Serum Pro-N-Cadherin Is a Marker of Subclinical Heart Failure in the General Population.

Background We recently reported aberrant processing and localization of the precursor PNC (pro-N-cadherin) protein in failing heart tissues and detected elevated PNC products in the plasma of patients with heart failure. We hypothesize that PNC mislocalization and subsequent circulation is an early event in the pathogenesis of heart failure, and therefore circulating PNC is an early biomarker of heart failure. Methods and Results In collaboration with the Duke University Clinical and Translational Science Institute's MURDOCK (Measurement to Understand Reclassification of Disease of Cabarrus and Kannapolis) study, we queried enrolled individuals and sampled 2 matched cohorts: a cohort of individuals with no known heart failure at the time of serum collection and no heart failure development in the following 13 years (n=289, cohort A) and a matching cohort of enrolled individuals who had no known heart failure at the time of serum collection but subsequently developed heart failure within the following 13 years (n=307, cohort B). Serum PNC and NT-proBNP (N-terminal pro B-type natriuretic peptide) concentrations in each population were quantified by ELISA. We detected no significant difference in NT-proBNP rule-in or rule-out statistics between the 2 cohorts at baseline. In participants who developed heart failure, serum PNC is significantly elevated relative to those who did not report development of heart failure (P<0.0001). Receiver operating characteristic analyses of PNC demonstrate diagnostic value for subclinical heart failure. Additionally, PNC has diagnostic potential when comparing participants with no reported heart failure risk factors from cohort A to at-risk participants from cohort B over the 13-year follow-up. Participants whose PNC levels measure >6 ng/mL have a 41% increased risk of all-cause mortality independent of age, body mass index, sex, NT-proBNP, blood pressure, previous heart attack, and coronary artery disease (P=0.044, n=596). Conclusions These data suggest that PNC is an early marker of heart failure and has the potential to identify patients who would benefit from early therapeutic intervention.

Physiological Roles of the Autoantibodies to the 78-Kilodalton Glucose-Regulated Protein (GRP78) in Cancer and Autoimmune Diseases.

The 78 kDa glucose-regulated protein (GRP78), a member of the 70 kDa heat-shock family of molecular chaperones (HSP70), is essential for the regulation of the unfolded protein response (UPR) resulting from cellular endoplasmic reticulum (ER) stress. During ER stress, GRP78 evades retention mechanisms and is translocated to the cell surface (csGRP78) where it functions as an autoantigen. Autoantibodies to GRP78 appear in prostate, ovarian, gastric, malignant melanoma, and colorectal cancers. They are also found in autoimmune pathologies such as rheumatoid arthritis (RA), neuromyelitis optica (NMO), anti-myelin oligodendrocyte glycoprotein antibody-associated disorder (AMOGAD), Lambert-Eaton myasthenic syndrome (LEMS), multiple sclerosis (MS), neuropsychiatric systemic lupus erythematosus (NPSLE) and type 1 diabetes (T1D). In NMO, MS, and NPSLE these autoantibodies disrupt and move across the blood-brain barrier (BBB), facilitating their entry and that of other pathogenic antibodies to the brain. Although csGRP78 is common in both cancer and autoimmune diseases, there are major differences in the specificity of its autoantibodies. Here, we discuss how ER mechanisms modulate csGRP78 antigenicity and the production of autoantibodies, permitting this chaperone to function as a dual compartmentalized receptor with independent signaling pathways that promote either pro-proliferative or apoptotic signaling, depending on whether the autoantibodies bind csGRP78 N- or C-terminal regions.

Pathologic Proteolytic Processing of N-Cadherin as a Marker of Human Fibrotic Disease.

Prior research has implicated the involvement of cell adhesion molecule N-cadherin in tissue fibrosis and remodeling. We hypothesize that anomalies in N-cadherin protein processing are involved in pathological fibrosis. Diseased tissues associated with fibrosis of the heart, lung, and liver were probed for the precursor form of N-cadherin, pro-N-cadherin (PNC), by immunohistochemistry and compared to healthy tissues. Myofibroblast cell lines were analyzed for cell surface pro-N-cadherin by flow cytometry and immunofluorescent microscopy. Soluble PNC products were immunoprecipitated from patient plasmas and an enzyme-linked immunoassay was developed for quantification. All fibrotic tissues examined show aberrant PNC localization. Cell surface PNC is expressed in myofibroblast cell lines isolated from cardiomyopathy and idiopathic pulmonary fibrosis but not on myofibroblasts isolated from healthy tissues. PNC is elevated in the plasma of patients with cardiomyopathy (p ≤ 0.0001), idiopathic pulmonary fibrosis (p ≤ 0.05), and nonalcoholic fatty liver disease with cirrhosis (p ≤ 0.05). Finally, we have humanized a murine antibody and demonstrate that it significantly inhibits migration of PNC expressing myofibroblasts. Collectively, the aberrant localization of PNC is observed in all fibrotic tissues examined in our study and our data suggest a role for cell surface PNC in the pathogenesis of fibrosis.

Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion.

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

Targeting cell surface GRP78 enhances pancreatic cancer radiosensitivity through YAP/TAZ protein signaling.

Ionizing radiation (IR) can promote migration and invasion of cancer cells, but the basis for this phenomenon has not been fully elucidated. IR increases expression of glucose-regulated protein 78kDa (GRP78) on the surface of cancer cells (CS-GRP78), and this up-regulation is associated with more aggressive behavior, radioresistance, and recurrence of cancer. Here, using various biochemical and immunological methods, including flow cytometry, cell proliferation and migration assays, Rho activation and quantitative RT-PCR assays, we investigated the mechanism by which CS-GRP78 contributes to radioresistance in pancreatic ductal adenocarcinoma (PDAC) cells. We found that activated α2-Macroglobulin (α2M*) a ligand of the CS-GRP78 receptor, induces formation of the AKT kinase (AKT)/DLC1 Rho-GTPase-activating protein (DLC1) complex and thereby increases Rho activation. Further, CS-GRP78 activated the transcriptional coactivators Yes-associated protein (YAP) and tafazzin (TAZ) in a Rho-dependent manner, promoting motility and invasiveness of PDAC cells. We observed that radiation-induced CS-GRP78 stimulates the nuclear accumulation of YAP/TAZ and increases YAP/TAZ target gene expressions. Remarkably, targeting CS-GRP78 with C38 monoclonal antibody (Mab) enhanced radiosensitivity and increased the efficacy of radiation therapy by curtailing PDAC cell motility and invasion. These findings reveal that CS-GRP78 acts upstream of YAP/TAZ signaling and promote migration and radiation-resistance in PDAC cells. We therefore conclude that, C38 Mab is a promising candidate for use in combination with radiation therapy to manage PDAC.

Activated α2-Macroglobulin Regulates Transcriptional Activation of c-MYC Target Genes through Cell Surface GRP78 Protein.

Activated α2-macroglobulin (α2M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α2M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α2M* requires ligation of the GRP78 primary amino acid sequence (Leu(98)-Leu(115)). After stimulation with α2M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser(62) Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser(10) In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α2M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression.

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. Pretreatment of cells with α2M* antagonist antibody directed against the carboxyl-terminal domain of GRP78 blocks these α2M*-mediated effects, and silencing GRP78 expression by RNAi inhibits up-regulation of ATP citrate lyase and fatty-acid synthase. α2M* induces a 2-3-fold increase in lipogenesis as determined by 6-[(14)C]glucose or 1-[(14)C]acetate incorporation into free cholesterol, cholesterol esters, triglycerides, free fatty acids, and phosphatidylcholine, which is blocked by inhibitors of fatty-acid synthase, PI 3-kinase, mTORC, or an antibody against the carboxyl-terminal domain of GRP78. We also assessed the incorporation of [(14)CH3]choline into phosphatidylcholine and observed similar effects. Lipogenesis is significantly affected by pretreatment of prostate cancer cells with fatostatin A, which blocks sterol regulatory element-binding protein proteolytic cleavage and activation. This study demonstrates that α2M* functions as a growth factor, leading to proliferation of prostate cancer cells by promoting insulin-like responses. An antibody against the carboxyl-terminal domain of GRP78 may have important applications in prostate cancer therapy.

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. Silencing the expression, not only of PDK1, but also Raptor by RNAi nearly abolished Akt phosphorylation at Akt(Thr308) in Raptor-immunoprecipitates of α2M*-stimulated prostate cancer cells. Immunodepleting Raptor or PDK from cell lysates of cells treated with α2M* drastically reduced Akt phosphorylation at Thr(308), which was recovered by adding the supernatant of Raptor- or PDK1-depleted cell lysates, respectively. Studies of insulin binding to its receptor on prostate cancer cells yielded similar results. We thus demonstrate that phosphorylating the T-loop Akt residue Thr(308) by PDK1 requires Raptor of the mTORC1 complex as a platform or scaffold protein.

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. Despite its four binding sites, Scatchard analysis of binding of α2M* to cells does not yield a cooperative plot. We, therefore, hypothesize that a monomeric cloned and expressed α2M receptor binding domain (RBD) should trigger comparable signaling events. Indeed, RBD or its K1370A mutant that binds to GRP78 but cannot bind to LRP regulates DNA and protein synthesis by human prostate cancer cells in a manner comparable to that of α2M*. Akt and mTORC1 activation and signaling are also comparably upregulated by α2M*, RBD, or mutant K1370A. Antibodies directed against the carboxyl-terminal domain of GRP78 are antagonists that block α2M*-mediated effects on pro-proliferative and anti-apoptotic signaling cascades and protein and DNA synthesis. The effects of RBD and its mutant were similarly blocked by these antibodies. Finally, proteolysis of α2M at pH values from 5.7 to 7.0 causes production of free RBD and RBD-containing fragments. Thus, while α2M* ligates only one GRP78 receptor molecule per α2M*, it may potentially serve as a reservoir for release of up to four binding fragments per molecule.

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. RNAi targeting Epac1, Raptor, and Rictor was also employed in these studies. RESULTS: 8-CPT-2Me-cAMP treatment caused a 2-2.5-fold increase of p-cPLA2(S505), COX-2, and PGE2 levels in human prostate cancer cell lines. Pretreatment of cells with the COX-2 inhibitor SC-58125 or the EP4 antagonist AH-23848, or with an inhibitor of mTORC1 and mTORC2, Torin1, significantly reduced the Epac1-dependent increase of p-cPLA2 and COX-2, p-S6-kinase(T389), and p-AKT(S473). In addition, Epac1-induced protein and DNA synthesis were greatly reduced upon pretreatment of cells with either COX-2, EP4, or mTOR inhibitors. Transfection of prostate cancer cells with Epac1 dsRNA, Raptor dsRNA, or Rictor dsRNA profoundly reduced Epac1-dependent increases in p-cPLA2 and COX-2. CONCLUSION: We show that Epac1, a downstream effector of cAMP, functions as a pro-inflammatory modulator in prostate cancer cells and promotes cell proliferation and survival by upregulating Ras-MAPK, and PI 3-kinase-Akt-mTOR signaling.

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. By study of phosphorylation-dependent activation, we demonstrate that EPAC-mediated cellular effects require activation of the B-Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3-kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B-Raf/ERK and mTOR signaling play an essential role in cAMP-dependent, but PKA-independent, proliferation of 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. These antibodies are receptor antagonists blocking autophosphorylation and activation of GRP78. Using 1-LN and DU145 prostate cancer cell lines and A375 melanoma cells, which express GRP78 on their cell surface, we show that antibodies directed against the COOH-terminal domain of GRP78 up-regulate the tumor suppressor protein p53. By contrast, antibody directed against the NH(2)-terminal domain of GRP78 shows negligible effects on p53 expression. PC-3 prostate cancer cells, which do not express GRP78 on their cell surface, are refractory to the effects of anti-GRP78 antibodies directed against either the COOH- or NH(2)-terminal domains. However, overexpression of GRP78 in PC-3 cells causes translocation of GRP78 to the cell surface and promotes apoptosis when these cells are treated with antibody directed against its COOH-terminal domain. Silencing GRP78 or p53 expression by RNA interference significantly blocked the increase in p53 induced by antibodies. Antibodies directed against the COOH-terminal domain may play a therapeutic role in cancer patients whose tumors trigger the production of autoantibodies directed against the NH(2)-terminal domain of GRP78.

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. When GRP78 was immunoprecipitated from plasma membranes of macrophages stimulated with alpha(2)M*, Galphaq11, and MTJ-1 were co-precipitated. Likewise Galphaq11 and GRP78 co-immunoprecipitated with MTJ-1 while GRP78 and MTJ-1 co-immunoprecipitated with Galphaq11. Silencing GRP78 expression with GRP78 dsRNA or MTJ-1 with MTJ-1 dsRNA greatly reduced the levels of Galphaq11 co-precipitated with GRP78 or MTJ-1. In conclusion, we show here that plasma membrane-associated GRP78 is coupled to pertussis toxin-insensitive Galphaq11 and forms a ternary signaling complex with MTJ-1.

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. Akt protein levels increased by approximately 1.5-fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t(1/2) approximately 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 microM forskolin. Forskolin-dependent Akt1 synthesis was abolished by pretreating the cells with CREB-directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [(35)S]-labeled Akt1 protein. The PKA inhibitor H-89, greatly attenuated forskolin-induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin-stimulated [(14)C]leucine-labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin-stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter.

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. ERK1/2 and p38 MAPK were also activated, but there was only a marginal effect on JNK activation. Treatment of 1-LN cells with alpha2M* down-regulated apoptosis and promoted NF-kappaB activation as shown by increases of Bcl-2, p-Bad(Ser-136), p-FOXO1(Ser-253), p-GSK3beta(Ser-9), XIAP, NF-kappaB, cyclin D1, GADD45beta, p-ASK1(Ser-83), and TRAF2 in a time of incubation-dependent manner. alpha2M* 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, IRE1alpha, XBP-1, ATF4, ATF6, p-PERK, p-eIF2alpha, and GADD34 and reduced levels of GADD153. Silencing of GRP78 gene expression by RNAi suppressed activation of Akt(Thr-308), Akt(Ser-473), and IkappaB kinase alpha kinase. The effects of alpha2M* on the NF-kappaB activation, antiapoptotic signaling, unfolded protein response signaling, and proapoptotic signaling were also reversed by this treatment. In conclusion, alpha2M* 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.

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. In the current study, we examined the ability of alpha2M* to activate PAK-1 and PAK-2. Upon macrophage stimulation with alpha2M*, PAK-2 is autophosphorylated, resulting in increased kinase activity; however, PAK-1 is negligibly affected. Alpha2M*-stimulated macrophages showed a marked elevation in the levels of Rac x GTP. Receptor tyrosine phosphorylation upon binding of alpha2M* to GRP78, recruits PAK-2 to the plasma membrane via the adaptor protein NCK. Consistent with this hypothesis, silencing of GRP78 gene expression greatly attenuated the levels of membrane-associated PAK-2 and NCK. PAK-2 activity was markedly decreased by inhibition of tyrosine kinases and PI3K before alpha2M* stimulation. We further demonstrate that phosphorylation of Lin-11, Isl-1, Mec-3 (LIM) kinase and cofilin is promoted by treating macrophages with alpha2M*. Thus, alpha2M* regulates activation of the PAK-2-dependent motility mechanism in these cells.

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. In the current study, we have examined the ability of alpha2M* to activate PAK-1 and PAK-2. Exposure of 1-LN cells to alpha2M* caused a 2- to 3-fold increase in phosphorylated PAK-2 and a similar increase in its kinase activity toward myelin basic protein. By contrast, the phosphorylation of PAK-1 was only negligibly affected. Silencing the expression of the GRP78 gene, using either of two different mRNA sequences, greatly attenuated the appearance of phosphorylated PAK-2 in alpha2M*-stimulated cells. Treatment of 1-LN cells with alpha2M* caused translocation of PAK-2 in association with NCK to the cell surface as evidenced by the co-immunoprecipitation of PAK-2 and NCK in the GRP78 immunoprecipitate from plasma membranes. alpha2M*-induced activation of PAK-2 was inhibited by prior incubation of the cells with specific inhibitors of tyrosine kinases and phosphatidylinositol 3-kinase. PAK-2 activation was accompanied by significant increases in the levels of phosphorylated LIMK and phosphorylated cofilin. Silencing the expression of the PAK-2 gene greatly attenuated the phosphorylation of LIMK. In conclusion, we show for the first time the activation of PAK-2 in 1-LN prostate cancer cells by a proteinase inhibitor, alpha2-macroglobulin. These studies suggest a mechanism by which alpha2M* enhances the metastatic potential of these cells.

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. Exposure of cells to insulin activated UPR, as evidenced by an increased expression of GRP78, XBP-1, phosphorylated PERK (p-PERK), and p-eIF2alpha. The insulin-induced, elevated expression of GRP78 was comparable with that observed with tunicamycin, a classical inducer of ER stress. Concomitantly, insulin also up-regulated prosurvival mechanisms by elevating GADD34 and elements of the antiapoptotic pathway including Bcl-2, X-linked inhibitor of apoptosis, and phosphorylated forkhead transcription factor. In conclusion, we show here that insulin treatment does cause ER stress in macrophages, but insulin-dependent mechanisms overcome this ER stress by up-regulating UPR and the antiapoptotic pathway to promote cell survival.

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. GRP78 also serves as the receptor for alpha2-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 alpha2-macroglobulin to macrophages was greatly reduced, and activated and receptor-recognized form of alpha2-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.

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*. Concomitantly, alpha2M*-induced increase in [3H]thymidine uptake was abolished in these transfected cells. Insulin treatment significantly upregulates alpha2MSR and it also caused a marked increase in Grp78 expression which could be blocked by RNA interference. alpha2M* treatment of cells activates the Ras- and PI 3-kinase-dependent signalling pathways. Suppressing Grp78 expression leads to the loss of these activation events in transfected macrophages. We thus conclude that Grp78 is the alpha2M* signalling receptor.