Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice.

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.

Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-kappaB activation.

Bcl10, a caspase recruitment domain (CARD)-containing protein identified from a breakpoint in mucosa-associated lymphoid tissue (MALT) B lymphomas, is essential for antigen-receptor-mediated nuclear factor kappaB (NF-kappaB) activation in lymphocytes. We have identified a novel CARD-containing protein and interaction partner of Bcl10, named Carma1. Carma1 is predominantly expressed in lymphocytes and represents a new member of the membrane-associated guanylate kinase family. Carma1 binds Bcl10 via its CARD motif and induces translocation of Bcl10 from the cytoplasm into perinuclear structures. Moreover, expression of Carma1 induces phosphorylation of Bcl10 and activation of the transcription factor NF-kappaB. We propose that Carma1 is a crucial component of a novel Bcl10-dependent signaling pathway in T-cells that leads to the activation of NF-kappaB.

Bcl10 controls TCR- and FcgammaR-induced actin polymerization.

Bcl10 plays an essential role in the adaptive immune response, because Bcl10-deficient lymphocytes show impaired Ag receptor-induced NF-kappaB activation and cytokine production. Bcl10 is a phosphoprotein, but the physiological relevance of this posttranslational modification remains poorly defined. In this study, we report that Bcl10 is rapidly phosphorylated upon activation of human T cells by PMA/ionomycin- or anti-CD3 treatment, and identify Ser(138) as a key residue necessary for Bcl10 phosphorylation. We also show that a phosphorylation-deficient Ser(138)/Ala mutant specifically inhibits TCR-induced actin polymerization yet does not affect NF-kappaB activation. Moreover, silencing of Bcl10, but not of caspase recruitment domain-containing MAGUK protein-1 (Carma1) induces a clear defect in TCR-induced F-actin formation, cell spreading, and conjugate formation. Remarkably, Bcl10 silencing also impairs FcgammaR-induced actin polymerization and phagocytosis in human monocytes. These results point to a key role of Bcl10 in F-actin-dependent immune responses of T cells and monocytes/macrophages.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors.

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-gamma (IFN-gamma)-producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X(7) purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1beta (IL-1beta). The priming of IFN-gamma-producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3(-/-)) or caspase-1-deficient (Casp-1(-/-)) mice unless exogenous IL-1beta is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7(-/-) or Nlrp3(-/-) or Casp1(-/-) hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade.

The family of death domain (DD)-containing proteins are involved in many cellular processes, including apoptosis, inflammation and development. One of these molecules, the adapter protein MyD88, is a key factor in innate and adaptive immunity that integrates signals from the Toll-like receptor/interleukin (IL)-1 receptor (TLR/IL-1R) superfamily by providing an activation platform for IL-1R-associated kinases (IRAKs). Here we show that the DD-containing protein Unc5CL (also known as ZUD) is involved in a novel MyD88-independent mode of IRAK signaling that culminates in the activation of the transcription factor nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase. Unc5CL required IRAK1, IRAK4 and TNF receptor-associated factor 6 but not MyD88 for its ability to activate these pathways. Interestingly, the protein is constitutively autoproteolytically processed, and is anchored by its N-terminus specifically to the apical face of mucosal epithelial cells. Transcriptional profiling identified mainly chemokines, including IL-8, CXCL1 and CCL20 as Unc5CL target genes. Its prominent expression in mucosal tissues, as well as its ability to induce a pro-inflammatory program in cells, suggests that Unc5CL is a factor in epithelial inflammation and immunity as well as a candidate gene involved in mucosal diseases such as inflammatory bowel disease.

BIRO1, a cell-permeable BH3 peptide, promotes mitochondrial fragmentation and death of retinoblastoma cells.

Retinoblastoma is the most common pediatric intraocular neoplasm. While retinoblastoma development requires the inactivation of both alleles of the retinoblastoma tumor suppressor gene (RB1) in the developing retina, additional genomic changes are involved in tumor progression, which progressively lead to resistance of tumor cells to death. Therapeutics acting at very downstream levels of death signaling pathways should therefore be interesting in killing retinoblastoma cells. The BH3-only proteins promote apoptosis by modulating the interaction between the pro- and antiapoptotic members of the BCL2 protein family, and this effect can be recapitulated by the BH3 domains. This report analyzes the effect of various BH3 peptides, corresponding to different BH3-only proteins, on two retinoblastoma cell lines, Y79 and WERI-Rb, as well as on the photoreceptor cell line 661W. The BH3 peptide BIRO1, derived from the BCL2L11 death domain, was very effective in promoting Y79 and WERI-Rb cell death without affecting the 661W photoreceptor cells. This cell death was efficient even in absence of BAX and was shown to be caspase independent. While ROS production or AIF release was not detected from mitochondria of treated cells, BIRO1 initiated mitochondria fragmentation in a short period of time following treatment. IMPLICATIONS: The BIRO1 peptide is highly effective at killing retinoblastoma cells and has potential as a peptidomimetic.

p53-induced protein with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase-2 in response to genotoxic stress

Cells respond to DNA damage in a complex way and the fate of damaged cells depends on the balance between pro- and antiapoptotic signals. This is of crucial importance in cancer as genotoxic stress is implied both in oncogenesis and in classical tumor therapies. p53-induced protein with a death domain (PIDD), initially described as a p53-inducible gene, is one of the molecular switches able to activate a survival or apoptotic program. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and we describe here a third isoform. These three isoforms are differentially expressed in tissues and cell lines. Genotoxic stress only affects PIDD isoform 3 mRNA levels, whereas isoforms 1 and 2 mRNA levels remain unchanged. All isoforms are capable of activating nuclear factor-kappaB in response to genotoxic stress, but only isoform 1 interacts with RIP-associated ICH-1/CED-3 homologous protein with a death domain and activates caspase-2. Isoform 2 counteracts the pro-apoptotic function of isoform 1, whereas isoform 3 enhances it. Thus, the differential splicing of PIDD mRNA leads to the formation of at least three proteins with antagonizing/agonizing functions, thereby regulating cell fate in response to DNA damage

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis.

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.

A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex.

Death receptors, such as Fas and tumor necrosis factor-related apoptosis-inducing ligand receptors, recruit Fas-associated death domain and pro-caspase-8 homodimers, which are then autoproteolytically activated. Active caspase-8 is released into the cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in apoptosis. The cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein long form (FLIP(L)), a structural homologue of caspase-8 lacking caspase activity because of several mutations in the active site, is a potent inhibitor of death receptor-induced apoptosis. FLIP(L) is proposed to block caspase-8 activity by forming a proteolytically inactive heterodimer with caspase-8. In contrast, we propose that FLIP(L)-bound caspase-8 is an active protease. Upon heterocomplex formation, a limited caspase-8 autoprocessing occurs resulting in the generation of the p43/41 and the p12 subunits. This partially processed form but also the non-cleaved FLIP(L)-caspase-8 heterocomplex are proteolytically active because they both bind synthetic substrates efficiently. Moreover, FLIP(L) expression favors receptor-interacting kinase (RIP) processing within the Fas-signaling complex. We propose that FLIP(L) inhibits caspase-8 release-dependent pro-apoptotic signals, whereas the single, membrane-restricted active site of the FLIP(L)-caspase-8 heterocomplex is proteolytically active and acts on local substrates such as RIP.

Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

FIST/HIPK3: a Fas/FADD-interacting serine/threonine kinase that induces FADD phosphorylation and inhibits fas-mediated Jun NH(2)-terminal kinase activation.

Fas is a cell surface death receptor that signals apoptosis. Several proteins have been identified that bind to the cytoplasmic death domain of Fas. Fas-associated death domain (FADD), which couples Fas to procaspase-8, and Daxx, which couples Fas to the Jun NH(2)-terminal kinase pathway, bind independently to the Fas death domain. We have identified a 130-kD kinase designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fas-interacting protein. Binding to Fas is mediated by a conserved sequence in the COOH terminus of the protein. FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm. In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction. Although Fas ligand-induced activation of Jun NH(2)-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected. These results suggest that Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas.

The SM protein Vps33 and the t-SNARE H(abc) domain promote fusion pore opening.

Intracellular membrane fusion proceeds via distinct stages of membrane docking, hemifusion and fusion pore opening and depends on interacting families of Rab, SNARE and SM proteins. Trans-SNARE complexes dock the membranes in close apposition. Efficient fusion requires further SNARE-associated proteins. They might increase the number of trans-SNARE complexes or the fusogenic potential of a single SNARE complex. We investigated the contributions of the SM protein Vps33 to hemifusion and pore opening between yeast vacuoles. Mutations in Vps33 that weaken its interactions with the SNARE complex allowed normal trans-SNARE pairing and lipid mixing but retarded content mixing. Deleting the H(abc) domain of the vacuolar t-SNARE Vam3, which interacts with Vps33, had the same effect. This suggests that SM proteins promote fusion pore opening by enhancing the fusogenic activity of a SNARE complex. They should thus be considered integral parts of the fusion machinery.

PIDD death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling.

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.