Over-Expression of Rose RrLAZY1 Negatively Regulates the Branch Angle of Transgenic Arabidopsis Inflorescence

Branch angle is a key shoot architecture trait that strongly influences the ornamental and economic value of garden plants. However, the mechanism underlying the control of branch angle, an important aspect of tree architecture, is far from clear in roses. In the present study, we isolated the RrLAZY1 gene from the stems of Rosa rugosa ‘Zilong wochi’. Sequence analysis showed that the encoded RrLAZY1 protein contained a conserved GΦL (A/T) IGT domain, which belongs to the IGT family. Quantitative real-time PCR (qRT-PCR) analyses revealed that RrLAZY1 was expressed in all tissues and that expression was highest in the stem. The RrLAZY1 protein was localized in the plasma membrane. Based on a yeast two-hybrid assay and bimolecular fluorescence complementation experiments, the RrLAZY1 protein was found to interact with auxin-related proteins RrIAA16. The over-expression of the RrLAZY1 gene displayed a smaller branch angle in transgenic Arabidopsis inflorescence and resulted in changes in the expression level of genes related to auxin polar transport and signal transduction pathways. This study represents the first systematic analysis of the LAZY1 gene family in R. rugosa. The results of this study will provide a theoretical basis for the improvement of rose plant types and molecular breeding and provide valuable information for studying the regulation mechanism of branch angle in other woody plants.     

HIF-1α Negatively Regulates Irisin Expression Which Involves in Muscle Atrophy Induced by Hypoxia

Exposure to high altitude environment leads to skeletal muscle atrophy. As a hormone secreted by skeletal muscles after exercise, irisin contributes to promoting muscle regeneration and ameliorating skeletal muscle atrophy, but its role in hypoxia-induced skeletal muscle atrophy is still unclear. Our results showed that 4 w of hypoxia exposure significantly reduced body weight and gastrocnemius muscle mass of mice, as well as grip strength and the duration time of treadmill exercise. Hypoxic treatment increased HIF-1α expression and decreased both the circulation level of irisin and its precursor protein FNDC5 expression in skeletal muscle. In in vitro, CoCl₂-induced chemical hypoxia and 1% O₂ ambient hypoxia both reduced FNDC5, along with the increase in HIF-1α. Moreover, the decline in the area and diameter of myotubes caused by hypoxia were rescued by inhibiting HIF-1α via YC-1. Collectively, our research indicated that FNDC5/irisin was negatively regulated by HIF-1α and could participate in the regulation of muscle atrophy caused by hypoxia.     

αN-Acetyl β-Endorphin Is an Endogenous Ligand of σ1Rs That Regulates Mu-Opioid Receptor Signaling by Exchanging G Proteins for σ2Rs in σ1R Oligomers

The opioid peptide β-endorphin coexists in the pituitary and brain in its ^αN-acetylated form, which does not bind to opioid receptors. We now report that these neuropeptides exhibited opposite effects in in vivo paradigms, in which ligands of the sigma type 1 receptor (σ1R) displayed positive effects. Thus, ^αN-acetyl β-Endorphin reduced vascular infarct caused by permanent unilateral middle cerebral artery occlusion and diminished the incidence of N-methyl-D-aspartate acid-promoted convulsive syndrome and mechanical allodynia caused by unilateral chronic constriction of the sciatic nerve. Moreover, ^αN-acetyl β-Endorphin reduced the analgesia of morphine, β-Endorphin and clonidine but enhanced that of DAMGO. All these effects were counteracted by β-Endorphin and absent in σ1R^−/− mice. We observed that σ1Rs negatively regulate mu-opioid receptor (MOR)-mediated morphine analgesia by binding and sequestering G proteins. In this scenario, β-Endorphin promoted the exchange of σ2Rs by G proteins at σ1R oligomers and increased the regulation of G proteins by MORs. The opposite was observed for the ^αN-acetyl derivative, as σ1R oligomerization decreased and σ2R binding was favored, which displaced G proteins; thus, MOR-regulated transduction was reduced. Our findings suggest that the pharmacological β-Endorphin-specific epsilon receptor is a σ1R-regulated MOR and that β-Endorphin and ^αN-acetyl β-Endorphin are endogenous ligands of σ1R.     

New Therapeutic Targets for Hepatic Fibrosis in the Integrin Family, α8β1 and α11β1, Induced Specifically on Activated Stellate Cells

A huge effort has been devoted to developing drugs targeting integrins over 30 years, because of the primary roles of integrins in the cell-matrix milieu. Five αv-containing integrins, in the 24 family members, have been a central target of fibrosis. Currently, a small molecule against αvβ1 is undergoing a clinical trial for NASH-associated fibrosis as a rare agent aiming at fibrogenesis. Latent TGFβ activation, a distinct talent of αv-integrins, has been intriguing as a therapeutic target. None of the αv-integrin inhibitors, however, has been in the clinical market. αv-integrins commonly recognize an Arg-Gly-Asp (RGD) sequence, and thus the pharmacophore of inhibitors for the 5-integrins is based on the same RGD structure. The RGD preference of the integrins, at the same time, dilutes ligand specificity, as the 5-integrins share ligands containing RGD sequence such as fibronectin. With the inherent little specificity in both drugs and targets, “disease specificity” has become less important for the inhibitors than blocking as many αv-integrins. In fact, an almighty inhibitor for αv-integrins, pan-αv, was in a clinical trial. On the contrary, approved integrin inhibitors are all specific to target integrins, which are expressed in a cell-type specific manner: αIIbβ3 on platelets, α4β1, α4β7 and αLβ2 on leukocytes. Herein, “disease specific” integrins would serve as attractive targets. α8β1 and α11β1 are selectively expressed in hepatic stellate cells (HSCs) and distinctively induced upon culture activation. The exceptional specificity to activated HSCs reflects a rather “pathology specific” nature of these new integrins. The monoclonal antibodies against α8β1 and α11β1 in preclinical examinations may illuminate the road to the first medical agents.     

LRP5 Regulates HIF-1α Stability via Interaction with PHD2 in Ischemic Myocardium

Low-density lipoprotein receptor-related protein 5 (LRP5) has been studied as a co-receptor for Wnt/β-catenin signaling. However, its role in the ischemic myocardium is largely unknown. Here, we show that LRP5 may act as a negative regulator of ischemic heart injury via its interaction with prolyl hydroxylase 2 (PHD2), resulting in hypoxia-inducible factor-1α (HIF-1α) degradation. Overexpression of LRP5 in cardiomyocytes promoted hypoxia-induced apoptotic cell death, whereas LRP5-silenced cardiomyocytes were protected from hypoxic insult. Gene expression analysis (mRNA-seq) demonstrated that overexpression of LRP5 limited the expression of HIF-1α target genes. LRP5 promoted HIF-1α degradation, as evidenced by the increased hydroxylation and shorter stability of HIF-1α under hypoxic conditions through the interaction between LRP5 and PHD2. Moreover, the specific phosphorylation of LRP5 at T1492 and S1503 is responsible for enhancing the hydroxylation activity of PHD2, resulting in HIF-1α degradation, which is independent of Wnt/β-catenin signaling. Importantly, direct myocardial delivery of adenoviral constructs, silencing LRP5 in vivo, significantly improved cardiac function in infarcted rat hearts, suggesting the potential value of LRP5 as a new target for ischemic injury treatment.     

Cobalt Regulates Activation of Camk2α in Neurons by Influencing Fructose 1,6-Bisphosphatase 2 Quaternary Structure and Subcellular Localization

Fructose 1,6-bisphosphatase 2 (Fbp2) is a gluconeogenic enzyme and multifunctional protein modulating mitochondrial function and synaptic plasticity via protein-protein interactions. The ability of Fbp2 to bind to its cellular partners depends on a quaternary arrangement of the protein. NAD⁺ and AMP stabilize an inactive T-state of Fbp2 and thus, affect these interactions. However, more subtle structural changes evoked by the binding of catalytic cations may also change the affinity of Fbp2 to its cellular partners. In this report, we demonstrate that Fbp2 interacts with Co²⁺, a cation which in excessive concentrations, causes pathologies of the central nervous system and which has been shown to provoke the octal-like events in hippocampal slices. We describe for the first time the kinetics of Fbp2 in the presence of Co²⁺, and we provide a line of evidence that Co²⁺ blocks the AMP-induced transition of Fbp2 to the canonical T-state triggering instead of a new, non-canonical T-state. In such a state, Fbp2 is still partially active and may interact with its binding partners e.g., Ca²⁺/calmodulin-dependent protein kinase 2α (Camk2α). The Fbp2-Camk2α complex seems to be restricted to mitochondria membrane and it facilitates the Camk2α autoactivation and thus, synaptic plasticity.     

Targeting Protein Kinases in Blood Cancer: Focusing on CK1α and CK2

Disturbance of protein kinase activity may result in dramatic consequences that often lead to cancer development and progression. In tumors of blood origin, both tyrosine kinases and serine/threonine kinases are altered by different types of mutations, critically regulating cancer hallmarks. CK1α and CK2 are highly conserved, ubiquitously expressed and constitutively active pleiotropic kinases, which participate in multiple biological processes. The involvement of these kinases in solid and blood cancers is well documented. CK1α and CK2 are overactive in multiple myeloma, leukemias and lymphomas. Intriguingly, they are not required to the same degree for the viability of normal cells, corroborating the idea of “druggable” kinases. Different to other kinases, mutations on the gene encoding CK1α and CK2 are rare or not reported. Actually, these two kinases are outside the paradigm of oncogene addiction, since cancer cells’ dependency on these proteins resembles the phenomenon of “non-oncogene” addiction. In this review, we will summarize the general features of CK1α and CK2 and the most relevant oncogenic and stress-related signaling nodes, regulated by kinase phosphorylation, that may lead to tumor progression. Finally, we will report the current data, which support the positioning of these two kinases in the therapeutic scene of hematological cancers.     

TaNBR1, a Novel Wheat NBR1-like Domain Gene Negatively Regulates Drought Stress Tolerance in Transgenic Arabidopsis

Drought stress is an important factor that severely affects crop yield and quality. Autophagy has a crucial role in the responses to abiotic stresses. In this study, we explore TaNBR1 in response to drought stress. Expression of the TaNBR1 gene was strongly induced by NaCl, PEG, and abscisic acid treatments. The TaNBR1 protein is localized in the Golgi apparatus and autophagosome. Transgenic Arabidopsis plants overexpressing TaNBR1 exhibited reduced drought tolerance. When subjected to drought stress, compared to the wild-type (WT) lines, the transgenic overexpressing TaNBR1 plants had a lower seed germination rate, relative water content, proline content, and reduced accumulation of antioxidant enzymes, i.e., superoxide dismutase, peroxidase, and catalase, as well as higher chlorophyll losses, malondialdehyde contents, and water loss. The transgenic plants overexpressing TaNBR1 produced much shorter roots in response to mannitol stress, in comparison to the WT plants, and they exhibited greater sensitivity to abscisic acid treatment. The expression levels of the genes related to stress in the transgenic plants were affected in response to drought stress. Our results indicate that TaNBR1 negatively regulates drought stress responses by affecting the expression of stress-related genes in Arabidopsis.     

Collagen I Modifies Connexin-43 Hemichannel Activity via Integrin α2β1 Binding in TGFβ1-Evoked Renal Tubular Epithelial Cells

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFβ1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2β1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFβ1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2β1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFβ1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2β1 signaling, ahead of establishing Peptide 5 as a potential intervention.     

Neuronal ApoE Regulates the Cell-to-Cell Transmission of α-Synuclein

The presence of protein inclusions, called Lewy bodies (LBs) and Lewy neurites (LNs), in the brain is the main feature of Parkinson’s disease (PD). Recent evidence that the prion-like propagation of α-synuclein (α-syn), as a major component of LBs and LNs, plays an important role in the progression of PD has gained much attention, although the molecular mechanism remains unclear. In this study, we evaluated whether neuronal ApoE regulates the cell-to-cell transmission of α-syn and explored its molecular mechanism using in vitro and in vivo model systems. We demonstrate that neuronal ApoE deficiency attenuates both α-syn uptake and release by downregulating LRP-1 and LDLR expression and enhancing chaperone-mediated autophagy activity, respectively, thereby contributing to α-syn propagation. In addition, we observed that α-syn propagation was attenuated in ApoE knockout mice injected with pre-formed mouse α-syn fibrils. This study will help our understanding of the molecular mechanisms underlying α-syn propagation.     

Indomethacin Disrupts the Formation of β-Amyloid Plaques via an α2-Macroglobulin-Activating lrp1-Dependent Mechanism

Epidemiological studies have implied that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the development and progression of Alzheimer’s disease (AD). However, the underlying mechanisms are notably understudied. Using a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) (APP/PS1) expressing transgenic (Tg) mice and neuroblastoma (N) 2a cells as in vivo and in vitro models, we revealed the mechanisms of indomethacin in ameliorating the cognitive decline of AD. By screening AD-associated genes, we observed that a marked increase in the expression of α₂-macroglobulin (A2M) was markedly induced after treatment with indomethacin. Mechanistically, upregulation of A2M was caused by the inhibition of cyclooxygenase-2 (COX-2) and lipocalin-type prostaglandin D synthase (L-PGDS), which are responsible for the synthesis of prostaglandin (PG)H₂ and PGD₂, respectively. The reduction in PGD₂ levels induced by indomethacin alleviated the suppression of A2M expression through a PGD₂ receptor 2 (CRTH2)-dependent mechanism. Highly activated A2M not only disrupted the production and aggregation of β-amyloid protein (Aβ) but also induced Aβ efflux from the brain. More interestingly, indomethacin decreased the degradation of the A2M receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which facilitated the brain efflux of Aβ. Through the aforementioned mechanisms, indomethacin ameliorated cognitive decline in APP/PS1 Tg mice by decreasing Aβ production and clearing Aβ from the brains of AD mice.     

The α2δ Calcium Channel Subunit Accessorily and Independently Affects the Biological Function of Ditylenchus destructor

The α₂δ subunit is a high-voltage activated (HVA) calcium channel (Ca_v1 and Ca_v2) auxiliary subunit that increases the density and function of HVA calcium channels in the plasma membrane of mammals. However, its function in plant parasitic nematodes remains unknown. In this study, we cloned the full-length cDNA sequence of the voltage-gated calcium channel (VGCC) α₂δ subunit (named DdCa_vα₂δ) in Ditylenchus destructor. We found that DdCa_vα₂δ tends to be expressed in the egg stage, followed by the J3 stage. RNA-DIG in situ hybridization experiments showed that the DdCa_vα₂δ subunit was expressed in the body wall, esophageal gland, uterus, post uterine, and spicules of D. destructor. The in vitro application of RNA interference (RNAi) affected the motility, reproduction, chemotaxis, stylet thrusting, and protein secretion of D. destructor to different degrees by targeting DdCα1D, DdCα1A, and DdCa_vα₂δ in J3 stages, respectively. Based on the results of RNAi experiments, it was hypothesized that L-type VGCC may affect the motility, chemotaxis, and stylet thrusting of D. destructor. Non-L-type VGCC may affect the protein secretion and reproduction of D. destructor. The DdCa_vα₂δ subunit gene also affected the motility, chemotaxis, and reproduction of D. destructor. These findings reveal the independent function of the VGCC α₂δ subunit in D. destructor as well as give a theoretical foundation for future research on plant parasitic nematode VGCC.     

ATP13A2 Regulates Cellular α-Synuclein Multimerization,Membrane Association,and Externalization

ATP13A2, a late endo-/lysosomal polyamine transporter, is implicated in a variety of neurodegenerative diseases, including Parkinson’s disease and Kufor–Rakeb syndrome, an early-onset atypical form of parkinsonism. Loss-of-function mutations in ATP13A2 result in lysosomal deficiency as a consequence of impaired lysosomal export of the polyamines spermine/spermidine. Furthermore, accumulating evidence suggests the involvement of ATP13A2 in regulating the fate of α-synuclein, such as cytoplasmic accumulation and external release. However, no consensus has yet been reached on the mechanisms underlying these effects. Here, we aimed to gain more insight into how ATP13A2 is linked to α-synuclein biology in cell models with modified ATP13A2 activity. We found that loss of ATP13A2 impairs lysosomal membrane integrity and induces α-synuclein multimerization at the membrane, which is enhanced in conditions of oxidative stress or exposure to spermine. In contrast, overexpression of ATP13A2 wildtype (WT) had a protective effect on α-synuclein multimerization, which corresponded with reduced αsyn membrane association and stimulation of the ubiquitin-proteasome system. We also found that ATP13A2 promoted the secretion of α-synuclein through nanovesicles. Interestingly, the catalytically inactive ATP13A2 D508N mutant also affected polyubiquitination and externalization of α-synuclein multimers, suggesting a regulatory function independent of the ATPase and transport activity. In conclusion, our study demonstrates the impact of ATP13A2 on α-synuclein multimerization via polyamine transport dependent and independent functions.     

The Role of Natural Polymorphic Variants of DNA Polymerase β in DNA Repair

DNA polymerase β (Polβ) is considered the main repair DNA polymerase involved in the base excision repair (BER) pathway, which plays an important part in the repair of damaged DNA bases usually resulting from alkylation or oxidation. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that protein–protein interactions of Polβ with enzymes from the BER pathway increase the efficiency of damaged base repair in DNA. However natural single-nucleotide polymorphisms can lead to a substitution of functionally significant amino acid residues and therefore affect the catalytic activity of the enzyme and the accuracy of Polβ action. Up-to-date databases contain information about more than 8000 SNPs in the gene of Polβ. This review summarizes data on the in silico prediction of the effects of Polβ SNPs on DNA repair efficacy; available data on cancers associated with SNPs of Polβ; and experimentally tested variants of Polβ. Analysis of the literature indicates that amino acid substitutions could be important for the maintenance of the native structure of Polβ and contacts with DNA; others affect the catalytic activity of the enzyme or play a part in the precise and correct attachment of the required nucleotide triphosphate. Moreover, the amino acid substitutions in Polβ can disturb interactions with enzymes involved in BER, while the enzymatic activity of the polymorphic variant may not differ significantly from that of the wild-type enzyme. Therefore, investigation regarding the effect of Polβ natural variants occurring in the human population on enzymatic activity and protein–protein interactions is an urgent scientific task.     

Yohimbine,an α2-Adrenoceptor Antagonist,Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.     

Type I Interferon α/β Receptor-Mediated Signaling Negatively Regulates Antiviral Cytokine Responses in Murine Bone-Marrow-Derived Mast Cells and Protects the Cells from Virus-Induced Cell Death

Mast cells (MCs) are critical for initiating inflammatory responses to pathogens including viruses. Type I interferons (IFNs) that exert their antiviral functions by interacting with the type I IFN receptor (IFNAR) play a central role in host cellular responses to viruses. Given that virus-induced excessive toxic inflammatory responses are associated with aberrant IFNAR signaling and considering MCs are an early source of inflammatory cytokines during viral infections, we sought to determine whether IFNAR signaling plays a role in antiviral cytokine responses of MCs. IFNAR-intact, IFNAR-blocked, and IFNAR-knockout (IFNAR^−/−) bone-marrow-derived MCs (BMMCs) were treated in vitro with a recombinant vesicular stomatitis virus (rVSVΔm51) to assess cytokine production by these cells. All groups of MCs produced the cytokines interleukin-6 and tumor necrosis factor-α in response to rVSVΔm51. However, production of the cytokines was lowest in IFNAR-intact cells as compared with IFNAR^−/− or IFNAR-blocked cells at 20 h post-stimulation. Surprisingly, rVSVΔm51 was capable of infecting BMMCs, but functional IFNAR signaling was able to protect these cells from virus-induced death. This study showed that BMMCs produced pro-inflammatory cytokines in response to rVSVΔm51 and that IFNAR signaling was required to down-modulate these responses and protect the cells from dying from viral infection.     

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.