Uma Kant Misra

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Activation and Cross-talk between Akt, NF-κB, and Unfolded Protein Response Signaling in 1-LN Prostate Cancer Cells Consequent to Ligation of Cell Surface-associated GRP78

Binding of activated forms of the proteinase inhibitor α2-macroglobulin (α2M*) to cell surface-associated GRP78 on 1-LN human prostate cancer cells causes their proliferation. We have now examined the interplay between Akt activation, regulation of apoptosis, the unfolded protein response, and activation of NF-κBin α2M*-induced proliferation of 1-LN cells. Exposure of cells to α2M* (50 pm) induced phosphatidylinositol 3-kinase-dependent activation of Akt by phosphorylation at Thr-308 and Ser-473 with a concomitant 60-80% increase in Akt-associated kinase activity. ERK1/2 and p38 MAPK were also activated, but there was only a marginal effect on JNK activation. Treatment of 1-LN cells with α2M* down-regulated apoptosis and promoted NF-κB activation as shown by increases of Bcl-2, p-BadSer-136, p-FOXO1Ser-253, p-GSK3βSer-9, XIAP, NF-κB, cyclin D1, GADD45β, p-ASK1Ser-83, and TRAF2 in a time of incubation-dependent manner. α2M* treatment of 1-LN cells, however, showed no increase in the activation of caspase -3, -9, or -12. Under these conditions, we observed increased unfolded protein response signaling as evidenced by elevated levels of GRP78, IRE1α, XBP-1, ATF4, ATF6, p-PERK, p-eIF2α, and GADD34 and reduced levels of GADD153. Silencing of GRP78 gene expression by RNAi suppressed activation of AktThr-308, AktSer-473, and IκB kinase α kinase. The effects of α2M* on the NF-κB activation, antiapoptotic signaling, unfolded protein response signaling, and proapoptotic signaling were also reversed by this treatment. In conclusion, α2M* promotes cellular proliferation of 1-LN prostate cancer cells by activating MAPK and Akt-dependent signaling, down-regulating apoptotic signaling, and activating unfolded protein response signaling.

Cadmium-induced DNA synthesis and cell proliferation in macrophages: the role of intracellular calcium and signal transduction mechanisms.

Cd(2+) exposure increases the risk of cancer in humans and animals. In this report, we have studied the effect of Cd(2+) on signal transduction and Ca(2+) mobilization in murine macrophages. At micromolar concentrations, Cd(2+) significantly increased cell division as judged by [3H]thymidine uptake and cell counts. Cd(2+)-treated cells continued to proliferate even after more than 4 weeks in culture. Cd(2+) (1 microM) treatment induced a 1.5- to 2-fold increase in cytosolic free Ca(2+), [Ca(2+)](i), which was transitory and/or oscillatory. The sources of this Ca(2+) included both inositol 1,4,5-trisphosphate (IP(3))-sensitive and -insensitive stores. Macrophage treatment with 1-(6-((17beta-3-methoxyestra-1,2,5(10)-triene-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), an inhibitor of phosphatidylinositol-specific phospholipase C (PLC), decreased Cd(2+)-induced formation of IP(3) in a concentration-dependent manner (K(d) about 2 microM). This caused a concomitant, partial decrease in the effect of Cd(2+) on [Ca(2+)](i). Cd(2+) itself crosses the macrophage membrane in part via L-type Ca(2+) channels, but it also interacts with a cell surface membrane protein(s) coupled to a pertussis toxin-sensitive G protein. Use of selective inhibitors of signal transduction and the quantitation of the levels of phosphorylated MAPK/ERK-activating kinase-1 (MEK1), extracellular signal-regulated kinase-1 (ERK1), and p38 mitogen-activated protein kinase (MAPK) suggests that the effects of Cd(2+) are mediated by the p21(ras)-dependent MAPK, but not the phosphoinositide 3 (PI 3)-kinase signalling pathway. The effect of activating these pathways includes increased availability of the transcription factor NFkappaB as well as activation of the early genes c-fos and c-myc.

The Role of Grp 78 in α2-Macroglobulin-induced Signal Transduction EVIDENCE FROM RNA INTERFERENCE THAT THE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN IS ASSOCIATED WITH, BUT NOT NECESSARY FOR, GRP 78-MEDIATED SIGNAL TRANSDUCTION

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of α2-Macroglobulin (α2M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni2+ nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block α2M*-induced signal transduction. In the current study, we employed α2-macroglobulin (α2M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to α2M* with high affinity (K d ∼150 pm). A monoclonal antibody directed against Grp 78 both abolished α2M*-induced signal transduction and co-precipitated LRP. Ligand blotting with α2M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on α2M*-mediated signaling. We conclude that Grp 78 is essential for α2M*-induced signal transduction and that a “co-receptor” relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.

Coordinate Regulation of Forskolin-induced Cellular Proliferation in Macrophages by Protein Kinase A/cAMP-response Element-binding Protein (CREB) and Epac1-Rap1 Signaling EFFECTS OF SILENCING CREB GENE EXPRESSION ON Akt ACTIVATION

In this study, we have examined the role of two cAMP downstream effectors protein kinase A (PKA) and Epac, in forskolin-induced macrophage proliferation. Treatment of macrophages with forskolin enhanced [3H]thymidine uptake and increased cell number, and both were profoundly reduced by prior treatment of cells with H-89, a specific PKA inhibitor. Incubation of macrophages with forskolin triggered the activation of Akt, predominantly by phosphorylation of Ser-473, as measured by Western blotting and assay of its kinase activity. Akt activation was significantly inhibited by LY294002 and wortmannin, specific inhibitors of phosphatidylinositol 3-kinase, but not by H-89. Incubation of macrophages with forskolin also increased Epac1 and Rap1·GTP. Immunoprecipitation of Epac1 in forskolin-stimulated cells co-immunoprecipitated Rap1, p-AktThr-308, and p-AktSer-473. Silencing of CREB gene expression by RNA interference prior to forskolin treatment not only decreased CREB protein and its phosphorylation at Ser-133, but also phosphorylation of Akt at Ser-473, and Thr-308. Concomitantly, this treatment inhibited [3H]thymidine uptake and reduced forskolin-induced proliferation of macrophages. Forskolin treatment also inhibited activation of the apoptotic mechanism while promoting up-regulation of the anti-apoptotic pathway. We conclude that forskolin mediates cellular proliferation via cAMP-dependent activation of both PKA and Epac.

The Role of MTJ-1 in Cell Surface Translocation of GRP78, a Receptor for α2-Macroglobulin-Dependent Signaling

MTJ-1 associates with a glucose-regulated protein of Mr ∼78,000(GRP78) in the endoplasmic reticulum and modulates GRP78 activity as a chaperone. GRP78 also exists on the cell surface membrane, where it is associated with a number of functions. MHC class I Ags on the cell surface are complexed to GRP78. GRP78 also serves as the receptor for α2-macroglobulin-dependent signaling and for uptake of certain pathogenic viruses. The means by which GRP78, lacking a transmembrane domain, can fulfill such functions is unclear. In this study we have examined the question of whether MTJ-1, a transmembrane protein, is involved in the translocation of GRP78 to the cell surface. MTJ-1 and GRP78 coimmunoprecipitated from macrophage plasma membrane lysates. Silencing of MTJ-1 gene expression greatly reduced MTJ-1 mRNA and protein levels, but also abolished cell surface localization of GRP78. Consequently, binding of the activated and receptor-recognized form of α2-macroglobulin to macrophages was greatly reduced, and activated and receptor-recognized form of α2-macroglobulin-induced calcium signaling was abolished in these cells. In conclusion, we show that in addition to assisting the chaperone GRP78 in protein quality control in the endoplasmic reticulum, MTJ-1 is essential for transport of GRP78 to the cell surface, which serves a number of functions in immune regulation and signal transduction.

Ligation of cancer cell surface GRP78 with antibodies directed against its COOH-terminal domain up-regulates p53 activity and promotes apoptosis

Binding of activated α2-macroglobulin to GRP78 on the surface of human prostate cancer cells promotes proliferation by activating signaling cascades. Autoantibodies directed against the activated α2-macroglobulin binding site in the NH2-terminal domain of GRP78 are receptor agonists, and their presence in the sera of cancer patients is a poor prognostic indicator. We now show that antibodies directed against the GRP78 COOH-terminal domain inhibit [3H]thymidine uptake and cellular proliferation while promoting apoptosis as measured by DNA fragmentation, Annexin V assay, and clonogenic assay. These antibodies are receptor antagonists blocking autophosphorylation and activation of GRP78. Using 1-LN and DU145 prostate cancer cell lines and A375 melanoma cells, which express GRP78 on their cell surface, we show that antibodies directed against the COOH-terminal domain of GRP78 up-regulate the tumor suppressor protein p53. By contrast, antibody directed against the NH2-terminal domain of GRP78 shows negligible effects on p53 expression. PC-3 prostate cancer cells, which do not express GRP78 on their cell surface, are refractory to the effects of anti-GRP78 antibodies directed against either the COOH- or NH2-terminal domains. However, overexpression of GRP78 in PC-3 cells causes translocation of GRP78 to the cell surface and promotes apoptosis when these cells are treated with antibody directed against its COOH-terminal domain. Silencing GRP78 or p53 expression by RNA interference significantly blocked the increase in p53 induced by antibodies. Antibodies directed against the COOH-terminal domain may play a therapeutic role in cancer patients whose tumors trigger the production of autoantibodies directed against the NH2-terminal domain of GRP78. [Mol Cancer Ther 2009;8(5):1350–62]

Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages

Apolipoprotein E (ApoE) is a 34‐kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca2+ stores. This cascade is inhibited by pretreatment with receptor‐associated protein and Ni2+, and it is mediated by a pertussis toxin‐sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor‐related protein. A peptide derived from the receptor‐binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE‐mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE‐dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells.

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP782 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 up-regulates and activates p53 causing decreased cell proliferation and up-regulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, AktThr308 and AktSer473 kinase activities and reduces phosphorylation of FOXO, and GSK3β. This treatment also suppresses activation of ERK1/2, p38 MAPK, and MKK3/6; however, it up-regulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX, and BAK expression as well as cleaved caspases-3, -7, -8, and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Ligation of Prostate Cancer Cell Surface GRP78 Activates a Proproliferative and Antiapoptotic Feedback Loop: A ROLE FOR SECRETED PROSTATE-SPECIFIC ANTIGEN*

GRP78, a well characterized chaperone in the endoplasmic reticulum, is critical to the unfolded protein response. More recently, it has been identified on the cell surface, where it has many roles. On cancer cells, it functions as a signaling receptor coupled to proproliferative/antiapoptotic and promigratory mechanisms. In the current study, we demonstrate that ligation of prostate cancer cell surface GRP78 by its natural ligand, activated α2-macroglobulin (α2M*), results in a 2–3-fold up-regulation in the synthesis of prostate-specific antigen (PSA). The PSA is secreted into the medium as an active proteinase, where it binds to native α2M. The resultant α2M·PSA complexes bind to GRP78, causing a 1.5–2-fold increase in the activation of MEK1/2, ERK1/2, S6K, and Akt, which is coupled with a 2–3-fold increase in DNA and protein synthesis. PSA is a marker for the progression of prostate cancer, but its mechanistic role in the disease is unclear. The present studies suggest that PSA may be involved in a signal transduction-dependent feedback loop, whereby it promotes a more aggressive behavior by human prostate cancer cells.