Enrichment of sweat-derived extracellular vesicles of human and bacterial origin for biomarker identification.

Sweat contains biomarkers for real-time non-invasive health monitoring, but only a few relevant analytes are currently used in clinical practice. In the present study, we investigated whether sweat-derived extracellular vesicles (EVs) can be used as a source of potential protein biomarkers of human and bacterial origin. Methods: By using ExoView platform, electron microscopy, nanoparticle tracking analysis and Western blotting we characterized EVs in the sweat of eight volunteers performing rigorous exercise. We compared the presence of EV markers as well as general protein composition of total sweat, EV-enriched sweat and sweat samples collected in alginate skin patches. Results: We identified 1209 unique human proteins in EV-enriched sweat, of which approximately 20% were present in every individual sample investigated. Sweat derived EVs shared 846 human proteins (70%) with total sweat, while 368 proteins (30%) were captured by medical grade alginate skin patch and such EVs contained the typical exosome marker CD63. The majority of identified proteins are known to be carried by EVs found in other biofluids, mostly urine. Besides human proteins, EV-enriched sweat samples contained 1594 proteins of bacterial origin. Bacterial protein profiles in EV-enriched sweat were characterized by high interindividual variability, that reflected differences in total sweat composition. Alginate-based sweat patch accumulated only 5% proteins of bacterial origin. Conclusion: We showed that sweat-derived EVs provide a rich source of potential biomarkers of human and bacterial origin. Use of commercially available alginate skin patches selectively enrich for human derived material with very little microbial material collected.

Generation of novel in vitro flexible kidney organoid model to investigate the role of extracellular vesicles in induction of nephrogenesis.

BACKGROUND: During kidney organogenesis, metanephric mesenchyme (MM) and ureteric bud (UB) interact reciprocally to form nephrons. Signaling stimuli involved in these interactions include Wnts, growth factors and nano/micro particles. How UB and MM are interacting is not completely understood. Our study investigated the signaling and communication via extracellular vesicles (EVs) during nephrogenesis. Embryonic day (E) 11.5 mouse kidney UB and MM produce very low number of primary cells that have limited ability for proliferation in culture. Such limitations obstruct studying the role of EVs in induction of nephrogenesis. These issues necessitate to generate a nephrogenesis model allowing to study the comprehensive role of EVs during nephrogenesis. RESULTS: Our study generated a UB derived cell line-based in vitro flexible model of nephrogenesis allowing expandable cell culturing, in addition to performing characterization, tracking and blocking of EVs. UB cell line aggregation with E11.5 MM cells induced the formation of segmented nephrons. Most efficient nephrogenesis was obtained by the co-culturing of 30,000 cells of UB cell line with 50,000 MM cells. Results revealed that both the UB and the MM secrete EVs during nephrogenesis. UB cell line derived EVs were characterized by their size, morphology and expression of markers (CD63, TSG101, CD9 and CD81). Furthermore, proteomics data of UB cell line-derived EVs revealed large number of proteins involved in nephrogenesis-related signaling pathways. Palmitoylated GFP-tagged EVs from UB cell line were found in the nephron formation zone in the developing kidney organoid. UB cell line derived EVs did not induce nephrogenesis in MM cells but significantly contributed to the survival and nephrogenesis-competency of MM cells. The secretion of EVs was continuously inhibited during the ongoing nephrogenesis by the knockdown of RalA and RalB gene expression using short hairpin RNAs. This inhibition partially impaired the ability of UB cell line to induce nephrogenesis. Moreover, impaired nephrogenesis was partially rescued by the addition of EVs. CONCLUSION: Our study established a novel in vitro flexible model of nephrogenesis that solved the limitations of primary embryonic kidney cells and mouse embryonic stem cell kidney organoids for the EV research. EVs were found to be an integral part of nephrogenesis process. Video Abstract.

Maternal microbiota communicates with the fetus through microbiota-derived extracellular vesicles.

BACKGROUND: Reports regarding the presence of bacteria in the fetal environment remain limited and controversial. Recently, extracellular vesicles secreted by the human gut microbiota have emerged as a novel mechanism for host-microbiota interaction. We aimed to investigate the presence of bacterial extracellular vesicles in the fetal environment during healthy pregnancies and determine whether extracellular vesicles derived from the gut microbiota can cross biological barriers to reach the fetus. RESULTS: Bacterial extracellular vesicles were detectable in the amniotic fluid of healthy pregnant women, exhibiting similarities to extracellular vesicles found in the maternal gut microbiota. In pregnant mice, extracellular vesicles derived from human maternal gut microbiota were found to reach the intra-amniotic space. CONCLUSIONS: Our findings reveal maternal microbiota-derived extracellular vesicles as an interaction mechanism between the maternal microbiota and fetus, potentially playing a pivotal role in priming the prenatal immune system for gut colonization after birth. Video Abstract.

Purification of Bacterial-Enriched Extracellular Vesicle Samples from Feces by Density Gradient Ultracentrifugation.

Commensal microbiota has huge impact on the maintenance of human health, its dysregulation being associated with the development of a plethora of diseases. Release of bacterial extracellular vesicles (BEVs) is a fundamental mechanism of systemic microbiome influence on the host organism. Nevertheless, due to the technical challenges of isolation methods, BEV composition and functions remain poorly characterized. Hereby, we describe the up-to-date protocol for isolation of BEV-enriched samples from human feces. Fecal extracellular vesicles (EVs) are purified through the orthogonal implementation of filtration, size-exclusion chromatography (SEC), and density gradient ultracentrifugation. EVs are first separated from bacteria, flagella, and cell debris by size. In the next steps, BEVs are separated from host-derived EVs by density. The quality of vesicle preparation is estimated via immuno-TEM (transmission electron microscopy) for the presence of vesicle-like structures expressing EV markers and via NTA (nanoparticle tracking analysis) for assaying particle concentration and size. Distribution of EVs of human origin in gradient fractions is estimated using antibodies against human exosomal markers with Western blot and ExoView R100 imaging platform. The enrichment for BEVs in vesicle preparation is estimated by Western blot for the presence of bacterial OMVs (outer membrane vesicles) marker and OmpA (outer membrane protein A). Taken together, our study describes a detailed protocol for EV preparation with enrichment for BEVs from feces with a purity level suitable for bioactivity functional assays.

Biogenesis of Mesoporous Silica Nanoparticles Enclosed in Extracellular Vesicles by Mouse Renal Adenocarcinoma Cells.

Integrating the versatility of synthetic nanoparticles to natural biomaterials, such as cells or cell membranes, has gained considerable attention as promising alternative cargo delivery platforms in recent years. Extracellular vesicles (EVs), natural nanomaterials composed of a protein-rich lipid bilayer secreted by cells, have also shown advantages and great potential as a nano delivery platform in combination with synthetic particles due to their specific natural properties in overcoming several biology hurdles possessed in the recipient cell. Therefore, the preservation of EV's origin properties is critical for their application as nanocarriers. This chapter will describe the encapsulation procedure of MSN encapsulated in EV membrane derived from mouse renal adenocarcinoma (Renca) cells through biogenesis. The FMSN-enclosed EVs produced through this approach still contain preserved EV's natural membrane properties.

Human Adult Astrocyte Extracellular Vesicle Transcriptomics Study Identifies Specific RNAs Which Are Preferentially Secreted as EV Luminal Cargo.

Astrocytes are central nervous system (CNS)-restricted glial cells involved in synaptic function and CNS blood flow regulation. Astrocyte extracellular vesicles (EVs) participate in neuronal regulation. EVs carry RNAs, either surface-bound or luminal, which can be transferred to recipient cells. We characterized the secreted EVs and RNA cargo of human astrocytes derived from an adult brain. EVs were isolated by serial centrifugation and characterized with nanoparticle tracking analysis (NTA), Exoview, and immuno-transmission electron microscopy (TEM). RNA from cells, EVs, and proteinase K/RNase-treated EVs was analyzed by miRNA-seq. Human adult astrocyte EVs ranged in sizes from 50 to 200 nm, with CD81 as the main tetraspanin marker and larger EVs positive for integrin ß1. Comparison of the RNA between the cells and EVs identified RNA preferentially secreted in the EVs. In the case of miRNAs, enrichment analysis of their mRNA targets indicates that they are good candidates for mediating EV effects on recipient cells. The most abundant cellular miRNAs were also abundant in EVs, and the majority of their mRNA targets were found to be downregulated in mRNA-seq data, but the enrichment analysis lacked neuronal specificity. Proteinase K/RNase treatment of EV-enriched preparations identified RNAs secreted independently of EVs. Comparing the distribution of cellular and secreted RNA identifies the RNAs involved in intercellular communication via EVs.

The modulatory role of internet-supported mindfulness-based cognitive therapy on extracellular vesicles and psychological distress in people who have had cancer: a protocol for a two-armed randomized controlled study.

BACKGROUND: Mindfulness-based interventions (MBIs) have been used in oncology contexts as a promising tool with numerous benefits for various health-related and psychosocial outcomes. Despite the increasing popularity of MBIs, few randomized controlled trials (RCTs) have examined their effects upon biological parameters. Specifically, no previous study has examined the effects of MBIs on extracellular vesicles (EVs), which are potentially important markers of health, disease, and stress. Moreover, the lack of RCTs is even more limited within the context of technology-mediated MBIs and long-term effects. METHODS: The current study protocol presents a two-arm, parallel, randomized controlled study investigating the effects of internet-supported mindfulness-based cognitive therapy (MBCT) compared with treatment as usual (TAU). Primary outcomes are psychological distress and EV cargo of distressed participants with previous breast, colorectal, or prostate cancer diagnoses. Secondary outcomes are self-reported psychosocial and health-related measures, and additional biological markers. Outcomes will be assessed at baseline, 4 weeks after baseline (mid-point of the intervention), 8 weeks after baseline (immediately post-intervention), 24 weeks after baseline (after booster sessions), and 52 weeks after baseline. Our goal is to recruit at least 111 participants who have been diagnosed with breast, prostate, or colorectal cancer (cancer stage I to III), are between 18 and 65 years old, and have had primary cancer treatments completed between 3 months and 5 years ago. Half of the participants will be randomized to the TAU group, and the other half will participate in an 8-week online MBCT intervention with weekly group sessions via videoconference. The intervention also includes asynchronous homework, an online retreat after the fifth week, and 4 monthly booster sessions after completion of the 8-week programme. DISCUSSION: This study will allow characterizing the effects of internet-based MBCT on psychosocial and biological indicators in the context of cancer. The effects on circulating EVs will also be investigated, as a possible neurobiological pathway underlying mind-body intervention effects. TRIAL REGISTRATION: ClinicalTrials.gov NCT04727593 (date of registration: 27 January 2021; date of record verification: 6 October 2021).

Time-gated Raman spectroscopy and proteomics analyses of hypoxic and normoxic renal carcinoma extracellular vesicles.

Extracellular vesicles (EVs) represent a diverse group of small membrane-encapsulated particles involved in cell-cell communication, but the technologies to characterize EVs are still limited. Hypoxia is a typical condition in solid tumors, and cancer-derived EVs support tumor growth and invasion of tissues by tumor cells. We found that exposure of renal adenocarcinoma cells to hypoxia induced EV secretion and led to notable changes in the EV protein cargo in comparison to normoxia. Proteomics analysis showed overrepresentation of proteins involved in adhesion, such as integrins, in hypoxic EV samples. We further assessed the efficacy of time-gated Raman spectroscopy (TG-RS) and surface-enhanced time-gated Raman spectroscopy (TG-SERS) to characterize EVs. While the conventional continuous wave excitation Raman spectroscopy did not provide a notable signal, prominent signals were obtained with the TG-RS that were further enhanced in the TG-SERS. The Raman signal showed characteristic changes in the amide regions due to alteration in the chemical bonds of the EV proteins. The results illustrate that the TG-RS and the TG-SERS are promising label free technologies to study cellular impact of external stimuli, such as oxygen deficiency, on EV production, as well as differences arising from distinct EV purification protocols.

Characterization of nucleic acids from extracellular vesicle-enriched human sweat.

BACKGROUND: The human sweat is a mixture of secretions from three types of glands: eccrine, apocrine, and sebaceous. Eccrine glands open directly on the skin surface and produce high amounts of water-based fluid in response to heat, emotion, and physical activity, whereas the other glands produce oily fluids and waxy sebum. While most body fluids have been shown to contain nucleic acids, both as ribonucleoprotein complexes and associated with extracellular vesicles (EVs), these have not been investigated in sweat. In this study we aimed to explore and characterize the nucleic acids associated with sweat particles. RESULTS: We used next generation sequencing (NGS) to characterize DNA and RNA in pooled and individual samples of EV-enriched sweat collected from volunteers performing rigorous exercise. In all sequenced samples, we identified DNA originating from all human chromosomes, but only the mitochondrial chromosome was highly represented with 100% coverage. Most of the DNA mapped to unannotated regions of the human genome with some regions highly represented in all samples. Approximately 5 % of the reads were found to map to other genomes: including bacteria (83%), archaea (3%), and virus (13%), identified bacteria species were consistent with those commonly colonizing the human upper body and arm skin. Small RNA-seq from EV-enriched pooled sweat RNA resulted in 74% of the trimmed reads mapped to the human genome, with 29% corresponding to unannotated regions. Over 70% of the RNA reads mapping to an annotated region were tRNA, while misc. RNA (18,5%), protein coding RNA (5%) and miRNA (1,85%) were much less represented. RNA-seq from individually processed EV-enriched sweat collection generally resulted in fewer percentage of reads mapping to the human genome (7-45%), with 50-60% of those reads mapping to unannotated region of the genome and 30-55% being tRNAs, and lower percentage of reads being rRNA, LincRNA, misc. RNA, and protein coding RNA. CONCLUSIONS: Our data demonstrates that sweat, as all other body fluids, contains a wealth of nucleic acids, including DNA and RNA of human and microbial origin, opening a possibility to investigate sweat as a source for biomarkers for specific health parameters.

Secreted Extracellular Vesicle Molecular Cargo as a Novel Liquid Biopsy Diagnostics of Central Nervous System Diseases.

Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs' role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context.

MicroRNAs in Extracellular Vesicles in Sweat Change in Response to Endurance Exercise.

BACKGROUND: To date, microRNAs (miRs) carried in extracellular vesicles (EVs) in response to exercise have been studied in blood but not in non-invasively collectable body fluids. In the present study, we examined whether six exercise-responsive miRs, miRs-21, -26, -126, -146, -221, and -222, respond to acute endurance exercise stimuli of different intensities in sweat. METHODS: We investigated the response of miRs isolated from sweat and serum EVs to three endurance exercise protocols: (1) maximal aerobic capacity (VO2 max ), (2) anaerobic threshold (AnaT), and (3) aerobic threshold (AerT) tests. Sauna bathing was used as a control test to induce sweating through increased body temperature in the absence of exercise. All protocols were performed by the same subjects (n = 8, three males and five females). The occurrence of different miR carriers in sweat and serum was investigated via EV markers (CD9, CD63, and TSG101), an miR-carrier protein (AGO2), and an HDL-particle marker (APOA1) with Western blot. Correlations between miRs in sweat and serum (post-sample) were examined. RESULTS: Of the studied miR carrier markers, sweat EV fractions expressed CD63 and, very weakly, APOA1, while the serum EV fraction expressed all the studied markers. In sweat EVs, miR-21 level increased after AerT and miR-26 after all the endurance exercise tests compared with the Sauna (p < 0.050). miR-146 after AnaT correlated to sweat and serum EV samples (r = 0.881, p = 0.004). CONCLUSION: Our preliminary study is the first to show that, in addition to serum, sweat EVs carry miRs. Interestingly, we observed that miRs-21 and -26 in sweat EVs respond to endurance exercise of different intensities. Our data further confirmed that miR responses to endurance exercise in sweat and serum were triggered by exercise and not by increased body temperature. Our results highlight that sweat possesses a unique miR carrier content that should be taken into account when planning analyses from sweat as a substitute for serum.

Extracellular ATP activates hyaluronan synthase 2 (HAS2) in epidermal keratinocytes via P2Y2, Ca2+ signaling, and MAPK pathways.

Extracellular nucleotides are used as signaling molecules by several cell types. In epidermis, their release is triggered by insults such as ultraviolet radiation, barrier disruption, and tissue wounding, and by specific nerve terminals firing. Increased synthesis of hyaluronan, a ubiquitous extracellular matrix glycosaminoglycan, also occurs in response to stress, leading to the attractive hypothesis that nucleotide signaling and hyaluronan synthesis could also be linked. In HaCaT keratinocytes, ATP caused a rapid and strong but transient activation of hyaluronan synthase 2 (HAS2) expression via protein kinase C-, Ca2+/calmodulin-dependent protein kinase II-, mitogen-activated protein kinase-, and calcium response element-binding protein-dependent pathways by activating the purinergic P2Y2 receptor. Smaller but more persistent up-regulation of HAS3 and CD44, and delayed up-regulation of HAS1 were also observed. Accumulation of peri- and extracellular hyaluronan followed 4-6 h after stimulation, an effect further enhanced by the hyaluronan precursor glucosamine. AMP and adenosine, the degradation products of ATP, markedly inhibited HAS2 expression and, despite concomitant up-regulation of HAS1 and HAS3, inhibited hyaluronan synthesis. Functionally, ATP moderately increased cell migration, whereas AMP and adenosine had no effect. Our data highlight the strong influence of adenosinergic signaling on hyaluronan metabolism in human keratinocytes. Epidermal insults are associated with extracellular ATP release, as well as rapid up-regulation of HAS2/3, CD44, and hyaluronan synthesis, and we show here that the two phenomena are linked. Furthermore, as ATP is rapidly degraded, the opposite effects of its less phosphorylated derivatives facilitate a rapid shut-off of the hyaluronan response, providing a feedback mechanism to prevent excessive reactions when more persistent signals are absent.

Exosomes as secondary inductive signals involved in kidney organogenesis.

The subfraction of extracellular vesicles, called exosomes, transfers biological molecular information not only between cells but also between tissues and organs as nanolevel signals. Owing to their unique properties such that they contain several RNA species and proteins implicated in kidney development, exosomes are putative candidates to serve as developmental programming units in embryonic induction and tissue interactions. We used the mammalian metanephric kidney and its nephron-forming mesenchyme containing the nephron progenitor/stem cells as a model to investigate if secreted exosomes could serve as a novel type of inductive signal in a process defined as embryonic induction that controls organogenesis. As judged by several characteristic criteria, exosomes were enriched and purified from a cell line derived from embryonic kidney ureteric bud (UB) and from primary embryonic kidney UB cells, respectively. The cargo of the UB-derived exosomes was analysed by qPCR and proteomics. Several miRNA species that play a role in Wnt pathways and enrichment of proteins involved in pathways regulating the organization of the extracellular matrix as well as tissue homeostasis were identified. When labelled with fluorescent dyes, the uptake of the exosomes by metanephric mesenchyme (MM) cells and the transfer of their cargo to the cells can be observed. Closer inspection revealed that besides entering the cytoplasm, the exosomes were competent to also reach the nucleus. Furthermore, fluorescently labelled exosomal RNA enters into the cytoplasm of the MM cells. Exposure of the embryonic kidney-derived exosomes to the whole MM in an ex vivo organ culture setting did not lead to an induction of nephrogenesis but had an impact on the overall organization of the tissue. We conclude that the exosomes provide a novel signalling system with an apparent role in secondary embryonic induction regulating organogenesis.

Reconstructed Serine 288 in the Left Flipper Region of the Rat P2X7 Receptor Stabilizes Nonsensitized States.

Serine 275, a conserved residue of the left flipper region of ATP-gated P2X3 receptors, plays a key role in both agonist binding and receptor desensitization. It is conserved in most of the P2X receptors except P2X7 and P2X6. By combining experimental patch-clamp and modeling approaches, we explored the role of the corresponding residue in the rat P2X7 receptor (rP2X7) by replacing the phenylalanine at position 288 with serine and characterizing the membrane currents generated by either the wild-type (WT) or the mutated rP2X7 receptor. F288S, an rP2X7 mutation, slowed the deactivation subsequent to 2 and 20 s applications of 1 mM ATP. F288S also prevented sensitization (a progressive current growth) observed with the WT in response to a 20 s application of 1 mM ATP. Increasing the ATP concentration to 5 mM promoted sensitization also in the mutated rP2X7 receptor, accelerating the deactivation rate to typical WT values. YO-PRO1 uptake in cells expressing either the WT or the F288S P2X7 receptor was consistent with recorded membrane current data. Interestingly, in the human P2X7 (hP2X7) receptor, substitution Y288S did not change the deactivation rate, while the Y288F mutant generated a "rat-like" phenotype with a fast deactivation rate. Our combined experimental, kinetic, and molecular modeling data suggest that the rat F288S novel phenotype is due to a slower rate of ATP binding and/or unbinding and stabilization of nonsensitized receptor states.

Selective Calcium-Dependent Inhibition of ATP-Gated P2X3 Receptors by Bisphosphonate-Induced Endogenous ATP Analog ApppI.

Pain is the most unbearable symptom accompanying primary bone cancers and bone metastases. Bone resorptive disorders are often associated with hypercalcemia, contributing to the pathologic process. Nitrogen-containing bisphosphonates (NBPs) are efficiently used to treat bone cancers and metastases. Apart from their toxic effect on cancer cells, NBPs also provide analgesia via poorly understood mechanisms. We previously showed that NBPs, by inhibiting the mevalonate pathway, induced formation of novel ATP analogs such as ApppI [1-adenosin-5'-yl ester 3-(3-methylbut-3-enyl) triphosphoric acid diester], which can potentially be involved in NBP analgesia. In this study, we used the patch-clamp technique to explore the action of ApppI on native ATP-gated P2X receptors in rat sensory neurons and rat and human P2X3, P2X2, and P2X7 receptors expressed in human embryonic kidney cells. We found that although ApppI has weak agonist activity, it is a potent inhibitor of P2X3 receptors operating in the nanomolar range. The inhibitory action of ApppI was completely blocked in hypercalcemia-like conditions and was stronger in human than in rat P2X3 receptors. In contrast, P2X2 and P2X7 receptors were insensitive to ApppI, suggesting a high selectivity of ApppI for the P2X3 receptor subtype. NBP, metabolite isopentenyl pyrophosphate, and endogenous AMP did not exert any inhibitory action, indicating that only intact ApppI has inhibitory activity. Ca2+-dependent inhibition was stronger in trigeminal neurons preferentially expressing desensitizing P2X3 subunits than in nodose ganglia neurons, which also express nondesensitizing P2X2 subunits. Altogether, we characterized previously unknown purinergic mechanisms of NBP-induced metabolites and suggest ApppI as the endogenous pain inhibitor contributing to cancer treatment with NBPs.

Human Keratinocytes Respond to Extracellular UTP by Induction of Hyaluronan Synthase 2 Expression and Increased Hyaluronan Synthesis.

The release of nucleotides into extracellular space is triggered by insults like wounding and ultraviolet radiation, resulting in stimulatory or inhibitory signals via plasma membrane nucleotide receptors. As similar insults are known to activate hyaluronan synthesis we explored the possibility that extracellular UTP or its breakdown products UDP and UMP act as mediators for hyaluronan synthase (HAS) activation in human epidermal keratinocytes. UTP increased hyaluronan both in the pericellular matrix and in the culture medium of HaCaT cells. 10-100 µm UTP strongly up-regulated HAS2 expression, although the other hyaluronan synthases (HAS1, HAS3) and hyaluronidases (HYAL1, HYAL2) were not affected. The HAS2 response was rapid and transient, with the maximum stimulation at 1.5 h. UDP exerted a similar effect, but higher concentrations were required for the response, and UMP showed no stimulation at all. Specific siRNAs against the UTP receptor P2Y2, and inhibitors of UDP receptors P2Y6 and P2Y14, indicated that the response to UTP was mediated mainly through P2Y2 and to a lesser extent via UDP receptors. UTP increased the phosphorylation of p38, ERK, CREB, and Ser-727 of STAT3 and induced nuclear translocation of pCaMKII. Inhibitors of PKC, p38, ERK, CaMKII, STAT3, and CREB partially blocked the activation of HAS2 expression, confirming the involvement of these pathways in the UTP-induced HAS2 response. The present data reveal a selective up-regulation of HAS2 expression by extracellular UTP, which is likely to contribute to the previously reported rapid activation of hyaluronan metabolism in response to tissue trauma or ultraviolet radiation.

Pro-nociceptive migraine mediator CGRP provides neuroprotection of sensory, cortical and cerebellar neurons via multi-kinase signaling.

Background Blocking the pro-nociceptive action of CGRP is one of the most promising approaches for migraine prophylaxis. The aim of this study was to explore a role for CGRP as a neuroprotective agent for central and peripheral neurons. Methods The viability of isolated rat trigeminal, cortical and cerebellar neurons was tested by fluorescence vital assay. Engagement of Nrf2 target genes was analyzed by qPCR. The neuroprotective efficacy of CGRP in vivo was tested in mice using a permanent cerebral ischemia model. Results CGRP prevented apoptosis induced by the amino acid homocysteine in all three distinct neuronal populations. Using a set of specific kinase inhibitors, we show the role of multi-kinase signaling pathways involving PKA and CaMKII in neuronal survival. Forskolin triggered a very similar signaling cascade, suggesting that cAMP is the main upstream trigger for multi-kinase neuroprotection. The specific CGRP antagonist BIBN4096 reduced cellular viability, lending further support to the proposed neuroprotective function of CGRP. Importantly, CGRP was neuroprotective against permanent ischemia in mice. Conclusion Our data show an unexpected 'positive' role for the endogenous pro-nociceptive migraine mediator CGRP, suggesting more careful examination of migraine prophylaxis strategy based on CGRP antagonism although it should be noted that homocysteine induced apoptosis in primary neuronal cell culture might not necessarily reproduce all the features of cell loss in the living organism.

The organic osmolyte betaine induces keratin 2 expression in rat epidermal keratinocytes - A genome-wide study in UVB irradiated organotypic 3D cultures.

The moisturizing and potentially protective properties of the organic osmolyte betaine (trimethylglycine) have made it an attractive component for skin care products. Its wide use despite the lack of comprehensive studies addressing its specific effects in skin led us to characterize the molecular targets of betaine in keratinocytes and to explore, whether it modifies the effects of acute UVB exposure. Genome-wide expression analysis was performed on organotypic cultures of rat epidermal keratinocytes, treated either with betaine (10mM), UVB (30 mJ/cm(2)) or their combination. Results were verified with qRT-PCR, western blotting and immunohistochemistry. Additionally, cell proliferation and differentiation were analyzed. Among the 89 genes influenced by betaine, the differentiation marker keratin 2 showed the highest upregulation, which was also confirmed at protein level. Expression of Egr1, a transcription factor, and Purkinje cell protein 4, a regulator of Ca(2+)/calmodulin metabolism, also increased, while downregulated genes included several ion-channel components, such as Fxyd2. Bioinformatics analyses suggest that genes modulated by betaine are involved in DNA replication, might counteract UV-induced processes, and include many targets of transcription factors associated with cell proliferation and differentiation. Our results indicate that betaine controls unique gene expression pathways in keratinocytes, including some involved in differentiation.

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion.

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanoma cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells.

Fluorescence resonance energy transfer (FRET) and proximity ligation assays reveal functionally relevant homo- and heteromeric complexes among hyaluronan synthases HAS1, HAS2, and HAS3.

In vertebrates, hyaluronan is produced in the plasma membrane from cytosolic UDP-sugar substrates by hyaluronan synthase 1-3 (HAS1-3) isoenzymes that transfer N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in alternative positions in the growing polysaccharide chain during its simultaneous extrusion into the extracellular space. It has been shown that HAS2 immunoprecipitates contain functional HAS2 homomers and also heteromers with HAS3 (Karousou, E., Kamiryo, M., Skandalis, S. S., Ruusala, A., Asteriou, T., Passi, A., Yamashita, H., Hellman, U., Heldin, C. H., and Heldin, P. (2010) The activity of hyaluronan synthase 2 is regulated by dimerization and ubiquitination. J. Biol. Chem. 285, 23647-23654). Here we have systematically screened in live cells, potential interactions among the HAS isoenzymes using fluorescence resonance energy transfer (FRET) and flow cytometric quantification. We show that all HAS isoenzymes form homomeric and also heteromeric complexes with each other. The same complexes were detected both in Golgi apparatus and plasma membrane by using FRET microscopy and the acceptor photobleaching method. Proximity ligation assays with HAS antibodies confirmed the presence of HAS1-HAS2, HAS2-HAS2, and HAS2-HAS3 complexes between endogenously expressed HASs. C-terminal deletions revealed that the enzymes interact mainly via uncharacterized N-terminal 86-amino acid domain(s), but additional binding site(s) probably exist in their C-terminal parts. Of all the homomeric complexes HAS1 had the lowest and HAS3 the highest synthetic activity. Interestingly, HAS1 transfection reduced the synthesis of hyaluronan obtained by HAS2 and HAS3, suggesting functional cooperation between the isoenzymes. These data indicate a general tendency of HAS isoenzymes to form both homomeric and heteromeric complexes with potentially important functional consequences on hyaluronan synthesis.