Govind Gawdi

Prostate Cancer Cell Proliferation In vitro Is Modulated by Antibodies against Glucose-Regulated Protein 78 Isolated from Patient Serum

Circulating autoantibodies against the glucose-regulated protein of 78 kDa (GRP78) are present at high levels in prostate cancer patients and are a biomarker of aggressive tumor behavior. We purified the anti-GRP78 IgGs and examined their effect on 1-LN, PC-3, DU145, and LnCap human prostate cancer cells. We also evaluated its effects on the breast cancer MDA-MB231 and melanoma DM413 cell lines. The anti-GRP78 antibody binds only to cells expressing GRP78 on the surface, to a site also recognized by its physiologic agonist, activated alpha(2)-macroglobulin (alpha(2)M*). This antibody is completely specific for a peptide, including the primary amino acid sequence CNVKSDKSC, which contains a tertiary structural motif mimicking an epitope in GRP78. Tertiary structural analysis suggested the linear GRP78 primary amino acid sequence LIGRTWNDPSVQQDIKFL (Leu(98)-Leu(115)) as the putative binding site, containing the tertiary structual arrangement described above, which was confirmed experimentally. The anti-GRP78 antibodies from prostate cancer patients recognize almost exclusively this epitope. We produced animal antibodies against both these peptides, and they are able to mimic the effects of the human antibody. Our experiments also suggest this epitope as highly immunogenic, thereby explaining the specificity of the immune response against this epitope in GRP78, observed in humans. Using 1-LN cells as a model, we show that anti-GRP78 IgG purified from the sera of these patients mimics the proproliferative effects induced by alpha(2)M* via the common receptor, GRP78. Furthermore, increasing concentrations of human anti-GRP78 IgG show a dose-dependent protective effect on apoptosis induced by tumor necrosis factor alpha.

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.

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.

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.

Plasminogen Structural Domains Exhibit Different Functions When Associated with Cell Surface GRP78 or the Voltage-dependent Anion Channel

Both the voltage-dependent anion channel and the glucose-regulated protein 78 have been identified as plasminogen kringle 5 receptors on endothelial cells. In this study, we demonstrate that kringle 5 binds to a region localized in the N-terminal domain of the glucose-regulated protein 78, whereas microplasminogen does so through the C-terminal domain of the glucose-regulated protein 78. Both plasminogen fragments induce Ca2+ signaling cascades; however, kringle 5 acts through voltage-dependent anion channel and microplasminogen does so via the glucose-regulated protein 78. Because trafficking of voltage-dependent anion channel to the cell surface is associated with heat shock proteins, we investigated a possible association between voltage-dependent anion channel and glucose-regulated protein 78 on the surface of 1-LN human prostate tumor cells. We demonstrate that these proteins co-localize, and changes in the expression of the glucoseregulated protein 78 affect the expression of voltage-dependent anion channel. To differentiate the functions of these receptor proteins, either when acting singly or as a complex, we employed human hexokinase I as a specific ligand for voltage-dependent anion channel, in addition to kringle 5. We show that kringle 5 inhibits 1-LN cell proliferation and promotes caspase-7 activity by a mechanism that requires binding to cell surface voltage-dependent anion channel and is inhibited by human hexokinase I.

Induction of mitogenic signalling in the 1LN prostate cell line on exposure to submicromolar concentrations of cadmium

Cadmium exposure increases the risk of prostate cancer. We now describe the effects of Cd2+ on signalling and proliferation in 1LN prostate cells. Cd2+ increased [3H]thymidine uptake and cell number twofold. Cd2+ elevated intracellular IP3, cytosolic-free Ca2+, phosphorylated MEK1/2, ERK1/2, p38 MAPK and JNK two- to threefold. Increased PDK1 and phosphorylation of the 85-kDa regulatory subunit of PI 3-kinase, Akt and p70s6k were also observed. Cd2+ treatment increased transcription factors NFkappaB and CREB, and the expression of c-fos and c-myc. Cd2+-induced increased uptake of [3H]thymidine was abolished by translational and transcriptional inhibitors, and Ca2+ channel blockers. Inhibition of phospholipase C and of Ca2+ binding to IP3 receptors inhibited Cd2+-induced DNA synthesis as did inhibition of tyrosine kinases, protein kinase C, PI 3-kinase, farnesyl transferase, MEK1/2, ERK1/2 and p38MAPK. Thus signalling events, which are triggered on exposure of 1LN cells to submicromolar concentrations of Cd2+, induce increased proliferation of these cells.

Chloroquine, quinine and quinidine inhibit calcium release from macrophage intracellular stores by blocking inositol 1,4,5‐trisphosphate binding to its receptor

The binding of many ligands to cellular receptors induces a signaling cascade which generates inositol 1,4,5‐trisphosphate (IP3). IP3 binding to its receptors in various internal compartments causes a rapid Ca2+ efflux into the cytosol. We now demonstrate that chloroquine blocks ligand‐induced Ca2+ mobilization without affecting IP3 synthesis. The effect is independent of the ligand employed and occurred with five unrelated ligands; namely, α2‐macroglobulin‐methylamine, angiotensin II, bradykinin, carbachol, and epidermal growth factor. Chloroquine, quinidine, and quinine, however, block binding of [3H]IP3 to its receptors by 90%, 88%, and 71%, respectively. These observations suggest a previously undetected mechanism by which these agents may in part function as antimalarials. J. Cell. Biochem. 64:225–232.

Interaction of plasminogen with dipeptidyl peptidase IV initiates a signal transduction mechanism which regulates expression of matrix metalloproteinase-9 by prostate cancer cells.

Both plasminogen (Pg) activation and matrix metalloproteinases (MMPs) are involved in the proteolytic degradation of extracellular matrix components, a requisite event for malignant cell metastasis. The highly invasive 1-LN human prostate tumour cell line synthesizes and secretes large amounts of Pg activators and MMPs. We demonstrate here that the Pg type 2 (Pg 2) receptor in these cells is composed primarily of the membrane glycoprotein dipeptidyl peptidase IV (DPP IV). Pg 2 has six glycoforms that differ in their sialic acid content. Only the highly sialylated Pg 2gamma, Pg 2delta and Pg 2epsilon glycoforms bind to DPP IV via their carbohydrate chains and induce a Ca(2+) signalling cascade; however, Pg 2epsilon alone is also able to significantly stimulate expression of MMP-9. We further demonstrate that the Pg-mediated invasive activity of 1-LN cells is dependent on the availability of Pg 2epsilon. This is the first demonstration of a direct association between the expression of MMP-9 and the Pg activation system.

Coordinate Regulation of the α2-Macroglobulin Signaling Receptor and the Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor by Insulin

We have studied insulin-dependent regulation of macrophage α2-macroglobulin signaling receptors (α2MSR) and low density lipoprotein receptor-related protein/α2M receptors (LRP/α2MR) employing cell binding of 125I-α2M*, inhibition of binding by receptor-associated protein (RAP) or Ni2+, LRP/α2MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of α2M* high (α2MSR) and low (LRP/α2MR) affinity binding sites from 1,600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni2+ blocked the binding of125I-α2M* to α2MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/α2MR. Insulin significantly increased LRP/α2MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented125I-α2M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in α2MSR receptor synthesis was reflected by augmented generation of IP3 and increased [Ca2+] i levels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and α2M* stimulation significantly reduced the insulin-augmented increase in IP3 and [Ca2+] i levels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new α2MSR. In conclusion, we show here that insulin coordinately regulates macrophage α2MSR and LRP/α2MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.