Development of an in vitro system for screening the ligands of a membrane glycoprotein CD36

It has well been known that human and rodents exhibit a preference for fats. This suggests the existence of an orosensory system responsible for the detection of dietary fats. A plasma membrane glycoprotein CD36, besides the role in the uptake of long-chain fatty acids (LCFAs) as well as oxidized low-density lipoprotein (OxLDL) in a variety of cells, has been postulated to be a candidate fat taste receptor on the tongue. Therefore, molecules that bind with CD36 to cause intracellular signaling but have fewer calories could be substitutes for dietary fats. In the present study, we developed an in vitro system for the screening of CD36 ligands using Chinese hamster ovary-K1 cells (CHO-K1) stably transfected with human or mouse CD36. When incubated with OxLDL labeled with fluorescence dye, the fluorescence was much higher in the transfected CHO-K1 cells than in non-transfected CHO-K1 cells. Incubation of the transfected cells with OxLDL caused a rapid phosphorylation of extracellular signal regulated kinase, and the degree was significantly higher compared with that in non-transfected CHO-K1 cells. The expression system using CHO-K1 cells could be a convenient tool to screen the novel ligands of CD36.     

Silencing CD36 gene expression results in the inhibition of latent-TGF-β1 activation and suppression of silica-induced lung fibrosis in the rat

Background

The biologically active form of transforming growth factor-β1 (TGF-β1) plays a key role in the development of lung fibrosis. CD36 is involved in the transformation of latent TGF-β1 (L-TGF-β1) to active TGF-β1. To clarify the role of CD36 in the development of silica-induced lung fibrosis, a rat silicosis model was used to observe both the inhibition of L-TGF-β1 activation and the antifibrotic effect obtained by lentiviral vector silencing of CD36 expression.

Methods

The rat silicosis model was induced by intratracheal injection of 10 mg silica per rat and CD36 expression was silenced by administration of a lentiviral vector (Lv-shCD36). The inhibition of L-TGF-β1 activation was examined using a CCL-64 mink lung epithelial growth inhibition assay, while determination of hydroxyproline content along with pathological and immunohistochemical examinations were used for observation of the inhibition of silica-induced lung fibrosis.

Results

The lentiviral vector (Lv-shCD36) silenced expression of CD36 in alveolar macrophages (AMs) obtained from bronchoalveolar lavage fluid (BALF) and the activation of L-TGF-β1 in the BALF was inhibited by Lv-shCD36. The hydroxyproline content of silica+Lv-shCD36 treated groups was significantly lower than in other experimental groups. The degree of fibrosis in the silica+Lv-shCD36-treated groups was less than observed in other experimental groups. The expression of collagen I and III in the silica+Lv-shCD36-treated group was significantly lower than in the other experimental groups.

Conclusion

These results indicate that silencing expression of CD36 can result in the inhibition of L-TGF-β1 activation in a rat silicosis model, thus further preventing the development of silica-induced lung fibrosis.     

Profiling of the tetraspanin CD151 web and conspiracy of CD151/integrin β1 complex in the progression of hepatocellular carcinoma

Tetraspanin CD151 has been implicated in metastasis through forming complexes with different molecular partners. In this study, we mapped tetraspanin web proteins centered on CD151, in order to explore the role of CD151 complexes in the progression of hepatocellular carcinoma (HCC). Immunoprecipitation was used to isolate tetraspanin complexes from HCCLM3 cells using a CD151 antibody, and associated proteins were identified by mass spectrometry. The interaction of CD151 and its molecular partners, and their roles in invasiveness and metastasis of HCC cells were assayed through disruption of the CD151 network. Finally, the clinical implication of CD151 complexes in HCC patients was also examined. In this study, we identified 58 proteins, characterized the tetraspanin CD151 web, and chose integrin β1 as a main partner to further investigate. When the CD151/integrin β1 complex in HCC cells was disrupted, migration, invasiveness, secretion of matrix metalloproteinase 9, and metastasis were markedly influenced. However, both CD151 and integrin β1 expression were untouched. HCC patients with high expression of CD151/integrin β1 complex had the poorest prognosis of the whole cohort of patients. Together, our data show that CD151 acts as an important player in the progression of HCC in an integrin β1-dependent manner.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

CD36与脂类味觉感受

CD36是一种脂类结合蛋白,在大鼠和小鼠的舌上皮细胞中均被发现,与长链脂肪酸（LCFA）具有高度亲和力,是啮齿类动物感受脂类的重要因子之一。研究表明这种蛋白质受体在口腔的脂类感受中起着至关重要的作用。CD36位于啮齿类口腔味蕾部分神经感受细胞的顶端部分,任轮廓状乳头中有特别高的表达,在叶状乳头中有少量表达,而在菌状乳头中几乎没有表达。     

CD36 expression and brain function: does CD36 deficiency impact learning ability?

This article first presents an overview of published literature documenting the role of the scavenger receptor CD36 in activation of brain microglia with reference to brain pathologies such as Alzheimer's and malaria. Second, the possibility that CD36 may play a role in brain FA metabolism is discussed. Long-chain polyunsaturated fatty acids (PUFAs) are important for brain function and are mostly derived from the plasma. Based on its role in facilitating FA uptake in several tissues and cell types, CD36 expressed on microvascular endothelial cells in the brain may facilitate local uptake of PUFAs. Alternatively, CD36 may influence brain FA supply indirectly via impacting utilization of dietary FA or their metabolism in tissues such as the liver. We examined the possibility that CD36 expression impacts brain function by evaluating the behavior of CD36 null mice using a battery of standard tests. Our data indicate that CD36 deficient mice have normal patterns of activity, anxiety and exploration of novel environments. However they appear to have a significant impairment in learning ability. These findings could provide a new perspective regarding the regulation of brain lipid metabolism.     

Expression of Estrogen Receptor α 36 (ESR36) in the Hamster Ovary throughout the Estrous Cycle: Effects of Gonadotropins

Estradiol-17β (E) plays an important role in ovarian follicular development. Evidence indicates that some of the effect of E is mediated by the transmembrane estrogen receptor. In this study, we examined the spatio-temporal expression of recently discovered ERα36 (ESR36), a splice variant of Esr1 and a receptor for non-genomic E signaling, in the hamster ovary during the estrous cycle and the role of gonadotropins and ovarian steroid hormones in ESR36 expression. ESR36 expression was high on estrus (D1∶0900 h) and declined precipitously by proestrus (D4∶0900 h) and remained low up to D4∶1600 h. Immunofluorescence findings corroborated immunoblot findings and revealed that ESR36 was expressed only in the cell membrane of both follicular and non-follicular cells, except the oocytes. Ovarian ESR36 was capable of binding to the E-affinity matrix, and have different molecular weight than that of the ESR1 or GPER. Hypophysectomy (Hx) resulted in a marked decline in ESR36 protein levels. FSH and LH, alone or combined, markedly upregulated ESR36 protein in Hx hamsters to the levels observed in D1 hamsters, but neither E nor P had any effect. Inhibition of the gonadotropin surge by phenobarbital treatment on D4∶1100 h attenuated ESR36 expression in D1∶0900 h ovaries, but the decline was restored by either FSH or LH replacement on D4 afternoon. This is the first report to show that ESR36, which is distinct from ESR1 or GPER is expressed in the plasma membrane of ovarian follicular and non-follicular cells, binds to E and its expression is regulated directly by the gonadotropins. In light of our previous findings, the results suggest that ovarian cells contain at least two distinct membrane estrogen receptors, such as GPER and ESR36, and strongly suggest for a non-genomic action of E regulating ovarian follicular functions.     

Roles of the Adenosine Receptor and CD73 in the Regulatory Effect of γδ T Cells

The adenosine A2A receptor (A2AR), the main functional adenosine receptor on murine T cells, plays a unique role in the attenuation of inflammation and tissue damage in vivo. Here, we showed that, of the immune cell types tested, activated γδ T cells expressed the highest levels of A2AR mRNA and that A2AR ligation inhibited αβ T cell activation, but enhanced γδ T cell activation. We also showed that the inhibitory effect of an adenosine receptor agonist on autoreactive T cells was prevented by addition of a low percentage of activated γδ T cells. Furthermore, compared to resting cells, activated γδ T cells expressed significantly lower levels of CD73, an enzyme involved in the generation of extracellular adenosine. Exogenous AMP had a significant inhibitory effect on autoreactive T cell responses, but only in the presence of CD73⁺ γδ T cells, and this effect was abolished by a CD73 inhibitor. Our results show that expression of increased amounts of A2AR allows γδ T cells to bind adenosine and thereby attenuate its suppressive effect, while decreased expression of CD73 results in less generation of adenosine in the inflammatory site. Together, these events allow activated γδ T cells to acquire increased proinflammatory activity, leading to augmented autoimmune responses.     

The Gene Expression Profile of CD11c+CD8α? Dendritic Cells in the Pre-Diabetic Pancreas of the NOD Mouse

Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c⁺CD8α⁻ DCs (strong CD4+ T cell proliferation inducers) and the CD8α⁺CD103⁺ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c⁺CD8α⁻ DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c⁺CD8α⁻ DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c⁺CD8α⁻ DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c⁺CD8α⁻ DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c⁺CD8α⁻ DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.     

oxHDL decreases the expression of CD36 on human macrophages through PPARγ and p38 MAP kinase dependent mechanisms

CD36, belongs to class B scavenger receptor family, is a macrophage receptor for oxidized low-density lipoprotein (oxLDL) and has been proven to play a critical role in atherosclerotic foam cell formation. In addition, CD36 expression is regulated by many factors including oxLDL and HDL. A recent study suggests that CD36 can also bind with oxidized high-density lipoprotein (oxHDL). However, the direct role of oxHDL in atherosclerosis is still not clear and it is not known whether oxHDL has any influence on the expression of CD36 in macrophages. Here, we performed experiments to investigate the effect of oxHDL on the expression of CD36 on human peripheral blood monocytes–macrophages and the possible mechanisms. Our results suggest that the uptake of oxHDL by CD36 on macrophages accelerates foam cell formation. In addition, oxHDL can down-regulate both the mRNA and surface protein expression of CD36 on human peripheral macrophages in vitro. oxHDL increased the mRNA expression and protein phosphorylation of peroxisome proliferators-activated receptor-γ (PPARγ). Using different mitogen-activated protein kinase (MAPK) inhibitors, we demonstrated that oxHDL regulated CD36 and PPARγ expression in a p38-MAP kinase dependent mechanism.     

Chromosomal locations of the gene coding for the CD3 (T3) γ subunit of the human and mouse CD3/T-cell antigen receptor complexes

The gene coding for the M _r 26000 chain of the human CD3 (T3) antigen/T-cell antigen receptor complex was mapped to chromosome band 11q23 by using a cDNA clone (pJ6T3 -2), by in situ hybridization to metaphase chromosomes and by Southern blot analysis of a panel of human-rodent somatic cell hybrids. The mouse homolog, here termed Cdg-3, was mapped to chromosome 9 using the mouse cDNA clone pB10.AT3 -1 and a panel of mouse-hamster somatic cell hybrids. Similar locations for the CD3 genes have been described previously. Thus, the corporate results indicate that the CD3 and genes have remained together since they duplicated about 200 million years ago.     

TNFα regulates the localization of CD40 in lipid rafts of glioma cells

Resistance of glioblastoma multiforme (GBM) to TNFα induced apoptosis is attributed to NFκB activation. As TNF-receptor family member CD40 regulates NFκB activation, we investigated the role of CD40 in NFκB activation in GBM. We observed elevated CD40 levels in human glioma samples as compared to the surrounding normal tissue. Treatment with TNFα elevated CD40 levels in glioma cells and inhibition of CD40 signaling failed to abrogate TNFα induced NFκΒ activity. While TNFα increased the interaction between TRAF2/6, IκBα, IKKα/β in the CD40 signalosome, the level of CD40 in the signalosome remained unaffected upon TNFα treatment. Interestingly, TNFα decreased the spatial localization of CD40 and increased TRAF2/6 co-localization with lipid raft marker Caveolin. As localization of CD40 signalosome in lipid raft is crucial for NFκB activation, TNFα mediated decreased clustering of CD40 in lipid rafts could have possibly contributed to its non-involvement in NFκB activation.     

Photo effect on the CD1d-binding ability of azobenzene-attached analogues of α-GalCer

α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states.     

Advances in the understanding of the structure and function of ER-α36,a novel variant of human estrogen receptor-alpha

Estrogen receptors (ERs) belong to the nuclear receptor superfamily, whose members include ER-α66, ER-α36, ER-α46 and ER-β. Each receptor performs specific functions through binding with a specific ligand, such as estrogen. Recently, ER-α36, a novel variant of human estrogen receptor-alpha (ER-α), was identified and cloned. ER-α36 inhibits, in a dominant-negative manner, the transactivation of both the wild-type ER-α (ER-α66) and ER-β. As a predominantly membrane-based ER, ER-α36 mediates nongenomic estrogen signaling and is involved in the resistance of breast cancer to endocrine therapy, i.e., tamoxifen. This review summarizes recent studies on the structure and function of ER-α36 and the relationship of ER-α36 with cancer, with special emphasis on its function in the resistance of breast cancer to endocrine therapy.     

Assignment of the gene encoding the catalytic subunit Cβ of CAMP-dependent protein kinase to the p36 band on chromosome

Summary A cDNA for the human catalytic subunit (C) of cAMP-dependent protein kinase (PKA) has been cloned from a testis cDNA library. In the present study, we have determined the chromosomal localization of this gene using a cDNA for C as a probe. Southern blot analysis of genomic DNA from human/mouse cell hybrids revealed that the presence or absence of a 20-kbXbaI fragment, which hybridized with the C probe, was concordant with the presence of human chromosome 1.In situ hybridization to metaphase chromosome confirmed the somatic cell hybrid data and regionally mapped the C gene of PKA to the p36 band on chromosome 1.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

Interleukin-21 maintains the expression of CD16 on monocytes via the production of IL-10 by human naïve CD4+ T cells

Interleukin 21 exerts a variety of regulatory effects on both innate and adaptive immune cells. Although the suppressive effect of IL-21 via the induction of IL-10 in mouse model has been defined, the inhibitory effect of IL-21 in humans is not well understood. In the present study, we showed that IL-21 induced IL-10 production by human naive CD4⁺ T cells. Most of the IL-10-producing CD4⁺ T cells did not co-express IFN-γ. IL-21 increased the expression of IL-21R on activated naïve CD4⁺ T cells. Further analysis indicated that IL-21 induced phosphorylation of STAT1, STAT3 and STAT5 in activated naïve CD4⁺ T cells. In addition, IL-21 maintained the expression of CD16 on monocytes via the production of IL-10 by human naïve CD4⁺ T cells. Taken together, our data indicated that IL-21 had a modulating effect on monocytes at least in part by inducing IL-10 production.