High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding.

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein ß-dystroglycan (ß-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. ß-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.

O-glycosylation regulates LNCaP prostate cancer cell susceptibility to apoptosis induced by galectin-1.

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.

Interaction of CD44 with different forms of hyaluronic acid. Its role in adhesion and migration of tumor cells.

Interaction between hyaluronic acid (HA) and CD44 has been considered a key event in tumor invasion and metastasis. HA is a linear, high molecular weight glycosaminoglycan in its native state, but fragmented low molecular forms are found at sites ofneoplastic or inflammatory infiltrates. Both high and low molecular weights HA are involved in diverse biological functions. In this study, we used two clonal variants of a T cell murine lymphoma designated LBLa and LBLc. These cell lines were found to differ in their in vivo and in vitro growth rates. LBLa grew faster and exhibited an enhanced invasive capacity as compared to LBLc. In contrast, cell lines did not differ in the expression of surface markers (CD8, CD24, CD25, CD44, and CD18), or in their capacity to bind HA. However, LBLa cells exhibited higher capacity to migrate to low molecular weight HA than did LBLc. Migration was mediated by CD44 since it was abrogated by anti-CD44 monoclonal antibody as well as by hyaluronidase. We suggest that interaction between CD44 and low molecular weight HA may trigger migration mechanisms in LBLa cells, thus contributing to enhanced invasive cell capacity.

Haploinsufficiency of C2GnT-I glycosyltransferase renders T lymphoma cells resistant to cell death.

Neoplastic T cells in mycosis fungoides (MF) are resistant to apoptotic agents, including galectin-1 that is abundant in skin. Although MF cells are typically CD7-, and thus galectin-1 resistant, CD7+ HH cells, derived from a patient with MF, were also resistant to galectin-1. HH cells demonstrate altered cell surface glycosylation, with loss of core 2 O-glycan ligands for galectin-1 created by core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I). Loss of core 2 O-glycans on tumor cells was also seen in primary CD7+ MF lesions. Surprisingly, HH cells are heterozygous for a C2GnT-I point mutation, yet this mutation resulted in a dramatic reduction in cellular glycosyltransferase activity. Expression of wild-type C2GnT-I in human HH cells, or murine lymphoma cells that lack C2GnT-I, restored core 2 O-glycan expression and susceptibility to galectin-1, whereas mutant enzyme lacked activity and did not restore core 2 O-glycan expression or susceptibility to galectin-1. Mutant enzyme did not have a dominant negative effect by affecting dimerization or activity of wild-type enzyme; rather, C2GnT-I haploinsufficiency is sufficient for loss of core 2 O-glycan expression and galectin-1 resistance. Thus, glycosyltransferase haploinsufficiency results in altered cellular glycosylation and resistance to cell death, identifying a new survival mechanism for T-lymphoma cells.

Coelomocyte locomotion in the sipunculan Themiste petricola induced by exogenous and endogenous chemoattractants: role of a CD44-like antigen--HA interaction.

Cell migration is a key event in the invertebrate immuno-defense system. Microbial products like lipopolysacharide (LPS) and formyl-methyl-leucyl-phenylalanine (fMLP) promote cell recruitment to sites of infection. In mammals, complement activation by factors such as zymosan induces C5a production, which influences leukocyte migration. The endogenous factor hyaluronic acid (HA), an extracellular matrix component, also promotes cell migration through its receptor CD44. We evaluated whether coelomocytes from the sipunculan worm T. petricola migrated towards LPS, fMLP, or zymosan treated plasma (ZTP) and if HA was involved in coelomocyte migration and adhesion. We also evaluated if antibodies specific for mouse HA receptor CD44 inhibited any of the effects induced by HA. Using microchemotaxis chambers we found that coelomocytes migrated towards exogenously and endogenously derived chemoattractants. We also observed that HA was a potent chemotactic signal and that coelomocytes adhered strongly to plates coated with LMW-HA but not with HMW-HA. In addition we found that these HA mediated effects were blocked by the monoclonal antibody IM7 directed to mouse CD44, suggesting that a CD44-like cross-reactive antigen might play a role in HA mediated coelomocyte locomotion.

Modulación de isoformas de la molécula de adhesión CD44 en un modelo murino de isquemia y reperfusión intestinal / Isoforms modulation of CD44 adhesion molecule in a murine model of ischemia and intestinal reperfusion

Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.

Modulación de isoformas de la molécula de adhesión CD44 en un modelo murino de isquemia y reperfusión intestinal / Isoforms modulation of CD44 adhesion molecule in a murine model of ischemia and intestinal reperfusion

Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.(Au)

Modulación de isoformas de la molécula de adhesión CD44 en un modelo murino de isquemia y reperfusión intestinal / Isoforms modulation of CD44 adhesion molecule in a murine model of ischemia and intestinal reperfusion

Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity. (AU)

Unión diferencial de ácido hialurónico en dos sublíneas celulares CD44+. Posible implicancia en la infiltración tumoral / Differential binding of hyaluronic acid in 2 CD44+ sublines. Relationship with tumor infiltration

We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.

Unión diferencial de ácido hialurónico en dos sublíneas celulares CD44+. Posible implicancia en la infiltración tumoral / Differential binding of hyaluronic acid in 2 CD44+ sublines. Relationship with tumor infiltration

We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors. (AU)

Unión diferencial de ácido hialurónico en dos sublíneas celulares CD44+. Posible implicancia en la infiltración tumoral / Differential binding of hyaluronic acid in 2 CD44+ sublines. Relationship with tumor infiltration

We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.(Au)