PDCD10，一个新的多功能信号转导调节分子

人程序性细胞死亡分子10(Homo sapiens programmed cell death 10, PDCD10)，最初被称为TFAR15(TF-1 cell apoptosis related gene 15)，是由撤除粒细胞 巨噬细胞集落刺激因子诱导凋亡的人红白血病细胞系TF-1中克隆得到的1个凋亡相关基因. 后来发现它的突变可引起散发性或家族性颅内海绵状血管瘤(cerebral cavernous malformations，CCMs)的发生，为CCMs的第3个致病基因，所以又被叫做CCM3.近年来研究发现,PDCD10能够和GCKⅢ蛋白、γ-PCDH、CCM2、VEGFR2、ERM等众多蛋白相互作用，并能调控ERK/MAPK通路，增加MST4/VEGFR2的稳定性，增强相应的信号转导，促进细胞的增殖、分化和中枢神经系统的发育，与癌症的发生相关，还能调节细胞的凋亡.以上研究证明了PDCD10 的多种生物学效应，并提示其在血管生成、氧化应激、肿瘤中发挥重要作用.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations

Cerebral cavernous malformations (CCMs) are hamartomatous vascular malformations characterized by abnormally enlarged capillary cavities without intervening brain parenchyma. They cause seizures and cerebral hemorrhages, which can result in focal neurological deficits. Three CCM loci have been mapped, and loss-of-function mutations were identified in the KRIT1 (CCM1) and MGC4607 (CCM2) genes. We report herein the identification of PDCD10 (programmed cell death 10) as the CCM3 gene. The CCM3 locus has been previously mapped to 3q26-27 within a 22-cM interval that is bracketed by D3S1763 and D3S1262. We hypothesized that genomic deletions might occur at the CCM3 locus, as reported previously to occur at the CCM2 locus. Through high-density microsatellite genotyping of 20 families, we identified, in one family, null alleles that resulted from a deletion within a 4-Mb interval flanked by markers D3S3668 and D3S1614. This de novo deletion encompassed D3S1763, which strongly suggests that the CCM3 gene lies within a 970-kb region bracketed by D3S1763 and D3S1614. Six additional distinct deleterious mutations within PDCD10, one of the five known genes mapped within this interval, were identified in seven families. Three of these mutations were nonsense mutations, and two led to an aberrant splicing of exon 9, with a frameshift and a longer open reading frame within exon 10. The last of the six mutations led to an aberrant splicing of exon 5, without frameshift. Three of these mutations occurred de novo. All of them cosegregated with the disease in the families and were not observed in 200 control chromosomes. PDCD10, also called "TFAR15," had been initially identified through a screening for genes differentially expressed during the induction of apoptosis in the TF-1 premyeloid cell line. It is highly conserved in both vertebrates and invertebrates. Its implication in cerebral cavernous malformations strongly suggests that it is a new player in vascular morphogenesis and/or remodeling.     

Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening

CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.     

人程序性细胞死亡因子10(PDCD10)：不仅与细胞凋亡相关

人程序性细胞死亡因子10(programmed cell death 10,PDCD10)最初被称为TFAR15(TF-1 cell apoptosis related gene 15),是在1999年运用cDNA-RDA技术首先克隆得到的一个新基因,早期研究提示与凋亡抑制功能相关.近期国外多项研究证明,PDCD10基因的缺失和突变与颅内海绵状血管瘤(cerebral cavernous malformations,CCM)的发生密切相关,CCM的第三个致病基因CCM3即为PDCD10.此外,其他研究表明,PDCD10受到严格的表达调控,在多种肿瘤组织中表达明显上调,提示可能在肿瘤的信号转导通路中起重要作用.最近通过对PDCD10相互作用蛋白的分析和研究,首次证实了PDCD10可以和Ste20激酶家族成员MST4相互作用,增强其激酶活性,并进而通过对ERK-MAPK通路的调控,促进细胞增殖和转化.以上研究证明了PDCD10的多种生物学效应,并提示其在血管生成和肿瘤中发挥重要作用.     

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.     

PDCD10 interacts with Ste20-related kinase MST4 to promote cell growth and transformation via modulation of the ERK pathway

PDCD10 (programmed cell death 10, TFAR15), a novel protein associated with cell apoptosis has been recently implicated in mutations associated with Cerebral Cavernous Malformations (CCM). Yeast two-hybrid screening revealed that PDCD10 interacts with MST4, a member of Ste20-related kinases. This interaction was confirmed by coimmunoprecipitation and colocalization assays in mammalian cells. Furthermore, the co-overexpression of PDCD10 and MST4 promoted cell proliferation and transformation via modulation of the extracellular signal-regulated kinase (ERK) pathway. Potent short interfering RNAs (siRNAs) against PDCD10 (siPDCD10) and MST4 (siMST4) were designed to specifically inhibit the expression of PDCD10 and MST4 mRNA, respectively. The induction of siPDCD10 or siMST4 resulted in decreased expression of endogenous PDCD10 or MST4, which was accompanied by reduced ERK activity and attenuated cell growth and anchorage-independent growth. On the other hand, siMST4 had similar effects in PDCD10-overexpressed cells. And more importantly, we confirmed that either overexpressing or endogenous PDCD10 can increase the MST4 kinase activity in vitro. Our results demonstrated that PDCD10 modulation of ERK signaling was mediated by MST4, and PDCD10 could be a regulatory adaptor necessary for MST4 function, suggesting a link between cerebral cavernous malformation pathogenesis and the ERK-MAPK cascade via PDCD10/MST4.     

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.     

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 (α1–3 and α4–6) 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 α5 helix. We generated a recombinant wild-type (WT) and three PDCD10 mutants that have two (Δ2KA), three (Δ3KA), and five (Δ5KA) K to A mutations. Δ2KA and Δ3KA mutants hypothetically lack binding residues to PtdIns(3,4,5)P3 at the beginning and the end of predicted helix, while Δ5KA completely lacks all predicted binding residues. The WT, Δ2KA, and Δ3KA mutants maintain their binding to PtdIns(3,4,5)P3. Only the Δ5KA abolishes binding to PtdIns(3,4,5)P3. Both Δ5KA and WT show similar secondary and tertiary structures; however, Δ5KA does not bind to OSM. When WT and Δ5KA 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 Δ5KA 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.     

Patterns of expression of the three cerebral cavernous malformation (CCM) genes during embryonic and postnatal brain development

Cerebral Cavernous Malformation (CCM) is a disease characterized by capillary-venous lesions mostly located in the central nervous system. It occurs both as a sporadic and hereditary autosomal dominant condition. Three CCM genes have been identified and shown to encode the KRIT1 (CCM1), MGC4607 (CCM2) and PDCD10 (CCM3) proteins whose functions are so far unknown. In an attempt to get some insight into the role of the 3 CCM genes, we used in situ hybridization to conduct a comparative analysis of their expression pattern at several time points during murine embryonic, postnatal and adult stages particularly within the central nervous system. A strong expression of the 3 Ccm genes was detected in the various neuronal cell layers of the brain, cerebellum and spinal cord, from embryonic to adult life. By E14.5 a moderate labelling was observed in the heart, arterial and venous large vessels with all 3 Ccm probes. Ccm2 and Ccm3 mRNAs, but not Ccm1, were clearly detected within meningeal and parenchymal cortical vessels at P8. This expression was no more detected by P19 and in adult murine brain, strongly suggesting a role for these 2 proteins in the intensive angiogenesis process occuring within the central nervous system during this period.     

Regulation of REGγ cellular distribution and function by SUMO modification

Discovery of emerging REGγ-regulated proteins has accentuated the REGγ-proteasome as an important pathway in multiple biological processes, including cell growth, cell cycle regulation, and apoptosis. However, little is known about the regulation of the REGγ-proteasome pathway. Here we demonstrate that REGγ can be SUMOylated in vitro and in vivo by SUMO-1, SUMO-2, and SUMO-3. The SUMO-E3 protein inhibitor of activated STAT (PIAS)1 physically associates with REGγ and promotes SUMOylation of REGγ. SUMOylation of REGγ was found to occur at multiple sites, including K6, K14, and K12. Mutation analysis indicated that these SUMO sites simultaneously contributed to the SUMOylation status of REGγ in cells. Posttranslational modification of REGγ by SUMO conjugation was revealed to mediate cytosolic translocation of REGγ and to cause increased stability of this proteasome activator. SUMOylation-deficient REGγ displayed attenuated ability to degrade p21(Waf//Cip1) due to reduced affinity of the REGγ SUMOylation-defective mutant for p21. Taken together, we report a previously unrecognized mechanism regulating the activity of the proteasome activator REGγ. This regulatory mechanism may enable REGγ to function as a more potent factor in protein degradation with a broader substrate spectrum.     

Mitochondrial adenine nucleotide translocator 3 is regulated by IL-4 and IFN-gamma via STAT-dependent pathways

IL-4 and IFN-γ are prototypical Th2 and Th1 cytokines, respectively. They reciprocally regulate a number of genes involved in Th1 vs Th2 immune balance. Using DD-PCR analysis, adenine nucleotide translocase (ANT) 3, an enzyme which exchanges ATP and ADP through mitochondrial membrane, has been identified as a novel target counter-regulated by IL-4 and IFN-γ. We have observed that IL-4 and IFN-γ each up-regulates ANT3 in T cells both at mRNA and protein levels, while cotreatment of IL-4 and IFN-γ counter-regulates ANT3 expression. In contrast, other isoforms of ANT were not affected by IL-4 or IFN-γ. Emplyoing transfection and overexpression of STAT6 and STAT1 in STAT-deficient cells, we demonstrate that induction of ANT3 by IL-4 and IFN-γ proceeds via pathways involving STAT6 and STAT1, respectively. Furthermore, regulation of ANT3 expression by IL-4 and IFN-γ correlated with the modulation T cell survival by these cytokines from dex-induced apoptosis. Considering the critical role of mitochondrial ANTs in energy metabolism and apoptosis, ANT3 regulation by IL-4 and IFN-γ may have a functional implication in cytokine-mediated T cell survival.     

IFN-γ-induced apoptosis of human embryonic stem cell derived oligodendrocyte progenitor cells is restricted by CXCR2 signaling

Engraftment of human embryonic stem cell (hESC)-derived OPCs in animal models of demyelination results in remyelination and clinical recovery, supporting the feasibility of cell replacement therapies in promoting repair of damaged neural tissue. A critical gap in our understanding of the mechanisms associated with repair revolves around the effects of the local microenvironment on transplanted cell survival. We have determined that treatment of human ESC-derived OPCs with the pleiotropic cytokine IFN-γ promotes apoptosis that is associated with mitochondrial cytochrome c released into the cytosol with subsequent caspase 3 activation. IFN-γ-induced apoptosis is mediated, in part, by secretion of the CXC chemokine ligand 10 (CXCL10) from IFN-γ-treated cells. Signaling through the chemokine receptor CXCR2 by the ligand CXCL1 functions in a tonic manner by muting apoptosis and this is associated with reduced levels of cytosolic cytochrome c and impaired cleavage of caspase 3. These findings support a role for both IFN-γ and CXCL10 in contributing to neuropathology by promoting OPC apoptosis. In addition, these data suggest that hOPCs used for therapeutic treatment for human neurologic disease/damage are susceptible to death through exposure to local inflammatory cytokines present within the inflammatory milieu.     

Estrogen-related receptor (ERR) γ protects against puromycin aminonucleoside-induced podocyte apoptosis by targeting PI3K/Akt signaling

Accumulating evidence has shown that podocyte apoptosis is of vital importance for the development of glomerulosclerosis and progressive loss of renal function. However, the molecular mechanisms leading to podocyte apoptosis are still elusive. In this study, we investigated the role of estrogen-related receptor (ERR) γ in podocyte apoptosis, as well as the underlying mechanisms. Treatment of PAN caused a dose- and time-dependent podocyte apoptosis in line with a significant downregulation of ERRγ. Interestingly, the occurrence of ERRγ downregulation appeared earlier than the onset of cell apoptosis, suggesting a potential that ERRγ reduction triggered apoptotic response in podocyte. To test this hypothesis, ERRγ siRNA was administered to the podocytes. Strikingly, ERRγ silencing resulted in a significant cell apoptosis accompanied with increased injury markers of B7-1 and cathepsin L and decreased podocyte protein nephrin. In contrast, overexpression of ERRγ remarkably attenuated PAN-induced cell apoptosis. Moreover, ERRγ overexpression stimulated PI3K/Akt signaling pathway evidenced by increased expression of PI3K subunits p85α and p110α and phosphorylated Akt. Importantly, a specific PI3K inhibitor LY294002 entirely reversed the anti-apoptotic effect of ERRγ following PAN treatment. Finally, we observed a striking downregulation of ERRγ in PAN-treated rat kidneys, suggesting that our cell model replicated the in vivo condition. Taken together, these data highly suggested that ERRγ played a novel role in modulating podocyte apoptosis by targeting PI3K/Akt signaling pathway.     

Activation of phospholipase-Cγ and protein kinase C signal pathways helps the survival of spinal motoneurons injured by root avulsion

The signaling transduction processes involved in avulsion-induced motoneuron (MN) death have not been elucidated. Using the brachial plexus root avulsion rat model, we showed that avulsion-activated phosphorylation of phospholipase-Cγ (PLCγ) and protein kinase C (PKC) occurred in injured spinal MNs within 72?h of injury. Moreover, some MNs positive for PLCγ and PKC are also positive for avulsion-induced neuronal nitric oxide synthase (nNOS). Inhibition of PLCγ/PKC signal pathway, either with PLCγ inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione, or with PLCγ siRNA augmented avulsion-induced MN death. 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione also inhibited PKC phosphorylation and exacerbated avulsion-induced reductions in the nNOS protein level in injured spinal segments. Moreover, activation of PLCγ/PKC signal pathway with PKC activator, phorbol-12-myristate-13-acetate, decreased avulsion-induced MN death. The temporal profile of PLCγ/PKC signaling appears to be crucial for the survival of spinal MNs after root avulsion. Our data suggest that PLCγ mediates, while PKC and nNOS are associated with, the avulsion-induced MN death in brachial plexus root avulsion.     

一中国脑海绵状血管瘤家系中发现krit1基因新的缺失突变

脑海绵状血管瘤（CCM）是多定位于中枢神经系统的一种脑部血管异常,少数在皮肤和视网膜处有并发症。依据致病基因在染色体上的不同位置分为CCM1、CCM2和CCM33种类型。目前,CCM1、CCM2和CCM3的致病基因已经被克隆,分别为krit1、MGC4607和细胞程序性死亡10基因（PDCD10）。利用连锁分析发现内蒙古的一个家系属于CCM1,突变检测发现患者CCM1基因（krit1）第9内含子和第10外显子拼接位点处存在一“GTA”缺失,该突变导致终止密码子提前出现,产生截短蛋白。实验结果支持krit1为CCM1致病基因。     

Molecular basis for T cell response induced by altered peptide ligand of type II collagen

Mounting evidence from animal models has demonstrated that alterations in peptide-MHC interactions with the T cell receptor (TCR) can lead to dramatically different T cell outcomes. We have developed an altered peptide ligand of type II collagen, referred to as A9, which differentially regulates TCR signaling in murine T cells leading to suppression of arthritis in the experimental model of collagen-induced arthritis. This study delineates the T cell signaling pathway used by T cells stimulated by the A9·I-A(q) complex. We have found that T cells activated by A9 bypass the requirement for Zap-70 and CD3-ζ and signal via FcRγ and Syk. Using collagen-specific T cell hybridomas engineered to overexpress either Syk, Zap-70, TCR-FcRγ, or CD3-ζ, we demonstrate that A9·I-A(q) preferentially activates FcRγ/Syk but not CD3-ζ/Zap-70. Moreover, a genetic absence of Syk or FcRγ significantly reduces the altered peptide ligand induction of the nuclear factor GATA3. By dissecting the molecular mechanism of A9-induced T cell signaling we have defined a new alternate pathway that is dependent upon FcRγ and Syk to secrete immunoregulatory cytokines. Given the interest in using Syk inhibitors to treat patients with rheumatoid arthritis, understanding this pathway may be critical for the proper application of this therapy.     

Programmed Cell Death 6 (PDCD6) Protein Interacts with Death-Associated Protein Kinase 1 (DAPk1): Additive Effect on Apoptosis via Caspase-3 Dependent Pathway

Programmed cell death 6 (PDCD6) protein is a 22 kDa EF-hand type Ca²⁺-binding protein involved in apoptosis. To define the regulating mechanism of PDCD6 activity in the apoptotic pathway, we searched a human ovary cDNA library for a novel PDCD6 binding protein using a yeast two-hybrid system. The selected protein was the human death-associated protein kinase 1 (DAPk1), another protein that functions as a positive mediator of apoptosis. Co-transfection of PDCD6 and DAPk1 cDNA into a tumor cell line accelerated apoptosis via caspase-3 dependent pathway.J.H. Lee and S.B. Rho contributed equally to this workRevisions requested 4 March 2005; Revisions received 10 May 2005