Chaperone BiP: Master regulator of the unfolded protein response (UPR) and its role in regulation of angiogenesis

Verschiedene Krankheiten gehen mit einer fehlerhaften Vaskularisierung einher. Allerdings ist der Erfolg der derzeitig vorhandenen Therapieansätze, die sich z.B. auf VEGF fokussieren, beschränkt. Aus diesem Grund ist es wichtig, neue Strategien zur Regulation der Angiogenese zu entwickeln. Hierbei stehen neue Signaltransduktions-wege im Fokus, die sich als vielversprechend erweisen, um Angiogenese zu fördern oder zu inhibieren. Die Blutgefäßneubildung ist ein hochregulierter Prozess, der mit einer hohen Proteinsyntheserate verknüpft ist. Die Angiogenese wurde bereits mit dem ER-Stress Signaltransduktionsweg, der Unfolded Protein Response (UPR), in Verbindung gebracht (Zeng et al., 2013; Bouvier et al., 2012). Eine im Rahmen der vorliegenden Studie durchgeführte histologische Untersuchung konnte eine Fehlregulierung der Expression von UPR beteiligten Proteinen in vivo unter pathologischen Bedingungen gezeigt werden. Bemerkenswerter Weise war BiP, der Hauptsensor der UPR, in Endothelzellen von Angiosarkomen sehr stark exprimiert. In in vitro Experimenten wurde gezeigt, dass das Herunterregulieren von BiP mittels RNAi Einfluss auf die inflammatorische Antwort und die Bildung angiogener Strukturen in Endothelzellen nimmt. Das Herunterregulieren des Proteins BiP verstärkte die inflammatorische Antwort von HUVEC, was sich in einer gesteigerten Bildung von IL-8 und ICAM-1 äußerte und wurde auf die Aktivierung der UPR durch die verringerte Menge an BiP zurückgeführt. Der Phänotyp BiP-herunterregulierter Zellen entsprach dem untransfizierter Zellen, welcher durch das Cytoskelett und die Expression des endothelspezifischen Markers CD31 charakterisiert wurde. Im Gegensatz dazu änderte sich der Grad der Glykosylierung in transfizierten Zellen. Im Hinblick auf die Blutgefäßbildung, zeigten sich eine gehemmte Migration und eine inhibierte Bildung Gefäß-ähnlicher Strukturen in BiP-herunterregulierten Zellen. In diesen Zellen war die Expression von KDR auffallend stark inhibiert, wohingegen die Flt-1 Expression sich als gleichbleibend herausstellte, was ebenfalls auf die Aktivierung der UPR zurückgeführt werden konnte. Alternativ wäre der reduzierte Level des Proteins BiP im Hinblick auf die Funktion als Helferenzym in der Proteinfaltung eine mögliche Erklärung für die gehemmte Expression von KDR. Die Ergebnisse dieser Studie deuten darauf hin, dass stabile Spiegel von BiP die Regulierung der Angiogenese durch die Kontrolle der UPR in physiologischen Prozessen unterstützen könnte. Eine Fehlregulierung von BiP durch Unterdrückung der UPR, wie z.B. in malignen Tumoren, könnte Tumorzellen und beteiligten Endothelzellen einen Vorteil verschaffen und zu einer gestörten Vaskularisierung führen. Somit stellt das Stresssensorprotein BiP und die UPR einen potentiellen Angriffspunkt für die Regulation der Angiogenese dar.Several diseases are accompanied by dysregulated vascularization. Commonly employed therapies, involving, for example, the targeting of VEGF, present several limitations. Therefore, new strategies for regulation of angiogenesis need to be evolved. Targeting novel signaling pathways to trigger or inhibit angiogenesis could prove to be promising. Angiogenesis is a highly regulated process associated with a high protein synthesis rate. Previously, angiogenesis was linked with the ER stress signaling pathway, the Unfolded Protein Response (UPR) (Zeng et al., 2013; Bouvier et al., 2012). In a histological examination in the present study, UPR components were shown to be dysregulated under pathological conditions in vivo. Remarkably, BiP, the major stress sensor protein of the UPR, was shown to be highly up-regulated in endothelial cells in angiosarcoma. Down-regulation of BiP in vitro with the help of the RNAi technique was shown to influence inflammatory responses and tube formation in endothelial cells. BiP down-regulation strengthened the inflammatory response of HUVEC, reflected in a significant increase in IL-8 release and ICAM-1 expression, this being attributable to UPR signaling, activated by BiP down-regulation. Moreover, the endothelial cell phenotype of BiP down-regulated cells was similar to non-transfected endothelial cells, analyzed by the investigation of the cytoskeleton and the expression of the endothelial cell-specific marker, CD31. In turn, glycosylation of membrane proteins in BiP down-regulated cells differed. The level of glycosylation resembled the status of UPR activation. Focussing on angiogenesis, reduced cell migration and inhibition of the formation of capillary-like structures in BiP down-regulated cells was very prominent. Importantly, KDR expression was strongly reduced in BiP down-regulated cells, while the expression of another VEGF receptor, Flt-1, remained unaltered. Characterization of the BiP down-regulated cells indicated that decreased expression of KDR could be attributed to UPR activation following BiP down-regulation. Alternatively, a deficiency of chaperone functionality in BiP down-regulated cells could be responsible for down-regulation of KDR. The results of this study indicate that stable levels of BiP contribute to regulated angiogenesis by controlling UPR signaling in physiological processes. Dysregulation of BiP levels, as in malignant tumors, could provide an advantage to tumor cells and involved endothelial cells by strong inhibition of UPR, eventually leading to aberrant vascularization. Therefore, BiP and the UPR pathway represent potential targets for the regulation of angiogenesis

Function and mutual interaction of BiP-, PERK-, and IRE1 alpha-dependent signalling pathways in vascular tumours

Spontaneously regressing infantile haemangiomas and aggressive angiosarcomas are vascular tumours with excessive angiogenesis. When analysing haemangiomas and angiosarcomas immunohistochemically with respect to their chaperone profiles we found that angiosarcomas have significantly elevated protein levels of binding immunoglobulin protein (BIP) and PERK with concomitant attenuated IRE1 alpha levels, whereas haemangioma tissue exhibits the same pattern as embryonal skin tissue. We show that BiP is essential for the maintenance of VEGFR2 protein, which is expressed in the endothelium of both tumour types. When studying the effects of BiP, the IRE1 alpha/Xbp1 -, and PERK/ATF4-signalling pathways on the migration and tube-forming potential of endothelial cells, we show that downregulation of BiP, as well as inhibition of the kinase activity of IRE1 alpha, inhibit in vitro angiogenesis. Downregulation of PERK (PKR-like kinase; PKR = protein kinase R) levels promotes Xbp1 splicing in endoplasmic reticulum (ER)-stressed cells, indicating that in angiosarcoma the elevated PERK levels might result in high levels of unspliced Xbp1, which have been reported to promote cell proliferation and increase tumour malignancy. The data presented in this study revealed that in addition to BiP or PERK, the kinase domains of IRE1 alpha and Xbp1 could be potential targets for the development of novel therapeutic approaches for treating angiosarcomas and to control tumour angiogenesis. (c) 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Honeybees Produce Millimolar Concentrations of Non-Neuronal Acetylcholine for Breeding: Possible Adverse Effects of Neonicotinoids.

The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh) for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4-8 mM). ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75-90%) in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM) than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb]) or thiacloprid (8,800 ppb). Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb) or clothianidin (1 and 10 ppb) reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh content in the brood food

Impact of polymer-modified gold nanoparticles on brain endothelial cells: exclusion of endoplasmic reticulum stress as a potential risk factor

<p>A library of polymer-coated gold nanoparticles (AuNPs) differing in size and surface modifications was examined for uptake and induction of cellular stress responses in the endoplasmic reticulum (ER stress) in human brain endothelial cells (hCMEC/D3). ER stress is known to affect the physiology of endothelial cells (ECs) and may lead to inflammation or apoptosis. Thus, even if applied at non-cytotoxic concentrations ER stress caused by nanoparticles should be prevented to reduce the risk of vascular diseases and negative effects on the integrity of barriers (e.g. blood–brain barrier). We exposed hCMEC/D3 to twelve different AuNPs (three sizes: 18, 35, and 65 nm, each with four surface-modifications) for various times and evaluated their effects on cytotoxicity, proinflammatory mediators, barrier functions and factors involved in ER stress. We demonstrated a time-dependent uptake of all AuNPs and no cytotoxicity for up to 72 h of exposure. Exposure to certain AuNPs resulted in a time-dependent increase in the proinflammatory markers IL-8, MCP-1, sVCAM, sICAM. However, none of the AuNPs induced an increase in expression of the chaperones and stress sensor proteins BiP and GRP94, respectively, or the transcription factors ATF4 and ATF6. Furthermore, no upregulation of the UPR stress sensor receptor PERK, no active splicing product of the transcription factor XBP1 and no upregulation of the transcription factor CHOP were detectable. In conclusion, the results of the present study indicate that effects of different-sized gold nanoparticles modified with various polymers were not related to the induction of ER stress in brain microvascular endothelial cells or led to apoptosis.</p

Content of acetylcholine (ACh) and choline and the ratio between both com-pounds in samples obtained from control bees (A; outside control) and from bees kept under semi-field condition to investigate the effect of neonicotinoids (B).

<p>Content of acetylcholine (ACh) and choline and the ratio between both com-pounds in samples obtained from control bees (A; outside control) and from bees kept under semi-field condition to investigate the effect of neonicotinoids (B).</p

ChAT activity and ACh release obtained with hypopharyngeal glands isolated from outside control bees.

<p>ChAT activity and ACh release obtained with hypopharyngeal glands isolated from outside control bees.</p

HPLC measurement of ACh/choline in royal jelly (A) and brood food (B).

<p>A: Typical chromatograms after injections of 1 pmol ACh and choline (standard) or of an aliquot (20 μl) of 12 mg royal jelly diluted in 1 ml HPLC buffer and further diluted by a factor of 1000. Chromatograms on the right hand show the same samples using an analytical column packed with acetylcholinesterase, i.e. ACh is already hydrolyzed during its passage along the analytical column; thus the ACh peak disappears and the choline peak increases. Retention times are indicated in brackets. B: Chromatograms of brood food collected from semi-field control (4.5 mg, diluted by 500) or from a hive exposed to 1ppb (1.6 mg brood in 500μl, diluted by 200), 100 ppb clothianidin (1.4 mg; diluted by 100) or to 8,800 thiacloprid (1.3 mg; diluted by 100); 2 x zoom out compared to 1A.</p

HPLC measurement of ACh and choline in royal jelly and water after incubation at different pH values and application of esterase.

<p>Aliquots of royal jelly were stored at the conditions indicated and analysed by HPLC. For comparison, a water solution containing 5 mM ACh was also investigated. The first peak corresponds to ACh and the second to choline. Reduction of the first peak and increase of the second one reflects hydrolysis of ACh. Typical chromatograms are presented (n = 3).</p

HPLC measurement of ACh release from hypopharyngeal glands under control conditions (FC) or after long term exposure of honeybees to neonicotinoids.

<p>The release of ACh from hypopharyngeal glands (3 pairs per individual sample) dissected from semi-field control bees (FC) or neonicotinoid-exposed bees is shown (cloth: clothianidin; thia: thiacloprid). An aliquot (10 μl) was removed from the assay buffer at the start and end of the 30 min period. Given are means±sem of the ACh content at both time periods A: Experiments were performed during September-October 2014; n = 9 for control and n = 6 for each neonicotinoid. B: Experiments were performed during April-September 2015; n = 15 for control and n = 7–11 for each neonicotinoid.). Significance of difference from the FC: * p<0.05;** p<0.005.</p