Beta(2)-microglobulin as a negative growth regulator of myeloma cells

High beta(2)-microglobulin (beta(2)m) levels in myeloma correlate with poor prognosis. We hypothesized that beta(2)m may affect myeloma cell growth and survival. In this study, we examined the in vitro effects of beta(2)m on myeloma cells. Primary myeloma cells freshly isolated from patients and myeloma cell lines were used, cultured in the presence of beta(2)m, and monitored for growth and survival. Beta(2)m suppressed the growth of primary tumour cells and myeloma cell lines (ARK-RS, ARP-1, RPMI-8226, U266, ARH-77 and IM-9). High concentrations of beta(2)m induced apoptosis and cell cycle arrest. Beta(2)m-induced apoptosis was dependent on activation of a caspase cascade, inhibited by interleukin 6, and did not involve the surface death receptors, as receptor-neutralizing antibodies had no inhibitory effect. Beta(2)m-induced growth arrest was associated with downregulation of cyclins A and D2. Surprisingly, anti-beta(2)m antibodies did not block the effect of beta(2)m but were synergistic with beta(2)m, resulting in 90% growth inhibition and 70% apoptosis of myeloma cells. Whereas beta(2)m treatment resulted in slight upregulation of surface beta(2)m and major histocompatibility complex class I alpha-chain expression, treatment of myeloma cells with anti-beta(2)m antibodies alone or with beta(2)m resulted in significant downregulation of surface beta(2)m and class I molecules, suggesting that class I molecules may be involved in signal transduction. Our data demonstrate that beta(2)m plays an important role in regulating the growth and survival of myeloma cells in vitro and warrants further investigation to delineate the mechanisms of beta(2)m and anti-beta(2)m antibody-induced growth regulation of myeloma cells.     

Identification of low density lipoprotein as a regulator of Fc receptor-mediated phagocytosis.

Optimal expression of the high-affinity Fc receptor for IgG (FcRI) by the human monocyte cell line U-937 requires the presence of low density lipoprotein (LDL), and neither cholesterol nor high density lipoprotein can provide the component necessary for optimal FcRI expression. Here we show that FcR-mediated phagocytosis also requires LDL. U-937 cells were cultured in medium containing interferon gamma and either fetal calf serum (FCS) or delipidated FCS (DLFCS). The phagocytosis of IgG-coated erythrocytes was measured by a colorimetric assay. U-937 cells cultured in DLFCS medium had less than 16% of the phagocytic activity of cells cultured in normal FCS medium. Phagocytosis of IgG-coated erythrocytes could be inhibited 85% by the addition of murine IgG2a myeloma protein (5 micrograms/ml). U-937 cells cultured in DLFCS medium supplemented with pure cholesterol in ethanol (10 micrograms/ml) had only 30% of the phagocytic activity of cells grown in FCS medium. Addition of very low density lipoprotein (0.2 mg of protein per ml) to DLFCS medium also failed to increase phagocytosis. However, the addition of LDL (0.2 mg of protein per ml) to DLFCS medium restored 90% of the phagocytic activity. Since neither pure cholesterol nor very low density lipoprotein restored normal phagocytic function to U-937 cells despite a normalization of cellular cholesterol content, the restoration of phagocytosis observed with LDL replacement cannot be explained by mere delivery of cholesterol by LDL. Thus, LDL is required for the expression of FcRI and FcR-mediated phagocytosis by U-937 cells and may be an important regulator of phagocytic activity of monocytes and macrophages in vivo.     

Borrelia burgdorferi--induced oxidative burst, calcium mobilization, and phagocytosis of human neutrophils are complement dependent

When Borrelia burgdorferi, the spirochete causing Lyme disease, is transmitted to a human, the complement system is among the first challenges facing the bacterium. Neutrophils are crucial leukocytes in the first line of host defense against bacterial infections. To investigate the role of complement in the Borrelia-induced activation of human neutrophils, oxidative burst, calcium mobilization, and phagocytosis induced by three subspecies of B. burgdorferi were studied. Each subspecies induced all observed neutrophil functions in a complement-dependent manner. Serum-derived factors bound to the surface of B. burgdorferi were found to be essential for the induction of the oxidative burst. The CD11b chain of CR3 was found to participate in the oxidative burst and calcium mobilization induced by B. burgdorferi.     

The A-kinase anchor protein AKAP121 is a negative regulator of cardiomyocyte hypertrophy

Pathologic cardiac hypertrophy imposes a significant clinical burden on patients, yet the precise intracellular mechanisms responsible for its induction are only partially understood. We examined a potential role for AKAP121 to regulate cardiomyocyte hypertrophy, since recent reports have implicated other AKAPs in this process. We report here that knockdown of AKAP121 expression in isolated neonatal rat cardiomyocytes results in pronounced cellular hypertrophy. Loss of AKAP121 expression is associated with dephosphorylation and nuclear localization of NFATc3, a downstream effector of the hypertrophic phosphatase calcineurin. We also demonstrate that over-expression of AKAP121 in cardiac myocytes reduces basal cell size, and blocks hypertrophy induced by isoproterenol, indicating that AKAP121 negatively regulates the hypertrophic process. Co-immunoprecipitation data indicates that AKAP121 and calcineurin directly interact. Our findings are consistent with a model in which loss of AKAP121 expression leads to the release of an active pool of calcineurin, in turn causing nuclear translocation of NFATc3 and activation of the hypertrophic gene program. These results are the first to identify AKAP121 as a negative regulator of cardiomyocyte hypertrophy, and highlight AKAP121 as a potential target for therapeutic exploitation.     

IRA2, an upstream negative regulator of RAS in yeast, is a RAS GTPase-activating protein.

The ras GTPase-activating protein (GAP), identified and characterized in mammalian cells, stimulates the intrinsic GTPase activity of ras proteins. We have previously proposed that the IRA genes, negative regulators of RAS genes in Saccharomyces cerevisiae, encode yeast homologs of the mammalian GAP. In this paper, we present the following evidence that a product of the IRA2 gene exhibits GAP activity similar to that of the mammalian GAP protein. (i) Extracts of yeast cells overexpressing IRA2 stimulated the GTPase activity of the yeast RAS2 protein. (ii) An epitope for a monoclonal antibody (12CA5) was added to the N terminus of the IRA2 protein. The GAP activity of extracts prepared from cells expressing this fusion protein was shown to be immunoprecipitable by 12CA5. (iii) An IRA2 protein fused to glutathione S-transferase (GST) was produced and partially purified from Escherichia coli cells. GAP activity was detected with this purified GST-IRA2 fusion protein. (iv) The GAP activity of IRA2 proteins described above did not stimulate the GTPase activity of the RAS2Val19 protein, a protein having an amino acid alteration analogous to that found in mammalian oncogenic ras proteins. This result parallels studies showing that mammalian GAP is incapable of stimulating the GTPase activity of mammalian oncogenic proteins. The remarkable conservation between the GAP activity in mammalian and yeast cells supports the idea that the function of GAP is to negatively regulate ras proteins in mammalian cells.     

Lats2 is a negative regulator of myocyte size in the heart

Mammalian sterile 20-like kinase (Mst)1 plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Because Hippo, a Drosophila homolog of Mst1, forms a signaling complex with Warts, a serine/threonine kinase, which in turn stimulates cell death and inhibits cell proliferation, mammalian homologs of Warts, termed Lats1 and Lats2, may mediate the function of Mst1. We here show that Lats2, but not Lats1, dose-dependently increased apoptosis in cultured cardiac myocytes. Lats2 also dose-dependently reduced [(3)H]phenylalanine incorporation and cardiac myocyte size, whereas dominant negative Lats2 (DN-Lats2) increased them, suggesting that endogenous Lats2 negatively regulates myocyte growth. DN-Lats2 significantly attenuated induction of apoptosis and inhibition of hypertrophy by Mst1, indicating that Lats2 mediates the function of Mst1 in cardiac myocytes. Cardiac specific overexpression of Lats2 in transgenic mice significantly reduced the size of left and right ventricles, whereas that of DN-Lats2 caused hypertrophy in both ventricles. Overexpression of Lats2 reduced left ventricular systolic and diastolic function without affecting baseline levels of myocardial apoptosis. Expression of endogenous Lats2 was significantly upregulated in response to transverse aortic constriction. Overexpression of DN-Lats2 significantly enhanced cardiac hypertrophy and inhibited cardiac myocyte apoptosis induced by transverse aortic constriction. These results suggest that Lats2 is necessary and sufficient for negatively regulating ventricular mass in the heart. Although Lats2 is required for cardiac myocyte apoptosis in response to pressure overload, it was not sufficient to induce apoptosis at baseline. In conclusion, Lats2 affects both growth and death of cardiac myocytes, but it primarily regulates the size of the heart and acts as an endogenous negative regulator of cardiac hypertrophy.     

The murine stanniocalcin 2 gene is a negative regulator of postnatal growth

Stanniocalcin (STC), a secreted glycoprotein, was first studied in fish as a classical hormone with a role in regulating serum calcium levels. There are two closely related proteins in mammals, STC1 and STC2, with functions that are currently unclear. Both proteins are expressed in numerous mammalian tissues rather than being secreted from a specific endocrine gland. No phenotype has been detected yet in Stc1-null mice, and to investigate whether Stc2 could have compensated for the loss of Stc1, we have now generated Stc2(-/-) and Stc1(-/-) Stc2(-/-) mice. Although Stc1 is expressed in the ovary and lactating mouse mammary glands, like the Stc1(-/-) mice, the Stc1(-/-) Stc2(-/-) mice had no detected decrease in fertility, fecundity, or weight gain up until weaning. Serum calcium and phosphate levels were normal in Stc1(-/-) Stc2(-/-) mice, indicating it is unlikely that the mammalian stanniocalcins have a major physiological role in mineral homeostasis. Mice with Stc2 deleted were 10-15% larger and grew at a faster rate than wild-type mice from 4 wk onward, and the Stc1(-/-) Stc2(-/-) mice had a similar growth phenotype. This effect was not mediated through the GH/IGF-I axis. The results are consistent with STC2 being a negative regulator of postnatal growth.