Activated α2-macroglobulin binding to human prostate cancer cells triggers insulin-like responses.

Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis. α2M*-treated human prostate cancer cells exhibit a 2-3-fold increase in glucose uptake and lactate secretion, an effect similar to insulin treatment. In both α2M* and insulin-treated cells, the mRNA levels of SREBP1-c, SREBP2, fatty-acid synthase, acetyl-CoA carboxylase, ATP citrate lyase, and Glut-1 were significantly increased together with their protein levels, except for SREBP2.

Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with Raptor.

PDK1 phosphorylates multiple substrates including Akt by PIP3-dependent mechanisms. In this report we provide evidence that in prostate cancer cells stimulated with activated α2-macroglobulin (α2M*) PDK1 phosphorylates Akt in the T-loop at Thr(308) by using Raptor in the mTORC1 complex as a scaffold protein. First we demonstrate that PDK1, Raptor, and mTOR co-immunoprecipitate.

The monomeric receptor binding domain of tetrameric α2-macroglobulin binds to cell surface GRP78 triggering equivalent activation of signaling cascades.

α2-Macroglobulin (α2M) is a broad spectrum proteinase inhibitor that when activated by proteinases (α2M*) undergoes a major conformational change exposing receptor recognition sites in each of its four subunits. These complexes bind to two distinct receptors, namely, the low-density lipoprotein receptor-related protein (LRP) and cell surface glucose-regulated protein [Mr ∼ 78000 (GRP78)]. The latter is a very high affinity receptor (Kd = 50-100 pM) whose ligation triggers pro-proliferative and anti-apoptotic signaling cascades.

Evidence for a pro-proliferative feedback loop in prostate cancer: the role of Epac1 and COX-2-dependent pathways.

Objective: In human prostate cancer cells, a selective Epac agonist, 8-CPT-2Me-cAMP, upregulates cell proliferation and survival via activation of Ras-MAPK and PI- 3-kinase-Akt-mTOR signaling cascades. Here we examine the role of inflammatory mediators in Epac1-induced cellular proliferation by determining the expression of the pro-inflammatory markers p-cPLA2, COX-2, and PGE2 in prostate cancer cells treated with 8-CPT-2Me-cAMP.

Methods: We employed inhibitors of COX-2, mTORC1, and mTORC2 to probe cyclic AMP-dependent pathways in human prostate cancer cells.

Epac1-induced cellular proliferation in prostate cancer cells is mediated by B-Raf/ERK and mTOR signaling cascades.

cAMP-dependent, PKA-independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8-CPT-2-O-Me-cAMP to study binding to EPAC and subsequent activation of B-Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1-LN prostate cancer cells.

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

Binding of activated α(2)-macroglobulin to GRP78 on the surface of human prostate cancer cells promotes proliferation by activating signaling cascades. Autoantibodies directed against the activated α(2)-macroglobulin binding site in the NH(2)-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 [(3)H]thymidine uptake and cellular proliferation while promoting apoptosis as measured by DNA fragmentation, Annexin V assay, and clonogenic assay.

Heterotrimeric Galphaq11 co-immunoprecipitates with surface-anchored GRP78 from plasma membranes of alpha2M*-stimulated macrophages.

We have previously shown that a fraction of newly expressed GRP78 is translocated to the cell surface in association with the co-chaperone MTJ-1. Proteinase and methylamine-activated alpha(2)M (alpha(2)M*) bind to cell surface-associated GRP78 activating phosphoinositide-specific phospholipase C coupled to a pertussis toxin-insensitive heterotrimeric G protein, generating IP(3)/calcium signaling. We have now studied the association of pertussis toxin-insensitive Galphaq11, with GRP78/MTJ-1 complexes in the plasma membranes of alpha(2)M*-stimulated macrophages.

Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter.

Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin-induced activation of Akt1. We now provide evidence that forskolin-treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [(14)C]leucine or [(35)S] incorporation into newly synthesized Akt1 protein.

Activation and cross-talk between Akt, NF-kappaB, 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 alpha2-macroglobulin (alpha2M*) 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-kappaB in alpha2M*-induced proliferation of 1-LN cells. Exposure of cells to alpha2M* (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.

Ligation of cell surface-associated glucose-regulated protein 78 by receptor-recognized forms of alpha 2-macroglobulin: activation of p21-activated protein kinase-2-dependent signaling in murine peritoneal macrophages.

Previous studies of the plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) demonstrated that alpha2M-proteinase complexes (alpha2M*) modulate immune responses and promotes macrophage locomotion and chemotaxis. Alpha2M* binds to cell surface-associated glucose-regulated protein 78 (GRP78), which activates downstream signaling events. The role of p21-activated protein kinase-1 and -2 (PAK-1 and -2) in promoting cellular motility is well documented.

Binding of activated alpha2-macroglobulin to its cell surface receptor GRP78 in 1-LN prostate cancer cells regulates PAK-2-dependent activation of LIMK.

Two characteristics of highly malignant cells are their increased motility and secretion of proteinases allowing these cells to penetrate surrounding basement membranes and metastasize. Activation of 21-kDa activated kinases (PAKs) is an important mechanism for increasing cell motility. Recently, we reported that binding of receptor-recognized forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M*) to GRP78 on the cell surface of 1-LN human prostate cancer cells induces mitogenic signaling and cellular proliferation.

Up-regulation of GRP78 and antiapoptotic signaling in murine peritoneal macrophages exposed to insulin.

The unfolded protein response pathway (UPR) compensates for excessive protein accumulation in the endoplasmic reticulum (ER). As insulin induces global protein synthesis, it may cause accumulation of unfolded proteins in the ER, thus triggering UPR. We assessed UPR activation in insulin-treated murine peritoneal macrophages using a number of markers including 78 kDa glucose response protein (GRP78), X-box-binding protein (XBP)-1, pancreatic ER kinase (PERK), eukaryotic initiation factor 2 (eIF2)alpha, and growth arrest and DNA damage (GADD)34.

The role of MTJ-1 in cell surface translocation of GRP78, a receptor for alpha 2-macroglobulin-dependent signaling.

MTJ-1 associates with a glucose-regulated protein of Mr approximately 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.

A novel receptor function for the heat shock protein Grp78: silencing of Grp78 gene expression attenuates alpha2M*-induced signalling.

The activated proteinase inhibitor alpha2-macroglobulin (alpha2M*) binds to two receptors, the low density lipoprotein receptor-related protein (LRP-1) and the alpha2M* signalling receptor (alpha2MSR). Silencing LRP-1 gene expression in macrophages by RNA interference does not block alpha2M* activation of signalling cascades. We now demonstrate that transfection of macrophages with a double-stranded RNA homologous in sequence to the Grp78 gene markedly decreased induction of inositol 1,4,5-trisphosphate (IP3) and subsequent IP3-dependent elevation of [Ca2+]i induced by alpha2M*.

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.

The role of cAMP-dependent signaling in receptor-recognized forms of alpha 2-macroglobulin-induced cellular proliferation.

Ligation of alpha(2)-macroglobulin receptors by receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) activates various signaling cascades and promotes cell proliferation. It also elevates cAMP in murine peritoneal macrophages. We now report that a significant elevation of cAMP-response element-binding protein (CREB) occurs in alpha(2)M*-stimulated cells, and this effect is potentiated by isobutylmethylxanthine, dibutyryl-cAMP, or forskolin.

Beryllium fluoride-induced cell proliferation: a process requiring P21(ras)-dependent activated signal transduction and NF-kappaB-dependent gene regulation.

We studied the effect of beryllium fluoride on murine peritoneal macrophages and determined its effects on signal transduction and genetic regulation. At low concentration (1-5 nM), BeF(2) caused an approximate twofold increase in [(3)H]thymidine uptake and cell number, but above 5 nM, it showed cytotoxic effects. BeF(2) increased cellular inositol (1,4,5)trisphosphate (IP(3)) and [Ca(2)(+)](i) about twofold.

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.

Regulation of cytosolic phospholipase A2 activity in macrophages stimulated with receptor-recognized forms of alpha 2-macroglobulin: role in mitogenesis and cell proliferation.

Macrophages exposed to receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*) demonstrate increased DNA synthesis and cell division. In the current study, we have probed the role of cytosolic phospholipase A(2) (cPLA(2)) activity in the cellular response to alpha(2)M*. Ligation of the alpha(2)M* signaling receptor by alpha(2)M*, or its receptor binding fragment, increased cPLA(2) activity 2-3-fold in a concentration and time-dependent manner.