Protein kinase C-alpha-mediated regulation of low-density lipoprotein receptor related protein and urokinase increases astrocytoma invasion.
ABSTRACT: Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor-related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-alpha (PKC-alpha) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-alpha small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate-induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-alpha/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-alpha and PI3K could provide a new treatment paradigm for glioblastomas.
Project description:The low-density lipoprotein receptor-related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, collagen synthesis, proteolysis, and migration in pleural mesothelial cells (PMCs). When PMCs were treated with the proinflammatory cytokines TNF-? and IL-1?, LRP-1 significantly decreased at the mRNA and protein levels (70 and 90%, respectively; P < 0.05). Consequently, uPA-mediated uPAR internalization was reduced by 80% in the presence of TNF-? or IL-1? (P < 0.05). In parallel studies, LRP-1 neutralization with receptor-associated protein (RAP) significantly reduced uPA-dependent uPAR internalization and increased uPAR stability in PMCs. LRP-1-deficient cells demonstrated increased uPAR t(1/2) versus LRP-1-expressing PMCs. uPA enzymatic activity was also increased in LRP-1-deficient and neutralized cells, and RAP potentiated uPA-dependent migration in PMCs. Collagen expression in PMCs was also induced by uPA, and the effect was potentiated in RAP-treated cells. These studies indicate that TNF-? and IL-1? regulate LRP-1 in PMCs and that LRP-1 thereby contributes to a range of pathophysiologically relevant responses of these cells.
Project description:Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.
Project description:Urokinase plasminogen activator (uPA) and PA inhibitor type 1 (PAI-1) are elevated in acute lung injury, which is characterized by a loss of endothelial barrier function and the development of pulmonary edema. Two-chain uPA and uPA-PAI-1 complexes (1-20 nM) increased the permeability of monolayers of human pulmonary microvascular endothelial cells (PMVECs) in vitro and lung permeability in vivo. The effects of uPA-PAI-1 were abrogated by the nitric-oxide synthase (NOS) inhibitor L-NAME (N(D)-nitro-L-arginine methyl ester). Two-chain uPA (1-20 nM) and uPA-PAI-1 induced phosphorylation of endothelial NOS-Ser(1177) in PMVECs, which was followed by generation of NO and the nitrosylation and dissociation of ?-catenin from VE-cadherin. uPA-induced phosphorylation of eNOS was decreased by anti-low density lipoprotein receptor-related protein-1 (LRP) antibody and an LRP antagonist, receptor-associated protein (RAP), and when binding to the uPA receptor was blocked by the isolated growth factor-like domain of uPA. uPA-induced phosphorylation of eNOS was also inhibited by the protein kinase A (PKA) inhibitor, myristoylated PKI, but was not dependent on PI3K-Akt signaling. LRP blockade and inhibition of PKA prevented uPA- and uPA-PAI-1-induced permeability of PMVEC monolayers in vitro and uPA-induced lung permeability in vivo. These studies identify a novel pathway involved in regulating PMVEC permeability and suggest the utility of uPA-based approaches that attenuate untoward permeability following acute lung injury while preserving its salutary effects on fibrinolysis and airway remodeling.
Project description:The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.
Project description:We reported previously that a signaling pathway consisting of G(i)-Ras-NF-kappaB mediates lysophosphatidic acid (LPA)-induced urokinase plasminogen activator (uPA) upregulation in ovarian cancer cells. However, it is not clear what signaling components link Ras to nuclear factor (NF)-kappaB for this LPA-induced event. In the present study, we found that treatment of protein kinase C (PKC) inhibitors including conventional PKC (cPKC) inhibitor Gö6976 abolished LPA-induced uPA upregulation in ovarian cancer cell lines tested, indicating the importance of cPKC activity in this LPA-induced event. Indeed, LPA stimulation led to the activation of PKCalpha and Ras-PKCalpha interaction. Although constitutively active mutants of PKCalpha (a cPKC), PKCtheta (a novel PKC (nPKC)) and PKCzeta (an atypical PKC (aPKC)) were all able to activate NF-kappaB and upregulate uPA expression, only dominant-negative PKCalpha mutant attenuated LPA-induced NF-kappaB activation and uPA upregulation. These results suggest that PKCalpha, rather than PKC isoforms in other PKC classes, participates in LPA-induced NF-kappaB activation and uPA upregulation in ovarian cancer cells. To determine the signaling components downstream of PKCalpha mediating LPA-induced uPA upregulation, we showed that forced expression of dominant-negative CARMA3 or silencing CARMA3, Bcl10 and MALT1 with specific siRNAs diminished these LPA-induced events. Furthermore, we demonstrated that PKCalpha/CARMA3 signaling axis is important in LPA-induced ovarian cancer cell in vitro invasion.
Project description:BACKGROUND:Glioblastoma is a malignant brain tumor characterized by rapid growth, diffuse invasion and therapeutic resistance. We recently used microRNA expression profiles to subclassify glioblastoma into five genetically and clinically distinct subclasses, and showed that microRNAs both define and contribute to the phenotypes of these subclasses. Here we show that miR-29a activates a multi-faceted growth and invasion program that promotes glioblastoma aggressiveness. METHODS:microRNA expression profiles from 197 glioblastomas were analyzed to identify the candidate miRNAs that are correlated to glioblastoma aggressiveness. The candidate miRNA, miR-29a, was further studied in vitro and in vivo. RESULTS:Members of the miR-29 subfamily display increased expression in the two glioblastoma subclasses with the worst prognoses (astrocytic and neural). We observed that miR-29a is among the microRNAs that are most positively-correlated with PTEN copy number in glioblastoma, and that miR-29a promotes glioblastoma growth and invasion in part by targeting PTEN. In PTEN-deficient glioblastoma cells, however, miR-29a nevertheless activates AKT by downregulating the metastasis suppressor, EphB3. In addition, miR-29a robustly promotes invasion in PTEN-deficient glioblastoma cells by repressing translation of the Sox4 transcription factor, and this upregulates the invasion-promoting protein, HIC5. Indeed, we identified Sox4 as the most anti-correlated predicted target of miR-29a in glioblastoma. Importantly, inhibition of endogenous miR-29a decreases glioblastoma growth and invasion in vitro and in vivo, and increased miR-29a expression in glioblastoma specimens correlates with decreased patient survival. CONCLUSIONS:Taken together, these data identify miR-29a as a master regulator of glioblastoma growth and invasion.
Project description:The complex of the type-1 plasminogen activator inhibitor (PAI-1) and its target proteinases, the urokinase and tissue-type plasminogen activators (uPA and tPA), but not the free components, bind with high affinity to the endocytosis receptors alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP) and very-low-density lipoprotein receptor (VLDLR). To characterize the molecular interaction between the complexes and the receptors, alanine codons were introduced into the human PAI-1 cDNA to replace the four basic residues, Arg-78, Lys-82, Arg-120 and Lys-124, as double mutations. The purified recombinant mutant proteins, rPAI-1/R78A-K124A and rPAI-1/K82A-R120A, produced by the yeast Pichia pastoris, were indistinghuisable from wild-type recombinant and natural human PAI-1 with respect to inhibitory activity against uPA, stability of SDS-resistant complexes with uPA, and vitronectin binding. Radiolabelled mutant uPA.PAI-1 complexes bound with a 10- to 20-fold, and 3- to 7-fold reduced affinity to purified alpha2MR/LRP and VLDLR respectively. alpha2MR/LRP-mediated endocytosis of the mutant complexes by COS-1 cells was reduced to 48 and 38% of the level of endocytosis of wild-type PAI-1. Binding of the mutant complexes to the uPA receptor was not affected. These findings suggest that the binding mode of the uPA.PAI-1 complex to both alpha2MR/LRP and VLDLR is similar. The four residues are surface exposed in the region defined by alpha-helix D and beta-strand 1A in the serine protease inhibitor (serpin) structure. Our study represents the first identification of residues in a surface region implicated in molecular recognition of protease.serpin complexes by endocytosis receptors of the low-density lipoprotein receptor family.
Project description:Despite the functional role of serglycin as an intracellular proteoglycan, a variety of malignant cells depends on its expression and constitutive secretion to advance their aggressive behavior. Serglycin arose to be a biomarker for glioblastoma, which is the deadliest and most treatment-resistant form of brain tumor, but its role in this disease is not fully elucidated. In our study we suppressed the endogenous levels of serglycin in LN-18 glioblastoma cells to decipher its involvement in their malignant phenotype. Serglycin suppressed LN-18 (LN-18<sup>shSRGN</sup>) glioblastoma cells underwent astrocytic differentiation characterized by induced expression of GFAP, SPARCL-1 and SNAIL, with simultaneous loss of their stemness capacity. In particular, LN-18<sup>shSRGN</sup> cells presented decreased expression of glioma stem cell-related genes and ALDH1 activity, accompanied by reduced colony formation ability. Moreover, the suppression of serglycin in LN-18<sup>shSRGN</sup> cells retarded the proliferative and migratory rate, the invasive potential in vitro and the tumor burden in vivo. The lack of serglycin in LN-18<sup>shSRGN</sup> cells was followed by G2 arrest, with subsequent reduction of the expression of cell-cycle regulators. LN-18<sup>shSRGN</sup> cells also exhibited impaired expression and activity of proteolytic enzymes such as MMPs, TIMPs and uPA, both in vitro and in vivo. Moreover, suppression of serglycin in LN-18<sup>shSRGN</sup> cells eliminated the activation of pro-tumorigenic signal transduction. Of note, LN-18<sup>shSRGN</sup> cells displayed lower expression and secretion levels of IL-6, IL-8 and CXCR-2. Concomitant, serglycin suppressed LN-18<sup>shSRGN</sup> cells demonstrated repressed phosphorylation of ERK1/2, p38, SRC and STAT-3, which together with PI3K/AKT and IL-8/CXCR-2 signaling control LN-18 glioblastoma cell aggressiveness. Collectively, the absence of serglycin favors an astrocytic fate switch and a less aggressive phenotype, characterized by loss of pluripotency, block of the cell cycle, reduced ability for ECM proteolysis and pro-tumorigenic signaling attenuation.
Project description:Urokinase-type plasminogen activator (uPA) participates in diverse (patho)physiological processes through intracellular signaling events that affect cell adhesion, migration, and proliferation, although the mechanisms by which these occur are only partially understood. Here we report that upon cell binding and internalization, single-chain uPA (scuPA) translocates to the nucleus within minutes. Nuclear translocation does not involve proteolytic activation or degradation of scuPA. Neither the urokinase receptor (uPAR) nor the low-density lipoprotein-related receptor (LRP) is required for nuclear targeting. Rather, translocation involves the binding of scuPA to the nucleocytoplasmic shuttle protein nucleolin through a region containing the kringle domain. RNA interference and mutational analysis demonstrate that nucleolin is required for the nuclear transport of scuPA. Furthermore, nucleolin is required for the induction smooth muscle alpha-actin (alpha-SMA) by scuPA. These data reveal a novel pathway by which uPA is rapidly translocated to the nucleus where it might participate in regulating gene expression.
Project description:<h4>Background</h4>Glioblastoma is one of the deadliest forms of cancer, in part because of its highly invasive nature. The tumor suppressor PTEN is frequently mutated in glioblastoma and is known to contribute to the invasive phenotype. However the downstream events that promote invasion are not fully understood. PTEN loss leads to activation of the atypical protein kinase C, PKC?. We have previously shown that PKC? is required for glioblastoma cell invasion, primarily by enhancing cell motility. Here we have used time-lapse videomicroscopy to more precisely define the role of PKC? in glioblastoma.<h4>Results</h4>Glioblastoma cells in which PKC? was either depleted by shRNA or inhibited pharmacologically were unable to coordinate the formation of a single leading edge lamellipod. Instead, some cells generated multiple small, short-lived protrusions while others generated a diffuse leading edge that formed around the entire circumference of the cell. Confocal microscopy showed that this behavior was associated with altered behavior of the cytoskeletal protein Lgl, which is known to be inactivated by PKC? phosphorylation. Lgl in control cells localized to the lamellipod leading edge and did not associate with its binding partner non-muscle myosin II, consistent with it being in an inactive state. In PKC?-depleted cells, Lgl was concentrated at multiple sites at the periphery of the cell and remained in association with non-muscle myosin II. Videomicroscopy also identified a novel role for PKC? in the cell cycle. Cells in which PKC? was either depleted by shRNA or inhibited pharmacologically entered mitosis normally, but showed marked delays in completing mitosis.<h4>Conclusions</h4>PKC? promotes glioblastoma motility by coordinating the formation of a single leading edge lamellipod and has a role in remodeling the cytoskeleton at the lamellipod leading edge, promoting the dissociation of Lgl from non-muscle myosin II. In addition PKC? is required for the transition of glioblastoma cells through mitosis. PKC? therefore has a role in both glioblastoma invasion and proliferation, two key aspects in the malignant nature of this disease.