Defining the functional domain of programmed cell death 10 through its interactions with phosphatidylinositol-3,4,5-trisphosphate.

Cerebral cavernous malformations (CCM) are vascular abnormalities of the central nervous system predisposing blood vessels to leakage, leading to hemorrhagic stroke. Three genes, Krit1 (CCM1), OSM (CCM2), and PDCD10 (CCM3) are involved in CCM development. PDCD10 binds specifically to PtdIns(3,4,5)P3 and OSM. Using threading analysis and multi-template modeling, we constructed a three-dimensional model of PDCD10. PDCD10 appears to be a six-helical-bundle protein formed by two heptad-repeat-hairpin structures (alpha1-3 and alpha4-6) sharing the closest 3D homology with the bacterial phosphate transporter, PhoU. We identified a stretch of five lysines forming an amphipathic helix, a potential PtdIns(3,4,5)P3 binding site, in the alpha5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Delta2KA), three (Delta3KA), and five (Delta5KA) K to A mutations. Delta2KA and Delta3KA mutants hypothetically lack binding residues to PtdIns(3,4,5)P3 at the beginning and the end of predicted helix, while Delta5KA completely lacks all predicted binding residues. The WT, Delta2KA, and Delta3KA mutants maintain their binding to PtdIns(3,4,5)P3. Only the Delta5KA abolishes binding to PtdIns(3,4,5)P3. Both Delta5KA and WT show similar secondary and tertiary structures; however, Delta5KA does not bind to OSM. When WT and Delta5KA are co-expressed with membrane-bound constitutively-active PI3 kinase (p110-CAAX), the majority of the WT is co-localized with p110-CAAX at the plasma membrane where PtdIns(3,4,5)P3 is presumably abundant. In contrast, the Delta5KA remains in the cytoplasm and is not present in the plasma membrane. Combining computational modeling and biological data, we propose that the CCM protein complex functions in the PI3K signaling pathway through the interaction between PDCD10 and PtdIns(3,4,5)P3.

Dexamethasone-induced inositol 1,4,5-trisphosphate receptor elevation in murine lymphoma cells is not required for dexamethasone-mediated calcium elevation and apoptosis.

Glucocorticosteroid hormones, including dexamethasone, have diverse effects on immature lymphocyte function that ultimately lead to cell death. Previous studies established that glucocorticoid-induced alterations in intracellular calcium homeostasis promote apoptosis, but the mechanism by which glucocorticoids disrupt calcium homeostasis is unknown. Through gene expression array analysis, we found that dexamethasone induces a striking elevation of inositol 1,4,5-trisphosphate receptor (IP(3)R) levels in two murine lymphoma cell lines, WEHI7.2 and S49.A2. IP(3)R elevation was confirmed at both mRNA and protein levels. However, there was not a strong correlation between IP(3)R elevation and altered calcium homeostasis in terms of either kinetics or dose response. Moreover, IP(3)R knockdown, by either antisense or small interfering RNA, did not prevent either calcium disruption or apoptosis. Finally, DT40 lymphoma cells lacking all three IP(3)R isoforms were just as sensitive to dexamethasone-induced apoptosis as wild-type DT40 cells expressing all three IP(3)R isoforms. Thus, although alterations in intracellular calcium homeostasis contribute to glucocorticoid-induced apoptosis, these calcium alterations are not directly attributable to IP(3)R elevation.

Hyperosmotic induction of mitogen-activated protein kinase scaffolding.

Eukaryotic cells respond to hyperosmotic conditions by expunging water from the cell, leading to cell shrinkage. This is counteracted by adaptive responses that restore cell volume and strengthen the cytoskeletal architecture. In the budding yeast Saccharomyces cerevisiae, this response is mediated primarily by the mitogen-activated protein kinase (MAPK) cascade CDC42-STE50-STE11-Pbs2-Hog1. In mammalian cells, MAPK scaffold proteins facilitate the efficiency of signaling within the cascade by placing a kinase near its substrate and also regulate the subcellular localization of the signaling. Our laboratory has discovered a scaffold that coordinates the analogous Hog1 signal in mammalian cells, termed OSM (osmosensing scaffold for MEKK3). OSM organizes a complex consisting of the small GTPase Rac, MEKK3, and MKK3 for the activation of p38 MAPK. Interactions among OSM, Rac, and MEKK3 are augmented in response to sorbitol and are also localized to membrane ruffles, sites of rapid actin turnover. Suppression of the expression of OSM or MEKK3 by RNA interference strongly inhibits the sorbitol-dependent activation of p38. Furthermore, mutations in OSM were concurrently found to cause cerebral cavernous malformations (CCM), a disease of the central nervous system characterized by thin-walled, leaky blood vessels that become hemorrhagic. Our laboratory has also demonstrated that Krit1, another gene harboring mutations that lead to CCM, binds OSM and its interaction is enhanced in response to sorbitol in a similar manner as the MEKK3-OSM interaction. This chapter describes the cell biological and biochemical methods used for assaying protein-protein interactions in live cells using fluorescence resonance energy transfer, in vitro kinase assays for MEKK3-MKK3-p38 pathway members, and gene suppression by RNA interference to study hyperosmotic stress-dependent signaling.

Proteomic identification of the cerebral cavernous malformation signaling complex.

Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217-->A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.

Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae.

BACKGROUND: The zebrafish Danio rerio is an important model system for drug discovery and to study cardiovascular development. Using a laser-scanning confocal microscope, we have developed a non-invasive method of measuring cardiac performance in zebrafish embryos and larvae that obtains cardiovascular parameters similar to those obtained using Doppler echocardiography in mammals. A laser scan line placed parallel to the path of blood in the dorsal aorta measures blood cell velocity, from which cardiac output and indices of vascular resistance and contractility are calculated. RESULTS: This technique, called laser-scanning velocimetry, was used to quantify the effects of pharmacological, developmental, and genetic modifiers of cardiac function. Laser-scanning velocimetry was applied to analyze the cardiovascular effects of morpholino knockdown of osmosensing scaffold for MEKK3 (OSM), which when mutated causes the human vascular disease cerebral cavernous malformations. OSM-deficient embryos had a constricted aortic arch and markedly increased peak cell velocity, a characteristic indicator of aortic stenosis. CONCLUSION: These data validate laser-scanning velocimetry as a quantitative tool to measure cardiovascular performance for pharmacological and genetic analysis in zebrafish, which requires no specialized equipment other than a laser-scanning confocal microscope.

The glucocorticoid-induced gene tdag8 encodes a pro-apoptotic G protein-coupled receptor whose activation promotes glucocorticoid-induced apoptosis.

The apoptotic action of glucocorticoids on lymphocytes makes them effective therapeutics for many lymphoid malignancies. Although it is clear that glucocorticoid-induced apoptosis requires transcription, the gene products that induce apoptosis remain unknown. Using gene expression profiles of lymphoma cell lines and primary thymocytes treated with the synthetic glucocorticoid dexamethasone, we discovered that induction of tdag8 (T-cell death-associated gene 8) was a common event in each model system investigated. Activation of TDAG8 by its agonist psychosine markedly enhanced dexamethasone-induced apoptosis in a TDAG8-dependent manner. Expression of a TDAG8-GFP fusion protein was sufficient to induce apoptosis, and repression of endogenous TDAG8 using RNA interference partially inhibited dexamethasone-induced apoptosis. Together, these data suggest that TDAG8 is a regulator of glucocorticoid-induced apoptosis and that agonists of TDAG8 may be promising agents to improve the efficacy of glucocorticoids for the treatment of leukemia and lymphoma.

Dexamethasone-induced gene 2 (dig2) is a novel pro-survival stress gene induced rapidly by diverse apoptotic signals.

Glucocorticoid hormones induce apoptosis in lymphoid cells. This process requires de novo RNA/protein synthesis. Here we report the identification and cloning of a novel dexamethasone-induced gene designated dig2. Using Affymetrix oligonucleotide microarray analysis of approximately 10,000 genes and expressed sequence tags, we found that the expression of dig2 mRNA is significantly induced not only in the murine T cell lymphoma lines S49.A2 and WEHI7.2 but also in normal mouse thymocytes following dexamethasone treatment. This result was confirmed by Northern blot analysis. The induction of dig2 mRNA by dexamethasone appears to be mediated through the glucocorticoid receptor as it is blocked in the presence of RU486, a glucocorticoid receptor antagonist. Furthermore, we demonstrated that dig2 is a novel stress response gene, as its mRNA is induced in response to a variety of cellular stressors including thapsigargin, tunicamycin, and heat shock. In addition, the levels of dig2 mRNA were up-regulated after treatment with the apoptosis-inducing chemotherapeutic drug etoposide. Though the function of dig2 is unknown, dig2 appears to have a pro-survival function, as overexpression of dig2 reduces the sensitivity of WEHI7.2 cells to dexamethasone-induced apoptosis.

Microarray analysis uncovers the induction of the proapoptotic BH3-only protein Bim in multiple models of glucocorticoid-induced apoptosis.

Despite being one of the earliest recognized and most clinically relevant forms of apoptosis, little is known about the transcriptional events that mediate glucocorticoid-induced apoptosis. Therefore, we used oligonucleotide microarrays to identify the pattern of dexamethasone-induced changes in gene expression in two well characterized models of glucocorticoid-induced apoptosis, the murine lymphoma cell lines S49.A2 and WEHI7.2. Dexamethasone treatment induced a diverse set of gene changes that evolved over a 24-h period preceding the onset of cell death. These include previously reported changes in the expression of genes regulating prosurvival signals mediated by c-Myc and NFkappaB. Unexpectedly, we discovered that glucocorticoid treatment increases expression of the gene encoding Bim, a BH3-only member of the Bcl-2 family that is capable of directly activating the apoptotic cascade. Induction of Bim was confirmed by immunoblotting not only in S49.A2 and WEHI7.2 cells but also in the human leukemia cell line CEM-C7 and in primary murine thymocytes. All three prototypical isoforms of Bim (BimEL, BimL, and BimS) were induced by dexamethasone. Because elevated expression of Bim initiates the execution phase of cell death, this report that Bim is induced by dexamethasone provides novel insight into the mechanism through which glucocorticoid-mediated changes in gene expression induce apoptosis in lymphoid cells.