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We previously identified a missense single nucleotide polymorphism rs2228315 (G>A, Met62Ile) in the selectin‐P‐ligand gene (SELPLG), encoding P‐selectin glycoprotein ligand 1 (PSGL‐1), to be associated with increased susceptibility to acute respiratory distress syndrome (ARDS). These earlier studies demonstrated that SELPLG lung tissue expression was increased in mice exposed to lipopolysaccharide (LPS)‐ and ventilator‐induced lung injury (VILI) suggesting that inflammatory and epigenetic factors regulate SELPLG promoter activity and transcription. In this report, we used a novel recombinant tandem PSGL1 immunoglobulin fusion molecule (TSGL‐Ig), a competitive inhibitor of PSGL1/P‐selectin interactions, to demonstrate significant TSGL‐Ig‐mediated decreases in SELPLG lung tissue expression as well as highly significant protection from LPS‐ and VILI‐induced lung injury. In vitro studies examined the effects of key ARDS stimuli (LPS, 18% cyclic stretch to simulate VILI) on SELPLG promoter activity and showed LPS‐mediated increases in SELPLG promoter activity and identified putative promoter regions associated with increased SELPLG expression. SELPLG promoter activity was strongly regulated by the key hypoxia‐inducible transcription factors, HIF‐1α, and HIF‐2α as well as NRF2. Finally, the transcriptional regulation of SELPLG promoter by ARDS stimuli and the effect of DNA methylation on SELPLG expression in endothelial cell was confirmed. These findings indicate SELPLG transcriptional regulation by clinically‐relevant inflammatory factors with the significant TSGL‐Ig‐mediated attenuation of LPS and VILI highly consistent with PSGL1/P‐selectin as therapeutic targets in ARDS. [ABSTRACT FROM AUTHOR]

Abstract We previously identified a missense single nucleotide polymorphism rs2228315 (G>A, Met62Ile) in the selectin‐P‐ligand gene (SELPLG), encoding P‐selectin glycoprotein ligand 1 (PSGL‐1), to be associated with increased susceptibility to acute respiratory distress syndrome (ARDS). These earlier studies demonstrated that SELPLG lung tissue expression was increased in mice exposed to lipopolysaccharide (LPS)‐ and ventilator‐induced lung injury (VILI) suggesting that inflammatory and epigenetic factors regulate SELPLG promoter activity and transcription. In this report, we used a novel recombinant tandem PSGL1 immunoglobulin fusion molecule (TSGL‐Ig), a competitive inhibitor of PSGL1/P‐selectin interactions, to demonstrate significant TSGL‐Ig‐mediated decreases in SELPLG lung tissue expression as well as highly significant protection from LPS‐ and VILI‐induced lung injury. In vitro studies examined the effects of key ARDS stimuli (LPS, 18% cyclic stretch to simulate VILI) on SELPLG promoter activity and showed LPS‐mediated increases in SELPLG promoter activity and identified putative promoter regions associated with increased SELPLG expression. SELPLG promoter activity was strongly regulated by the key hypoxia‐inducible transcription factors, HIF‐1α, and HIF‐2α as well as NRF2. Finally, the transcriptional regulation of SELPLG promoter by ARDS stimuli and the effect of DNA methylation on SELPLG expression in endothelial cell was confirmed. These findings indicate SELPLG transcriptional regulation by clinically‐relevant inflammatory factors with the significant TSGL‐Ig‐mediated attenuation of LPS and VILI highly consistent with PSGL1/P‐selectin as therapeutic targets in ARDS.

Expression of recombinant glycoproteins carrying the sialyl-LewisX epitope requires host cells equipped with appropriate glycosyltransferases. Chinese hamster ovary cells transfected with tricistronic vectors and encoding the resistance marker gene, neoR, in the third cistron and core 2 N-acetylglucosaminyltransferase or α1,3-fucosyltransferase III or IV in either the first or second cistron, respectively, produced both enzyme activities in a constant ratio. A representative clone was transfected with PSGL-1 (P-selectin glycoprotien ligand 1) and conferred P-selectin-binding activity to PSGL-1. [ABSTRACT FROM AUTHOR]