Receptor-recognized α2-macroglobulin binds to cell surface-associated GRP78 and activates mTORC1 and mTORC2 signaling in prostate cancer cells.

OBJECTIVE: Tetrameric α(2)-macroglobulin (α(2)M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2)M (α(2)M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2)M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells. METHODS: Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies. RESULTS: Stimulation of cells with α(2)M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308), and Akt(S473) in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2)M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2)M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2)M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473) phosphorylation and levels of p-Akt(S473) in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2)M*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates. CONCLUSION: Binding of α(2)M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.

Upregulation of mTORC2 activation by the selective agonist of EPAC, 8-CPT-2Me-cAMP, in prostate cancer cells: assembly of a multiprotein signaling complex.

Ligation of cell surface-associated GRP78 by activated α(2) -macroglobulin triggers pro-proliferative cellular responses. In part, this results from activation of adenylyl cyclase leading to an increase in cAMP. We have previously employed the cAMP analog 8-CPT-2Me-cAMP to probe these responses. Here we show in 1-LN prostate cancer cells that 8-CPT-2Me-cAMP causes a dose-dependent increase in Epac1, p-Akt(T308) , p-Akt(S473) , but not p-CREB. By contrast, the PKA activator 6-Benz-cAMP caused a dose-dependent increase in p-CREB, but not Epac1. We measured mTORC2-dependent Akt phosphorylation at S473 in immunoprecipitates of mTOR or Rictor from 1-LN cells. 8-CPT-2Me-cAMP caused a two-threefold increase in p-Akt(S473) and Akt(S473) kinase activity in Rictor immunoprecipitates. By contrast, there was only a negligible effect on p-Akt(T308) in Rictor immunoprecipitates. Silencing Rictor gene expression by RNAi significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of Akt at Ser(473) . These studies represent the first report that Epac1 mediates mTORC2-dependent phosphorylation of Akt(S473) . Pretreatment of these cells with the PI 3-Kinase inhibitor LY294002 significantly suppressed 8-CPT-2Me-cAMP-dependent p-Akt(S473) and p-Akt(S473) kinase activities, and both effects were rapamycin insensitive. This treatment caused a two to threefold increase in S6 Kinase and 4EBP1 phosphorylation, indices of mTORC1 activation. Pretreatment of the cells with LY294002 and rapamycin significantly suppressed 8-CPT-2Me-cAMP-induced phosphorylation of S6 Kinase and 4EBP1. We further demonstrate that in 8-CPT-2Me-cAMP-treated cells, Epac1 co-immunoprecipitates with AKAP, Raptor, Rictor, PDE3B, and PDE4D suggesting thereby that during Epac1-induced activation of mTORC1 and mTORC2, Epac1 may have an additional function as a "scaffold" protein.

Modulation of the unfolded protein response by GRP78 in prostate cancer.

The unfolded protein response (UPR) is an adaptive survival mechanism through which cells can weather the stress of misfolded protein accumulation induced by a wide variety of pathophysiologic and pharmacologic insults. The ER chaperone GRP78 is a central modulator of the UPR both through its protein-binding capacity and its direct regulation of the UPR signaling molecules IRE1α, PERK, and ATF6. Recent reports have revealed the presence of GRP78 on the surface of cancer cells. Biological roles for cell-surface GRP78 include competing NH(2)-domain and COOH-domain agonist receptor activities that induce opposite effects on proliferation and apoptosis. Modulation of the UPR impacts both of these processes directly and indirectly. Here, we outline methods that we use to investigate UPR modulation via direct ligation of cell-surface GRP78. Specifically, we review methods of cell culture, cell-signaling analysis with emphasis on UPR components, and ultimately, the impact that these have on cell proliferation, survival, and apoptosis.

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 α(2)-macroglobulin (α(2)M*), 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 α(2)M. The resultant α(2)M·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.

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 GRP78 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 upregulates and activates p53 causing decreased cell proliferation and upregulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, Akt(Thr308) and Akt(Ser473) kinase activities and reduces phosphorylation of FOXO, and GSK3beta. This treatment also suppresses activation of ERK1/2, p38 MAPK and MKK3/6; however, it upregulates 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.

The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages.

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.

Interaction between TCL1 and Epac1 in the activation of Akt kinases in plasma membranes and nuclei of 8-CPT-2-O-Me-cAMP-stimulated macrophages.

Epac1 is a cAMP-stimulated guanine exchange factor that activates Rap1. The protein product of the T cell leukemia 1 (TCL1) proto-oncogene binds to Akt enhancing its kinase activity. TCL1 and Epac promote cellular proliferation because of their activating effects on Akt. Employing macrophages, we have studied the mechanisms whereby these proteins function in the regulation of Akt kinase activity. Cells were treated with 8-CPT-2-O-Me-cAMP, a cAMP analog which acts selectively and specifically via Epac1. Epac1 co-immunoprecipitated with TCL1 in plasma membrane and nuclear fractions of 8-CPT-2-O-Me-cAMP-stimulated macrophages. Interaction of TCL1 and Epac1 was also observed in a [125I]GST-Epac1 pulldown assay. A two-threefold increase in Akt Thr-308 and Akt Ser-473 protein kinase activities and their phosphoprotein levels was observed in TCL1 immunoprecipitates of plasma membranes and nuclei of the treated cells. Elevated Akt Thr-308 protein kinase activity and its phosphoprotein levels were significantly reduced in TCL1 immunoprecipitates of plasma membranes of 8-CPT-2-O-Me-cAMP-treated cells where Epac1 gene expression was silenced. In contrast, Akt Ser-473 protein kinase activity and its phosphoprotein levels were reduced only in plasma membranes. Our studies suggest that a ternary complex of TCL1, Epac1, and Akt forms in activated macrophages both promoting Akt activation and regulating intracellular distribution of Akt.

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 [(3)H]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-Akt(Thr-308), and p-Akt(Ser-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 [(3)H]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.

Association of plasminogen with dipeptidyl peptidase IV and Na+/H+ exchanger isoform NHE3 regulates invasion of human 1-LN prostate tumor cells.

Binding of plasminogen type II (Pg 2) to dipeptidyl peptidase IV (DPP IV) on the surface of the highly invasive 1-LN human prostate tumor cell line induces an intracellular Ca2+ ([Ca2+]i) signaling cascade accompanied by a rise in intracellular pH (pHi). In endothelial cells, Pg 2 regulates intracellular pH via Na+/H+ exchange (NHE) antiporters; however, this mechanism has not been demonstrated in any other cell type including prostate cancer cells. Because the Pg 2 receptor DPP IV is associated with NHE3 in kidney cell plasma membranes, we investigated a similar association in 1-LN human prostate cancer cells and a mechanistic explanation for changes in [Ca2+]i or pHi induced by Pg 2 in these cells. Our results suggest that the signaling cascade initiated by Pg 2 and its receptor proceeds via activation of phospholipase C, which promotes formation of inositol 3,4,5-trisphosphate, an inducer of Ca2+ release from endoplasmic reticulum stores. Furthermore, our results suggest that Pg 2 may regulate pHi via an association with NHE3 linked to DPP IV in these cells. These associations suggest that Pg has the potential to simultaneously regulate calcium signaling pathways and Na+/H+ exchanges necessary for tumor cell proliferation and invasiveness.

Role of lysophospholipid growth factors in the modulation of aqueous humor outflow facility.

PURPOSE: To investigate the role of lysophospholipid growth factors in the regulation of aqueous humor outflow in the trabecular meshwork (TM). METHODS: The expression profile of the endothelial differentiation gene (Edg) family of G-protein coupled receptors was determined by RT-PCR of human TM (HTM) cell-derived total RNA and by PCR amplification of HTM cell-derived and tissue-derived cDNA libraries. The effects of lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) on actin cytoskeleton and focal adhesions and on myosin light-chain (MLC) phosphorylation in HTM cells were evaluated by immunofluorescence microscopy and Western blot analysis, respectively. Activation of Rho GTPase in HTM cells was quantified by "pull-down" assays. Mobilization of intracellular calcium in HTM cells was determined using spectrofluorometric digital-imaging microscopy. The effects of LPA and S1P on aqueous humor outflow facility were evaluated by perfusion of enucleated porcine eyes. RESULTS: Each of the receptor isoforms Edg1, -2, -3, and -4 was readily detectable in three of four HTM cell-derived libraries, whereas Edg2 was detectable in the HTM tissue library. LPA (20 microM) and S1P (1 microM) stimulated actin stress fiber and focal adhesion formation, increased MLC phosphorylation, and induced marked activation of Rho GTPase in HTM cells. Both LPA (20 microM) and S1P (10 microM) also stimulated increases in intracellular calcium concentration in HTM cells. LPA- and S1P-induced effects on MLC phosphorylation in HTM cells were markedly inhibited by pretreatment with the Rho kinase-specific inhibitor Y-27632 (5 microM). Perfusion of LPA (50 microM) and S1P (5 microM) in enucleated porcine eyes produced a significant decrease in aqueous humor outflow facility from baseline of 37% (n = 6) and 31% (n = 5), respectively. CONCLUSIONS: These studies demonstrate that LPA and S1P, the physiological agonists of Edg receptors, decrease outflow facility in perfused porcine eyes in association with increased MLC phosphorylation and Rho guanosine triphosphatase (GTPase) activation. These data provide evidence for a novel mechanism for negative regulation of outflow facility, which may contribute to overall physiological homeostasis of aqueous humor outflow facility.

The role of Grp 78 in alpha 2-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 alpha(2)-Macroglobulin (alpha(2)M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni(2+) nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block alpha(2)M*-induced signal transduction. In the current study, we employed alpha(2)-macroglobulin (alpha(2)M)-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 alpha(2)M* with high affinity (K(d) approximately 150 pm). A monoclonal antibody directed against Grp 78 both abolished alpha(2)M*-induced signal transduction and co-precipitated LRP. Ligand blotting with alpha(2)M* 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 alpha(2)M*-mediated signaling. We conclude that Grp 78 is essential for alpha(2)M*-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.