Epigenetic inactivation of endothelin-2 and endothelin-3 in colon cancer.

Endothelin-1 (ET-1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET-2 and ET-3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET-2 and ET-3 expression and determine the underlying mechanisms. Human primary colon cancers and carcinogen-induced rat colon tumors were subjected to real-time RT-PCR, immunoblotting and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation-specific PCR, bisulfite sequencing and pyrosequencing; and forced expression of ET-2 and ET-3 was conducted in human colon cancer cells followed by real-time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET-2 and ET-3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers and in a panel of human colon cancer cell lines. Forced expression of ET-2 and ET-3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET-2 and ET-3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re-expression of ET-2 and ET-3 as natural antagonists of ET-1 in colon cancer.

Identification of gold nanoparticle-resistant mutants of Saccharomyces cerevisiae suggests a role for respiratory metabolism in mediating toxicity.

Positively charged gold nanoparticles (0.8-nm core diameter) reduced yeast survival, but not growth, at a concentration of 10 to 100 µg/ml. Among 17 resistant deletion mutants isolated in a genome-wide screen, highly significant enrichment was observed for respiration-deficient mutants lacking genes encoding proteins associated with the mitochondrion.

CRP1, a protein localized in filopodia of growth cones, is involved in dendritic growth.

The cysteine-rich protein (CRP) family is a subgroup of LIM domain proteins. CRP1, which cross-links actin filaments to make actin bundles, is the only CRP family member expressed in the CNS with little known about its function in nerve cells. Here, we report that CRP1 colocalizes with actin in the filopodia of growth cones in cultured rat hippocampal neurons. Knockdown of CRP1 expression by short hairpin RNA interference results in inhibition of filopodia formation and dendritic growth in neurons. Overexpression of CRP1 increases filopodia formation and neurite branching, which require its actin-bundling activity. Expression of CRP1 with a constitutively active form of Cdc42, a GTPase involved in filopodia formation, increases filopodia formation in COS-7 cells, suggesting cooperation between the two proteins. Moreover, we demonstrate that neuronal activity upregulates CRP1 expression in hippocampal neurons via Ca²âº influx after depolarization. Ca²âº/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein mediate the Ca²âº-induced upregulation of CRP1 expression. Furthermore, CRP1 is required for the dendritic growth induced by Ca²âº influx or CaMKIV. Together, these data are the first to demonstrate a role for CRP1 in dendritic growth.

Calpain 2 is required for the invasion of glioblastoma cells in the zebrafish brain microenvironment.

Glioblastoma is an aggressive primary brain tumor with a 5-year survival rate of less than 5%. The ability of glioblastoma cells to invade surrounding brain tissue presents the primary challenge for the success of focal therapeutic approaches. We previously reported that the calcium-activated protease calpain 2 is critical for glioblastoma cell invasion in vitro. Here, we show that expression of calpain 2 is required for the dispersal of glioblastoma cells in a living brain microenvironment. Knockdown of calpain 2 resulted in a 2.9-fold decrease in the invasion of human glioblastoma cells in zebrafish brain. Control cells diffusely migrated up to 450 µm from the site of injection, whereas knockdown cells remained confined in clusters. The invasion study was repeated in organotypic mouse brain tissues, and calpain 2 knockdown cells demonstrated a 2.3-fold lower area of dispersal compared with control cells. In zebrafish brain, glioblastoma cells appeared to migrate in part along the blood vessels of the host. Furthermore, angiogenesis was detected in 27% of zebrafish injected with control cells, whereas only 12.5% of fish receiving knockdown cells showed the formation of new vessels, suggesting a role for calpain 2 in tumor cell angiogenesis. Consistent with the progression of glioblastoma in humans, transplanted tumor cells were not observed to metastasize outside the brain of zebrafish. This study demonstrates that calpain 2 expression is required for the dispersal of glioblastoma cells within the dynamic microenvironment of the brain, identifying zebrafish as a valuable orthotopic system for studying glioblastoma cell invasion.

Calpain 2 is required for glioblastoma cell invasion: regulation of matrix metalloproteinase 2.

Invasion of glioblastoma cells significantly reduces the effectiveness of current treatments, highlighting the importance of understanding dispersal mechanisms and characteristics of the invasive population. Induction of calcium fluxes into glioblastoma cells by autocrine glutamate is critical for invasion. However, the target(s) by which calcium acts to stimulate the dispersal of glioblastoma cells is not clear. In this study, we tested the hypothesis that the calcium-activated protease calpain 2 is required for glioblastoma cell invasion. Knockdown of calpain 2 expression using shRNA or chemical inhibition of calpain activity reduced glioblastoma cell invasion by 90%. Interestingly, decreased expression of calpain 2 did not influence morphology or migration, suggesting regulation of invasion specific mechanisms. Consistent with this idea, 39% less extracellular MMP2 was measured from knockdown cells identifying one mechanism by which calpain 2 mediates glioblastoma cell invasion. This is the first report demonstrating that calpain 2 is required for glioblastoma cell invasion.

Glycine-rich region regulates cysteine-rich protein 1 binding to actin cytoskeleton.

Cysteine-rich protein 1 (CRP1) has a unique structure with two well separated LIM domains, each followed by a glycine-rich region. Although CRP1 has been shown to interact with actin-binding proteins and actin filaments, the mechanism regulating localization to the actin cytoskeleton in cells is not clear. Experiments using truncated forms showed that the first LIM domain and glycine-rich region are necessary for CRP1 bundling of actin filaments and localization to the actin cytoskeleton. Furthermore, domain swapping experiments replacing the first glycine-rich region with the second resulted in the loss of CRP1 bundling activity and localization to the actin cytoskeleton, identifying seven critical amino acid residues. These results highlight the importance of the first glycine-rich region for CRP1 bundling activity and localization to the actin cytoskeleton. In addition, this work identifies the first LIM domain and glycine-rich region as a distinct actin filament bundling module.

Phosphoinositide binding regulates alpha-actinin CH2 domain structure: analysis by hydrogen/deuterium exchange mass spectrometry.

alpha-Actinin is an actin bundling protein that regulates cell adhesion by directly linking actin filaments to integrin adhesion receptors. Phosphatidylinositol (4,5)-diphosphate (PtdIns (4,5)-P(2)) and phosphatidylinositol (3,4,5)-triphosphate (PtdIns (3,4,5)-P(3)) bind to the calponin homology 2 domain of alpha-actinin, regulating its interactions with actin filaments and integrin receptors. In this study, we examine the mechanism by which phosphoinositide binding regulates alpha-actinin function using mass spectrometry to monitor hydrogen-deuterium (H/D) exchange within the calponin homology 2 domain. The overall level of H/D exchange for the entire protein showed that PtdIns (3,4,5)-P(3) binding alters the structure of the calponin homology 2 domain increasing deuterium incorporation, whereas PtdIns (4,5)-P(2) induces changes in the structure decreasing deuterium incorporation. Analysis of peptic fragments from the calponin homology 2 domain showed decreased local H/D exchange within the loop region preceding helix F with both phosphoinositides. However, the binding of PtdIns (3,4,5)-P(3) also induced increased exchange within helix E. This suggests that the phosphate groups on the fourth and fifth position of the inositol head group of the phosphoinositides constrict the calponin homology 2 domain, thereby altering the orientation of actin binding sequence 3 and decreasing the affinity of alpha-actinin for filamentous actin. In contrast, the phosphate group on the third position of the inositol head group of PtdIns (3,4,5)-P(3) perturbs the calponin homology 2 domain, altering the interaction between the N and C terminus of the full-length alpha-actinin antiparallel homodimer, thereby disrupting bundling activity and interaction with integrin receptors.

Cysteine-rich protein 1 (CRP1) regulates actin filament bundling.

BACKGROUND: Cysteine-rich protein 1 (CRP1) is a LIM domain containing protein localized to the nucleus and the actin cytoskeleton. CRP1 has been demonstrated to bind the actin-bundling protein alpha-actinin and proposed to modulate the actin cytoskeleton; however, specific regulatory mechanisms have not been identified. RESULTS: CRP1 expression increased actin bundling in rat embryonic fibroblasts. Although CRP1 did not affect the bundling activity of alpha-actinin, CRP1 was found to stabilize the interaction of alpha-actinin with actin bundles and to directly bundle actin microfilaments. Using confocal and photobleaching fluorescence resonance energy transfer (FRET) microscopy, we demonstrate that there are two populations of CRP1 localized along actin stress fibers, one associated through interaction with alpha-actinin and one that appears to bind the actin filaments directly. Consistent with a role in regulating actin filament cross-linking, CRP1 also localized to the membrane ruffles of spreading and PDGF treated fibroblasts. CONCLUSION: CRP1 regulates actin filament bundling by directly cross-linking actin filaments and stabilizing the interaction of alpha-actinin with actin filament bundles.

Phosphoinositides differentially regulate alpha-actinin flexibility and function.

Alpha-actinin is a cell-adhesion and cytoskeletal protein that bundles actin microfilaments and links these filaments directly to integrin-adhesion receptors. Phosphoinositides bind to and regulate the interaction of a-actinin with actin filaments and integrin receptors. In the present study, we demonstrate that PtdIns(3,4,5)P3 inhibits and disrupts a-actinin-bundling activity, whereas PtdIns(4,5)P2 can only inhibit activity. In addition, a protease-sensitivity assay was developed to examine the flexibility of the linker region between the actin-binding domain and the spectrin repeats of a-actinin. Both phosphoinositides influenced the extent of proteolysis and the cleavage sites. PtdIns(4,5)P2 binding decreased the proteolysis of a-actinin, suggesting a role in stabilizing the structure of the protein. In contrast, PtdIns(3,4,5)P3 binding enhanced a-actinin proteolysis, indicating an increase in the flexibility of the protein. Furthermore, phosphoinositide binding influenced the proteolysis of the N- and C-terminal domains of a-actinin, indicating regulation of structure within both domains. These results support the hypothesis that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 differentially regulate a-actinin function by modulating the structure and flexibility of the protein.

Coibamide A induces mTOR-independent autophagy and cell death in human glioblastoma cells.

Coibamide A is an N-methyl-stabilized depsipeptide that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups (ICBG) program based in Panama. Previous testing of coibamide A in the NCI in vitro 60 cancer cell line panel revealed a potent anti-proliferative response and "COMPARE-negative" profile indicative of a unique mechanism of action. We report that coibamide A is a more potent and efficacious cytotoxin than was previously appreciated, inducing concentration- and time-dependent cytotoxicity (EC50<100 nM) in human U87-MG and SF-295 glioblastoma cells and mouse embryonic fibroblasts (MEFs). This activity was lost upon linearization of the molecule, highlighting the importance of the cyclized structure for both anti-proliferative and cytotoxic responses. We show that coibamide A induces autophagosome accumulation in human glioblastoma cell types and MEFs via an mTOR-independent mechanism; no change was observed in the phosphorylation state of ULK1 (Ser-757), p70 S6K1 (Thr-389), S6 ribosomal protein (Ser-235/236) and 4EBP-1 (Thr-37/46). Coibamide A also induces morphologically and biochemically distinct forms of cell death according to cell type. SF-295 glioblastoma cells showed caspase-3 activation and evidence of apoptotic cell death in a pattern that was also seen in wild-type and autophagy-deficient (ATG5-null) MEFs. In contrast, cell death in U87-MG glioblastoma cells was characterized by extensive cytoplasmic vacuolization and lacked clear apoptotic features. Cell death was attenuated, but still triggered, in Apaf-1-null MEFs lacking a functional mitochondria-mediated apoptotic pathway. From the study of ATG5-null MEFs we conclude that a conventional autophagy response is not required for coibamide A-induced cell death, but likely occurs in dying cells in response to treatment. Coibamide A represents a natural product scaffold with potential for the study of mTOR-independent signaling and cell death mechanisms in apoptotic-resistant cancer cells.

Phosphoinositide binding to the substrate regulates susceptibility to proteolysis by calpain.

Calpain-mediated proteolysis regulates cytoskeletal dynamics and is altered during aging and the progression of numerous diseases or pathological conditions. Although several cytoskeletal proteins have been identified as substrates, how localized calpain activity is regulated and the mechanisms controlling substrate recognition are not clear. In this study, we report that phosphoinositide binding regulates the susceptibility of the cytoskeletal adhesion protein alpha-actinin to proteolysis by calpains 1 and 2. At first, alpha-actinin did not appear to be a substrate for calpain 2; however, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) binding to alpha-actinin resulted in nearly complete proteolysis of the full-length protein, producing stable breakdown products. Calpain 1 was able to cleave alpha-actinin in the absence of phosphoinositide binding; however, PtdIns(3,4,5)P(3) binding increased the rate of proteolysis, and phosphatidylinositol 4,5-diphosphate (PtdIns(4,5)P(2)) binding significantly inhibited cleavage. Phosphoinositide binding appeared to regulate calpain proteolysis of alpha-actinin by modulating the exposure of a highly sensitive cleavage site within the calponin homology 2 domain. In U87MG glioblastoma cells, which contain elevated levels of PtdIns(3,4,5)P(3), alpha-actinin colocalized with calpain within dynamic actin cytoskeletal structures. Furthermore, proteolysis of alpha-actinin producing stable breakdown products was observed in U87MG cells treated with calcium ionophore to activate the calcium-dependent calpains. Additional evidence of PtdIns(3,4,5)P(3)-mediated calpain proteolysis of alpha-actinin was observed in rat embryonic fibroblasts. These results suggest that PtdIns(3,4,5)P(3) binding is a critical determinant for alpha-actinin proteolysis by calpain. In conclusion, phosphoinositide binding to the substrate is a potential mechanism for regulating susceptibility to proteolysis by calpain.

Phospholipase C-beta3 and -beta1 form homodimers, but not heterodimers, through catalytic and carboxyl-terminal domains.

Phospholipase C-beta (PLC-beta) isoenzymes are key effectors in G protein-coupled signaling pathways. Prior research suggests that some isoforms of PLC-beta may exist and function as dimers. Using coimmunoprecipitation assays of differentially tagged PLC-beta constructs and size-exclusion chromatography of native PLC-beta, we observed homodimerization of PLC-beta3 and PLC-beta1 isoenzymes but failed to detect heterodimerization of these isoenzymes. Size-exclusion chromatography data suggest that PLC-beta3 and PLC-beta1 form higher affinity homodimers than PLC-beta2. Evidence supportive of limited PLC-beta monomer-homodimer equilibrium appears at < or =100 nM. Further assessment of homodimerization status by coimmunoprecipitation assays with differentially tagged PLC-beta3 fragments demonstrated that at least two subdomains of PLC-beta3 are involved in dimer formation, one in the catalytic X and Y domains and the other in the G protein-regulated carboxyl-terminal domain. In addition, we provide evidence consistent with the existence of PLC-beta homodimers in a whole-cell context, using fluorescent protein-tagged constructs and microscopic fluorescence resonance energy transfer assays.

Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling.

The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of alpha-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-alpha-actinin and a GFP-alpha-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039-24045). In fibroblasts, recovery of the mutant alpha-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, alpha-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of alpha-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant alpha-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the alpha-actinin mutant inhibited actin reorganization with the appearance of enlarged alpha-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of alpha-actinin with actin filaments and integrin adhesion receptors and that the dynamics of alpha-actinin is important for PI 3-kinase-induced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of alpha-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.

Phosphoinositide binding inhibits alpha-actinin bundling activity.

alpha-Actinin is an abundant actin-bundling and adhesion protein that directly links actin filaments to integrin receptors. Previously, in platelet-derived growth factor-treated fibroblasts, we demonstrated that phosphoinositides bind to alpha-actinin, regulating its localization (Greenwood, J. A., Theibert, A. B., Prestwich, G. D., and Murphy-Ullrich, J. E. (2000) J. Cell Biol. 150, 627- 642). In this study, phosphoinositide binding and regulation of alpha-actinin function is further characterized. Phosphoinositide binding specificity, determined using a protein-lipid overlay procedure, suggests that alpha-actinin interacts with phosphates on the 4th and 5th position of the inositol head group. Binding assays and mutational analyses demonstrate that phosphoinositides bind to the calponin homology domain 2 of alpha-actinin. Phosphoinositide binding inhibited the bundling activity of alpha-actinin by blocking the interaction of the actin-binding domain with actin filaments. Consistent with these results, excessive bundling of actin filaments was observed in fibroblasts expressing an alpha-actinin mutant with decreased phosphoinositide affinity. We conclude that the interaction of alpha-actinin with phosphoinositides regulates actin stress fibers in the cell by controlling the extent to which microfilaments are bundled.