Expression of 67-Kda Laminin Receptor (67LR) Relates to the Aggressiveness and Poor Prognosis of AML: Modulation of the Expression of GM-CSF Receptor by 67LR.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 926-926
Author(s):  
Koji Ando ◽  
Yasushi Miyazaki ◽  
Daisuke Imanishi ◽  
Masako Iwanaga ◽  
Hideki Tsushima ◽  
...  
Keyword(s):  
Cell Surface ◽  
Poor Prognosis ◽  
Surface Expression ◽  
Laminin Receptor ◽  
Cell Surface Receptor ◽  
High Expression ◽  
Clinical Samples ◽  
Α Subunit ◽  
Gm Csf ◽  
Significant Difference

Abstract Background: The 67-kDa laminin receptor (67LR) is a non-integrin cell surface receptor for laminin, one of the major components of extracellular matrix. Previously (at ASH 2007, PS 2845), we reported that the high expression of 67LR on CD34+AML cells was related with a poor prognosis of AML, and that the forced expression of 67LR resulted in the enhanced proliferation and the resistance against apoptosis of leukemia cells by increasing phosphorylated STAT5. However, there was no information about how the expression of 67LR modulated the signaling of STAT5 pathway. Objective: To elucidate how 67LR enhances the signaling of STAT5 pathway and the role of 67LR in clinical course of AML. Methods: The cDNA or short interfering RNA for LR cloned into various kinds of plasmids was expressed in TF-1 and AML193 leukemia cell lines via stable transfection. As overexpressors of LR, TF-1LR and AML193LR were generated, and as a suppressor of LR, TF-1si. Because it is well known that the signaling of STAT5 pathway was enhancing by GM-CSF signaling, the expression of the GM-CSF receptor α subunit (GM-CSFRα) was analyzed by flowcytometry. To test the interaction between 67LR with V5-tag and GM-CSFRα with Flag-tag protein, we transiently transfected expression plasmids containing cDNA for these protein into human 293T cells. Lysates of these transfected cells were subjected to the immunoprecipitation using anti-V5 or anti-Flag Ab, followed by immunoblotting with HRP conjugated antibodies. From 44 AML patients (M0[2], M1[4], M2[18], M4[11], M5[3], M6[3], MDS/AML[3]), CD34-positive cells were isolated by column method to examine the surface expression of 67LR with flowcytometry. To assess the clinical significance of 67LR expression, 44 pattients with AML were divided into two groups by the surface-expression level of 67LR: 20 cases in the high-expression group (positive in >25% of cells, LR-H) and 24 cases in the low-expression group (LRL). Clinical factors including WBC, LDH, and overall survival were compared between two groups. Results: The surface expression of 67LR was 64% in parental TF-1, 92% in TF-1LR, and 36% in TF-1si, and that was 65% in AML193 and 91% in AML193LR. The surface expression of GM-CSFRα was lower in TF-1si (MFI: 5.1) than its control (MFI:7.3), whereas that was higher in TF-1LR (MFI:19.2) than its control (MFI:7.3). In AML193LR, GM-CSFRα was also higher than the control (MFI:34.7 and 18.9, respectively). These results demonstrated that the modulation of 67LR expression contributed to changes in the level of GM-CSFRα on cell surface. Immunoprecipitation assays indicated that 67LR and GM-CSFRα protein were present in the same protein complex in vivo. In clinical samples, median surface expression of 67LR on CD34 positive AML cells was 17%, and the median intensity of GM-CSFRα was 5.8. We found a significant positive relationship between the surface expression of 67LR and the median intensity of GM-CSFRα on CD34 positive AML cells (chi-square value was 0.04). WBC counts and LDH levels at diagnosis were significantly higher in LR-H group than that in LH-L (p=0.04, p=0.02, respectively). There was a significant difference in survival between LR-L and LR-H groups (median survival 803 and 239 days, respectively, p=0.009). Conclusion: We found the level of 67LR could modulate the expression of GM-CSFRα, thereby it might enhance the phosphorylation of STAT5. Among AML patients, high level of 67LR expression was related to the higher WBC count, elevated LDH and shorter survival. The expression of 67LR and GM-CSFRα in clinical samples also showed significant correlation in amount. These data suggested that the high expression of 67LR resulted in the proliferation of AML cells by increasing the expression of GM-CSFRα. These features could contribute, at least in part, to a poor prognosis of AML.

Expression of the 67-kDa Laminin Receptor Is Associated with Increased AML Cells through the Enhanced GM-CSF/Stat5 Signaling.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2845-2845
Author(s):  
Koji Ando ◽  
Yasushi Miyazaki ◽  
Yasushi Sawayama ◽  
Kensuke Horio ◽  
Emi Matsuo ◽  
...  
Keyword(s):  
Cell Growth ◽  
Molecular Mechanisms ◽  
Hematopoietic Cells ◽  
Leukemia Cell ◽  
Surface Expression ◽  
Laminin Receptor ◽  
Cell Surface Receptor ◽  
Leukemia Cell Line ◽  
Clinical Samples ◽  
Gm Csf

Abstract [Background] The 67-kDa laminin receptor (67LR) is a non-integrin cell surface receptor that mediates high-affinity interactions with laminin. Although 67LR was reported to interact with GM-CSF receptor (GM-CSFR) modulating the signaling pathway in hematopoietic cells, little report regarding its expression and the characteristics of AML. [Objective] We addressed whether the surface expression of 67LR be related to the characteristics of AML, and its molecular mechanisms. [Methods] The surface expression of 67LR on CD34-positive AML cells was assessed since it is widely expressed on the mature hematopoietic cells. After purification of CD34 positive cells, flowcytometric analysis of 67LR in 27 AML (M0 [1], M1 [3], M2 [11], M4 [6], M5 [2], M6 [2], MDS/AML [2]) was performed. A GM-CSF dependent TF-1 leukemia cell line (established from M6) was used for the in vitro analysis. cDNA or short interfering RNA of LR was transfected into TF-1 (TF-1LR and TF-1si). Using these cell lines, cell growth, colony formation, cell cycle distribution, and protein phosphorylation were tested. [Results] AML cases were divided into two groups by the surface-expression of 67LR: 9cases in the high-expression group (positive in >25% of cells, LR-H) and 18 in the low-expression group (LR-L).The median WBC was significantly higher in LR-H than LH-L (36,700 and 3,250/μl, respectively, p=0.015). Nucleated cell count of bone marrow was also higher in LR-H (p=0.01). Overall survival of LR-H was significantly poor than LR-L (20% and 54%, respectively, p=0.01). Since the expression of 67LR was related to the increased AML cells in clinical samples, we hypothesized that the expression of 67LR influenced the growth of leukemia cells. Using TF-1, the surface-expression of 67LR was modulated by the over expression or interfering of mRNA of 67LR. The surface expression of 67LR on wild type TF-1, TF-1LR and TF-1si was 64%, 92% and 36%, respectively. In WST1 assay, the expression of 67LR was related to the proliferation of TF-1 cells: TF-1LR proliferated rapidly than control (absorbance 1.71±0.06 and 1.24±0.10, respectively. p=0.0023), whereas TF-1si cells grew slowly (absorbance of TF-1si, 0.790±0.004; control, 1.149±0.052, p=0.0018). The number of colony in semi-solid media was related to the expression of 67LR: TF-1LR formed 300±10, the control cells made 91±8colonies/5000cells (p<0.001), and TF-1si made less colonies than control (46±3 and 76±6 colonies, respectively, p=0.002). The cells in S-phase increased in TF-1LR than control by BrdU assay (38±1% and 32±2%, respectively, p=0.01). These results demonstrated the surface expression of 67LR was associated with the enhanced cell growth in TF-1. Since 67LR was reported to interact with GM-CSFR, we tested the signaling though GM-CSFR. After 16 hours of serum-free culture, cells were treated with GM-CSF and FBS. The phosphorylation of both ERK1/2 and Stat5 was diminished in TF-1si than control. In contrast, that of Stat5 increased in TF-1LR than control (MFI of phspho-Stat5, 265±39 and 158±54, respectively, p=0.05). [Conclusion] These results suggested that the surface expression of 67LR contributed, at least, to the proliferation of AML through the enhancing the signaling of GM-CSF/Stat5 pathway. This may relate to the poor prognosis of LR-H group.

Biochemical characterization of PRV-1, a novel hematopoietic cell surface receptor, which is overexpressed in polycythemia rubra vera

Blood ◽  
2002 ◽  
Vol 100 (7) ◽  
pp. 2441-2448 ◽  
Author(s):  
Steffen Klippel ◽  
Elisabeth Strunck ◽  
Christian E. Busse ◽  
Dirk Behringer ◽  
Heike L. Pahl
Keyword(s):  
Cell Surface ◽  
Polycythemia Vera ◽  
Biochemical Characterization ◽  
Mrna Level ◽  
Alternative Polyadenylation ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Rich Domain ◽  
Significant Difference ◽  
Polycythemia Rubra Vera

The cDNA for polycythemia rubra vera 1 (PRV-1), a novel hematopoietic receptor, was recently cloned by virtue of its overexpression in patients with polycythemia vera. PRV-1 is a member of the uPAR/CD59/Ly6 family of cell surface receptors, which share a common cysteine-rich domain and are tethered to the cell surface via a glycosylphosphatidylinositol (GPI) link. We have determined the intron-exon structure of the PRV1gene and show that the locus is structurally intact in patients with polycythemia vera. Thus, PRV-1 overexpression in these patients is not due to rearrangement or structural alteration of the gene. Northern blot analysis detects multiple PRV-1 transcripts. Here we show that these transcripts arise from alternative polyadenylation and encode the same protein. Biochemical analysis reveals that PRV-1 isN-glycosylated and embedded in the cell membrane by a lipid anchor, like other members of this family. Moreover, PRV-1 is shed from the cell surface because soluble protein can be detected in cell supernatants. Fluorescence-activated cell sorting analysis of stably transfected cells revealed that PRV-1 is recognized by antibodies directed against the neutrophil antigen NB1/CD177. Flow cytometry of bone marrow and peripheral blood of both healthy donors and patients with polycythemia vera showed that PRV-1 protein is expressed on myeloid cells of the granulocytic lineage. However, unlike the significant difference in PRV-1 expression observed on the mRNA level, the amount of PRV-1 protein on the cell surface is not consistently elevated in patients with polycythemia vera compared with healthy controls. Therefore, quantification of PRV-1 surface expression cannot be used for the diagnosis of polycythemia vera.

scholarly journals Dectin-1-Mediated Production of Pro-Inflammatory Cytokines Induced by Yeast β-Glucans in Bovine Monocytes

2021 ◽  
Vol 12 ◽  
Author(s):  
Ana R. V. Pedro ◽  
Tânia Lima ◽  
Ricardo Fróis-Martins ◽  
Bárbara Leal ◽  
Isabel C. Ramos ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Inflammatory Cytokines ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Dependent Manner ◽  
Feed Supplements ◽  
Surface Receptor ◽  
Dose Dependent ◽  
Pro Inflammatory Cytokines

Yeast-derived products containing β-glucans have long been used as feed supplements in domesticated animals in an attempt to increase immunity. β-glucans are mainly recognized by the cell surface receptor CLEC7A, also designated Dectin-1. Although the immune mechanisms elicited through Dectin-1 activation have been studied in detail in mice and humans, they are poorly understood in other species. Here, we evaluated the response of bovine monocytes to soluble and particulate purified β-glucans, and also to Zymosan. Our results show that particulate, but not soluble β-glucans, can upregulate the surface expression of costimulatory molecules CD80 and CD86 on bovine monocytes. In addition, stimulated cells increased production of IL-8 and of TNF, IL1B, and IL6 mRNA expression, in a dose-dependent manner, which correlated positively with CLEC7A gene expression. Production of IL-8 and TNF expression decreased significantly after CLEC7A knockdown using two different pairs of siRNAs. Overall, we demonstrated here that bovine monocytes respond to particulate β-glucans, through Dectin-1, by increasing the expression of pro-inflammatory cytokines. Our data support further studies in cattle on the induction of trained immunity using dietary β-glucans.

Effect of aging on CD11b and CD69 surface expression by vesicular insertion in human polymorphonuclear leucocytes

1999 ◽  
Vol 97 (3) ◽  
pp. 323-329 ◽  
Author(s):  
J. M. NOBLE ◽  
G. A. FORD ◽  
T. H. THOMAS
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Elderly Subjects ◽  
Polymorphonuclear Leucocytes ◽  
Cd11b Expression ◽  
Cd69 Expression ◽  
Vesicle Exocytosis

The exocytosis of intracellular vesicles is an important function of the plasma membrane, which is responsible for hormone secretion, cell surface expression of antigens, ion transporters and receptors, and intracellular and intercellular signalling. Human aging is associated with many physiological and cellular changes, many of which are due to alterations in plasma membrane functioning. Alterations in vesicle externalization with age could account for many of these changes. We investigated whether alterations in vesicle exocytosis occur with increasing age by flow-cytometric determination of CD11b and CD69 expression on the surface of human polymorphonuclear leucocytes (PMN) stimulated with phorbol myristate acetate (PMA), a tumour promoter which binds to and activates protein kinase C (PKC) directly, or with formyl-Met-Leu-Phe (fMLP), which activates PKC indirectly via interactions with a cell surface receptor and G-protein, and subsequent inositol phosphate hydrolysis. Following stimulation with PMA, a decrease in the proportion of PMN expressing CD69 at high levels was observed in elderly compared with young subjects (young, 55.3%; elderly, 43.9%; P = 0.01). No aging-related differences in the proportion of PMN expressing CD11b (young, 73.7%; elderly, 68.4%; P = 0.15), or in the number of molecules of CD69 or CD11b expressed per cell, were observed. Stimulation with fMLP or low PMA concentrations resulted in full CD11b expression but minimal CD69 expression in both young and elderly subjects. Cells which expressed CD69 had no CD11b expression, while those cells expressing CD11b had minimal CD69 expression. Thus the PMA-induced expression of CD11b and CD69 in human PMN represents two separate processes, only one of which is affected in aging. CD11b expression appears to require a lesser degree of PKC stimulation compared with that required for CD69 expression. The age-associated reduction in PMA-stimulated CD69 expression may occur either at or distal to PKC activation. Such a decrease may contribute to the age-associated impairments in PMN function that contribute, in turn, to immunosenescence.

Vaccination with Leukemia Cells Expressing Cell-Surface-Associated GM-CSF Blocks Leukemia Induction in Immunocompetent Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2531-2531
Author(s):  
Xiaoyang Ling ◽  
Yan Wang ◽  
Ralph B. Arlinghaus
Keyword(s):  
Immune System ◽  
Cell Surface ◽  
Immune Tolerance ◽  
Leukemia Cells ◽  
Host Immune System ◽  
Over Expression ◽  
Gm Csf ◽  
Significant Difference ◽  
Specific Antigens ◽  
Immunocompetent Mice

Abstract The fundamental basis for immunotherapy of leukemia is that leukemia cells express specific antigens that are not expressed by normal hematopoietic cells. However, the host immune system appears to be tolerant of leukemia cells. To overcome this immune tolerance, we transduced WEHI-3B mouse monocyte leukemia cells (1) with a transmembrane form of GM-CSF (tmGM-CSF). The tmGM-CSF was constructed using the pDisplay vector for cell-surface targeting (Invitrogen) into the pLOX lentivirus gene transfer vector (2). After infection of WEHI-3B cells with a recombinant lentivirus encoding tmGM-CSF, nearly all the transduced cells expressed tmGM-CSF on the cell-surface, as determined by flow cytometry analysis using anti-GM-CSF. To determine whether vaccination with tmGM-CSF expressing WEHI-3B cells would prevent leukemia formation, immunocompetent BALB/c mice were immunized with lethally-irradiated WEHI-3B cells (106, 3 times 7 day intervals), which express tmGM-CSF, prior to challenging vaccinated mice with WEHI-3B cells (5x104) that express GFP as a marker. 100% of vaccinated mice were protected from leukemia. Non-vaccinated mice succumbed to leukemia within 50–55 days. Vaccination of mice with lethally-irradiated WEHI-3B cells expressing CD40L protected 80% of the mice from leukemia. In contrast, mice immunized with lethally-irradiated WEHI-3B/GFP cells lacking tmGM-CSF were not protected. Mice vaccinated three times at 5,12, 19 days after challenge with WEHI-3B/GFP cells had a significant increase in survival in that 60% of mice were alive and healthy at 16 days (to this date) after all control non-vaccinated mice had died. Similar vaccine studies were performed with BCR-ABL (b3a2)+ 32D cells (106) in immunocompetent C3H/HeJ mice (3). These mice die of leukemia within 35 days. After infection of BCR-ABL+ 32D cells with the lentivirus encoding tmGM-CSF/GFP, tmGM-CSF was expressed on the cell-surface. The C3H/HeJ mice challenged with BCR-ABL+32D/GFP cells (106) showed a significant level of protection by vaccination with lethally-irradiated tmGM-CSF+ 32D BCR-ABL cells (106, 2 times at 7 day intervals); 40% of the vaccinated mice remained healthy; all non-vaccinated mice died of leukemia. There was a significant difference in survival (P=0.03) between the vaccinated and non-vaccinated groups. Interestingly, the spleens of vaccinated C3H/HeJ mice that died of leukemia at the same time as non-vaccinated mice approached normal size whereas non-vaccinated mice had enlarged spleens. Our findings suggest that over-expression of cell-surface tmGM-CSF in leukemia cells can overcome immune tolerance, allowing the immune system to efficiently recognize and destroy the leukemia cells, providing extended survival of vaccinated mice. Because significant protection from death was achieved by vaccination after challenge with leukemia cells, tmGM-CSF expression in leukemia cells has potential as a therapeutic strategy for treatment of leukemia.

Regulation of Hematopoiesis by Cholesterol

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-53-SCI-53 ◽  
Author(s):  
Alan R. Tall
Keyword(s):  
Growth Factor ◽  
Cell Surface ◽  
Cholesterol Efflux ◽  
Surface Expression ◽  
Membrane Lipid ◽  
Cell Surface Expression ◽  
Atp Binding ◽  
Gm Csf ◽  
Atp Binding Cassette Transporters ◽  
Atp Binding Cassette

Abstract Leukocytosis is a risk factor for athero-thrombotic disease in humans, and develops in animal models of atherosclerosis in response to feeding high-fat, high-cholesterol diets. The ATP binding cassette transporters ABCA1 and ABCG1 promote cholesterol efflux to apoA-1 and high density lipoprotein (HDL), respectively and are targets of liver X receptor (LXR) transcription factors. Mice lacking ABCA1/G1 develop a dramatic myeloproliferative phenotype with monocytosis and neutrophilia, associated with expansion and proliferation of hematopoietic stem and myeloid progenitor populations (HSPCs). The transporters are highly expressed in HSPCs where they act to control proliferative responses to growth factors (IL-3, GM-CSF) by regulating plasma membrane lipid rafts and cell surface expression of the common β subunit of the IL-3/GM-CSF receptor. ABCG4 is closely related to ABCG1 but is expressed primarily in the megakaryocyte progenitor (MkP) population of the bone marrow. ABCG4-deficient mice have MkP proliferation and expansion, thrombocytosis, increased platelet/leukocyte aggregates and accelerated atherosclerosis. ABCG4 promotes cholesterol efflux onto HDL, and thereby reduces the cell surface expression of the thrombopoietin (TPO) receptor. This appears to involve membrane cholesterol enrichment and interruption of a negative feedback loop involving the TPO receptor and mediated by Lyn Kinase and c-CBL which mediate ubiquitination and internalization/degradation of the receptor. Overall results suggest that ATP binding cassette transporters promote cholesterol efflux, decrease membrane lipid raft formation and enhance the feedback downregulation of growth factor receptors in response to growth factor binding, with anti-proliferative responses that may be beneficial in atherosclerosis and myeloproliferative neoplasms. Disclosures No relevant conflicts of interest to declare.

scholarly journals Coupling of Osteopontin and Its Cell Surface Receptor CD44 to the Cell Survival Response Elicited by Interleukin-3 or Granulocyte-Macrophage Colony-Stimulating Factor

2000 ◽  
Vol 20 (8) ◽  
pp. 2734-2742 ◽  
Author(s):  
Yi-Hung Lin ◽  
Chang-Jen Huang ◽  
Jyh-Rong Chao ◽  
Shui-Tsung Chen ◽  
Shern-Fwu Lee ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Surface Receptor ◽  
Β Subunit ◽  
Colony Stimulating Factor ◽  
Adhesion Protein ◽  
Gm Csf ◽  
Surface Receptor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

ABSTRACT The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common β subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor β subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.

scholarly journals Granulocyte macrophage colony stimulating factor produced in lesioned peripheral nerves induces the up-regulation of cell surface expression of MAC-2 by macrophages and Schwann cells.

1996 ◽  
Vol 133 (1) ◽  
pp. 159-167 ◽  
Author(s):  
A Saada ◽  
F Reichert ◽  
S Rotshenker
Keyword(s):  
Cell Surface ◽  
Schwann Cells ◽  
Interleukin 1 ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Gm Csf ◽  
Molecular Events ◽  
Macrophage Colony

Peripheral nerve injury is followed by Wallerian degeneration which is characterized by cellular and molecular events that turn the degenerating nerve into a tissue that supports nerve regeneration. One of these is the removal, by phagocytosis, of myelin that contains molecules which inhibit regeneration. We have recently documented that the scavenger macrophage and Schwann cells express the galactose-specific lectin MAC-2 which is significant to myelin phagocytosis. In the present study we provide evidence for a mechanism leading to the augmented expression of cell surface MAC-2. Nerve lesion causes noneuronal cells, primarily fibroblasts, to produce the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF). In turn, GM-CSF induces Schwann cells and macrophages to up-regulate surface expression of MAC-2. The proposed mechanism is based on the following novel observations. GM-CSF mRNA was detected by PCR in in vitro and in vivo degenerating nerves, but not in intact nerves. The GM-CSF molecule was detected by ELISA in medium conditioned by in vitro and in vivo degenerating peripheral nerves as of the 4th h after injury. GM-CSF activity was demonstrated by two independent bioassays, and repressed by activity blocking antibodies. Significant levels of GM-CSF were produced by nerve derived fibroblasts, but neither by Schwann cells nor by nerve derived macrophages. Mouse rGM-CSF enhanced MAC-2 production in nerve explants, and up-regulated cell surface expression of MAC-2 by Schwann cells and macrophages. Interleukin-1 beta up-regulated GM-CSF production thus suggesting that injury induced GM-CSF production may be mediated by interleukin-1 beta. Our findings highlight the fact that fibroblasts, by producing GM-CSF and thereby affecting macrophage and Schwann function, play a significant role in the cascade of molecular events and cellular interactions of Wallerian degeneration.

scholarly journals Human epithelial Na+ channel missense variants identified in the GenSalt study alter channel activity

2016 ◽  
Vol 311 (5) ◽  
pp. F908-F914 ◽  
Author(s):  
Evan C. Ray ◽  
Jingxin Chen ◽  
Tanika N. Kelly ◽  
Jiang He ◽  
L. Lee Hamm ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Channel Activity ◽  
Dietary Salt ◽  
Α Subunit ◽  
Γ Subunit ◽  
Genes Encoding ◽  
Altered Sensitivity

Mutations in genes encoding subunits of the epithelial Na+ channel (ENaC) can cause early onset familial hypertension, demonstrating the importance of this channel in modulating blood pressure. It remains unclear whether other genetic variants resulting in subtler alterations of channel function result in hypertension or altered sensitivity of blood pressure to dietary salt. This study sought to identify functional human ENaC variants to examine how these variants alter channel activity and to explore whether these variants are associated with altered sensitivity of blood pressure to dietary salt. Six-hundred participants of the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study with salt-sensitive or salt-resistant blood pressure underwent sequencing of the genes encoding ENaC subunits. Functional effects of identified variants were examined in a Xenopus oocyte expression system. Variants that increased channel activity included three in the gene encoding the α-subunit (αS115N, αR476W, and αV481M), one in the β-subunit (βS635N), and one in the γ-subunit (γL438Q). One α-subunit variant (αA334T) and one γ-subunit variant (βD31N) decreased channel activity. Several α-subunit extracellular domain variants altered channel inhibition by extracellular Na+ (Na+ self-inhibition). One variant (αA334T) decreased and one (αV481M) increased cell surface expression. Association between these variants and salt sensitivity did not reach statistical significance. This study identifies novel functional human ENaC variants and demonstrates that some variants alter channel cell surface expression and/or Na+ self-inhibition.

scholarly journals The PAR2 signal peptide prevents premature receptor cleavage and activation

2019 ◽  
Author(s):  
Belinda Liu ◽  
Grace Lee ◽  
Jiejun Wu ◽  
Janise Deming ◽  
Chester Kuei ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Synthetic Peptide ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Peptide Ligand ◽  
Protease Activated Receptors ◽  
N Terminus

AbstractUnlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that these is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.

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