Glycosylation inhibitors efficiently inhibit P-selectin-mediated cell adhesion to endothelial cells.

Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD

Glycosylation inhibitors block thrombin-induced sRBC adhesion to HUVECs.

<p><b>A</b>. Thrombin induces sRBC adhesion to HUVECs. sRBCs were prepared from two sickle cell model mice (SCD 1 & SCD 2) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099363#pone.0099363-Ryan1" target="_blank">[26]</a>. HUVECs were treated with 1 U/mL thrombin for 5 minutes after which sRBCs were added. The level of sRBC adhesion to untreated HUVECs was set to 100%. **, p<0.0001 <b>B</b>. Glycosylation inhibitors decrease thrombin-induced sRBC adhesion to HUVECs. HUVECs were pretreated with glycosylation inhibitors for 48 hours and P-selectin expression was stimulated by 1 U/mL thrombin. sRBCs were then added to HUVECs for adhesion assays. The levels of sRBC adhesion to HUVECs that were not treated with glycosylation inhibitor but stimulated with thrombin were set to 100%. **, p<0.0001 <b>C</b>. Representative microscopic images (x10 magnification) showing sRBC adhesion to HUVECs. Note that both tunicamycin and castanospermine inhibit sRBC adhesion to thrombin-activated HUVECs.</p

Glycosylation inhibitors block hypoxia-induced P-selectin expression in HUVECs.

<p>P-selectin expression in HUVECs exposed to 1% O₂ was examined by Western blotting (A) and immunofluorescence (B). <b>A</b>. Western blotting analysis of P-selectin expression in HUVECs exposed to hypoxia. HUVECs were cultured in the absence or presence of 2.5 or 5.0 µg/mL tunicamycin or 0.5 to 1.0 mM castanospermine, treated with 1 U/mL thrombin, and then were exposed to 1% O₂ for 1 hour. Cell lysates (30 µg) prepared from HUVECs were analyzed by Western blotting as described in Materials and Methods. β-Actin was as the loading control. The ratio of the P-selectin protein level to that of β-actin was set to 100%. Note that this experiment focused on the expression of the 140 kDa band in response to hypoxia. **, p<0.0001 <b>B</b>. P-selectin expression on HUVECs by immunohistochemistry. HUVECs were permeabilized and fixed as described in Materials and Methods. Note that expression of P-selectin (green) was induced by exposure to 1-hour hypoxia (1% O₂), but by pretreatment of 5 µg/mL tunicamycin.</p

Molecular effects of glycosylation inhibitors on P-selectin mRNA expression (A) and the proteasomal degradation of P-selectin (B) on HUVECs.

<p><b>A</b>. Tunicamycin has no effect on P-selectin mRNA levels. HUVECs were incubated with 2.5 or 5 µg/mL tunicamycin for 48 hours before 1 U/mL thrombin was added. Total RNAs were prepared and P-selectin mRNA levels were determined by real time-PCR analysis by using 18S rRNA as an internal control. The P-selectin mRNA level of untreated HUVECs was set to 1. Lanes: 1, untreated HUVECs; 2, thrombin-stimulated HUVECs; 3, HUVECs pretreated with 2.5 µg/mL tunicamycin; 4, HUVECs pretreated with 2.5 µg/mL tunicamycin and then stimulated by 1 U/mL thrombin; 5, HUVECs pretreated with 5 µg/mL tunicamycin and then stimulated by 1 U/mL thrombin. **, p<0.0001. <b>B</b>. Effects of tunicamycin on proteasomal degradation of P-selectin. HUVECs were pretreated for 1 hour with or without 1 µM MG132, treated for 48 hours with no, 2.5, or 5 µg/mL tunicamycin, and then treated with or without 1 U/mL thrombin. Cell lysates (30 µg) prepared from HUVECs were analyzed by Western blotting as described in Materials and Methods. β-Actin was used for the loading control. The ratio of the P-selectin protein level to that of β-actin was set to 1. Lanes: 1, untreated HUVECs; 2, thrombin-stimulated HUVECs; 3, HUVECs pretreated with 2.5 µg/mL tunicamycin; 4, HUVECs pretreated with MG132 and then stimulated with thrombin; 5, HUVECs pretreated with MG132, then with 5 µg/mL tunicamycin, and stimulated with thrombin. Experiments were repeated three times that gave similar results and a representative blot is shown. **, p<0.0001 <b>C</b>. Effects of glycosylation inhibitors on AKT phosphorylation. HUVECs were cultured for 48 hours in the presence or absence of tunicamycin or castanospermine at various concentrations followed by activation with 1 U/mL thrombin. Western blot analysis was performed to determine the phosphorylation status of AKT (Ser473 and Thr308). β-Actin was the loading control. <b>D</b>. AKT inhibitor suppresses CD62P expression on HUVECs. Flow cytometric analysis of P-selectin (CD62P) expression on HUVECs treated with the AKT inhibitor LY-294002 for 1 hour and subsequently stimulated with 1 U/mL thrombin. CD59-FITC was used as the positive marker for ECs. Flow cytometry analyses were repeated three times and a representative result was shown.</p

Comparison of sRBC adhesion inhibition by glycosylation inhibitors and P-selectin monoclonal antibody.

<p>HUVECs that were cultured as described in Materials and Methods were pretreated with saline or glycosylation inhibitors for 48 hours and stimulated with 1/mL thrombin. sRBCs were isolated from SCD patients and added to HUVECs. To examine the inhibition of sRBC adhesion, P-selectin monoclonal antibody was added to HUVECs prior to the addition of sRBCs. To inactivate P-selectin monoclonal antibody, the antibody was premixed with blocking peptide. Lanes: 1, HUVECs stimulated with 1U/mL thrombin; 2, HUVECs pretreated with 5 µg/mL tunicamycin; 3, HUVECs pretreated with 1 mM castanospermine; 4, HUVECs added with P-selectin monoclonal antibody; 5, HUVECs added with P-selectin monoclonal antibody premixed with blocking peptide; 6, HUVECs pretreated with 5 µg/mL tunicamycin and further added with P-selectin monoclonal antibody. **, p<0.0001; ^##, p<0.01 (vs lanes 2 & 4).</p

PKCδ-Mediated Nox2 Activation Promotes Fluid-Phase Pinocytosis of Antigens by Immature Dendritic Cells

AimsMacropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved.MethodsVarious cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC macropinocytosis.ResultsConfocal laser scanning microscopy confirmed that DCs internalize fluorescent antigens (ovalbumin) using macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor-induced antigen macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-β. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC macropinocytosis of antigens downstream of PKCδ activation.ConclusionThe findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKCδ/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses

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Aims<p>Macropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved.</p>Methods<p>Various cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC macropinocytosis.</p>Results<p>Confocal laser scanning microscopy confirmed that DCs internalize fluorescent antigens (ovalbumin) using macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor-induced antigen macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-β. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC macropinocytosis of antigens downstream of PKCδ activation.</p>Conclusion<p>The findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKCδ/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses.</p

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Aims<p>Macropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved.</p>Methods<p>Various cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC macropinocytosis.</p>Results<p>Confocal laser scanning microscopy confirmed that DCs internalize fluorescent antigens (ovalbumin) using macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor-induced antigen macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-β. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC macropinocytosis of antigens downstream of PKCδ activation.</p>Conclusion<p>The findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKCδ/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses.</p

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Aims<p>Macropinocytosis is a major endocytic pathway by which dendritic cells (DCs) internalize antigens in the periphery. Despite the importance of DCs in the initiation and control of adaptive immune responses, the signaling mechanisms mediating DC macropinocytosis of antigens remain largely unknown. The goal of the present study was to investigate whether protein kinase C (PKC) is involved in stimulation of DC macropinocytosis and, if so, to identify the specific PKC isoform(s) and downstream signaling mechanisms involved.</p>Methods<p>Various cellular, molecular and immunological techniques, pharmacological approaches and genetic knockout mice were utilized to investigate the signaling mechanisms mediating DC macropinocytosis.</p>Results<p>Confocal laser scanning microscopy confirmed that DCs internalize fluorescent antigens (ovalbumin) using macropinocytosis. Pharmacological blockade of classical and novel PKC isoforms using calphostin C abolished both phorbol ester- and hepatocyte growth factor-induced antigen macropinocytosis in DCs. The qRT-PCR experiments identified PKCδ as the dominant PKC isoform in DCs. Genetic studies demonstrated the functional role of PKCδ in DC macropinocytosis of antigens, their subsequent maturation, and secretion of various T-cell stimulatory cytokines, including IL-1α, TNF-α and IFN-β. Additional mechanistic studies identified NADPH oxidase 2 (Nox2) and intracellular superoxide anion as important players in DC macropinocytosis of antigens downstream of PKCδ activation.</p>Conclusion<p>The findings of the present study demonstrate a novel mechanism by which PKCδ activation via stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKCδ/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses.</p