Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F 1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F 1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F 1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surfaceassociated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the ␣-and ␤-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelialsurface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

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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 ␣ 2 -macroglobulin (␣ 2 M*) 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 ␣ 2 M* to activate PAK-1 and PAK-2. Exposure of 1-LN cells to ␣ 2 M* 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 ␣ 2 M*-stimulated cells. Treatment of 1-LN cells with ␣ 2 M* caused translocation of PAK-2 in association with NCK to the cell surface as evidenced by the coimmunoprecipitation of PAK-2 and NCK in the GRP78 immunoprecipitate from plasma membranes. ␣ 2 M*-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, ␣ 2 -macroglobulin. These studies suggest a mechanism by which ␣ 2 M* enhances the metastatic potential of these cells.

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.

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

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 Rap1⅐GTP. 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.The binding of many hormones and growth factors to cells induces activation of adenylyl cyclase, which catalyzes synthesis of cAMP from ATP (1-2). cAMP regulates a wide range of processes through its downstream effectors PKA, 2 cyclic nucleotide-gated cation channels, and a small family of guanine nucleotide exchange factors (GEFs) involved in the regulation of Ras-related proteins (Refs. 3-5 and references therein). PKA-dependent pathways regulate cell proliferation and differentiation, microtubules dynamics, chromatin condensation and decondensation, nuclear envelop disassembly and reassembly, and exocytosis (3). The intracellular targeting and compartmentalization of PKA is controlled through association with A kinase-anchoring proteins (3). By binding to cyclic nucleotide-gated channels, cAMP mediates the transduction of olfactory and visual signals (3). Depending on the cell type, cAMP can either inhibit or stimulate cell growth and proliferation in a PKA-dependent and/or PKA-independent manner (4 -6). For example, intracellular cAMP-elevating agents promote the G 1 to S phase cell cycle transition in cells which include Swiss 3T3, hepatocytes, rat thyroid cells, bone cells, human prostate cancer cells, cardiac myocyte, and PC12 cells (4 -6) while inhibiting the proliferation of cells, which include Rat1 and NIH3T3 adipocytes and endothelial cells (4 -6). cAMP binds to the regulatory subunit of PKA, which causes dissociation of the catalytic subunit and its translocation to the nucleus, where it affects diverse cellular processes by reversible phosphorylation of enzymes and transcrip...

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 ␣ 2 -macroglobulin (␣ 2 M*), results in a 2-3-fold upregulation in the synthesis of prostate-specific antigen (PSA). The PSA is secreted into the medium as an active proteinase, where it binds to native ␣ 2 M. The resultant ␣ 2 M⅐PSA 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.Prostate-specific antigen (PSA) 2 is a serine proteinase that complexes with serum proteinase inhibitors, including ␣ 2 -macroglobulin (␣ 2 M) (1). Monitoring of PSA is generally recommended in men over 50 years old to screen for prostate cancer; however, there is significant controversy with respect to its use as a marker for the appearance of the disease (1-6). By contrast, substantial data support its use in monitoring progression and metastasis (1, 2, 5). Recently, a Phase II controlled trial suggested that immunization against PSA prolongs patient survival, suggesting a direct role for PSA in the pathobiology of the disease (7). Studies have shown that PSA produced by metastatic prostate cancer cells is involved in bone remodeling, a common feature in the bony metastasis of this disease (8). There is, however, little other evidence indicating a direct role for PSA in disease progression. ␣ 2 M is synthesized by many tissues, particularly the liver; however, it is also produced locally in prostate stromal tissue, where it is available to complex with PSA (9). When a proteinase attacks the so-called "bait region," thiol esters in each of the four ␣ 2 M subunits rupture, and the protein undergoes a very large conformational change, exposing receptor recognition sites in each subunit (10). Two receptors have been identified for activated forms of ␣ 2 M (␣ 2 M*), namely the LDL receptor-related protein (LRP) and cell surface-associated GRP78 (glucose-regulated protein of M r ϳ78,000) (10, 11). In addition to proteinases, exposure of ␣ 2 M to small primary amines or ammonia, by direct attack on the thiol esters, produces ␣ 2 M* (10). Although GRP78 is primarily known as a resident endoplasmic reticulum chaperone, it appears on the cell surface of many types of malignant cells, including human prostate cancer (12-16). Here it has many functions, including serving as an ␣ 2 M* signaling re...

Human plasma alpha 2-macroglobulin (alpha 2M) is a tetrameric proteinase inhibitor, which undergoes a conformational change upon reaction with either a proteinase or methylamine. As a result, a receptor recognition site is exposed on each subunit of the molecule enabling it to bind to its receptors on macrophages. We have used Fura-2-loaded murine peritoneal macrophages and digital video fluorescence microscopy to examine the effects of receptor binding on second messenger levels. alpha 2M-methylamine caused a rapid 2-4-fold increase in intracellular Ca2+ concentration ([Ca2+]i) within 5 s of binding to receptors. The agonists induced a focal increase in [Ca2+]i that spread out to other areas of the cell. The increase in [Ca2+]i was dependent on the alpha 2M-methylamine concentration and on the extracellular [Ca2+]. Both sinusoidal and transitory oscillations were observed, which varied from cell to cell. Neither alpha 2M nor boiled alpha 2M-methylamine, forms that are not recognized by the receptor, affected [Ca2+]i in peritoneal macrophages under identical conditions of incubation. The alpha 2M-methylamine-induced rise in [Ca2+]i was accompanied by a rapid and transient increase in macrophage inositol phosphates, including inositol tris- and tetrakis-phosphates. Native alpha 2M did not stimulate a rise in inositol phosphates. Finally, binding of alpha 2M-methylamine to macrophages increased cyclic AMP transiently. Thus receptor-recognized alpha-macroglobulins behave as agonists whose receptor binding causes stimulation of signal transduction pathways.