CDB15:0001337 SELPLG — SELE

ABSTRACT: Chemoresistance is the major obstacle in multiple myeloma (MM) management. We previously showed that macrophages protect myeloma cells, on a cell contact basis, from melphalan or dexamethasone-induced apoptosis in vitro. In this study, we found that macrophage-mediated myeloma drug resistance was also seen with purified macrophages from myeloma patients' bone marrow (BM) in vitro and was confirmed in vivo using the human myeloma-SCID (severe combined immunodeficient) mouse model. By profiling differentially regulated and paired plasma membrane protein genes, we showed that PSGL-1 (P-selectin glycoprotein ligand-1)/selectins and ICAM-1/CD18 played an important role in macrophage-mediated myeloma cell drug resistance, as blocking antibodies against these molecules or genetic knockdown of PSGL-1 or ICAM-1 in myeloma cells repressed macrophages' ability to protect myeloma cells. Interaction of macrophages and myeloma cells via these molecules activated Src and Erk1/2 kinases and c-myc pathways and suppressed caspase activation induced by chemotherapy drugs. Thus, our study sheds new light on the mechanism of drug resistance in MM and provides novel targets for improving the efficacy of chemotherapy in patients.

ABSTRACT: P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated, fucosylated O-linked glycans and tyrosine sulfate to bind P-selectin. Less is known about the determinants that PSGL-1 requires to bind E-selectin. To further define the modifications required for PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary (CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases. CHO cells synthesize only core 1 O-linked glycans (Galbeta1-3GalNAcalpha1-Se r/Thr); they lack core 2 O-linked glycans (Galbeta1-3(Galbeta1-4GlcNAcbeta1-6)GalNAcalpha1 -Ser/Thr) because they do not express the core 2 beta1 6-N-acetylglucosaminyltransferase (C2GnT). CHO cells also lack alpha1 3 fucosyltransferase activity. PSGL-1 expressed on transfected CHO cells bound P- and E-selectin only when it was co-expressed with both C2GnT and an alpha1 3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of beta-eliminated O-linked glycans from PSGL-1 co-expressed with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues on PSGL-1 expressed in CHO cells were shown to be sulfated. Phenylalanine replacement of three tyrosines within a consensus sequence for tyrosine sulfation abolished binding to P-selectin but not to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind P-selectin but not E-selectin.

ABSTRACT: Leukocyte adhesion to vascular endothelium under flow involves an adhesion cascade consisting of multiple receptor pairs that may function in an overlapping fashion. P-selectin glycoprotein ligand-1 (PSGL-1) and L-selectin have been implicated in neutrophil adhesion to P- and E-selectin under flow conditions. To study, in isolation, the interaction of PSGL-1 with P- and E-selectin under flow, we developed an in vitro model in which various recombinant regions of extracellular PSGL-1 were coupled to 10-microm-diameter microspheres. In a parallel plate chamber with well defined flow conditions, live time video microscopy analyses revealed that microspheres coated with PSGL-1 attached and rolled on 4-h tumor necrosis factor-alpha-activated endothelial cell monolayers, which express high levels of E-selectin, and CHO monolayers stably expressing E- or P-selectin. Further studies using CHO-E and -P monolayers demonstrate that the first 19 amino acids of PSGL-1 are sufficient for attachment and rolling on both E- and P-selectin and suggest that a sialyl Lewis x-containing glycan at Threonine-16 is critical for this sequence of amino acids to mediate attachment to E- and P-selectin. The data also demonstrate that a sulfated, anionic polypeptide segment within the amino terminus of PSGL-1 is necessary for PSGL-1-mediated attachment to P- but not to E-selectin. In addition, the results suggest that PSGL-1 has more than one binding site for E-selectin: one site located within the first 19 amino acids of PSGL-1 and one or more sites located between amino acids 19 through 148.