Choline Transporter-like Protein Research Articles

The Cs a and Cs b Red Cell Antigens of the Cost Blood Group Collection Correspond to the HNA-3a and HNA-3b Neutrophil Antigens: Unexpected Twins with Implications for Sickle Cell Anemia

Recent genome-wide association and murine studies identified the human neutrophil antigen 3a/b polymorphism (HNA-3a/b) in SLC44A2 (rs2288904-G/A) as a risk factor in venous thromboembolism (VTE). We tested whether this polymorphism associates with clinical features in SCA patients in whose VTE and stroke are frequently encountered complications. Incidentally, we discovered that rare subjects with the homozygous HNA-3b/b genotype (genotype frequency < 2%) also carry the uncommon Cs(a-) red cell phenotype. Despite its discovery more than 50 years ago, the molecular basis of this red cell phenotype and the presumptive protein carrier for the antigens of the COST blood group collection (Cs a and Cs b) has remained unknown. Inspired by this fortuitous discovery, we hypothesized that rs2288904 and COST antigens are closely associated. Thus, we genotyped this SNP in a cohort of 35 Cs(a-) subjects and found that all of them showed a HNA-3b/b genotype, indicating that the high-frequency allele of rs2288904 (HNA-3a; 461G) encoding Arg154 might encode the Cs a antigen (COST1). Interestingly, anti-Cs a does not react with SLC44A2 null RBCs (exceptional VER- blood type) and SLC44A2 knockout K562 cell line, confirming that the COST antigens are carried on this protein. In addition, using a panel of K562 cells expressing HNA-3a or 3b antigens developed by CRISPR/Cas9 genome editing, we confirmed that anti-Cs a recognizes the HNA-3a antigen. Overall, our results confirmed that HNA-3 and COST antigens have the same molecular basis. As a result, the Cs a and Cs b red cell antigens can now be included in the CTL2 blood group system, encoded by the SLC44A2 gene. This new finding could be of medical and biological value because anti-HNA-3a may be responsible for severe cases of neonatal neutropenia and transfusion-related acute lung injury (TRALI). Current testing for HNA-3a antibodies includes the granulocyte agglutination test and granulocyteindirect immunofluorescence test, as recommended by the ISBT Working Party on Granulocyte Immunobiology. These classical methods, however, are cumbersome because they require fresh granulocytes that have to be prepared daily and used immediately. Consequently, screening for HNA-3a antibodies is restricted to a few expert laboratories. Therefore, we propose to use the simple indirect antiglobulin red cell agglutination test (IAT) and Cs(a-) RBCs to screen for this antibody in the plasma of donors and patients. Finally, given that the starting point for this project was the study of a potential association of the HNA-3 polymorphism with clinical and biological features in SCA patients, we found that the HNA-3b allele was significantly associated with a higher monocyte and platelet count (p<0.02) and the occurrence of leg ulcer (p=0.01), suggesting an important role of the SLC44A2 protein in the SCD physiopathology.

Proteomic changes of the bilateral M1 and spinal cord in hemiplegic cerebral palsy mouse: Effects of constraint-induced movement therapy

Hemiplegic cerebral palsy (HCP) is a non-progressive movement and posture disorder that affects one side of the body. Constraint-induced movement therapy (CIMT) can improve the hand function of children with HCP. We used label-free proteomic quantification technology to evaluate proteomic changes in the bilateral M1 and spinal cord in HCP mouse induced by hypoxia/ischemia and CIMT. Nissl staining showed reduced neuron density in the HCP mice’s lesioned and contralesional M1. The rotarod test and grip strength test showed motor dysfunction in mice with HCP and improved motor ability after CIMT. A total of 5147 proteins were identified. Fifty-one, five, and sixty common differentially expressed proteins (DEPs), which were co-regulated by HCP and CIMT, were found in the lesioned M1, the contralesional M1 and the spinal cord respectively. The significant proteins included alpha-centractin, metaxin complex, PKC, septin 11, choline transporter-like proteins, protein 4.1, teneurin-4, and so on, which mainly related to synapse stability, neuronal development and maintenance, axon development, and myelin formation. The KEGG pathways of HCP-induced DEPs mainly related to lipid metabolism, synaptic remodeling, SNARE interactions in vesicular transport and axon formation. The CIMT-induced DEPs were mainly related to synaptic remodeling and axon formation in the lesioned M1 and spinal cord. This study investigated the proteomic changes of the bilateral M1 and spinal cord as well as the CIMT-induced proteomic changes in HCP mice, which might provide new insights into the therapy of HCP.

Carrier-Mediated Process of Putrescine Elimination at the Rat Blood-Retinal Barrier.

Putrescine is a bioactive polyamine. Its retinal concentration is strictly controlled to maintain a healthy sense of vision. The present study investigated putrescine transport at the blood-retinal barrier (BRB) to gain a better understanding of the mechanisms of putrescine regulation in the retina. Our microdialysis study showed that the elimination rate constant during the terminal phase was significantly greater (1.90-fold) than that of [14C]D-mannitol, which is a bulk flow marker. The difference in the apparent elimination rate constants of [3H]putrescine and [14C]D-mannitol was significantly decreased by unlabeled putrescine and spermine, suggesting active putrescine transport from the retina to the blood across the BRB. Our study using model cell lines of the inner and outer BRB showed that [3H]putrescine transport was time-, temperature-, and concentration-dependent, suggesting the involvement of carrier-mediated processes in putrescine transport at the inner and outer BRB. [3H]Putrescine transport was significantly reduced under Na+-free, Cl--free, and K+-replacement conditions, and attenuated by polyamines or organic cations such as choline, a choline transporter-like protein (CTL) substrate. Rat CTL1 cRNA-injected oocytes exhibited marked alterations in [3H]putrescine uptake, and CTL1 knockdown significantly reduced [3H]putrescine uptake in model cell lines, suggesting the possible participation of CTL1 in putrescine transport at the BRB.

104P Choline transporter-like protein 1 is a novel molecular target for the treatment of hepatocellular carcinoma

104P Choline transporter-like protein 1 is a novel molecular target for the treatment of hepatocellular carcinoma

Altered choline level in atherosclerotic lesions: Upregulation of choline transporter-like protein 1 in human coronary unstable plaque.

Inflammatory activity and hypoxia in atherosclerotic plaques are associated with plaque instability and thrombotic complications. Recent studies show that vascular cell metabolism affects atherogenesis and thrombogenicity. This study aimed to identify the metabolites in macrophage-rich unstable plaques that modulate atherogenesis and serve as potential markers of plaque instability. Atherosclerotic plaques were induced by balloon injury in the iliofemoral arteries of rabbits fed on a conventional or 0.5% cholesterol diet. At 3 months post-balloon injury, the arteries and cardiac tissues were subjected to histological, quantitative real-time polymerase chain reaction, and metabolomic analyses. The identified metabolite-related proteins were immunohistochemically analyzed in stable and unstable plaques from human coronary arteries. The factors modulating the identified metabolites were examined in macrophages derived from human peripheral blood mononuclear cells. Metabolomic analysis revealed that choline and guanine levels in macrophage-rich arteries were upregulated compared with those in non-injured arteries and cardiac tissues. Vascular choline levels, but not guanine levels, were positively correlated with the areas immunopositive for macrophages and tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP) 9 mRNA levels in injured arteries. In human coronary arteries, choline transporter-like protein (CTL) 1 was mainly localized to macrophages within plaques. The area that was immunopositive for CTL1 in unstable plaques was significantly higher than that in stable plaques. Intracellular choline levels were upregulated upon stimulation with TNF-α but were downregulated under hypoxia in cultured macrophages. Administration of choline upregulated the expression of TNF-α and CTL1 mRNA in cultured macrophages. The transfection of CTL1 small interfering RNA decreased CTL1, TNF-α, and MMP9 mRNA levels in cultured macrophages. These results suggest that choline metabolism is altered in macrophage-rich atherosclerotic lesions and unstable plaques. Thus, CTL1 may be potential markers of plaque instability.

Lack of the human choline transporter-like protein SLC44A2 causes hearing impairment and a rare red blood phenotype.

Blood phenotypes are defined by the presence or absence of specific blood group antigens at the red blood cell (RBC) surface, due to genetic polymorphisms among individuals. The recent development of genomic and proteomic approaches enabled the characterization of several enigmatic antigens. The choline transporter-like protein CTL2 encoded by the SLC44A2 gene plays an important role in platelet aggregation and neutrophil activation. By investigating alloantibodies to a high-prevalence antigen of unknown specificity, found in patients with a rare blood type, we showed that SLC44A2 is also expressed in RBCs and carries a new blood group system. Furthermore, we identified three siblings homozygous for a large deletion in SLC44A2, resulting in complete SLC44A2 deficiency. Interestingly, the first-ever reported SLC44A2-deficient individuals suffer from progressive hearing impairment, recurrent arterial aneurysms, and epilepsy. Furthermore, SLC44A2null individuals showed no significant platelet aggregation changes and do not suffer from any apparent hematological disorders. Overall, our findings confirm the function of SLC44A2 in hearing preservation and provide new insights into the possible role of this protein in maintaining cerebrovascular homeostasis.

Licochalcone E, a β-Amyloid Aggregation Inhibitor, Regulates Microglial M1/M2 Polarization via Inhibition of CTL1-Mediated Choline Uptake.

Alzheimer's disease (AD) is thought to be a series of neuroinflammatory diseases caused by abnormal deposits of amyloid-β (Aβ) and tau protein in the brain as part of its etiology. We focused on Aβ aggregation and M1 and M2 microglial polarity in microglia to search for novel therapeutic agents. It has been reported that the inhibition of choline uptake via choline transporter-like protein 1 (CTL1) in microglia preferentially induces M2 microglial polarity. However, the role of the choline transport system on the regulation of microglial M1/M2 polarity in AD is not fully understood. Licochalcones (Licos) A-E, flavonoids extracted from licorice, have been reported to have immunological anti-inflammatory effects, and Lico A inhibits Aβ aggregation. In this study, we compared the efficacy of five Licos, from Lico A to E, at inhibiting Aβ1-42 aggregation. Among the five Licos, Lico E was selected to investigate the relationship between the inhibition of choline uptake and microglial M1/M2 polarization using the immortalized mouse microglial cell line SIM-A9. We newly found that Lico E inhibited choline uptake and Aβ1-42 aggregation in SIM-A9 cells in a concentration-dependent manner, suggesting that the inhibitory effect of Lico E on choline uptake is mediated by CTL1. The mRNA expression of tumor necrosis factor (TNF-α), a marker of M1 microglia, was increased by Aβ1-42, and its effect was inhibited by choline deprivation and Lico E in a concentration-dependent manner. In contrast, the mRNA expression of arginase-1 (Arg-1), a marker of M2 microglia, was increased by IL-4, and its effect was enhanced by choline deprivation and Lico E. We found that Lico E has an inhibitory effect on Aβ aggregation and promotes polarity from M1 to M2 microglia via inhibition of the CTL1 function in microglia. Thus, Lico E may become a leading compound for a novel treatment of AD.

Functional Expression of Choline Transporters in Microglia and Their Regulation of Microglial M1/M2 Polarization.

Background: Microglia are key cells of the immune system in the central nervous system and are suggested to be deeply involved in the development of neurodegenerative diseases. It is well known that microglia have functional plasticity, with an inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Inhibition of choline transport in macrophages has been reported to suppress the secretion of inflammatory cytokines. However, the role of the choline transport system in regulating microglial M1/M2 polarization has not been fully elucidated to date. In this study, we investigated the mechanism of choline uptake in microglia, and its association with microglial M1/M2 polarization. Methods: The immortalized mouse microglial cell line SIM-A9 was used for [3H]choline uptake and expression analysis of choline transporters. The association between the choline uptake system and the M1/M2 polarization of microglia was also analyzed. Results: Choline transporter-like protein (CTL) 1 and CTL2 were highly expressed in SIM-A9 cells, and CTL1 and CTL2 were localized in the plasma membrane and mitochondria, respectively. Functional analysis of choline uptake demonstrated the existence of Na+-independent, pH-dependent, and intermediate-affinity choline transport systems. Choline uptake was concentration-dependently inhibited by hemicholinium-3 (HC-3), an inhibitor of choline uptake, and increased by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Expression of the mRNA of M1 microglia markers IL-1β and IL-6 was increased by LPS, and their effects were suppressed by choline deprivation and HC-3. In contrast, mRNA expression of the M2 microglial marker arginase-1 (Arg-1) was increased by IL-4, and the effect was enhanced by choline deprivation and HC-3. Conclusions: Our results suggest that inhibition of CTL1-mediated choline uptake in microglia preferentially induces M2 microglia polarization, which is a potential therapeutic approach for inflammatory brain diseases.

Phenotyping and Genotyping of HNA: Prevalence, Risk of Alloimmunization, and HNA Incompatibilities in Indians

Background: Antibodies to human neutrophil alloantigens (HNA) are involved in the pathophysiology of several clinical conditions including transfusion-related acute lung injury (TRALI), alloimmune and autoimmune neutropenia, and febrile nonhemolytic transfusion reactions leading to neutropenia. The cognate antigens are polymorphic structures expressed on several glycoproteins on the neutrophils, i.e., antigens HNA-1a, -1b, -1c, and -1d on Fc-γ-receptor IIIb; HNA-2 on CD177; HNA-3a and -3b on choline transporter-like protein 2; HNA-4a and -4b on CD11b/αM subunit of the αMβ2-integrin (CD11b/CD18, Mac-1, CR3); and HNA-5a and -5b on αL-subunit (CD11a) of the αLβ2 integrin (CD11a/CD18), leukocyte function associated molecule (LFA)-1. Currently, there is a lacuna of diagnostic methods for detection of HNA in India. This study aimed to determine the HNA frequencies in Indians, estimate the risk of alloimmunization, and prepare typed neutrophil panels, which can be used to detect HNA antibodies in neutropenia cases. Material and Methods: EDTA blood samples were collected from random 1,054 blood donors. HNA-2 was phenotyped on fresh EDTA samples using FITC labelled monoclonal anti-CD177 by flowcytometry. HNA-1 (FCGR3B) genotyping was carried out by DNA sequencing and PCR-RFLP. Antigens of HNA-3 (SLC44A2) and HNA-5 (ITGAL) were genotyped by PCR-RFLP using TaqαI and Bsp1286I restriction enzymes, respectively, while HNA-4 (ITGAM) was genotyped by PCR-SSP. Results: Allele frequencies of FCGR3B*01, FCGR3B*02, and FCGR3B*03 were found to be 0.433, 0.444, and 0.087, respectively. FCGR3B*01+*02+*03− was the most common genotype (33.78%). Ten individuals showed deficiency of FCGR3B individuals, while 23 showed hyperexpression, i.e., FCGR3B*01+*02+*03+. FCGR3B*04and *05 occurred with a frequency of 0.002 and 0.024. HNA-2 was found to be a high frequency antigen occurring in 98.8% population. Four percent individuals showed atypical expression of CD177 on their neutrophils. Allele frequencies of SLC44A2*01 and SLC44A2*02were 0.812 and 0.188, respectively, and that of ITGAM*01, ITGAM*02, ITGAL*01, and ITGAL*02 were 0.9546, 0.0454, 0.2372, and 0.7628, respectively. Conclusion: This is the first study in India to report the frequencies of HNA among blood donors. Typed neutrophil panels identified in the present study will enable us to investigate suspected cases of immune neutropenia in future.

SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR.

Background: Human neutrophil antigen-3A (HNA-3A) and human neutrophil antigen-3B (HNA-3B) are generated by a single-nucleotide polymorphism (rs2288904, c.461G > A) in exon 7 of the choline transporter-like protein-2 gene (CTL2, also known as SLC44A2). Antibodies to HNA-3 can be generated following blood transfusion or other factors resulting in exposure to HNA-3 antigens. These antibodies can cause transfusion-related acute lung injury (TRALI) or neonatal alloimmune neutropenia (NAIN). This study describes a sensitive and specific TaqMan real-time polymerase chain reaction (PCR) method to screen for the HNA-3 genotype using specific primers and probes designed to detect allelic polymorphisms. Considering the high sensitivity and accuracy of droplet digital PCR (ddPCR) in the identification of the rare SLC44A2*2 allele, we used this technique to identify blood donors with the rare HNA-3B antigen and calculate the allele frequency of SLC44A2 in mixed populations with different proportions. Methods: DNA samples purified from 208 donors in northwest China were subjected to TaqMan real-time PCR to detect allelic polymorphisms in SLC44A2. The results were confirmed by Sanger sequencing. The rare HNA-3B antigen was detected by ddPCR. SLC44A2 frequency was determined by two-channel ddPCR. Results: The genotypes of all DNA samples were detected by the TaqMan real-time PCR using specific probes for HNA-3, and the results were consistent with the Sanger sequencing results in respect to the HNA-3A and HNA-3B polymorphisms. The allele frequencies of SLC44A2*1 and SLC44A2*2 in the 208 donors in northwest China were 64.9% (95% confidence interval [CI], 59%–70.8%) and 35.1% (95% CI, 29.2%–41%), respectively. The ratio of SLC44A2*2 alleles was accurately detected in all blood pools by ddPCR but not by TaqMan real-time PCR. This allowed for the SLC44A2 frequency in the population to be accurately inferred. Conclusion: This new method of detecting SLC44A2 alleles was highly sensitive and specific, as confirmed by Sanger sequencing. ddPCR using the designed probes resulted in successful detection of the rare HNA-3B antigen. Furthermore, we successfully detected the rare HNA-3B antigen and inferred the SLC44A2 frequency by ddPCR using the probes that we designed.

Choline metabolism underpins macrophage IL-4 polarization in vitro and in vivo

Abstract Choline homeostasis in macrophage biology is important for LPS-polarized inflammation. In macrophages, choline mainly supports phosphatidylcholine (PC) synthesis, which is crucial for membrane production and cytokine secretion. Here, we examined choline metabolism in IL-4 polarized macrophages in vitro and in vivo. Like LPS, IL-4 increased choline transporter-like protein 1 (CTL1) expression, choline uptake and the incorporation of choline into PC. Targeted lipidomics analysis revealed increased PC content in IL-4-polarized macrophages, with an enrichment in low-saturated species. Pharmacological inhibition of choline uptake/choline kinase with hemicholinium-3 or RSM-932a showed no effect on certain hallmark IL-4-induced macrophage genes (Chil3, Mrc1, Arg1) but significantly reduced the transcript and protein expression of RELMα. Consistent with the function of RELMα in wound healing, 3T3-L1 cells healed more slowly in a scratch wound assay with conditioned media from IL-4 polarized macrophages in which choline metabolism was inhibited compared to vehicle-treated conditioned media. In addition, inhibiting choline metabolism completely prevented PD-L2 upregulation and increased PD-L1 expression on IL-4-polarized macrophages, together with suppressed cellular respiration and increased glycolysis. Furthermore, in vivo administration of RSM-932a (3 mg/kg i.p.) in C57BL/6J mice lowered RELMα in macrophages, decreased PD-L2 and increased PD-L1, and resulted in a loss of resident peritoneal F4/80hi macrophages. Choline represents an underappreciated regulator of macrophage immunometabolism and strategies to target macrophage choline uptake or choline metabolism may be therapeutically valuable.

A Novel Plant-Derived Choline Transporter-like Protein 1 Inhibitor, Amb544925, Induces Apoptotic Cell Death via the Ceramide/Survivin Pathway in Tongue Squamous Cell Carcinoma.

Simple SummaryIn many cancer cells, the choline uptake mechanism via the choline transporter-like protein 1 (CTL1) is involved in cell proliferation, and inhibiting its function is known to induce cell death by apoptosis. Therefore, CTL1 is attracting attention as a target molecule for cancer therapy. Here, we investigated the mechanism of the antitumor effect and metastasis inhibition of Amb544925, a novel CTL1 inhibitor discovered from plant-derived organic compounds in the tongue squamous cell carcinoma (TSCC) cell line, HSC-3. Amb544925 increased caspase-3/7 activity and caused apoptotic cell death. The antitumor effect of Amb544925 was brought about by suppressing the expression of survivin, an apoptosis inhibitor, through ceramide, an apoptosis-inducing factor (ceramide/survivin pathway). Furthermore, it was suggested that the inhibitory effect of Amb544925 on cell migration was also mediated by the ceramide/survivin pathway. Plant-derived Amb544925 is a lead compound in the treatment of TSCC that exerts antitumor effects via the ceramide/survivin pathway by targeting CTL1.Background: Despite recent advances in the early detection and treatment of TSCC patients, recurrence rates and survival rates have not improved. The high frequency of lymph node metastasis is one of the causes, and the drug development of new therapeutic mechanisms such as metastasis control is desired. Choline transporter-like protein 1 (CTL1) has attracted attention as a target molecule in cancer therapy. In this study, we examined the antitumor effects of Amb544925, a plant-derived CTL1 inhibitor. Methods: The TSCC cell line HSC-3 was used to measure [3H]choline uptake, cell survival, caspase activity, and cell migration. Xenograft model mice were prepared to verify the antitumor effect of Amb544925. Results: Amb544925 inhibited cell viability and increased caspase-3/7 activity at concentrations that inhibited choline uptake. Amb544925 and ceramide increased SMPD4 expression and suppressed surivivin expression. Furthermore, Amb544925 and ceramide inhibited the migration of HSC-3 cells. In the xenograft model mice, Amb544925 suppressed tumor growth and CTL1 mRNA expression. Conclusions: The plant-derived CTL1 inhibitor Amb544925 is a lead compound of a new anticancer agent exhibiting antitumor effects and inhibition of cell migration through the ceramide/survivin pathway.

Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development

Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, we demonstrate that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. Our findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein.

Anti-HNA-3a Alloantibodies Can Cause Differential Endothelial Damage through a Direct Neutrophil Activation Pathway

INTRODUCTIONThe human neutrophil antigen (HNA) 3a/b is associated with a single amino acid substitution (R 154Q polymorphism) on the first extracellular loop of choline transporter-like protein 2 (CTL2). Antibodies against HNA-3a have been associated with severe and fatal transfusion-related acute lung injury (TRALI). Epitope mapping suggests the existence of two types of HNA-3a antibodies. Type I antibodies only require first extracellular loop of the CTL2 for expression of the antigen, type II antibodies require at least the first 3 extracellular loops of CTL2 in a native configuration (i.e. expressed on a cell membrane). This study aimed to evaluate the activity of type I and type II anti-HNA-3a antibodies in an in vitro TRALI model.STUDY DESIGN & METHODS: The granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used to confirm anti-HNA-3a activity in two donor sera (Q49 and Q50). Flow cytometry was used to confirm antibody binding to freshly isolated human or mouse neutrophils. A rabbit polyclonal antibody against an epitope in the third extracellular loop of CTL2's was coated onto the wells of an ELISA plate and neutrophil HNA-3a antigen was captured and sera tested. For the TRALI model, human lung microvascular endothelial cells (HLMVECs) were grown to confluence before being treated with lipopolysaccharide (LPS). Freshly isolated neutrophils from HNA-3aa homozygote donors were then added with 10% Q49 or Q50. Microscopic counting of Trypan blue staining was used to measure rate HLMVEC death. Significance was determined using a one-way ANOVA (p<0.05), followed by Dunnett's post-hoc test. RESULTS AND DISCUSSIONGAT and GIFT confirmed that both Q49 and Q50 contained an anti-HNA-3a antibody, and that neither contained antibodies against any other HNA or HLA molecules. In flow cytometry, both Q49 and Q50 bound to human neutrophils, but only Q49 bound to mouse neutrophils. In the ELISA, signal was detected only for Q49. These data provided evidence that Q49 was a type I antibody and Q50 was a type II antibody. Within the TRALI model, HLVMEC death was observed with both Q49 and Q50 only in the presence of LPS and neutrophils (Table 1). Previously reported HLMVEC permeability in the absence of neutrophils (Bayat et al. Arterioscler Thromb Vasc Biol. 2013) was not observed as in this study the outcome measured was HLMVEC death. In the present study, the effect for Q49 (type I) was significantly larger than for Q50 (type II; Table 1; p-value <0.0001). CONCLUSIONSIn this model, LPS and neutrophils were required for anti-HNA-3a mediated HLMVEC death thought to reflect events involved in the initiation of TRALI. The observation that type I antibodies elicited a stronger response provides a direction for further research. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dietary Choline-Enhanced Skin Immune Response of Juvenile Grass Carp Might Be Related to the STAT3 and NF-kB Signaling Pathway (Ctenopharyngodon idella).

To investigate the effects and potential mechanisms of dietary choline on immune function in the skin of juvenile grass carp (Ctenopharyngodon idella), fish were fed different diets containing different levels of choline (142. 2, 407.4, 821.6, 1215.8, 1589.3, and 1996.6 mg/kg) for 70 d and then sampled after a 6-d challenge test. The results exhibited that dietary choline (1) advanced the contents of phosphatidylcholine (PC), betaine, and choline in grass carp skin (P < 0.05) and upregulated the mRNA abundance of choline transporter high-affinity choline transporter 1 (CHT1), choline transporter-like protein 1 (CTL1), and choline transporter-like protein 5 (CTL5), indicating that dietary choline could increase the contents of choline which might be connected with choline transporters in the grass carp skin; (2) receded skin rot symptom after infection with A. hydrophila (Aeromonas hydrophila), increased the levels of IgM, C4, and C3 and the activities of acid phosphatase (ACP) and lysozyme (LZ), raised mucin2, β-defensin, hepcidin, and LEAP-2B mRNA abundance (rather than LEAP-2A), downregulated pro-inflammatory cytokine mRNA abundance (IFN-γ2, IL-15, TNF-α, IL-6, IL-12P40, and IL-1β) in skin of juvenile grass carp (P < 0.05), and upregulated anti-inflammatory cytokine mRNA abundance (IL-10, IL-4/13A, TGF-β1, IL-11, and IL-4/13B) in grass carp skin (P < 0.05), demonstrating that choline enhanced the skin immune function; and (3) downregulated the mRNA abundance of IKKγ, NF-κBp52, IKKβ, c-Rel, NF-κBp65, STAT3b2, STAT3b1, JAK1, and JAK2 as well as protein level of NF-κBp65, p-STAT3 Tyr705, and p-STAT3 Ser727 in nucleus and inhibited the mRNA and protein level of IkBα (P < 0.05), indicating that choline-enhanced immune function might be relevant to the JAK1, 2 /STAT3, and NF-κB signaling pathway in fish skin. In conclusion, choline enhanced the skin immune function which might be related to JAK1, 2/STAT3, and NF-κB signaling molecules in fish. Furthermore, based on immune indices of grass carp (9.28–108.97 g) skin (C3 and IgM contents as well as ACP activities), the choline requirements were estimated to be 1475.81, 1364.24, and 1574.37 mg/kg diet, respectively.

Functional Expression of Choline Transporters in Human Neural Stem Cells and Its Link to Cell Proliferation, Cell Viability, and Neurite Outgrowth.

Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.

Choline transporter-like proteins 1 and 2 are newly identified plasma membrane and mitochondrial ethanolamine transporters

The membrane phospholipids phosphatidylcholine and phosphatidylethanolamine (PE) are synthesized de novo by the CDP-choline and CDP-ethanolamine (Kennedy) pathway, in which the extracellular substrates choline and ethanolamine are transported into the cell, phosphorylated, and coupled with diacylglycerol to form the final phospholipid product. Although multiple transport systems have been established for choline, ethanolamine transport is poorly characterized and there is no single protein assigned a transport function for ethanolamine. The solute carriers 44A (SLC44A) known as choline transporter-like proteins-1 and -2 (CTL1 and CTL2) are choline transporter at the plasma membrane and mitochondria. We report a novel function of CTL1 and CTL2 in ethanolamine transport. Using the lack or the gain of gene function in combination with specific antibodies and transport inhibitors we established two distinct ethanolamine transport systems of a high affinity, mediated by CTL1, and of a low affinity, mediated by CTL2. Both transporters are Na+-independent ethanolamine/H+ antiporters. Primary human fibroblasts with separate frameshift mutations in the CTL1 gene (M1= SLC44A1ΔAsp517 and M2= SLC44A1ΔSer126) are devoid of CTL1 ethanolamine transport but maintain unaffected CTL2 transport. The lack of CTL1 in M2 cells reduced the ethanolamine transport, the flux through the CDP-ethanolamine Kennedy pathway, and PE synthesis. In contrast, overexpression of CTL1 in M2 cells improved ethanolamine transport and PE synthesis. These data firmly establish that CTL1 and CTL2 are the first identified ethanolamine transporters in whole cells and mitochondria, with intrinsic roles in de novo PE synthesis by the Kennedy pathway and intracellular redistribution of ethanolamine.

Molecular and Functional Analysis of Choline Transporters and Antitumor Effects of Choline Transporter-Like Protein 1 Inhibitors in Human Hepatocellular Carcinoma Cells

Molecular and Functional Analysis of Choline Transporters and Antitumor Effects of Choline Transporter-Like Protein 1 Inhibitors in Human Hepatocellular Carcinoma Cells

Dietary choline deficiency aggravated the intestinal apoptosis in association with the MAPK signalling pathways of juvenile grass carp (Ctenopharyngodon idella)

To investigate the effects and potential mechanisms of apoptosis in the intestines of juvenile grass carp (Ctenopharyngodon idella) for dietary choline, fish were fed different choline-containing diets (142.2, 407.4, 821.6, 1215.8, 1589.3, and 1996.6 mg kg−1) for 70 days. Results revealed that dietary choline deficiency (1) decreased the contents of choline, phosphatidylcholine, betaine, and acetylcholine (not in distal intestine [DI]) in grass crap intestines; down-regulated the mRNA abundances of choline transportations, such as high-affinity choline transporter CHT1 (not in proximal intestine[PI] and DI), choline transporter-like proteins such as CTL-1, CTL-2, CTL-4 (not in DI), and CTL-5 in grass crap intestines; (2) enhanced the degree of DNA fragments. Moreover, it up-regulated the activities of caspase-3, −8, and-9; reduced mRNA levels of caspase-3, −7, −8 (not in DI), and − 9, Fas ligand (FASL) (not in PI), and apoptotic protease activating factor-1(Apaf1), as well as the protein level of cytochrome c (Cty c). Dietary choline deficiency also down-regulated myeloid cell leukemia-1 (Mcl-1) (not in PI), B-cell lymphoma-2 (Bcl-2), and the inhibitor of apoptosis protein (IAP) mRNA levels in grass carp intestines, suggesting that choline inhibited cell apoptosis in fish intestines; (3) increased p38MAPK and JNK(not in mid intestine [MI]) mRNA level, as well as p-p38MAPK and T-p38MAPK protein levels in the PI and the ratio of p-p38MAPK/T-p38MAPK in the MI and DI, indicating that choline deficiency aggravated cell apoptosis in association with the MAPK signalling pathway in fish intestines; In conclusion, choline insufficiency promotion of intestinal apoptosis might be related to MAPK signalling pathways in fish. Moreover, the requirements of choline for resist intestinal apoptosis indices (caspase-8 and capase-9 activities) of grass carp (9.28–108.97 g) were calculated to be (1162.13–1780.93) mg kg−1 diet, respectively.

Molecular and Functional Analysis of Choline Transporters and Antitumor Effects of Choline Transporter-Like Protein 1 Inhibitors in Human Pancreatic Cancer Cells.

Choline, an organic cation, is one of the biofactors that play an important role in the structure and the function of biological membranes, and it is essential for the synthesis of phospholipids. Choline positron emission tomography-computed tomography (PET/CT) provides useful information for the imaging diagnosis of cancers, and increased choline accumulation has been identified in a variety of tumors. However, the molecular mechanisms of choline uptake and choline transporters in pancreatic cancer have not been elucidated. Here, we examined molecular and functional analyses of choline transporters in human pancreatic-cancer cell line MIA PaCa-2 and the elucidation of the action mechanism behind the antitumor effect of novel choline-transporter-like protein 1 (CTL1) inhibitors, Amb4269951 and its derivative Amb4269675. CTL1 and CTL2 mRNAs were highly expressed in MIA PaCa-2 cells, and CTL1 and CTL2 proteins were localized in the plasma membrane and the intracellular compartments, respectively. Choline uptake was characterized by Na+-independence, a single-uptake mechanism, and inhibition by choline-uptake inhibitor HC-3, similar to the function of CTL1. These results suggest that the uptake of extracellular choline in MIA PaCa-2 cells is mediated by CTL1. Choline deficiency and HC-3 treatment inhibited cell viability and increased caspase 3/7 activity, suggesting that the inhibition of CTL1 function, which is responsible for choline transport, leads to apoptosis-induced cell death. Both Amb4269951 and Amb4269675 inhibited choline uptake and cell viability and increased caspase-3/7 activity. Ceramide, which is increased by inhibiting choline uptake, also inhibited cell survival and increased caspase-3/7 activity. Lastly, both Amb4269951 and Amb4269675 significantly inhibited tumor growth in a mouse-xenograft model without any adverse effects such as weight loss. CTL1 is a target molecule for the treatment of pancreatic cancer, and its inhibitors Amb4269951 and Amb4269675 are novel lead compounds.