HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 19, 13694-13707, May 12, 2006

This Article Abstract Full Text (PDF) All Versions of this Article: 281/19/13694    most recent M511694200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Misra, U. K. Articles by Pizzo, S. V. Search for Related Content PubMed PubMed Citation Articles by Misra, U. K. Articles by Pizzo, S. V. Social Bookmarking                      What's this?

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

From the Department of Pathology, Duke University, Medical Center, Durham, North Carolina 27710

Received for publication, October 28, 2005 , and in revised form, March 15, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Binding of activated forms of the proteinase inhibitor ₂-macroglobulin (₂M^*) 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-Bin ₂M^*-induced proliferation of 1-LN cells. Exposure of cells to ₂M^* (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 ₂M^* down-regulated apoptosis and promoted NF-B activation as shown by increases of Bcl-2, p-Bad^Ser-136, p-FOXO1^Ser-253, p-GSK3^Ser-9, XIAP, NF-B, cyclin D1, GADD45, p-ASK1^Ser-83, and TRAF2 in a time of incubation-dependent manner. ₂M^* 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, IRE1, XBP-1, ATF4, ATF6, p-PERK, p-eIF2, and GADD34 and reduced levels of GADD153. Silencing of GRP78 gene expression by RNAi suppressed activation of Akt^Thr-308, Akt^Ser-473, and IB kinase kinase. The effects of ₂M^* on the NF-B activation, antiapoptotic signaling, unfolded protein response signaling, and proapoptotic signaling were also reversed by this treatment. In conclusion, ₂M^* 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Prostate cancer is the most commonly diagnosed malignancy of men (1). Its progression may be accompanied by acquisition of androgen independence, a poor prognostic indicator associated with a more metastatic phenotype (2, 3, 5). Many growth factors play roles in the progression of androgen-independent prostate cancer. Ligand binding to growth factor receptors induces their autophosphorylation at specific tyrosine residues, resulting in the assembly of multiprotein complexes, which activate the Ras/MAPK² and PI 3-kinase signaling pathways, promoting tumor cell proliferation (4). Receptor-mediated activation of PI 3-kinase results in its recruitment to the inner surface of the plasma membrane, where it generates 3'-phosphorylated phosphoinositides. These lipids regulate downstream signal transduction by activating Akt, which is overexpressed in many forms of cancer, including prostate (5-7). Once activated, Akt both promotes cellular proliferation, and it phosphorylates the NF-B inhibitor IB, inducing translocation of NF-B to the nucleus, where it up-regulates the transcription of antiapoptotic genes, such as the inhibitors of apoptosis proteins (IAPs) and Bcl-2 (8-13).

The molecular chaperone GRP78 (glucose-regulated protein of 78 kDa) is important in the folding, maturation, and transport of proteins out of the cell. GRP78 is critical for the unfolded protein response (UPR) required to alleviate ER "stress," maintain ER function, and protect cells against death (14-17). Whereas GRP78 is constitutively expressed, its synthesis is induced by stressful conditions that perturb protein folding and assembly within the ER (14-17). A small pool of this newly synthesized GRP78 translocates to the cell surface from the ER in association with MTJ-1 (18), where it functions as a co-receptor for viruses and major histocompatibility complex Class I antigen presentation and as a receptor for activated forms of the plasma proteinase inhibitor ₂-macroglobulin (₂M^*) (see Refs. 18-21 and references therein). Activation of this receptor on the surface of 1-LN human prostate cancer cells by ₂M^* triggers proproliferative and antiapoptotic behavior; therefore, it could be argued that up-regulation of cell surface-associated GRP78 is part of the aggressive phenotype in prostate cancer (22). Consistent with this hypothesis, autoantibodies against GRP78 appear in the sera of prostate cancer patients, and they are a biomarker of aggressive behavior (23, 24). The circulating concentration of ₂M is about 2-5 µM, and its proteinase activated form, ₂M^*, may comprise approximately a 200-500 nM concentration of this pool (25). Prostate cancer cells themselves may produce prostate cancer-specific antigen, a proteinase that binds readily to ₂M, converting it to ₂M^* (26, 27). Aggressive prostate cancers also produce matrix metalloproteinases, which readily convert ₂M to ₂M^*. Therefore, it could be envisaged that under the conditions that exist in patients harboring prostate cancer, a substantial amount of ₂M^* is available to bind to cell surface-associated GRP78, thus triggering the activation of mitogenic signaling and promoting cellular proliferation.

The activation of all three MAPK cascades (namely extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK) occurs in cells undergoing ER stress (28, 28-33, 35-39). ASK1 (apoptosis signal-regulating kinase 1) is a Ser/Thr kinase that activates both the p38 MAPK and JNK pathways by directly phosphorylating MKK3/MKK6 and MKK4/MKK7, respectively (28, 39-42). ASK1 is activated by a variety of stimuli, including TNF, ER stress, and calcium signaling consequent to ligation of G-protein-coupled receptors (39-42). The antiapoptotic activity of Bcl-2 is reduced when it is phosphorylated by activated ASK1 (28, 41). On the other hand, overexpression of ASK 1 may induce not only apoptosis but cell differentiation and survival, depending on the cell type and cellular context (28, 41, 42). TNF is a potent activator of ASK1, and this activation is regulated by TRAF2 (TNF receptor-associated factor 2) (28, 40-42). TRAF2 is an adaptor protein that couples TNF receptor ligation to activation of the ASK1-JNK/p38 MAPK cascades (28, 40-42).

Prolonged ER stress can activate apoptosis both by mitochondria-dependent and -independent pathways (14-17). Bad, a proapoptotic member of the Bcl-2 family, triggers release of cytochrome c from mitochondria (see Ref. 43 and references therein), and this activates cytosolic apoptotic proteinase-activating factor 1, thus activating the downstream caspase-dependent proapoptotic cascade (43, 44). UPR-induced free calcium elevation activates calpain, which cleaves procaspase-12, an ER stress-specific caspase (14-17, 45, 50). Once activated during ER stress, the catalytic subunit of caspase-12 is released into the cytosol, where it activates the caspase-9 cascade in a cytochrome c-independent manner. Procaspase-12 can also be activated by caspase-7 or through IRE1-TRAF2-dependent pathways (16, 29, 49-51).

NF-B regulates the transcription of many genes involved in stress remediation, cell growth, and apoptosis (52-55). In the inert state, NF-B is present in the cytoplasm in association with the inhibitory protein, IB. Activation of NF-B occurs in response to a variety of stressful conditions, where impaired protein folding in the ER is characteristic (52-55). These stimuli activate the IB kinase (IKK) subunit, which phosphorylates NF-B-bound IB and targets it for ubiquitin-dependent degradation, allowing liberated NF-B dimers to translocate to the nucleus (53). In an alternative pathway, the IKK subunit of the IKK complex processes NF-B2 precursor protein, which preferentially binds RelB in the cytoplasm, resulting in the release of RelB-p52 dimers (55). Persistent NF-B activation is detected in many solid tumors, and its inhibition increases their sensitivity to chemotherapeutic agents and radiation (54). Overexpression of NF-B suppresses apoptosis, whereas IB- mutants, which can no longer be phosphorylated, are resistant to ubiquitin-mediated degradation and suppress NF-B activation, thus sensitizing cells to apoptotic insults (16, 52-55). IRE1 is also involved in the activation of NF-B induced by ER stress (52-55). Activation of NF-B by ER stress-inducing agents is inhibited by dominant negative IRE1 (50) or TRAF2 mutants (56). A recent report demonstrates that UV light-induced activation of NF-B is due to the PERK-induced phosphorylation of eIF2, which causes translational inhibition of new IB synthesis (57).

In the present report, we have examined the role of Akt activation, regulation of apoptosis, and the unfolded protein response in ₂M^*-induced growth of 1-LN prostate cancer cells. We report an increase in Akt^Thr-308 and Akt^Ser-473 kinase activities, up-regulation of UPR components, including IREI, XBP-1, ATF6, GRP78, p-PERK, p-eIF2, GADD34 (growth arrest and DNA damage-inducible 34), and ATF4, as well as up-regulation of components of the anti-apoptotic machinery Bcl-2, p-FOXO1, NF-B, GADD45, and XIAP in 1-LN prostate cancer cells treated with ₂M^*. We have also studied the role of GRP78 in ₂M^*-induced activation of cell survival and antiapoptotic signaling by silencing GRP78 gene expression employing RNAi.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Culture media were purchased from Invitrogen. ₂M^* was prepared as described previously (18, 19). Antibodies against Akt protein, p-Akt^Thr-308, p-Akt^Ser-473, phosphorylated and unphosphorylated ERK1/2, p38 MAPK, JNK, ASK1, MKK3/6, MKK4, MKK7, eIF2, FOXO, GSK3, p-PERK, TRAF2, NF-B (p65), NF-B₂ (p52), IKK/, IB-, IB-, NIK, Bcl-2, XIAP, p27^kip, cyclin D1, cleaved caspase-3, procaspase-9, and procaspase-12, used in this study were procured from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies against IREI, XBP1, ATF6, ATF4, GADD153, GADD34, GADD45, 14-3-3, and actin were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-GRP78 antibodies and glyceraldehyde-3-phosphate dehydrogenase antibodies were from Stressgen (Victoria, Canada). [-³³P]ATP (specific activity 3000 Ci mmol) was purchased from PerkinElmer Life Sciences. The peptide substrates for Akt^Ser-473 kinase (NH₂-RRPHFPQFSYSA-COOH) and for Akt^Thr-308 kinase (NH₂-KTFCGTPEYLAPEVRR-COOH) were synthesized by Genemed (South San Francisco, CA). Control substrate peptide Zak3tide and glutathione S-transferase-IB- substrate (amino acids 1-54) were purchased from Upstate Cell Signaling Solutions (Charlottesville, VA). Other reagents were of the highest available grade.

Western Blotting of Activated Akt in ₂M^*-stimulated 1-LN Prostate Cancer Cells—The highly metastatic prostate carcinoma cell line 1-LN, derived from less metastatic PC-3 cells, was a kind gift from Dr. Philip Walther (Duke University Medical Center, Durham, NC). 1-LN cells in 6-well plates (500 x 10³ cells/well) were allowed to grow to confluence in RPMI 1640 medium containing 10% FBS, penicillin (12.5 units/ml), streptomycin (6.5 µg/ml), 2 mM glutamine, and 10 nM insulin at 37 °C in a humidified CO₂ (5%) incubator. At about 90% confluence, the medium was aspirated, the monolayers were washed with ice-cold Hepes-buffered Hanks' basic salt solution, pH 7.4, and a fresh volume of medium was added to the monolayers. The cells were exposed to ₂M^* (50 pM) for varying periods of time. The reaction was stopped by aspirating the medium, and a volume of lysis buffer containing 50 mM Tris·HCl (pH 7.5), 120 mM NaCl, 1% (v/v) Nonidet P-40, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 1 mM benzamidine, and leupeptin (20 µg/ml) was added. The cells were lysed for 10 min over ice, scraped into tubes, and centrifuged at 8000 x g for 10 min at 4 °C, and their protein contents were determined (58). In each case an equal amount of protein was used for electrophoresis. The immunoblottting of membranes with antibodies specific for phosphorylation of Akt in Thr-308 or Ser-473 was performed according to the manufacturer's instructions. The detection and quantification of immunoblots were performed by ECF and phosphorimaging. The membranes were reprobed for the protein loading control actin.

Measurement of Phosphorylation of Akt at Thr-308 and Ser-473 and Their Kinase Activities in Cells Stimulated with ₂M^*—The p-Akt^Thr-308 and p-Akt^Ser-473 kinase activities in Akt immunoprecipitates of 1-LN prostate cancer cells stimulated with ₂M^* (50 pM/15 min) were assayed essentially according to Hill and Jennings (59). Briefly, 1-LN cells (in two 6-well plates, 4 x 10⁶ cells/well) were incubated as above until confluence. The monolayers were washed in Hepes-buffered Hanks' basic salt solution, pH 7.4, twice, and a fresh volume of incubation medium was added to each well. The monolayers were stimulated with buffer or ₂M^* (50 pM/15 min) in triplicate. The reactions were stopped by aspirating the medium, and a volume of lysis buffer containing 50 mM Tris·HCl (pH 7.5), 120 mM NaCl, 1% (v/v) Nonidet P-40, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 1 mM benzamidine, and leupeptin (20 µg/ml) was then added to each incubation. The cells were lysed for 10 min over ice, scraped into tubes, and centrifuged at 8000 x g for 10 min at 4 °C, and their protein contents were determined (58). Equal amounts of lysate proteins were immunoprecipitated with Akt antibodies (1:50) at 4 °C overnight with gentle rotations, according to the manufacturer's instruction. Akt immunoprecipitates were washed sequentially with lysis buffer supplemented with 0.5 M NaCl, lysis buffer, and 50 mM Tris·HCl (pH 7.4) supplemented with 1 mM dithiothreitol, 1 mM PMSF, and 1 mM benzamidine with centrifugation at 8000 x g for 5 min at 4 °C employed between each wash. To each immunoprecipitate, 40 µl of cold kinase buffer containing 50 mM Tris·HCl (pH 7.5), 10 mM MgCl₂,1 mM dithiothreitol, 1 mM PMSF, 1 mM benzamidine, and 20 µg/ml leupeptin was added, followed by the addition of 30 µM Akt^Thr-308 kinase substrate peptide (NH₂-RRPHFPQFSYSA-COOH) in respective tubes. Zak3tide (NH₂-GGEEEEYFELVKKKK-COOH) was used as the control peptide. The reaction was initiated by adding 50 µM ATP and 2 µCi of [-³³P]ATP in each tube, and the tubes were incubated for 30 min at 30 °C with shaking. The reaction was stopped by adding 5 µl of 0.5 M EDTA to each tube, the tubes were centrifuged at 3000 rpm for 3 min, and 40 µl of each supernatant was applied on p81 phosphocellulose paper (Whatman). Supernatants were allowed to dry, and the papers were washed four times each by immersing them in a liter of 1 N phosphoric acid for 3 min. The papers were rinsed with acetone, and their radioactivity was counted in a liquid scintillation counter (22).

Western Blotting of p-ERK1/2^{Thr-202/Tyr-204}, MKK3/6, p-MKK3/6^Ser-189/207, MKK4, p-MKK4^{Ser-258/Thr-261}, MKK^{Ser-271/Thr-275}, p-MKK7^{Thr-180/Tyr-182}, p-p38 MAPK, p-JNK, p-ASK^Ser-83, and TRAF2 in ₂M^*-stimulated 1-LN Prostate Cancer Cells—1-LN cells in 6-well plates (4 x 10⁶ cells/well) were allowed to grow to confluence in RPMI 1640 medium containing 10% FBS, penicillin (12.5 units/ml), streptomycin (6.5 µg/ml), 2 mM glutamine, and 10 nM insulin at 37 °C in a humidified CO₂ (5%) incubator as described above. At about 90% confluence, the medium was aspirated, the monolayers were washed with ice-cold Hepes-buffered Hanks' basic salt solution, pH 7.4, and a fresh volume of the medium was added to the monolayers. The cells were exposed to ₂M^* (50 pM) for varying periods of time. The reaction was stopped by aspirating the medium, and a volume of lysis buffer containing 50 mM Tris·HCl (pH 7.5), 120 mM NaCl, 1% (v/v) Nonidet P-40, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 1 mM PMSF, 1 mM benzamidine, and leupeptin (20 µg/ml) was added. The cells were lysed for 10 min over ice, scraped into tubes, and centrifuged at 8000 x g for 10 min at 4 °C, and their protein contents were determined. In each case, an equal amount of protein was used for electrophoresis. The immunoblotting of the respective membranes was done with antibodies specific for p-ERK1/2, p-MKK3/6, p-MKK4, p-MKK7, p-p38 MAPK, p-JNK, p-ASK1, and TRAF2, respectively, according to the manufacturer's instructions. The respective membranes were reprobed for unphosphorylated ERK1/2, MKK3/6, MKK4, MKK7, p38 MAPK, JNK, and ASK1 as well as the protein loading control actin according to the manufacturer's instruction. The detection and quantification of immunoblots were performed by ECF and phosphorimaging. The membranes were reprobed for protein loading control actin.

Measurement of the Effects of Treatment of 1-LN Prostate Cancer Cells with ₂M^* on Activation of Caspases by Western Blotting—The experimental details of incubating 1-LN prostate cancer cells with ₂M^*, their lysis, and electrophoresis were identical to that described above. The respective membranes were immunoblotted with antibodies specific for procaspase-12, procaspase-9, and cleaved caspase-3, respectively. Since the antibody is specific for mouse procaspase-12, we performed a study to demonstrate that this antibody cross-reacts with the human protein. For these studies, murine macrophages were compared with human 1-LN and PC-3 prostate cancer cells to demonstrate that the anti-murine antibody recognizes human procaspase-12. This study is shown as a control in Fig. 3. The membranes were reprobed for actin, a protein loading control. The detection of immunoblots was performed by ECF and phosphorimaging.

Measurement of the Effects of Treatment of 1-LN Prostate Cancer Cells with ₂M^* on Up-regulation of Bcl-2, p-Bad^Ser-136, p-FOXO1^Ser-256, p-GSK3^Ser-9, 14-3-3, XIAP, p27 ^kip, and Cyclin D1 by Western Blotting—The experimental details of incubating 1-LN prostate cancer cells with ₂M^*, their lysis, and electrophoresis were identical to those described above. The respective membranes were immunoblotted with antibodies specific for Bcl-2, p-Bad^Ser-136, p-FOXO1, p-GSK3, 14-3-3, XIAP, p27^kip, and cyclin D1, respectively. The membranes were reprobed for FOXO1, Bad, GSK3^Ser-9, and actin as the protein loading control. The detection and quantification of immunoblots were performed by ECF and phosphorimaging.

Measurement of Activation of NF-B (p65), NF-B (p52), p-IKK/ ^Ser-176/180, p-IB-^Ser-32, and NIK in 1-LN Cells Stimulated with ₂M^* by Western Blotting—In an experiment identical to the preceding one, cell lysates from buffer and ₂M^*-stimulated 1-LN cells were electrophoresed and transferred to membranes, and the respective membranes were probed for NF-B (p65), NF-B2 (p52), p-IKK/,IB-, p-IB-,IB-, p-IB-, and NIK, respectively by Western blotting. The detection and quantification of immunoblots were performed by ECF and phosphorimaging.

Measurement of NF-B1 (p65) and NF-B2 (p52) in Nuclei of 1-LN Cells Stimulated with ₂M^*—1-LN cells (6 x 10⁶ cells) were incubated and stimulated with ₂M^* (50 pM/15 min) as described above. Nuclei from 1-LN cells were isolated, and their purity was determined as described previously (18, 19). Nuclei were lysed in lysis buffer as described above, and their protein contents were determined. Equal amounts of protein were immunoprecipitated with antibodies against NF-B1 and NF-B2 as described above. The NF-B1 and NF-B2 immunoprecipitates were processed for electrophoresis and Western blotting as described above, and the p65 and p52 protein bands were visualized by ECF and phosphorimaging.

Assay of IKK Activation in 1-LN Cells Stimulated with ₂M^*—The IKK assay in 1-LN cells stimulated with ₂ M(50 pM/15 min) was performed essentially according to Takada et al. (52). 1-LN cells (4 x 10⁶/well in 6-well plates) were stimulated with ₂M^* (50 pM/15 min) and incubated as above. The reaction was terminated by aspirating the medium. The cells in the respective wells were lysed in lysis buffer as described above, and lysate protein contents were determined. Equal amounts of lysate protein from buffer and ₂M^*-stimulated cells were immunoprecipitated with 1 µg of IKK antibodies as described above. The protein A-Sepharose beads were washed three times with lysis buffer and then three times with kinase assay buffer containing 50 mM Hepes (pH 7.5), 20 mM MgCl₂,and 2 mM dithiothreitol, by centrifugation at 2500 rpm for 5 min at 4 °C. To the IKK immunoprecipitate was added 20 µl of kinase buffer containing 10 µM ATP, 10 µCi of [-³³P]ATP, and 2 µg of substrate glutathione S-transferase-IB- (amino acid residues 1-54), and the samples were incubated for 30 min at 30 °C with shaking. The reaction was terminated by adding 10 µl of 4x SDS sample buffer, and the samples were boiled for 5 min. The samples were electrophoresed on 10% gels, protein bands were transferred to membranes, the membranes were autoradiographed, and radioactive bands were visualized and quantified by phosphorimaging.

Measurement of the Effects of ₂M^* Treatment of 1-LN Prostate Cancer Cells on Up-regulation of the Components of the UPR Signaling Pathway by Western Blotting—The experimental details of incubating 1-LN prostate cancer cells with ₂M^*, their lysis, and electrophoresis were identical to those described above. The respective membranes were immunoblotted with antibodies specific for GRP78, IRE1, XBP-1, ATF6, p-PERK^Thr-980, p-eIF2^Ser-51, ATF4, GADD34, and GADD153, respectively (37). The membranes were reprobed for eIF2 and actin as the protein loading control. The detection of immunoblots was performed by ECF and phosphorimaging.

View larger version (38K): [in this window] [in a new window]   FIGURE 1. 2M* activates p-AktThr-308, p-AktSer-473 and p-ERK 1/2Thr-202/Tyr-204 in 1-LN cells. Experimental details are described under "Experimental Procedures." A, changes in p-AktThr-308 (white), p-AktSer-473 (gray), and p-ERK1/2Thr-202/Tyr-204 (black) over time. Values are inarbitrary units and are the mean ± S.E. from three individual experiments. Representative corresponding immunoblots as well as protein loading controls are shown below the graph. B, PI 3-kinase inhibition prevents Akt activation in 1-LN cells stimulated with 2M*. Lane 1, buffer; lane 2, 2M* (50 pm/15 min); lane 3, LY294002 (20 µM/20 min) and then 2M* (50 pM/15 min). Values are expressed in arbitrary units and are the mean ± S.E. from three or four independent experiments. White bars, p-AktThr-308; gray bars, p-AktSer-473;black bars, p-ASK1Ser-83. Representative corresponding immunoblots are shown below the graph. C, 2 M* stimulates p-AktThr-308 and p-AktSer-473 kinase activities in 1-LN cells. Bars 1, buffer; bars 2, 2M* (50 pM/15 min); bars 3, control peptide. The values are expressed as pmol of [-33P]ATP incorporated/mg of protein and are the mean ± S.E. from two independent experiments done in duplicate. *, p values significantly different from buffer- and inhibitor-treated cells at the 5% level. GADPH, glyceraldehyde-3-phosphate dehydrogenase.

Chemical Synthesis of dsRNA Homologous in Sequence to the Target GRP78 Gene—The chemical synthesis of dsRNA homologous in sequence to the target GRP78 peptide sequence ³⁷⁰KIQQLVK³⁷⁶, mRNA sequence 5'-AAA ATA CAG CAA TTA GTA AAG-3' (Swiss-Prot GRP primary sequence accession number P11021 [GenBank] ), was performed by Ambion (Austin, TX). For making dsRNA of the sense 5'-AAU ACA GCA AUU AGU AAA GTT-3' and antisense 5'-CUU UAC UAA UUG CUG UAU UTT-3', oligonucleotides were annealed according to the manufacturer's instructions. Throughout the studies, handling of reagents was performed in an RNase-free environment. The protocol is described in detail elsewhere (20, 22). Briefly, equal amounts of sense and antisense oligonucleotides were mixed in annealing buffer and heated at 90 °C for 1 min and then maintained for 1 h at 37 °C in an incubator. The dsRNA preparation was stored at -20 °C before use.

Transfection of 1-LN Cells with dsRNA Homologous in Sequence to the GRP78 Gene—Silencing of GRP78 gene expression was performed as previously described (18-22). We have used the following protocol for silencing the expression of the GRP78 gene in macrophages and 1-LN cells. This protocol results in 60-70% suppression of GRP78 gene expression as quantified by GRP78 mRNA and protein levels. This suppression of GRP78 gene expression also causes a concomitant loss of cell surface-anchored GRP78 as measured by receptor binding, as well as abrogation of ₂M^*-induced 1,4,5-trisphosphate synthesis, elevation of [Ca²⁺]_i, DNA synthesis, and mitogenic signaling (19-22). Confluent 1-LN cell monolayers (1.5 x 10⁶/well in 6-well plates) in quadruplicate, incubated as described above, were washed twice with Hanks' balanced salt solution, and 2 ml of DMEM containing 10% of FBS and antibiotics was added. The cells were incubated as above for 16 h. Just before each transfection, 25 µg of GRP78 dsRNA was diluted to 100 µl of serum- and antiobiotic-free DMEM in a tube. In another tube, 10 µl of Lipofectamine was diluted into 100 µl of serum- and antiobiotic-free medium. The two solutions were combined, mixed gently, and incubated for 45 min at room temperature, followed by the addition of 800 µl of serum- and antibiotic-free medium to each tube. The monolayers were washed twice with serum-antibiotic-free DMEM, layered in each well with 1 ml of Lipofectamine-DMEM or lipid dsRNA mixtures containing 25 µg of dsRNA of GRP78 target mRNA gently mixed, and incubated for 5 h at 37 °C in a humidified CO₂ incubator in separate experiments. At the end of the incubation, 1 ml of antibiotic-free DMEM containing 10% FBS was added to each well, and the cells were incubated for 16 h. The medium was replaced with DMEM containing antibiotics and 10% FBS 24 h following the start of the transfection. The monolayers were incubated for a further 24 h as above. At the end of the incubation, the medium was aspirated, and the monolayers were washed with the above medium once. A volume of the same medium was added, and the cells were used for the experiment outlined below. To demonstrate that the transfection of 1-LN prostate cancer cells with dsRNA homologous in sequence to the target GRP78 gene does not produce any nonspecific effects on target gene expression, the 1-LN cells were transfected with equimolar concentrations of scrambled small interference RNA (Silencer^TM negative control, catalog number 4610; Ambion) under identical conditions as described above for transfection with GRP78 dsRNA. At the end of the transfection period (48 h), the medium was aspirated, and a volume of DMEM was added. The cells were stimulated either with buffer or ₂M^* (50 pM) for 15 min. The reaction was stopped by aspirating the medium and adding a volume of lysis buffer as described above. As stated above, this protocol suppresses the expression of GRP78 gene expression by 60-70% as measured by GRP78 mRNA and protein levels (19-22). Equal amounts of lysate protein as determined by Bradford (58) were employed for the Akt kinase assay and IKK kinase assays as well as electrophoresis. We have previously demonstrated that this protocol does not significantly alter cell viability (20, 22).

Assay of Akt and IKK Kinase Activities in 1-LN Cells Transfected with GRP78 dsRNA and Stimulated with ₂M^*—p-Akt^Thr-308, p-Akt^Ser-473, and IKK kinase activities were determined in Akt or IKK immunoprecipitates from Lipofectamine plus buffer, Lipofectamine plus ₂M^* (50 pM/15 min), GPR78 dsRNA-transfected plus ₂M, or scrambled dsRNA + ₂M^*-treated cells as described.

Measurement of the Effects of Transfecting 1-LN Cells with GRP78 dsRNA on Activation of NF-B1, NF-B2, pIKK-, and p-IB- after ₂M^* Stimulation—Equal amounts of lysate protein from 1-LN cells treated with Lipofectamine plus buffer, Lipofectamine plus ₂M^* (50 pM/15 min), GRP78 dsRNA plus ₂M^* (50 pM/15 min), or GRP78 dsRNA plus ₂M^* were electrophoresed, and protein bands were transferred to membranes. After immunoblotting with the respective antibodies, the immunoblots were visualized and quantified by ECF and phosphorimaging. The immunoblots were reprobed for actin as a protein loading control.

Measurements of the Effects of Transfecting 1-LN Cells with GRP78 dsRNA on UPR Signaling Components after ₂M^*—Equal amounts of lysate protein from 1-LN cells treated with Lipofectamine plus buffer, Lipofectamine plus ₂M^* (50 pM/15 min), GRP78 dsRNA + ₂M^*, or scrambled dsRNA + ₂M^* were electrophoresed, and protein bands were transferred to membranes. The membranes were immunoblotted with antibodies against IRE1_, XBP-1, ATF6, p-PERK, p-eIF2, ATF4, GADD34, or GADD153, respectively. The immunoblots were visualized and quantified by ECF and phosphorimaging as described above. The immunoblots were reprobed for actin as a protein loading control.

Measurements of the Effects of Transfection of 1-LN Cells with GRP78 dsRNA on Expression of p-FOXO, p-GSK3, XIAP, Cyclin D1, and p27 ^kip upon ₂M^* Stimulation—Equal amounts of lysate protein from the respective experimental groups were electrophoresed, and protein bands on the gels were transferred to membranes. The immunoblots were probed with the respective antibodies, and the immunoblots were visualized and quantified by ECF and phosphorimaging as described above. The immunoblots were reprobed for actin as a protein loading control.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
₂M^* Up-regulates Activation of Akt in 1-LN Cells in a PI 3-Kinase-dependent Manner—Activation of Akt is dependent upon PI 3-kinase activation, and disregulation of Akt activation is implicated in the behavior of malignant tumors (5-7). Upon stimulation, cytosolic Akt is recruited to the plasma membrane through the binding of its NH₂-terminal plekstrin homology domain to the lipid product of PI 3-kinase activation (5-7). Akt is then activated by phosphorylation on two residues, namely Thr-308 in the activation loop and Ser-473 in the hydrophobic motif of the COOH-terminal tail. Phosphorylation of both residues is required for its full activation (60-62).3'-Phosphatidylinositol-dependent kinase 1 is a plekstrin homology domain-containing kinase that phosphorylates Akt at Thr-308 (5, 6); however, the identity of the kinase responsible for phosphorylating Akt at Ser-473 is disputed (60-62). We have previously demonstrated that expression of GRP78 on the cell surface is essential for ₂M^*-dependent signal transduction in both macrophages and human prostate cancer (18-22). We have characterized the identity of cell surface synthesis, cellular proliferation, and mitogenic signaling upon binding of ₂M^* to GRP78 in a variety of prostate cancer cells of differing metastatic potential as well as in macrophages (18-22). These postreceptor events are drastically inhibited by prior treatment of cells with antibodies against GRP78 or silencing GRP78 gene expression by RNA interference (18-22). Human prostate cancer cells lacking GRP78 on their cell surface do not demonstrate ₂M^*-dependent signal transduction (18, 20). This includes PC-3 cells, the parent line for 1-LN cells. Thus, PC-3 cells do not express GRP78 on the cell surface and do not demonstrate ₂M^*-dependent activation of signaling cascades. We therefore focused the present studies on the 1-LN cell line. Treatment of 1-LN prostate cancer cells with ₂M^* (50 pM) elevated both p-Akt^Thr-308 and p-Akt^Ser-473 at about 5 min, and these levels remained elevated during 60 min of incubation (Fig. 1A). The kinetics of activation of both p-Akt^Thr-308 and p-Akt^Ser-473 were comparable in ₂M^*-treated 1-LN prostate cancer cells (Fig. 1A). ₂M^*-induced phosphorylation of Akt at Thr-308 and Ser-473 was greatly reduced by prior treatment of cells with LY294002, a specific inhibitor ofPI3-kinase(Fig. 1B),demonstrating that activation of Akt is PI3-kinase-dependent. We further demonstrated ₂M^*-induced phosphorylation of Akt at Thr-308 and Ser-473 in 1-LN cells by assaying their kinase activities using specific kinase peptide substrates (Fig. 1C). ₂M^* treatment caused a nearly equal activation of both p-Akt^Thr-308 kinase and p-Akt^Ser-473 kinase which corroborates the Western blotting data (Fig. 1A). The results presented show that ₂M^*, like growth factors, induces mitogenic and cell survival signaling in 1-LN prostate cancer cells.

View larger version (50K): [in this window] [in a new window]   FIGURE 2. Effect of transfection of 1-LN cells with GRP78 dsRNA on 2M*-induced activation of Akt kinases and IKK kinase and up-regulation of NF-B activation. For experimental details, see "Experimental Procedures." A, AktThr-308 kinase (white) and AktSer-473 kinase (black) activities in 1-LN cells treated with Lipofectamine plus buffer (1),Lipofectamine plus 2M* (50 pM/15 min) (2), GRP78 dsRNA plus 2M* (3), and scrambled dsRNA and 2M* (4). The kinase activities are shown as the mean ± S.E. from two experiments performed in triplicate and are expressed as pmol of [-33P]ATP incorporated into substrate peptides/mg of protein. B, IKK kinase activity as measured by the phosphorylation of I- in 1-LN cells treated as above. The respective autoradiograph is shown below the graph. IKK kinase activity is in arbitrary units and is expressed as the mean ± S.E. from two experiments performed in duplicate. C, effect of silencing GRP78 gene expression on 2M*-induced activation of NF-B1 and NF-B2 in 1-LN prostate cancer cells. Shown are NF-B1 (white bars) and NF-B2 (black bars) from 1-LN cells treated as above. A representative immunoblot is shown below the graph. The results are expressed in arbitrary units and are the mean ± S.E. from triplicate experiments. D, 2M*-induced phosphorylation of IKK and IKK in 1-LN cells transfected with GRP78 dsRNA by Western blotting. Shown are p-IKK/ (white bars) and IB- (black bars) from cells treated as above. The results are expressed in arbitrary units and are the mean ± S.E. from triplicate experiments. A representative immunoblot is shown below the graph. *, values significantly different from corresponding buffer-treated and GRP78 dsRNA-transfected cells and cells treated with 2M* at the 5% level. Actin was used as a protein loading control in all experiments, but only a representative actin immunoblot is shown.

₂ M^* Up-regulates Activation of ERK1/2 in 1-LN Prostate Cancer Cells—The activation of surface receptors leads to the activation of Ras, a membrane-resident GTPase, which recruits Raf kinase from the cytosol to the cell membrane. Most tumors demonstrate sustained and elevated activation of the Raf-MEK-ERK pathway. This pathway promotes cell survival by activating antiapoptotic mechanisms. In the MEK-dependent pathway, activated ERK activates 90-kDa ribosomal S6 kinase, which phosphorylates and inactivates proapoptotic Bad (16, 33, 35, 64-70). 90-kDa ribosomal S6 kinase also activates the transcription factor CREB, which promotes cell survival by up-regulating Bcl-2 expression (33, 35, 64-70). In addition, Raf up-regulates the expression of the antiapoptotic transcription factor NF-B. Treatment of murine macrophages with ₂M^* causes activation of Ras·GTP and up-regulates activation of MEK1 and ERK (20, 71). In 1-LN prostate cancer cells, ₂M^* treatment promotes cell proliferation as evidenced by increased DNA synthesis and increased cell numbers (20). To assess the role of the MEK-ERK pathway in 1-LN cancer cells, we determined the effect of ₂M^* treatment of these cells on activation of ERK1/2. ₂M^*-induced the phosphorylation of ERK1/2 as early as 5 min of incubation, which remained nearly sustained up to 60 min of incubation (Fig. 1A). These data suggest that ₂M^*-induced cell growth is partly mediated by the ERK signaling pathway.

Affect on Caspase-12, Caspase-9, and Caspase-3 Activation by ₂M^* in 1-LN Prostate Cancer Cells—In response to intrinsic and extrinsic signals, caspases are activated through cleavage by upstream caspases, which leads to apoptotic cell death (see Ref. 16 and references therein). Activation of caspase-12 is specific to ER stress. Once activated during ER stress, the catalytic subunit of caspase-12 is released into the cytosol, where it activates the caspase-9 cascade (73). To assess the role of proapoptotic caspases in ₂M^*-induced cell proliferation in 1-LN prostate cancer cells, we quantified levels of caspase-12, caspase 9, and cleaved caspase-3 (Fig. 3) by Western blotting. Under our experimental conditions, ₂M^* did not activate the activation of caspase-12, caspase-9, and caspase-3.

View larger version (65K): [in this window] [in a new window]   FIGURE 3. Immunoblots showing that 2M* does not induce activation of caspase-12, caspase-9, and caspase-3 in 1-LN cells. Immunoblots shown are representative of two or three independent experiments. The protein loading control glyceraldehyde-3-phosphate dehydrogenase (GADPH) is also shown. As a control, the figure also demonstrates that the anti-murine procaspase-12 antibody reacts with not only murine procaspase-12 (lane 1) but also human procaspase-12 derived from human 1-LN cells (lane 2), MDA-MB231 breast cancer cells (lane 3), PC-3 prostate cancer cells (lane 4), and DM-6 melanoma cells (lane 5), respectively.

View larger version (68K): [in this window] [in a new window]   FIGURE 4. Induction of antiapoptotic signaling in 1-LN cells treated with2M*. A, up-regulation of Bcl-2 (white), XIAP (gray), and 14-3-3 (black)in 1-LN cells treated with 2M*. Values are expressed in arbitrary units and are the mean ± S.E. from three or four experiments. Representative corresponding immunoblots along with the protein loading control actin are shown below each graph. B, p-BadSer-136 (white bars), p-FOXO1Ser-256 (dark gray bars), p-GSk3Ser-9 (black bars), and cyclin D (light gray bars). Values are expressed in arbitrary units and are the mean ± S.E. from three or four experiments. Representative corresponding immunoblots along with protein loading controls are shown below the graph. C, regulation of p-FOXO1Ser-256 (white),p-GSk3Ser-9 (dark gray), p-BadSer-136 (black), and XIAP (light gray) by Akt. Values are expressed in arbitrary units and are the mean ± S.E. from two or three experiments. Representative corresponding immunoblots and protein loading controls are shown below the graph. Bars 1, buffer; bars 2, 2M* (50 pM/15 min); bars 3, LY294002 (20 µM/20 min) and then 2M*. *, p values significantly different from buffer and inhibitor-treated cells at the 5% level.

₂M^* Up-regulates the Phosphorylation of FOXO1 in 1-LN Cancer Cells—Akt activation positively regulates G₁/S cell cycle progression through inactivation of GSK3, leading to increased cyclin D1, inhibition of forkhead transcription factors including the FOXO subfamily, and the subsequent reduction of p27^kip (5, 74, 75). Phosphorylation of FOXO1 by activated Akt promotes its export from the nucleus to the cytosol, thus preventing FOXO1 interaction with DNA, which otherwise would up-regulate transcription factors involved in the apoptotic pathway. FOXO1 also interacts with the 14-3-3 protein, which serves to localize the p-FOXO1 in the cytoplasm, and it also facilitates nuclear export of FOXO1 (5, 74, 75). Treatment of 1-LN cancer cells with ₂M^* (50 pM/15 min) elevated p-FOXO1 by about 50-70% at about 10 min of incubation, and these levels remained elevated up to 60 min of incubation as compared with control 1-LN cells (Fig. 4B). Treatment of 1-LN cells with LY294002 (20 µM/20 min), before ₂M^* addition markedly reduced p-FOXO1 levels (Fig. 4C). An increased up-regulation of 14-3-3 parallel to that of p-FOXO1 was also observed in 1-LN cells treated with ₂M^* (Fig. 4A), which suggests Akt-mediated inactivation of proapoptotic FOXO1.

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Up-regulation of NF-B by the canonical (NF-B1) and noncanonical (NF-B2) pathways in 1-LN cells treated with2 M*. A, TRAF2 (), NF-B1 (), p-IKK/Ser-176/180 (), and p-IB-Ser-32(). B, changes in IKK- activity in 1-LN cells stimulated with 2M*. Bars 1, buffer-treated; bars 2, 2M*-stimulated(50 pM/15 min).The values are expression in arbitrary units as the mean ± S.E. from two experiments performed in triplicate. C, up-regulation of NF-B by the noncanonical pathway (NF-B2) in 1-LN cells treated with2M* as demonstrated by up-regulation of NIK () and NF-B2 (). Also shown in this panel is up-regulation of GADD45 (). D,PI3-kinase-dependent and -independent activation of NF-B1 and NF-B2 in 1-LN cells. Shown are NF-B1 (p65) (white bars), NF-B2 (p52) (dark gray bars), p-IB-Ser-32 (black bars), and NIK (light gray bars) in cells stimulated with buffer (1), 2M* (50 pM/15 min) (2), and LY294002 (20µM/20 min) and then2M* (3). Values in all of the panels are expressed in arbitrary units and are the mean ± S.E. from four experiments. Representative corresponding immunoblots of each panel along with protein loading controls are shown below the respective panel. E, NF-B1 and NF-B2 translocation to nuclei of 1-LN cells treated with 2M*. Lane 1, buffer-treated; lane 2, 2M*-treated (50 pM/15 min). *, p values significantly different from buffer and inhibitor-treated cells at the 5% level.

Activation of NF-B1 and NF-B2 in 1-LN Prostate Cancer Cells Treated with ₂M^*—NF-B is constitutively activated in many cancers where ER stress is a common occurrence. Treatment of 1-LN cancer cells with ₂M^* (50 pM) caused a severalfold increase in the NF-B1 as determined by Western blotting (Fig. 5A). The upstream activators of NF-B1 (namely IKK/ and IB-) also exhibited a 1.5-2-fold transitory activation at about 10-20 min of incubation (Fig. 5A), subsequently returning to the basal state. The Western blotting data with respect to p-IKK are also corroborated by the IKK kinase activity studies, which demonstrate a nearly parallel increase in ₂M^*-stimulated 1-LN cells, as compared with buffer-treated cells (Fig. 5B). Under these experimental conditions, the levels of phosphorylated IB- were not affected (data not shown). Activation of NF-B is also affected by NIK in the cytoplasm as well as in the modulation of its transactivation potential in the nucleus (55). NIK associates with TRAF2 and promotes the phosphorylation of IKK and IKK. Enhanced activity of NIK occurs in various tumors, which suggests its association with enhanced levels of p52 in breast cancer (53, 54). We therefore studied the effect of ₂M^* in 1-LN cancer cells with respect to activation of NF-B2 by the noncanonical pathway (Fig. 5C). Exposure of 1-LN cells to ₂M^* caused a transient increase in both TRAF2 (Fig. 5A) and NIK (Fig. 5C) at about 10-20 min of incubation. ₂M^* treatment of 1-LN cancer cells also caused a 50-60% sustained increase in NF-B2 (Fig. 5C). Furthermore, whereas activation of NF-B1 via the canonical pathway was PI 3-kinase-dependent, activation of NF-B2 via the noncanonical pathway was PI 3-kinase-independent (Fig. 5D). Incubation of 1-LN cells with LY294002 (20 µM/20 min) before the addition of ₂M^* inhibited the increase in the protein levels of NF-B1 and p-IB- but had no effect on NF-B2 and NIK protein levels compared with ₂M^*-treated cells (Fig. 5D). That activated NF-B1 and NF-B2 translocate to nuclei in₂M^*-stimulated 1-LN cells is shown in Fig. 4E, where these proteins were present at concentrations about 2-fold higher than the buffer-treated controls.

Attenuation of ₂M^*-induced Activation of IKK Kinase and NF-Bin 1-LN Cells Transfected with GRP78 dsRNA—Above, we have shown that ₂M^* treatment of 1-LN prostate cancer cells with ₂M^* increases activation of IKK and up-regulates both NF-B1 and NF-B2 in an Akt-dependent manner (Fig. 5). Transfection of 1-LN prostate cancer cells with GRP78 dsRNA nearly abolished ₂M^*-induced activation of IKK compared with cells treated with Lipofectamine and ₂M^* or cells treated with scrambled dsRNA and ₂M^* (Fig. 2). Since silencing GRP78 gene expression profoundly inhibited the activation of p-Akt^Thr-308 and p-Akt^Ser-473 kinases caused by ₂M^* stimulation (Fig. 2), we suggest that activation of IKK, hence NF-B activation, is mediated by activated Akt. These observations are further supported by inhibited expression of p-IKK/, p-IB, NF-B1, and NF-B2 in 1-LN cells transfected with GRP78 dsRNA and stimulated with ₂M^* as compared with cells treated with Lipofectamine and ₂M^* or cells transfected with scrambled dsRNA and treated with ₂M^* (Fig. 2).

₂M^* Treatment of 1-LN Cancer Cells Up-regulates the Expression of GADD45—GADD45 is a pivotal mediator of the cell-protective effects of NF-B against TNF and Fas-induced apoptosis (78, 79). NF-B induces GADD45 promoter activity, which down-regulates proapoptotic JNK. GADD45 associates tightly with MKK7, a selective activator of JNK, and inhibits its activation and apoptosis (80, 81). Treatment of 1-LN cancer cells within ₂M^* showed little or negligible activation of either MKK7 or JNK phosphorylation as determined by Western blotting (Fig. 6). Under our experimental conditions, JNK activation appears to be suppressed by Akt-dependent phosphorylation of ASK1 (Fig. 1B) and/or NF-B activation (Fig. 5, A and C). In the next series of experiments, we evaluated the role of GADD45 in JNK activation in 1-LN cancer cells treated with ₂M^* by Western blotting (Fig. 5C). ₂M^* treatment of 1-LN cells elevated the expression of GADD45 by about 2-fold at 10-20 min of incubation but returned to basal levels at 60 min (Fig. 5C). The results indicate that the very modest effect of JNK in ₂M^*-treated 1-LN cells occurs at the levels of Akt-dependent inhibition of ASK1 and GADD45 inhibition of MKKK7.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Suppression of 2M*-induced antiapoptotic factors in 1-LN cells transfected with GRP78 dsRNA. A, pFOXO (white bars), p-GSK3 (gray bars), and XIAP (black bars) from 1-LN cells treated with Lipofectamine plus buffer (1), Lipofectamine plus 2M* (2), GRP78 dsRNA plus 2M* (3), and scrambled dsRNA plus RNA and 2M* (4). A representative immunoblot is shown below the graph. The results are expressed in arbitrary units and are the mean ± S.E. from triplicate experiments. B, effect of transfecting 1-LN cells with GRP78 dsRNA on 2M*-induced changes in cyclin D1 (white) and p27kip (black). Bars 1, Lipofectamine plus buffer, bars 2, Lipofectamine plus 2M*; bars 3, GRP78 dsRNA plus 2M*; bars 4, scrambled dsRNA plus 2M*. A representative immunoblot is shown below the graph. The results are expressed in arbitrary units and are the mean ± S.E. from triplicate experiments. *, values significantly different from the corresponding buffer-treated, GRP78ds RNA-transfected, 2M*-treated, and scrambled dsRNA cells that were 2M*-treated; **, values significantly different from Lipofectamine plus 2M*-treated cells. Actin was used as a protein loading control in all experiments, but only a representative actin immunoblot is shown.