OBJECTIVEInhibition of the Na+-glucose cotransporter type 2 (SGLT2) is currently being pursued as an insulin-independent treatment for diabetes; however, the behavioral and metabolic consequences of SGLT2 deletion are unknown. Here, we used a SGLT2 knockout mouse to investigate the effect of increased renal glucose excretion on glucose homeostasis, insulin sensitivity, and pancreatic β-cell function.RESEARCH DESIGN AND METHODSSGLT2 knockout mice were fed regular chow or a high-fat diet (HFD) for 4 weeks, or backcrossed onto the db/db background. The analysis used metabolic cages, glucose tolerance tests, euglycemic and hyperglycemic clamps, as well as isolated islet and perifusion studies.RESULTSSGLT2 deletion resulted in a threefold increase in urine output and a 500-fold increase in glucosuria, as well as compensatory increases in feeding, drinking, and activity. SGLT2 knockout mice were protected from HFD-induced hyperglycemia and glucose intolerance and had reduced plasma insulin concentrations compared with controls. On the db/db background, SGLT2 deletion prevented fasting hyperglycemia, and plasma insulin levels were also dramatically improved. Strikingly, prevention of hyperglycemia by SGLT2 knockout in db/db mice preserved pancreatic β-cell function in vivo, which was associated with a 60% increase in β-cell mass and reduced incidence of β-cell death.CONCLUSIONSPrevention of renal glucose reabsorption by SGLT2 deletion reduced HFD- and obesity-associated hyperglycemia, improved glucose intolerance, and increased glucose-stimulated insulin secretion in vivo. Taken together, these data support SGLT2 inhibition as a viable insulin-independent treatment of type 2 diabetes.

Lysophosphatidic acid (LPA) is both a potential marker and a therapeutic target for ovarian cancer. It is critical to identify the sources of elevated LPA levels in ascites and blood of patients with ovarian cancer. We show here that human peritoneal mesothelial cells constitutively produce LPA, which accounts for a significant portion of the chemotactic activity of the conditioned medium from peritoneal mesothelial cells to ovarian cancer cells. Both production of LPA by peritoneal mesothelial cells and the chemotactic activity in the conditioned medium can be blocked by HELSS [an inhibitor of the calcium-independent phospholipase A 2 (iPLA 2 )] and AACOCF 3 [an inhibitor of both cytosolic PLA 2 (cPLA 2 ) and iPLA 2 ]. Moreover, cell-based enzymatic activity assays for PLA 2 indicate that peritoneal mesothelial cells have strong constitutive PLA 2 activity. Receptors for LPA, LPA 2 , and LPA 3 are involved in the conditioned medium-induced chemotactic activity. Invasion of ovarian cancer cells into peritoneal mesothelial cells has also been analyzed and shown to require PLA 2 , LPA receptors, and the mitogen-activated protein/ extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase signaling pathway. Thus, we show here, for the first time, that human peritoneal mesothelial cells constitutively produce bioactive lipid signaling molecules, such as LPA, via iPLA 2 and/or cPLA 2 activities. Conditioned medium from peritoneal mesothelial cells stimulate migration, adhesion, and invasion of ovarian cancer cells, and may play similar roles in vivo.

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCδ, -ε, -θ, and -η) and conventional PKC (including PKCα and -β), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCε, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCδ, -θ, or -η is required. Furthermore, rottlerin (at doses that inhibit PKCδ activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O⨪2 production at similar doses. In addition to pharmacological inhibitors, PKCδ-specific antisense oligodeoxyribonucleotides were used. PKCδ antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCδ in vitro. Furthermore, lysate-derived PKCδ from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCδ plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.

Calcium-independent phospholipase A 2 (iPLA 2 ) plays a pivotal role in phospholipid remodeling and many other biological processes, including inflammation and cancer development. iPLA 2 can be activated by caspase-3 via a proteolytic process in apoptotic cells. In this study we identify novel signaling and functional loops of iPLA 2 activation leading to migration of non-apoptotic human ovarian cancer cells. The extracellular matrix protein, laminin-10/11, but not collagen I, induces integrin-and caspase-3-dependent cleavage and activation of overexpressed and endogenous iPLA 2 . The truncated iPLA 2 (amino acids 514 -806) generates lysophosphatidic acid and arachidonic acid. Arachidonic acid is important for enhancing cell migration toward laminin-10/11. Lysophosphatidic acid activates Akt that in turn acts in a feedback loop to block the cleavage of poly-(ADP-ribose) polymerase and DNA fragmentation factor as well as prevent apoptosis. By using pharmacological inhibitors, blocking antibodies, and genetic approaches (such as point mutations, dominant negative forms of genes, and siRNAs against specific targets), we show that ␤ 1 , but not ␤ 4 , integrin is involved in iPLA 2 activation and cell migration to laminin-10/ 11. The role of caspase-3 in iPLA 2 activation and cell migration are supported by several lines of evidence. 1) Point mutation of Asp 513 (a cleavage site of caspase-3 in iPLA 2 ) to Ala blocks laminin-10/11-induced cleavage and activation of overexpressed iPLA 2 , whereas mutation of Asp 733 to Ala has no such effect, 2) treatment of inhibitors or a small interfering RNA against caspase-3 results in decreased cell migration toward laminin-10/11, and 3) selective caspase-3 inhibitor blocks cleavage of endogenous iPLA 2 induced by laminin-10/11. Importantly, small interfering RNA-mediated down-regulation of endogenous iPLA 2 expression in ovarian carcinoma HEY cells results in decreased migration toward laminin, suggesting that our findings are pathophysiologically important.

The online version of this article has a supplementary appendix.Background C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models. Design and MethodsC-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated. -stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemiaderived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to Ctype lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells. ConclusionsOur results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.Key words: C-type, lectin-like molecule-1, immunotherapy, acute myeloid leukemia.Citation: Zhao X, Singh S, Pardoux C, Zhao J, Hsi ED, Abo A, and Korver W. Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia. Haematologica. 2010;95:71-78. doi:10.3324/haematol.2009 This is an open-access paper.Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia