HSPGs appear to have no direct role in exosome internalization. On a functional level, exosome-induced ERK1/2 signaling activation was attenuated in PG-deficient mutant cells as well as in WT cells treated with xyloside. Importantly, exosome-mediated stimulation of cancer cell migration was significantly reduced in PG-deficient mutant cells, or by treatment of WT cells with heparin or xyloside. We conclude that cancer cell-derived exosomes use HSPGs for their internalization and functional activity, which significantly extends the emerging role of HSPGs as key receptors of macromolecular cargo.

  View details for DOI 10.1073/pnas.1304266110

  View details for Web of Science ID 000325943300052

  View details for PubMedID 24101524

  View details for PubMedCentralID PMC3808637

• Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kucharzewska, P., Christianson, H. C., Welch, J. E., Svensson, K. J., Fredlund, E., Ringner, M., Morgelin, M., Bourseau-Guilmain, E., Bengzon, J., Belting, M. 2013; 110 (18): 7312-7317

  Abstract

  Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.

  View details for DOI 10.1073/pnas.1220998110

  View details for Web of Science ID 000318682300049

  View details for PubMedID 23589885

  View details for PubMedCentralID PMC3645587

• Role of extracellular membrane vesicles in intercellular communication of the tumour microenvironment BIOCHEMICAL SOCIETY TRANSACTIONS Svensson, K. J., Belting, M. 2013; 41: 273-276

  Abstract

  Over the last few decades, extensive studies by several groups have introduced the concept of cell-derived secreted extracellular membrane vesicles as carriers of complex molecular information. Owing to their pleiotropic biological effects and involvement in a wide variety of biological processes, extracellular membrane vesicles have been implicated in physiological as well as pathological events, including tumour development and metastasis. In the present review, we discuss the role of secreted membrane vesicles in intercellular communication with a focus on tumour biology. Of particular interest is the potential role of extracellular vesicles as orchestrators of common features of the malignant tumour microenvironment, e.g. coagulation activation and angiogenesis.

  View details for DOI 10.1042/BST20120248

  View details for Web of Science ID 000314222900045

  View details for PubMedID 23356296

• Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1. J Biol Chem. Svensson, K. J., Christianson, H. C., Wittrup, A., Bourseau-Guilmain, E., Lindqvist, E., Svensson, L. M., Morgelin, M., Belting, M., et al 2013; 2013 Jun 14;288(24):17713-24.
• Standardization and Utilization of Biobank Resources in Clinical Protein Science with Examples of Emerging Applications JOURNAL OF PROTEOME RESEARCH Marko-Varga, G., Vegvari, A., Welinder, C., Lindberg, H., Rezeli, M., Edula, G., Svensson, K. J., Belting, M., Laurell, T., Fehniger, T. E. 2012; 11 (11): 5124-5134

  Abstract

  Biobanks are a major resource to access and measure biological constituents that can be used to monitor the status of health and disease, both in unique individual samples and within populations. Most "omic" activities rely on access to these collections of stored samples to provide the basis for establishing the ranges and frequencies of expression. Furthermore, information about the relative abundance and form of protein constituents found in stored samples provides an important historical index for comparative studies of inherited, epidemic, and developing disease. Standardizations of sample quality, form, and analysis are an important unmet need and requirement for gaining the full benefit from collected samples. Coupled to this standard is the provision of annotation describing clinical status and metadata of measurements of clinical phenotype that characterizes the sample. Today we have not yet achieved consensus on how to collect, manage, and build biobank archives in order to reach goals where these efforts are translated into value for the patient. Several initiatives (OBBR, ISBER, BBMRI) that disseminate best practice examples for biobanking are expected to play an important role in ensuring the need to preserve the sample integrity of biosamples stored for periods that reach one or several decades. These developments will be of great value and importance to programs such as the Chromosome Human Protein Project (C-HPP) that will associate protein expression in healthy and disease states with genetic foci along of each of the human chromosomes.

  View details for DOI 10.1021/pr300185k

  View details for Web of Science ID 000311190600002

  View details for PubMedID 22607352

• Dermatan Sulfate Is Involved in the Tumorigenic Properties of Esophagus Squamous Cell Carcinoma CANCER RESEARCH Thelin, M. A., Svensson, K. J., Shi, X., Bagher, M., Axelsson, J., Isinger-Ekstrand, A., van Kuppevelt, T. H., Johansson, J., Nilbert, M., Zaia, J., Belting, M., Maccarana, M., Malmstrom, A. 2012; 72 (8): 1943-1952

  Abstract

  Extracellular matrix, either produced by cancer cells or by cancer-associated fibroblasts, influences angiogenesis, invasion, and metastasis. Chondroitin/dermatan sulfate (CS/DS) proteoglycans, which occur both in the matrix and at the cell surface, play important roles in these processes. The unique feature that distinguishes DS from CS is the presence of iduronic acid (IdoA) in DS. Here, we report that CS/DS is increased five-fold in human biopsies of esophagus squamous cell carcinoma (ESCC), an aggressive tumor with poor prognosis, as compared with normal tissue. The main IdoA-producing enzyme, DS epimerase 1 (DS-epi1), together with the 6-O- and 4-O-sulfotransferases, were highly upregulated in ESCC biopsies. Importantly, CS/DS structure in patient tumors was significantly altered compared with normal tissue, as determined by sensitive mass spectrometry. To further understand the roles of IdoA in tumor development, DS-epi1 expression, and consequently IdoA content, was downregulated in ESCC cells. IdoA-deficient cells exhibited decreased migration and invasion capabilities in vitro, which was associated with reduced cellular binding of hepatocyte growth factor, inhibition of pERK-1/2 signaling, and deregulated actin cytoskeleton dynamics and focal adhesion formation. Our findings show that IdoA in DS influences tumorigenesis by affecting cancer cell behavior. Therefore, downregulation of IdoA by DS-epi1 inhibitors may represent a new anticancer therapy.

  View details for DOI 10.1158/0008-5472.CAN-11-1351

  View details for Web of Science ID 000302905700006

  View details for PubMedID 22350411

  View details for PubMedCentralID PMC3328612

• Chondroitin sulfate expression predicts poor outcome in breast cancer INTERNATIONAL JOURNAL OF ONCOLOGY Svensson, K. J., Christianson, H. C., Kucharzewska, P., Fagerstrom, V., Lundstedt, L., Borgquist, S., Jirstrom, K., Belting, M. 2011; 39 (6): 1421-1428

  Abstract

  Experimental studies have established that the sulfated glycosaminoglycans heparan sulfate and chondroitin sulfate act as co-receptors of cytokines and growth factors that drive the malignant cell phenotype and the remodelling of the surrounding tumor stroma. However, the clinical relevance of these studies remains ill-defined. The present study investigates the significance of chondroitin sulfate expression in malignant cells and the stroma, respectively, of tumors from two independent cohorts of breast cancer patients (cohort I: 144 patients, 130 evaluable samples; cohort II: 498 patients, 469 evaluable samples; ER-positive patients ~86% in both cohorts). Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the relationship between chondroitin sulfate and recurrence-free and overall survival. High chondroitin sulfate expression in malignant cells was shown to predict shorter recurrence-free survival (P=0.007, cohort I; P=0.024, cohort II) and overall survival (cohort I: P=0.044; cohort II: P<0.001) in both cohorts. In multivariate analysis, high chondroitin sulfate in malignant cells was shown to be an independent, predictive factor of poor overall survival (cohort I: hazard ratio 2.28: 95% confidence interval 1.08-4.81, P=0.031; cohort II: hazard ratio 1.71: 95% confidence interval 1.23-2.38, P=0.001). However, chondroitin sulfate in the stroma showed no correlation with known markers of tumor aggressiveness or with clinical outcome in either cohort. Our data suggest that high chondroitin sulfate expression in malignant cells is associated with an adverse outcome in patients with primary breast cancer, supporting the idea of a functional and potentially targetable role of chondroitin sulfate in tumor disease.

  View details for DOI 10.3892/ijo.2011.1164

  View details for Web of Science ID 000296315400010

  View details for PubMedID 21850370

• Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Svensson, K. J., Kucharzewska, P., Christianson, H. C., Skold, S., Lofstedt, T., Johansson, M. C., Morgelin, M., Bengzon, J., Ruf, W., Belting, M. 2011; 108 (32): 13147-13152

  Abstract

  Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

  View details for DOI 10.1073/pnas.1104261108

  View details for Web of Science ID 000293691400041

  View details for PubMedID 21788507

  View details for PubMedCentralID PMC3156184

• Heparan sulfate proteoglycan-mediated polyamine uptake. Methods in molecular biology (Clifton, N.J.) Welch, J., Svensson, K., Kucharzewska, P., Belting, M. 2011; 720: 327-338

  Abstract

  The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display-derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.

  View details for DOI 10.1007/978-1-61779-034-8_20

  View details for PubMedID 21318883

• Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells EXPERIMENTAL CELL RESEARCH Kucharzewska, P., Welch, J. E., Svensson, K. J., Belting, M. 2010; 316 (16): 2683-2691

  Abstract

  The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by alpha-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

  View details for DOI 10.1016/j.yexcr.2010.05.033

  View details for Web of Science ID 000281305800015

  View details for PubMedID 20594968

• Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wittrup, A., Zhang, S., Svensson, K. J., Kucharzewska, P., Johansson, M. C., Morgelin, M., Belting, M. 2010; 107 (30): 13342-13347

  Abstract

  An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intracellular vesicles of a nonclathrin-, noncaveolin-dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.

  View details for DOI 10.1073/pnas.1002622107

  View details for Web of Science ID 000280602800030

  View details for PubMedID 20624969

  View details for PubMedCentralID PMC2922147

• The polyamines regulate endothelial cell survival during hypoxic stress through PI3K/AKT and MCL-1 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Kucharzewska, P., Welch, J. E., Svensson, K. J., Belting, M. 2009; 380 (2): 413-418

  Abstract

  Hypoxia-dependent angiogenesis is an inherent feature of solid tumors, and a better understanding of the molecular mechanisms of hypoxic cell-death should provide additional targets for cancer therapy. Here, we show a novel role of the polyamines in endothelial cell (EC) survival during hypoxia. Polyamine depletion by specific inhibition of ornithine decarboxylase was shown to protect ECs from hypoxia-induced apoptosis. Inhibition of the polyamines resulted in a significant induction of PI3K/AKT and its down-stream target MCL-1, i.e. an anti-apoptotic member of the BCL-2 family. Specific inhibitors of PI3K reversed the decrease of hypoxia-induced apoptosis as well as the induction of MCL-1 in polyamine-deprived cells. Moreover, siRNA-mediated down-regulation of MCL-1 was found to counter-act the protective effect of polyamine inhibition. We conclude that the polyamines regulate hypoxia-induced apoptosis in ECs through PI3K/AKT and MCL-1 dependent pathways. Our results may have important implications for the modulation of hypoxia-driven neovascularization.

  View details for DOI 10.1016/j.bbrc.2009.01.097

  View details for Web of Science ID 000263742200039

  View details for PubMedID 19250631

• Hypoxia-Mediated Induction of the Polyamine System Provides Opportunities for Tumor Growth Inhibition by Combined Targeting of Vascular Endothelial Growth Factor and Ornithine Decarboxylase CANCER RESEARCH Svensson, K. J., Welch, J. E., Kucharzewska, P., Bengtson, P., Bjurberg, M., Pahlman, S., ten Dam, G. B., Persson, L., Belting, M. 2008; 68 (22): 9291-9301

  Abstract

  Hypoxia is a hallmark of solid tumors, which may offer opportunities for targeted therapies of cancer; however, the mechanisms that link hypoxia to malignant transformation and tumor progression are not fully understood. Here, we show that up-regulation of the polyamine system promotes cancer cell survival during hypoxic stress. Hypoxia was found to induce polyamine transport and the key enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC), in a variety of cancer cell lines. Increased ODC protein expression was shown in hypoxic, GLUT-1-expressing regions of tumor spheroids and experimental tumors, as well as in clinical tumor specimens. Hypoxic induction of the polyamine system was dependent on antizyme inhibitor (i.e., a key positive regulator of ODC and polyamine transport), as shown by RNA interference experiments. Interestingly, depletion of the polyamines during hypoxia resulted in increased apoptosis, which indicates an essential role of the polyamines in cancer cell adaptation to hypoxic stress. These results were supported by experiments in an in vivo glioma tumor model, showing significantly enhanced antitumor effects of the antiangiogenic, humanized anti-vascular endothelial growth factor (VEGF) antibody bevacizumab when used in combination with the well-established, irreversible inhibitor of ODC, alpha-difluoromethylornithine. Our results provide important insights into the hypoxic stress response in malignant cells and implicate combined targeting of VEGF and ODC as an alternative strategy to treat cancer disease.

  View details for DOI 10.1158/0008-5472.CAN-08-2340

  View details for Web of Science ID 000261136600025

  View details for PubMedID 19010902

• Single chain fragment anti-heparan sulfate antibody targets the polyamine transport system and attenuates polyamine-dependent cell proliferation INTERNATIONAL JOURNAL OF ONCOLOGY Welch, J. E., Bengtson, P., Svensson, K., Wittrup, A., Jenniskens, G. J., ten Dam, G. B., van Kuppevelt, T. H., Belting, M. 2008; 32 (4): 749-756

  Abstract

  The growth-promoting polyamines are polybasic compounds that efficiently enter cancer cells by as yet incompletely defined mechanisms. Strategies to inhibit their internalization may have important implications in the management of tumor disease. Here, we show that cellular binding and uptake of polyamines are inhibited by a single chain variable fragment anti-heparan sulfate (HS) antibody. Polyamine uptake was inhibited in a dose-dependent manner, and was associated with compensatory up-regulation of ornithine decarboxylase (ODC), i.e. the key enzyme of the polyamine biosynthesis pathway. Conversely, depletion of intracellular polyamines by the specific ODC-inhibitor alpha-difluoromethylornithine (DFMO) resulted in increased cellular binding of polyamine and anti-HS antibody. Importantly, anti-HS antibody also efficiently targeted DFMO-induced polyamine uptake, and combined polyamine biosynthesis inhibition by DFMO, and uptake inhibition by anti-HS antibody attenuated tumor cell proliferation in vitro. In conclusion, cell-surface HS proteoglycan is a relevant target for antibody-mediated inhibition of the uptake of polyamines, and polyamine-dependent cell proliferation.

  View details for Web of Science ID 000254357600003

  View details for PubMedID 18360702

• HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells MOLECULAR CANCER THERAPEUTICS Mani, K., Sandgren, S., Lilja, J., Cheng, F., Svensson, K., Persson, L., Belting, M. 2007; 6 (2): 782-788

  Abstract

  Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake-deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor alpha-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with alpha-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer.

  View details for DOI 10.1158/1535-7163.MCT-06-0370

  View details for Web of Science ID 000244262700039

  View details for PubMedID 17308074

• Synthesis and transfection efficiencies of new lipophilic polyamines JOURNAL OF MEDICINAL CHEMISTRY Gardner, R. A., Belting, M., Svensson, K., Phanstiel, O. 2007; 50 (2): 308-318

  Abstract

  A homologous series of lipophilic polyamines was synthesized and evaluated for DNA delivery and transfection efficiency. The series contained 1,4-butanediamine, 1,8-octanediamine, 2-[2-(2-amino-ethoxy)-ethoxy]-ethylamine, homospermidine, and homospermine covalently attached via their N1 terminus to a 3,4-bis(oleyloxy)-benzyl motif. In addition, homospermidine and homospermine were also attached via amide linkers. The homospermidine derivatives (i.e., benzyl tether 25 and benzamide tether 27) showed a 3-fold and 4-fold respective enhancement in delivery of AlexaFluor-488-labeled DNA over the butanediamine analogue 22. Homospermine derivative 26 was shown to inhibit 14C-spermine uptake (IC50 approximately 10 microM), which implied that 26 is able to compete effectively for polyamine recognition sites on the cell surface. This study demonstrated that the number and position of the positive charges along the polyamine scaffold plays a key role in DNA delivery and in determining the transfection efficiency.

  View details for DOI 10.1021/jm0607101

  View details for Web of Science ID 000243535600015

  View details for PubMedID 17228872