Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

Viruses are obligate parasites that depend on the cellular machinery for their propagation. Several viruses also incorporate cellular proteins that facilitate viral spread. Defining these cellular proteins is critical to decipher viral life cycles and delineate novel therapeutic strategies. While numerous studies have explored the importance of host proteins in coronavirus spread, information about their presence in mature virions is limited. In this study, we developed a protocol to highly enrich mature HCoV-OC43 virions and characterize them by proteomics. Recognizing that cells release extracellular vesicles whose content is modulated by viruses, and given our ability to separate virions from these vesicles, we also analyzed their protein content in both uninfected and infected cells. We uncovered 69 unique cellular proteins associated with virions including 31 high-confidence hits. These proteins primarily regulate RNA metabolism, enzymatic activities, vesicular transport, cell adhesion, metabolite interconversion, and translation. We further discovered that the virus had a profound impact on exosome composition, incorporating 47 novel cellular proteins (11 high confidence) and excluding 92 others (61 high confidence) in virus-associated extracellular vesicles compared to uninfected cells. Moreover, a dsiRNA screen revealed that 11 of 18 select targets significantly impacted viral yields, including proteins found in virions or extracellular vesicles. Overall, this study provides new and important insights into the incorporation of numerous host proteins into HCoV-OC43 virions, their biological significance, and the ability of the virus to modulate extracellular vesicles. IMPORTANCE: In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

Single cell RNA sequencing reveals emergent notochord-derived cell subpopulations in the postnatal nucleus pulposus.

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus (NP) hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native NP cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study, we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived NP cells in the postnatal mouse disc. Specifically, we established the existence of progenitor and mature NP cells, corresponding to notochordal and chondrocyte-like cells, respectively. Mature NP cells exhibited significantly higher expression levels of extracellular matrix (ECM) genes including aggrecan, and collagens II and VI, along with elevated transforming growth factor-beta and phosphoinositide 3 kinase-protein kinase B signaling. Additionally, we identified Cd9 as a novel surface marker of mature NP cells, and demonstrated that these cells were localized to the NP periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich matrix. Finally, we used a goat model to show that Cd9+ NP cell numbers decrease with moderate severity disc degeneration, suggesting that these cells are associated with maintenance of the healthy NP ECM. Improved understanding of the developmental mechanisms underlying regulation of ECM deposition in the postnatal NP may inform improved regenerative strategies for disc degeneration and associated low back pain.

Proteomic analysis and in vivo visualization of extracellular vesicles from mouse oviducts during pre-implantation embryo development.

Pre-implantation embryonic development occurs in the oviduct during the first few days of pregnancy. The presence of oviductal extracellular vesicles (oEVs, also called oviductosomes) is crucial for pre-implantation embryonic development in vivo as oEVs often contain molecular transmitters such as proteins. Therefore, evaluating oEV cargo during early pregnancy could provide insights into factors required for proper early embryonic development that are missing in the current in vitro embryo culture setting. In this study, we isolated oEVs from the oviductal fluid at estrus and different stages of early embryonic development. The 2306-3066 proteins in oEVs identified at the different time points revealed 58-60 common EV markers identified in exosome databases. Oviductal extracellular vesicle proteins from pregnant samples significantly differed from those in non-pregnant samples. In addition, superovulation changes the protein contents in oEVs compared to natural ovulation at estrus. Importantly, we have identified that embryo-protectant proteins such as high-mobility protein group B1 and serine (or cysteine) peptidase inhibitor were only enriched in the presence of embryos. We also visualized the physical interaction of EVs and the zona pellucida of 4- to 8-cell stage embryos using transmission electron microscopy as well as in vivo live imaging of epithelial cell-derived GFP-tagged CD9 mouse model. All protein data in this study are readily available to the scientific community in a searchable format at https://genes.winuthayanon.com/winuthayanon/oviduct_ev_proteins/. In conclusion, we identified oEVs proteins that could be tested to determine whether they can improve embryonic developmental outcomes in vivo and in vitro setting.

In-Depth Proteome Profiling of Small Extracellular Vesicles Isolated from Cancer Cell Lines and Patient Serum.

Extracellular vesicle (EV) secretion has been observed in many types of both normal and tumor cells. EVs contain a variety of distinctive cargoes, allowing tumor-derived serum proteins in EVs to act as a minimally invasive method for clinical monitoring. We have undertaken a comprehensive study of the protein content of the EVs from several cancer cell lines using direct data-independent analysis. Several thousand proteins were detected, including many classic EV markers such as CD9, CD81, CD63, TSG101, and Syndecan-1, among others. We detected many distinctive cancer-specific proteins, including several known markers used in cancer detection and monitoring. We further studied the protein content of EVs from patient serum for both normal controls and pancreatic cancer and hepatocellular carcinoma. The EVs for these studies have been isolated by various methods for comparison, including ultracentrifugation and CD9 immunoaffinity column. Typically, 500-1000 proteins were identified, where most of them overlapped with the EV proteins identified from the cell lines studied. We were able to identify many of the cell-line EV protein markers in the serum EVs, in addition to the large numbers of proteins specific to pancreatic and HCC cancers.

CD9 promotes TßR2-TßR1 association driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.

BACKGROUND: During wound healing, fibroblast to myofibroblast transition is required for wound contraction and remodeling. While hypoxia is an important biophysical factor in wound microenvironment, the exact regulatory mechanism underlying hypoxia and fibroblast-to-myofibroblast transition remains unclear. We previously found that tetraspanin CD9 plays an important role in oxygen sensing and wound healing. Herein, we investigated the effects of physiological hypoxia on fibroblast-to-myofibroblast transition and the biological function and mechanism of CD9 in it. METHODS: Human skin fibroblasts (HSF) and mouse dermis wounds model were established under physiological hypoxia (2% O2). The cell viability and contractility of HSF under hypoxia were evaluated by CCK8 and collagen gel retraction, respectively. The expression and distribution of fibroblast-to-myofibroblast transition markers and CD9 in HSF were detected by Western blotting and immunofluorescence. CD9 slicing and overexpressing HSFs were constructed to determine the role of CD9 by small interfering RNA and recombinant adenovirus vector. The association of TßR2 and TßR1 was measured by immunoprecipitation to explore the regulatory mechanism. Additionally, further validation was conducted on mouse dermis wounds model through histological analysis. RESULTS: Enhanced fibroblast-to-myofibroblast transition and upregulated CD9 expression was observed under hypoxia in vitro and in vivo. Besides, reversal of fibroblast-to-myofibroblast transition under hypoxia was observed when silencing CD9, suggesting that CD9 played a key role in this hypoxia-induced transition. Moreover, hypoxia increased fibroblast-to-myofibroblast transition by activating TGF-ß1/Smad2/3 signaling, especially increased interaction of TßR2 and TßR1. Ultimately, CD9 was determined to directly affect TßR1-TßR2 association in hypoxic fibroblast. CONCLUSION: Collectively, these findings suggest that CD9 promotes TßR2-TßR1 association, thus driving the transition of human dermal fibroblasts to myofibroblast under hypoxia.

Elucidating hippocampal proteome dynamics in moderate hepatic encephalopathy rats: insights from high-resolution mass spectrometry.

Hepatic encephalopathy (HE) is a debilitating neurological disorder associated with liver failure and characterized by impaired brain function. Decade-long studies have led to significant advances in our understanding of HE; however, effective therapeutic management of HE is lacking, and HE continues to be a significant cause of morbidity and mortality in patients, underscoring the need for continued research into its pathophysiology and treatment. Accordingly, the present study provides a comprehensive overview aimed at elucidating the molecular underpinnings of HE and identifying potential therapeutic targets. A moderate-grade HE model was induced in rats using thioacetamide, which simulates the liver damage observed in patients, and its impact on cognitive function, neuronal arborization, and cellular morphology was also evaluated. We employed label-free LC-MS/MS proteomics to quantitatively profile hippocampal proteins to explore the molecular mechanism of HE pathogenesis; 2175 proteins were identified, 47 of which exhibited significant alterations in moderate-grade HE. The expression of several significantly upregulated proteins, such as FAK1, CD9 and Tspan2, was further validated at the transcript and protein levels, confirming the mass spectrometry results. These proteins have not been previously reported in HE. Utilizing Metascape, a tool for gene annotation and analysis, we further studied the biological pathways integral to brain function, including gliogenesis, the role of erythrocytes in maintaining blood-brain barrier integrity, the modulation of chemical synaptic transmission, astrocyte differentiation, the regulation of organ growth, the response to cAMP, myelination, and synaptic function, which were disrupted during HE. The STRING database further elucidated the protein‒protein interaction patterns among the differentially expressed proteins. This study provides novel insights into the molecular mechanisms driving HE and paves the way for identifying novel therapeutic targets for improved disease management.

Increased circulating platelet-derived extracellular vesicles in severe COVID-19 disease.

Coagulation disturbances are major contributors to COVID-19 pathogenicity, but limited data exist on the involvement of extracellular vesicles (EVs) and residual cells (RCs). Fifty hospitalized COVID-19 patients stratified by their D-dimer levels into high (>1.5 mg/L, n = 15) or low (≤1.5 mg/l, n = 35) and 10 healthy controls were assessed for medium-sized EVs (mEVs; 200-1000 nm) and large EVs/RCs (1000-4000 nm) by high sensitivity flow cytometry. EVs were analyzed for CD61, CD235a, CD45, and CD31, commonly used to detect platelets, red blood cells, leukocytes or endothelial cells, respectively, whilst phosphatidyl serine EVs/RCs were detected by lactadherin-binding implicating procoagulant catalytic surface. Small EV detection (sEVs; 50-200 nm) and CD41a (platelet integrin) colocalization with general EV markers CD9, CD63, and CD81 were performed by single particle interferometric reflectance imaging sensor. Patients with increased D-dimer exhibited the highest number of RCs and sEVs irrespective of cell origin (p < .05). Platelet activation, reflected by increased CD61+ and lactadherin+ mEV and RC levels, associated with coagulation disturbances. Patients with low D-dimer could be discriminated from controls by tetraspanin signatures of the CD41a+ sEVs, suggesting the changes in the circulating platelet sEV subpopulations may offer added prognostic value during COVID progression.

What is the context? Coronavirus disease 19 (COVID-19) frequently leads to blood clotting disturbances, including thromboses.Particles smaller than cells, extracellular vesicles (EVs), and residual cells (RCs) affect blood clotting, but data on their role and diagnostic utility in COVID-19 are sparse.What is new? In this study, we assessed 50 hospitalized COVID-19 patients and 10 healthy controls for their different EV subpopulations and residual cells (50­4000 nm).Blood clotting marker D-dimer, which is elevated in severe COVID-19 infection, was used to characterize disease severity and stratify the patient subgroups. Fifteen patients (30%) with high D-dimer (>1.5 mg/L) were compared to controls, and 35 patients with lower D-dimer (≤1.5 mg/mL).The most topical state-of-the-art methods for detection of EV subpopulations, that is, high sensitivity flow cytometry (hsFCM) and single particle interferometric reflectance imaging sensor (SP-IRIS), were used with markers indicative of platelet, red blood cell, leukocyte or endothelial cells. The subpopulations differentiated by platelet and tetraspanin signatures by hsFCM and SP-IRIS, respectively.The main findings are Patients with high D-dimer systematically exhibited the highest number of platelet EVs in all subpopulations (p < .05).Small EVs subpopulations (differentiated by the tetraspanin signatures) could discriminate patients with low D-dimer (p < .001) from healthy controls.Differences between the two D-dimer groups were seen in the platelet-derived (large and medium EVs and RCs), RBC-derived mEVs and l EVs and RCs, and lactadherin-positive large EVs and RCs (p < .05).What is the impact? Platelet activation, reflected by increased EVs was associated with blood clotting disturbances. Small EVs signatures revealed changes in the EV subpopulations in association with blood clotting during COVID-19. Such signatures may enable identification of severely ill patients before the increase in coagulation is evident by coagulation parameters, for example, by high D-dimer.

Three-dimensional cell culture using CD9-positive cells isolated from marginal cell layer of intermediate lobe of rats sustains in vivo-like primary niche environment.

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

A shared, stochastic pathway mediates exosome protein budding along plasma and endosome membranes.

Exosomes are small extracellular vesicles of ∼30 to 150 nm that are secreted by all cells, abundant in all biofluids, and play important roles in health and disease. However, details about the mechanism of exosome biogenesis are unclear. Here, we carried out a cargo-based analysis of exosome cargo protein biogenesis in which we identified the most highly enriched exosomal cargo proteins and then followed their biogenesis, trafficking, and exosomal secretion to test different hypotheses for how cells make exosomes. We show that exosome cargo proteins bud from cells (i) in exosome-sized vesicles regardless of whether they are localized to plasma or endosome membranes, (ii) ∼5-fold more efficiently when localized to the plasma membrane, (iii) ∼5-fold less efficiently when targeted to the endosome membrane, (iv) by a stochastic process that leads to ∼100-fold differences in their abundance from one exosome to another, and (v) independently of small GTPase Rab27a, the ESCRT complex-associated protein Alix, or the cargo protein CD63. Taken together, our results demonstrate that cells use a shared, stochastic mechanism to bud exosome cargoes along the spectrum of plasma and endosome membranes and far more efficiently from the plasma membrane than the endosome. Our observations also indicate that the pronounced variation in content between different exosome-sized vesicles is an inevitable consequence of a stochastic mechanism of small vesicle biogenesis, that the origin membrane of exosome-sized extracellular vesicles simply cannot be determined, and that most of what we currently know about exosomes has likely come from studies of plasma membrane-derived vesicles.

Fibroblastic reticular cell response to dendritic cells requires coordinated activity of podoplanin, CD44 and CD9.

In adaptive immunity, CLEC-2+ dendritic cells (DCs) contact fibroblastic reticular cells (FRCs) inhibiting podoplanin-dependent actomyosin contractility, permitting FRC spreading and lymph node expansion. The molecular mechanisms controlling lymph node remodelling are incompletely understood. We asked how podoplanin is regulated on FRCs in the early phase of lymph node expansion, and which other proteins are required for the FRC response to DCs. We find that podoplanin and its partner proteins CD44 and CD9 are differentially expressed by specific lymph node stromal populations in vivo, and their expression in FRCs is coregulated by CLEC-2 (encoded by CLEC1B). Both CD44 and CD9 suppress podoplanin-dependent contractility. We find that beyond contractility, podoplanin is required for FRC polarity and alignment. Independently of podoplanin, CD44 and CD9 affect FRC-FRC interactions. Furthermore, our data show that remodelling of the FRC cytoskeleton in response to DCs is a two-step process requiring podoplanin partner proteins CD44 and CD9. Firstly, CLEC-2 and podoplanin binding inhibits FRC contractility, and, secondly, FRCs form protrusions and spread, which requires both CD44 and CD9. Together, we show a multi-faceted FRC response to DCs, which requires CD44 and CD9 in addition to podoplanin.

Unveiling a novel function of CD9 in surface compartmentalization of oocytes.

Gamete fusion is an indispensable process for bearing offspring. In mammals, sperm IZUMO1-oocyte JUNO recognition essentially carries out the primary step of this process. In oocytes, CD9 is also known to play a crucial role in gamete fusion. In particular, microvilli biogenesis through CD9 involvement appears to be a key event for successful gamete fusion, because CD9-disrupted oocytes produce short and sparse microvillous structures, resulting in almost no fusion ability with spermatozoa. In order to determine how CD9 and JUNO cooperate in gamete fusion, we analyzed the molecular profiles of each molecule in CD9- and JUNO-disrupted oocytes. Consequently, we found that CD9 is crucial for the exclusion of GPI-anchored proteins, such as JUNO and CD55, from the cortical actin cap region, suggesting strict molecular organization of the unique surface of this region. Through distinct surface compartmentalization due to CD9 governing, GPI-anchored proteins are confined to the appropriate fusion site of the oocyte.

Fluctuation of CD9/SOX2-positive cell populations during the turnover of GH- and TSH-producing cells in the adult anterior pituitary gland.

The adenohypophysis is comprised of the anterior and intermediate lobes (AL and IL, respectively). Cluster of differentiation 9 (CD9)- and sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor hormone-producing cells in the AL. They are located in the marginal cell layer (MCL) facing Rathke's cleft between the AL and IL (primary niche) and the parenchyma of the AL (secondary niche). We previously showed that, in rats, CD9/SOX2-positive cells in the IL side of the MCL (IL-side MCL) migrate to the AL side (AL-side MCL) and differentiate into prolactin-producing cells (PRL cells) in the AL parenchyma during pregnancy, lactation, and diethylstilbestrol treatment, all of which increase PRL cell turnover. This study examined the changes in CD9/SOX2-positive stem/progenitor cell niches and their proportions by manipulating the turnover of growth hormone (GH)- and thyroid-stimulating hormone (TSH)-producing cells (GH and TSH cells, respectively), which are Pit1 lineage cells, as well as PRL cells. After induction, the isolated CD9/SOX2-positive cells from the IL-side MCL formed spheres and differentiated into GH and TSH cells. We also observed an increased GH cell proportion upon treatment with GH-releasing hormone and recovery from continuous stress and an increased TSH cell proportion upon propylthiouracil treatment, concomitant with alterations in the proportion of CD9/SOX2-positive cells in the primary and secondary niches. These findings suggest that CD9/SOX2-positive cells have the potential to supply GH and TSH when an increase in GH and TSH cell populations is required in the adult pituitary gland.

Study on the expression patterns and function of JUNO and CD9 in bovine oocytes during in vitro maturation.

Fertilization proteins JUNO and CD9 play vital roles in sperm-egg fusion, but little is known about their expression patterns during in vitro maturation (IVM) and their function during in vitro fertilization (IVF) of bovine oocytes. In this study, qRT-PCR and immunofluorescence staining were used to detect the mRNA and protein expression levels of JUNO and CD9 genes in bovine oocytes and cumulus cells. Then, fertilization rate of MII oocytes treated with (i) JUNO antibody (1, 5 and 25 µg/ml) or (ii) CD9 antibody (1, 5 and 25 µg/ml) or (iii) CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) were recorded. Our results showed that the mRNA and protein expression levels of JUNO and CD9 genes significantly increased from bovine GV oocytes to MII oocytes, and similar mRNA expression patterns of JUNO and CD9 were also detected in cumulus cells. All groups of oocytes treated with CD9 antibody or JUNO antibody showed significantly decreased fertilization rates (p < .05). Particularly, the fertilization ability of oocytes treated with CD9 antibody (5 µg/ml) + JUNO antibody (5 µg/ml) sharply decreased to 3.48 ± 0.11%. In conclusion, our study revealed the expression levels of JUNO and CD9 genes in oocytes and cumulus cells increased during IVM of bovine oocytes, with JUNO protein playing a major role in the fertilization of bovine oocytes.

A bioorthogonal chemical reporter for fatty acid synthase-dependent protein acylation.

Mammalian cells acquire fatty acids (FAs) from dietary sources or via de novo palmitate production by fatty acid synthase (FASN). Although most cells express FASN at low levels, it is upregulated in cancers of the breast, prostate, and liver, among others, and is required during the replication of many viruses, such as dengue virus, hepatitis C, HIV-1, hepatitis B, and severe acute respiratory syndrome coronavirus 2, among others. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo FA synthesis contributes to host or viral protein acylation has been traditionally difficult to study. Here, we describe a cell-permeable and click chemistry-compatible alkynyl acetate analog (alkynyl acetic acid or 5-hexynoic acid [Alk-4]) that functions as a reporter of FASN-dependent protein acylation. In an FASN-dependent manner, Alk-4 selectively labels the cellular protein interferon-induced transmembrane protein 3 at its known palmitoylation sites, a process that is essential for the antiviral activity of the protein, and the HIV-1 matrix protein at its known myristoylation site, a process that is required for membrane targeting and particle assembly. Alk-4 metabolic labeling also enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.

How tetraspanin-mediated cell entry of SARS-CoV-2 can dysregulate the shedding of the ACE2 receptor by ADAM17.

COVID-19, the respiratory infection caused by the novel coronavirus SARS-CoV-2, presents a clinical picture consistent with the dysregulation of many of the pathways mediated by the metalloprotease ADAM17. ADAM17 is a sheddase that plays a key role in the modulation of ACE2, the receptor which also functions as the point of attachment leading to cell entry by the virus. This work investigates the possibility that ADAM17 dysregulation and attachment of the SARS-CoV-2 virion to the ACE2 receptor are linked events, with the latter causing the former. Tetraspanins, the transmembrane proteins that function as scaffolds for the construction of viral entry platforms, are mooted as key components in this connection.

The multiciliated cells in Rathke's cleft express CYP26A1 and respond to retinoic acid in the pituitary.

The adenohypophysis consists of the anterior and intermediate lobes (AL and IL). The marginal cell layer (MCL), including the ventral region of the IL and the dorsal region of the AL lining the Rathke's cleft, acts as the primary stem/progenitor cell niches in adult adenohypophysis. The cells of the MCL on the IL side consisted of cluster of differentiation 9 (CD9)-positive stem/progenitor cells with or without motile cilia. However, any additional cellular properties of multiciliated CD9-positive cells are not known. The present study aimed to identify the character of the multiciliated cells in stem cell niche of the pituitary gland. We observed the fine structure of the multiciliated cells in the MCL of male Wistar rats at an early stage after birth and in adulthood (P60) using scanning electron microscopy. Since the previous study showed that the MCL cells of adult rats synthesize retinoic acid (RA), the present study determined whether the multiciliated cells are involved in RA regulation by the expression of retinal aldehyde dehydrogenase 1 (RALDH1) and CYP26A1, an enzyme synthesizing and degrading RA, respectively. Results showed that 96% of multiciliated cells in adult male rats expressed CYP26A1, while 60% expressed RALDH1. Furthermore, the isolated CD9-positive cells from the IL side MCL responded to RA and activated the degradation system of RA by increasing Cyp26a1 expression. These findings indicated that multiciliated cells are involved in RA metabolism in the MCL. Our observations provide novel insights regarding the stem cell niche of the adult pituitary.

IKKß increases neuropilin-2 and promotes the inhibitory function of CD9+ Bregs to control allergic diseases.

Regulatory B cells (Bregs) potently suppress immune disorders, including allergic contact hypersensitivity (CHS). IKKß overactivation is prominent in various inflammatory diseases. However, its effect on Bregs has not been defined. This study is to investigate the new regulator and inhibitory mechanism of Bregs. IkkßC46A transgenic mice with a Cys46 mutation, resulting in increased IKKß activation, were employed for analysis. IL-10-competent CD9+ Bregs were expanded in IkkßC46A mice and B cell specific-IkkßC46A mutation mice. IkkßC46A mutant CD9+ Bregs had stronger suppressive effects on CD4+ and CD8+ T cells in vitro and CHS responses in vivo. The inhibitory CD9+ Bregs from IkkßC46A mice were characterized by upregulated Neuropilin 2 (Nrp2) and IL-10 in comparison with that of Ikkßwt mice. Interestingly, increased expression of Nrp2 was observed in CD9+ Bregs compared with that of CD9- B cells in wild-type mice. The suppressive activity of wild-type CD9+ Bregs in vitro was attenuated by inhibition of Nrp2 on Bregs or silencing its ligand Sema3f on CD4+ T cells. Our findings delineate a distinct role of IKKß activation in enhancing Bregs to disturb the immune balance. It identifies Nrp2 as a novel regulatory molecule of Bregs that partly contributes to B cell-mediated immune tolerance.

Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary.

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke's cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.

Neoadjuvant chemotherapy endows CD9 with prognostic value that differs between tumor and stromal areas in patients with pancreatic cancer.

BACKGROUND: The selective pressure imposed by chemotherapy creates a barrier to tumor eradication and an opportunity for metastasis and recurrence. As a newly discovered stemness marker of pancreatic ductal adenocarcinoma (PDAC), the impact of CD9 on tumor progression and patient's prognosis remain controversial. METHODS: A total of 179 and 211 PDAC patients who underwent surgical resection with or without neoadjuvant chemotherapy, respectively, were recruited for immunohistochemical analyses of CD9 expression in both tumor and stromal areas prior to statistical analyses to determine the prognostic impact and predictive accuracy of CD9. RESULTS: The relationship between CD9 and prognostic indicators was not significant in the non-neoadjuvant group. Nevertheless, CD9 expression in both tumor (T-CD9) and stromal areas (S-CD9) was significantly correlated with the clinicopathological features in the neoadjuvant group. High levels of T-CD9 were significantly associated with worse OS (p = 0.005) and RFS (p = 0.007), while positive S-CD9 showed the opposite results (OS: p = 0.024; RFS: p = 0.008). Cox regression analyses identified CD9 in both areas as an independent prognostic factor. The T&S-CD9 risk-level system was used to stratify patients with different survival levels. The combination of T&S-CD9 risk level and TNM stage were accurate predictors of OS (C-index: 0.676; AIC: 512.51) and RFS (C-index: 0.680; AIC: 519.53). The calibration curve of the nomogram composed of the combined parameters showed excellent predictive consistency for 1-year RFS. These results were verified using a validation cohort. CONCLUSION: Neoadjuvant chemotherapy endows CD9 with a significant prognostic value that differs between tumor and stromal areas in patients with pancreatic cancer.