Molecular Mechanism of Tetraspanin CD9 Mediated Cell Motility

CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas CD9, a member of the tetraspanin superfamily of proteins participates in the regulation of cell adhesive functions such as cell migration. The mechanisms underlying CD9 mediated cell migration are not known. In the current study, we investigated the molecular basis for the CD9 promoted cell migration. Our findings show that the phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortamannin and LY294002 inhibited CD9 promoted cell motility in Chinese hamster ovary (CHO) cells. In contrast, inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect on CD9 driven CHO cell motility. Furthermore, inhibition of PI-3K activity in CHO cells by dominant/negative PI-3K cDNA transfection abolished CD9 mediated pro-migratory effects. Consistent with these observations, CD9 expression in CHO cells and in the rat aortic smooth muscle (RASM) cells induced enhanced phosphorylation of PI-3K substrate, Akt. In CHO cells, CD9 expression also enhanced protein levels and tyrosine phosphorylation of the adaptor protein p130Cas. However, no significant changes in the CD9 enhanced migration were observed in CHO cells upon down regulation of p130Cas by siRNA transfection suggesting that p130Cas dependent pathways are not mandatory for CD9 mediated motility. To further understand the mechanisms by which CD9 regulates cell migration, we studied the relative contribution of the fibronectin (FN) receptor integrin, alpha-5-beta-1 in CD9 mediated cell motility. Our findings show that CD9 is in molecular complex with alpha-5-beta-1 and that CD9 promoted migration can be completely blocked by an alpha-5-beta-1 function blocking antibody. Further studies revealed that CD9 expression may stabilize the active conformer of the beta-1 integrin. Taken togeather, our study demonstrates key molecular mechanisms governing CD9 mediated haptotactic cell motility to FN in CHO cells. Our findings indicate that CD9 in concert with integrin alpha-5-beta-1 requires activation of the PI-3K pathway leading to enhanced haptotactic cell migration on FN

CD9 may contribute to the survival of human germinal center B cells by facilitating the interaction with follicular dendritic cells

AbstractThe germinal center (GC) is a dynamic microenvironment where antigen (Ag)-activated B cells rapidly expand and differentiate, generating plasma cells (PC) that produce high-affinity antibodies. Precise regulation of survival and proliferation of Ag-activated B cells within the GC is crucial for humoral immune responses. The follicular dendritic cells (FDC) are the specialized stromal cells in the GC that prevent apoptosis of GC-B cells. Recently, we reported that human GC-B cells consist of CD9+ and CD9− populations and that it is the CD9+ cells that are committed to the PC lineage. In this study, we investigated the functional role of CD9 on GC-B cells. Tonsillar tissue section staining revealed that in vivo CD9+ GC-B cells localized in the light zone FDC area. Consistent this, in vitro CD9+ GC-B cells survived better than CD9− GC-B cells in the presence of HK cells, an FDC line, in a cell–cell contact-dependent manner. The frozen tonsillar tissue section binding assay showed that CD9+ GC-B cells bound to the GC area of tonsillar tissues significantly more than the CD9− GC-B cells did and that the binding was significantly inhibited by neutralizing anti-integrin β1 antibody. Furthermore, CD9+ cells bound to soluble VCAM-1 more than CD9− cells did, resulting in activation and stabilization of the active epitope of integrin β1. All together, our data suggest that CD9 on GC-B cells contributes to survival by strengthening their binding to FDC through the VLA4/VCAM-1 axis

Characterisation of CD9 as a functional marker of cancer stem cells in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most common and most aggressive type of pancreatic cancer, with a five-year survival rate of approximately 5%. PDAC tumours are heterogeneous, containing morphologically, genetically and epigenetically distinct cancer cells, as well as large numbers of stromal and immune cells. To date there is no consensus on any well-defined cancer stem or tumour- initiating cell (CSC/TIC) in PDAC that might explain the tumour cell heterogeneity. Our laboratory recently identified the tetraspanin protein CD9 as a marker of PDAC CSCs/TICs. In this thesis, I further characterised CD9 as being a functional marker of these cells: in vitro, knocking down or overexpressing CD9 in mouse PDAC organoids decreased or increased organoid growth, respectively. These growth differences were recapitulated in tumour grafts in immuno-compromised mice. Furthermore, heterozygous deletion of CD9 in the embryonic pancreas in the commonly used Pdx1-Cre; LSL-KRasG12D; p53F/F and Pdx1-Cre; LSL-KRasG12D; p53F/+ mice prolonged overall survival. CD9 was therefore important in PDAC tumour initiation. Knocking out CD9 heterozygously in already-established tumours using a dual-recombinase genetic system showed that CD9 is also involved in tumour maintenance. Human PDAC patients with high levels of CD9 expression have a worse overall survival than patients with low levels of CD9 expression. Additionally, about 10% of patients have genomic amplifications of the CD9 locus. Mechanistically, CD9 interacted with several metabolite transporters in primary PDAC cells. In particular, CD9 promoted the plasma membrane localisation of the glutamine transporter ASCT2, which enhanced glutamine uptake. ASCT2 overexpression could rescue the growth defect of CD9-depleted PDAC cells, and CD9-depleted PDAC cells were more sensitive to a glutaminase inhibitor in vitro, supporting the functional link between CD9 and ASCT2. Overall, this work identifies the tetraspanin CD9 as an important functional marker of PDAC CSCs, which may have therapeutic implications in pancreatic cancer patients

Plasma Corticotropin-Releasing Factor Receptors and B7-2⁺ Extracellular Vesicles in Blood Correlate with Irritable Bowel Syndrome Disease Severity.

Extracellular vesicles (EVs) are composed of bilayer membranes that are released by different cell types and are present in bodily fluids, such as blood, urine, and bile. EVs are thought to play a key role in intracellular communication. Based on their size and density, EVs are classified into small, medium, or large EVs. Cargo composition in EVs reflects physiological changes in health and disease. Patients with irritable bowel syndrome (IBS) exhibit visceral hypersensitivity and mood disorders. Stressful episodes often precede disease symptoms in IBS patients. Stress-induced symptoms include, but are not limited to, abdominal pain and mood swings. Perceived stress responses are mediated by two known G protein-coupled receptors (GPCRs), corticotropin-releasing factor receptor 1 and 2 (CRFRs). CRFRs belong to the Class B secretin receptor family of GPCRs. Here, we show that CRFRs were present in human and murine plasma, and in EVs purified from mouse serum. CRFRs were present in plasma from IBS patients and healthy controls. EVs secreted from immune cells influence both adaptive and innate immune responses via exchange of EVs between different immune cell types. B7-2 (CD86), a plasma membrane antigen-presenting protein, is present on EVs secreted from dendritic, B-, and mast cells, whereas CD9 is present on EVs secreted from dendritic and intestinal epithelial cells. We found that plasma CRFR levels positively correlated with B7-2+ EVs (R = 0.8597, p < 0.0001), but no association was seen with CD9+ EVs. Plasma CRFRs expression negatively correlated with IBS severity scores. Our data suggests that plasma EVs from immune cells carry CRFRs as cargos and influence cell-cell communication in health and disease

Evaluation of human umbilical cord blood as a source of embryonic stem cells

Human umbilical cord blood (HUCB) has been poorly characterised as a source of embryonic stem cells (ESCs). The aim of this study, therefore, was to evaluate HUCB as source of mesenchymal stem cells (MSCs) with embryonic characteristics. HUCB was collected from consenting women undergoing elective caesarean sections. HUCB was meticulously explanted into MesenCult media and incubated. Qualitative and quantitative immunophenotyping of cells was achieved using fluorescein isothiocyanate (FITC) labelled antibodies (CD34, CD45, CD29, CD44, CD73 and CD105) phenotypic markers. Immunocytochemistry was carried out for the human ESC markers CD9, stage-specific embryonic antigen-1 and 4 (SSEA-1 and SSEA-4), E-cadherin, Podocalyxin (PODXL), sex-determining region Y-box 2 (SOX2), NANOG and Octamer (OCT3/4). MSCs were cultured to induce differentiation into adipogenic, osteogenic, chondrogenic and neurogenic cells. Immunocytochemistry was used to identify fatty acid binding protein-4 (FABP-4), osteocalcin, aggrecan, SOX2 and oligodendrocyte-4 (Olig-4) markers. The cells were strongly positive for the MSC markers CD29, CD44, CD73 and CD105; these cells also expressed the ESC markers CD9, SSEA-1 and SSEA-4, E-cadherin, PODXL, SOX2, NANOG and OCT3/4. Additionally, the MSCs expressed the adipogenic FABP-4, osteogenic osteocalcin, chondrogenic aggrecan and neural Olig-4 and SOX2 markers after differentiation. Therefore, HUCB is a rich source for MSCs with embryonic characteristics

The Tetraspanin CD9 Localizes to Platelet-Platelet Contacts and Regulates Thrombus Stability

CD9, a member of the tetraspanin family, is highly expressed on platelets (50,000-80,000 copies per platelet). Tetraspanins have been implicated in modulation of integrin function, and it is hypothesized that CD9 will modulate GPIIb-IIIa, the major platelet integrin. The association between CD9 and GPIIb-IIIa was analyzed using immunoprecipitations and confocal microscopy. These two proteins were found to associate with each other, particularly at areas of platelet-platelet contact and at the periphery. Confocal analysis revealed CD9 localization was most intense at platelet-platelet contact, as well as in platelet filopodia and lamellipodia, but there is a lack of CD9 at areas of platelet-matrix contact. Co-localization with F-actin decreased as platelets progressed through the stages of spreading. In order to analyze CD9 contributions to platelet function, a Fab fragment was generated from mAb7, an antibody which binds with high affinity to the large extracellular loop of CD9. Fab fragments were used to avoid complications of bound antibody crosslinking CD9 with the platelet FcγRII receptor. Confocal analysis of platelet spreading indicated that CD9 perturbation by mFab7 resulted in increased platelet spreading on a variety of matrices, although platelet adhesion was unaffected on all matrices except fibrin. Platelet-platelet interactions, including aggregation and disaggregation, were studied using light transmission aggregometry. CD9 perturbation increased the extent of platelet aggregation in response to threshold levels of agonist, with diminished effect with more potent agonists such as TRAP (Thrombin Receptor Agonist Peptide). CD9 perturbation also diminished, but did not completely inhibit, platelet disaggregation induced by eptifibatide. Interestingly, CD9 perturbation did not alter platelet-mediated clot retraction of platelets in PRP (Platelet rich plasma). In response to CD9 alteration by mFab7, both increased fibrinogen binding and platelet activation (as evidenced by CD63 and P-selectin expression) were elicited in the presence of threshold ADP levels. Both responses were dependent on presence of low-level agonist, as CD9 perturbation in the absence of agonist yielded no effect. Cytoskeletal isolation revealed that CD9 perturbation results in increased incorporation of actin and other cytoskeletal molecules into the platelet core cytoskeleton. Immunoprecipitations results showed increased CD63 incorporation into the tetraspanin web, although the level of CD9 coprecipitation with GPIIb-IIIa was unaffected. A preliminary analysis of tyrosine phosphorylation, particularly Akt, did not reveal alterations on tyrosine phosphorylation of signaling molecules. These results suggest that CD9 functions as a scaffold, organizing molecules into specific domains on the platelet surface. It appears to function as a “gatekeeper” for GPIIb-IIIa activation, particularly modulating activation and aggregate stability at low levels of agonist

MARCH1 protects the lipid raft and tetraspanin web from MHCII proteotoxicity in dendritic cells

Dendritic cells (DCs) produce major histocompatibility complex II (MHCII) in large amounts to function as professional antigen presenting cells. Paradoxically, DCs also ubiquitinate and degrade MHCII in a constitutive manner. Mice deficient in the MHCII-ubiquitinating enzyme membrane-anchored RING-CH1, or the ubiquitin-acceptor lysine of MHCII, exhibit a substantial reduction in the number of regulatory T (Treg) cells, but the underlying mechanism was unclear. Here we report that ubiquitin-dependent MHCII turnover is critical to maintain homeostasis of lipid rafts and the tetraspanin web in DCs. Lack of MHCII ubiquitination results in the accumulation of excessive quantities of MHCII in the plasma membrane, and the resulting disruption to lipid rafts and the tetraspanin web leads to significant impairment in the ability of DCs to engage and activate thymocytes for Treg cell differentiation. Thus, ubiquitin-dependent MHCII turnover represents a novel quality-control mechanism by which DCs maintain homeostasis of membrane domains that support DC's Treg cell-selecting function

Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes

Introduction: In recent years, there has been an exponential increase in the number of studies aiming to understand the biology of exosomes, as well as other extracellular vesicles. However, classification of membrane vesicles and the appropriate protocols for their isolation are still under intense discussion and investigation. When isolating vesicles, it is crucial to use systems that are able to separate them, to avoid cross-contamination. Method: EVs released from three different kinds of cell lines: HMC-1, TF-1 and BV-2 were isolated using two centrifugation-based protocols. In protocol 1, apoptotic bodies were collected at 2,000×g, followed by filtering the supernatant through 0.8 µm pores and pelleting of microvesicles at 12,200×g. In protocol 2, apoptotic bodies and microvesicles were collected together at 16,500×g, followed by filtering of the supernatant through 0.2 µm pores and pelleting of exosomes at 120,000×g. Extracellular vesicles were analyzed by transmission electron microscopy, flow cytometry and the RNA profiles were investigated using a Bioanalyzer®. Results: RNA profiles showed that ribosomal RNA was primary detectable in apoptotic bodies and smaller RNAs without prominent ribosomal RNA peaks in exosomes. In contrast, microvesicles contained little or no RNA except for microvesicles collected from TF-1 cell cultures. The different vesicle pellets showed highly different distribution of size, shape and electron density with typical apoptotic body, microvesicle and exosome characteristics when analyzed by transmission electron microscopy. Flow cytometry revealed the presence of CD63 and CD81 in all vesicles investigated, as well as CD9 except in the TF-1-derived vesicles, as these cells do not express CD9. Conclusions: Our results demonstrate that centrifugation-based protocols are simple and fast systems to distinguish subpopulations of extracellular vesicles. Different vesicles show different RNA profiles and morphological characteristics, but they are indistinguishable using CD63-coated beads for flow cytometry analysis

Exosomes: A Novel Zika Virus Vaccine Candidate

With the recent emergence of Zika virus (ZIKV) diseases, increasing global concern has driven the demand for a vaccine. One promising vaccine platform has presented itself in the form of exosomes: a subgroup of extracellular vesicles released by many human cell types that facilitate intercellular communication. The objective of this study is to engineer exosomes that incorporate ZIKV structural proteins into its phospholipid bilayer. Previous studies indicate that CD9 and CD63 proteins are highly enriched in exosomal membranes. From this, it was hypothesized that attaching ZIKV genes to CD9 or CD63 to produce a gene fusion may enable exosomes to act as antigen-presenting vesicles. These engineered exosomes may potentially stimulate T-cells to mount a strong immune response. The cDNA of the CD9, CD63, and the highly immunogenic ZIKV genes (envelope, precursor membrane, and NS1) were generated using RT-PCR. These products were used as a template for regular PCR, and cloned into pcDNA3.1/V5 vector. The chimeric gene fusion was assembled using the Gibson assembly kit, and transfected into human embryonic kidney epithelial (HEK293T) cells for expression. The exosomes were purified from the supernatant and subjected to immunoblotting and immunofluorescence assays to confirm the presence of ZIKV proteins. The results of this study are pending at the time of this abstract submission. A future study will be conducted using an in vitro activation assay to determine if the engineered exosomes induce T-cell activation. The potential candidates will be used in an animal study for immunity against ZIKV infection

Single-cell gene expression profiles define self-renewing, pluripotent, and lineage primed States of human pluripotent stem cells

Pluripotent stem cells display significant heterogeneity in gene expression, but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states, even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity, consistently high transcripts for all pluripotency-related genes studied, and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states, which ultimately become primed for lineage specification