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Circulating plasma extracellular vesicles (EVs) mostly originate from platelets and may promote organ dysfunction in sepsis. However, the role of platelet-derived EVs in sepsis-induced acute kidney injury (AKI) remains poorly understood. The present study extracted EVs from the supernatant of human platelets treated with phosphate buffer saline (PBS) or lipopolysaccharide (LPS). Then, we subjected PBS-EVs or LPS-EVs to cecal ligation and puncture (CLP) mice in vivo or LPS-stimulated renal tubular epithelial cells (RTECs) in vitro. Our results indicated that LPS-EVs aggravate septic AKI via promoting apoptosis, inflammation and oxidative stress. Further, ADP-ribosylation factor 6 (ARF6) was identified as a differential protein between PBS-EVs and LPS-EVs by quantitative proteomics analysis. Mechanistically, ARF6 activated ERK/Smad3/p53 signaling to exacerbate sepsis-induced AKI. LPS upregulated ARF6 in RTECs was dependent on TLR4/MyD88 pathway. Both genetically and pharmacologically inhibition of ARF6 attenuated septic AKI. Moreover, platelets were activated by TLR4 and its downstream mediator IKK controlled platelet secretion during sepsis. Inhibition of platelet secretion alleviated septic AKI. Collectively, our study demonstrated that platelet-derived EVs may be a therapeutic target in septic AKI.Circulating plasma extracellular vesicles (EVs) mostly originate from platelets and may promote organ dysfunction in sepsis. However, the role of platelet-derived EVs in sepsis-induced acute kidney injury (AKI) remains poorly understood. The present study extracted EVs from the supernatant of human platelets treated with phosphate buffer saline (PBS) or lipopolysaccharide (LPS). Then, we subjected PBS-EVs or LPS-EVs to cecal ligation and puncture (CLP) mice in vivo or LPS-stimulated renal tubular epithelial cells (RTECs) in vitro. Our results indicated that LPS-EVs aggravate septic AKI via promoting apoptosis, inflammation and oxidative stress. Further, ADP-ribosylation factor 6 (ARF6) was identified as a differential protein between PBS-EVs and LPS-EVs by quantitative proteomics analysis. Mechanistically, ARF6 activated ERK/Smad3/p53 signaling to exacerbate sepsis-induced AKI. LPS upregulated ARF6 in RTECs was dependent on TLR4/MyD88 pathway. Both genetically and pharmacologically inhibition of ARF6 attenuated septic AKI. Moreover, platelets were activated by TLR4 and its downstream mediator IKK controlled platelet secretion during sepsis. Inhibition of platelet secretion alleviated septic AKI. Collectively, our study demonstrated that platelet-derived EVs may be a therapeutic target in septic AKI.

The cytoplasmic tyrosine kinase SRC controls cell growth, proliferation, adhesion, and motility. The current view is that SRC acts primarily downstream of cell-surface receptors to control intracellular signaling cascades. Here we reveal that SRC functions in cell-to-cell communication by controlling the biogenesis and the activity of exosomes. Exosomes are viral-like particles from endosomal origin that can reprogram recipient cells. By gain- and loss-of-function studies, we establish that SRC stimulates the secretion of exosomes having promigratory activity on endothelial cells and that syntenin is mandatory for SRC exosomal function. Mechanistically, SRC impacts on syndecan endocytosis and on syntenin–syndecan endosomal budding, upstream of ARF6 small GTPase and its effector phospholipase D2, directly phosphorylating the conserved juxtamembrane DEGSY motif of the syndecan cytosolic domain and syntenin tyrosine 46. Our study uncovers a function of SRC in cell–cell communication, supported by syntenin exosomes, which is likely to contribute to tumor–host interactions.

Hypocotyl cell elongation related to photomorphogenesis in Arabidopsis seedlings is regulated by a network involving ethylene, auxin, and brassinosteroid signalling that is mediated by interactions among ERF72, ARF6, and BZR1, forming a revised BZR-ARF-PIF/DELLA-ERF (BAP/DE) module. Abstract The phytohormones brassinosteroid (BR), auxin, and gibberellin (GA) regulate photomorphogenesis-related hypocotyl elongation in Arabidopsis via the co-operative interaction of BZR-ARF-PIF/DELLA (BAP/D) transcription factors/regulators. In addition, ethylene activates the PIF3 or ERF1 pathway through EIN3/EIL1 to balance hypocotyl elongation in Arabidopsis seedlings. However, the mechanism by which ethylene is co-ordinated with other phytohormones to produce light-regulated hypocotyl growth remains elusive. In this study, we found that hypocotyl cell elongation is regulated by a network involving ethylene, auxin, and BR signalling, which is mediated by interactions among ERF72, ARF6, and BZR1. ERF72 interacted directly with ARF6 and BZR1 in vitro and in vivo, and it antagonised regulation by ARF6 and BZR1 of the transcription of BEE3 and XTH7. In addition, light modulated the subcellular localisation of ERF72 and transcription of ERF72 through the EIN2-EIN3/EIL1 pathway, facilitating the function of ERF72 in photomorphogenesis. The expression of BEE3 and XTH7 was also regulated by the EIN2-EIN3/EIL1 pathway. Our findings indicate that a revised BZR-ARF-PIF/DELLA-ERF (BAP/DE) module integrates light and hormone signals to regulate hypocotyl elongation in Arabidopsis.

Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6–AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5′-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5′-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor β (PDGFRβ) and enzymes of the mevalonate pathway (MVP). The ARF6–AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.

ADP-ribosylation factor 6 small GTPase plays an important role in cell migration, invasion and angiogenesis, which are the hallmarks of cancer. Although alterations in ARF6 expression and activity have been linked to metastatic cancer in one or two tissues, the expression of ARF6 in cancers over a wide range of tissues has not been studied so far. In this report, we analysed the expression of ARF6 mRNA in cancers and corresponding healthy controls from 17 different tissues by real-time qualitative polymerase chain reaction (RT-qPCR). We further evaluated ARF6 protein expression in oesophageal adenocarcinoma (EAC) tissue microarray cores by immunohistochemistry. The ARF6 gene expression levels are highly variable between healthy and cancer tissues. Our findings suggest that the ARF6 gene expression is up-regulated highest in oesophageal cancer. In EAC TMAs, ARF6 protein expression increase correlated with EAC progression. This is the first study to investigate ARF6 gene expression in a wide array of cancer tissues and demonstrate that ARF6 expression, at both mRNA and protein levels, is significantly upregulated in higher grades of EAC, which may be useful in targeting ARF6 for cancer diagnostic and therapeutic purposes.

Macropinocytosis is a form of endocytosis that brings large fluid-filled endosomes into the cell interior. Macrophages and dendritic cells are especially active in this process, but all cells can be stimulated to initiate this remarkable form of endocytosis. Although much is known about the membrane lipid and actin requirements for initiating macropinocytosis, less is known about the membrane that forms the macropinosome and the fate of that membrane once the macropinosome enters the cell interior. Since macropinocytosis is a specialized form of clathrin-independent endocytosis (CIE), studies of the constitutive internalization and trafficking of cargo proteins and membrane that enter cells independently of clathrin could reveal the types of membrane that form the macropinosome and the machinery that handles cargo sorting and recycling during the maturation of the macropinosome. This article is part of the Theo Murphy meeting issue ‘Macropinocytosis’.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants is threatening public health around the world. Endocytosis functions as an important way for viral infection, and SARS-CoV-2 bears no exception. However, the specific endocytic mechanism of SARS-CoV-2 remains unknown. In this study, we used endocytic inhibitors to evaluate the role of different endocytic routes in SARS-CoV-2 pseudovirus infection and found that the viral infection was associated with caveolar/lipid raft- and cytoskeleton-mediated endocytosis, but independent of the clathrin-mediated endocytosis and macropinocytosis. Meanwhile, the knockdown of CD147 and Rab5a in Vero E6 and Huh-7 cells inhibited SARS-CoV-2 pseudovirus infection, and the co-localization of spike protein, CD147, and Rab5a was observed in pseudovirus-infected Vero E6 cells, which was weakened by CD147 silencing, illustrating that SARS-CoV-2 pseudovirus entered the host cells via CD147-mediated endocytosis. Additionally, Arf6 silencing markedly inhibited pseudovirus infection in Vero E6 and Huh-7 cells, while little change was observed in CD147 knockout-Vero E6 cells. This finding indicated Arf6-mediated CD147 trafficking plays a vital role in SARS-CoV-2 entry. Taken together, our findings provide new insights into the CD147-Arf6 axis in mediating SARS-CoV-2 pseudovirus entry into the host cells, and further suggest that blockade of this pathway seems to be a feasible approach to prevent the SARS-CoV-2 infection clinically.

Auxin regulates the expression of diverse genes that affect plant growth and development. This regulation requires AUXIN RESPONSE FACTORS (ARFs) that bind to the promoter regions of these genes. ARF6 and ARF8 in Arabidopsis thaliana are required to promote inflorescence stem elongation and late stages of petal, stamen, and gynoecium development. All seed plants studied thus far have ARF6 and ARF8 orthologues as well as the microRNA miR167, which targets ARF6 and ARF8. Whether these genes have broadly conserved roles in flower development is not known. To address this question, the effects of down-regulation of ARF6 and ARF8 were investigated through transgenic expression of Arabidopsis MIR167a in tomato, which diverged from Arabidopsis before the radiation of dicotyledonous plants approximately 90–112 million years ago. The transgenic tomato plants overexpressing MIR167a exhibited reductions in leaf size and internode length as well as shortened petals, stamens, and styles. More significantly, the transgenic plants were female-sterile as a result of failure of wild-type pollen to germinate on the stigma surface and/or to grow through the style. RNA-Seq analysis identified many genes with significantly altered expression patterns, including those encoding products with functions in ‘transcription regulation’, ‘cell wall’ and ‘lipid metabolism’ categories. Putative orthologues of a subset of these genes were also differentially expressed in Arabidopsis arf6 arf8 mutant flowers. These results thus suggest that ARF6 and ARF8 have conserved roles in controlling growth and development of vegetative and flower organs in dicots.

The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host–pathogen interactions.

The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.