Candidate genes for Down syndrome, where NFATc1 occupancy on the promoter. ‘Weak ‘means
1. Introduction
Down syndrome is the most common genetic cause of mental retardation in humans, occurring in one out of 700 live births. Epidemiological studies suggest that although individuals with Down syndrome have an increased risk of infant cardiovascular malformation, muscle hypotonia, lymphatic edema, and leukemia, noteworthy they have a considerably reduced incidence of most solid tumor, atherosclerosis, and pathological angiogenesis-mediated diabetic retinopathy and kidney dysfunction.
Such data indicate that one or more of the 231 trisomic genes on chromosome 21 are responsible for protecting these individuals against cancer and vascular disease. We and others recently have identified the candidate genes are Down syndrome critical region (DSCR)-1, and A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1. In primary cultured endothelial cells, vascular endothelial cell growth factor (VEGF) resulted in rapid and profound upregulation of both genes, which in turn negatively feeds back to attenuate VEGF-mediated signaling and following the endothelial cell activation. In genome-wide screening, important regulatory transcription factor for many pathological features of Down syndrome, NFAT, bound more than 10,000 independent regions in VEGF-treated activated endothelial cells. Down syndrome trisomy model mice or endothelium-specific modest DSCR-1 increases in mice resulted in significant suppression of the vascular density in matrigel-plugs, inflammatory leukocyte infiltration, and tumor growth. In contrast, DSCR-1 null mice demonstrated markedly decreased vascular integrity and increased susceptibility to tumor metastasis. In a mouse model of endotoxemia, DSCR-1 null mice showed greater morbidity and mortality compared with wild-type littermate. Conversely, adenovirus-mediated overexpression of DSCR-1 resulted in marked attenuation of lipopolysaccharide (LPS) or VEGF-mediated inflammation. Collectively, these data provide that Down syndrome overexpressed protein; DSCR-1 serves to dampen the host response to infection and the tumor growth. The molecular research for Down syndrome with patients or model mice unexpectedly provide us a great hint for therapeutic targets in solid tumor and vasculopathic disease against all individuals.
2. Down Syndrome Critical Region (DSCR)-1 expression in activated endothelium
2.1. Foundation of the DSCR-1 from endothelial cell research
The endothelium is highly malleable cell layer, constantly responding to changes within the extracellular environment and responding in ways that are usually beneficial, but at times harmful to the organism. Several mediators, including growth factors (e.g. vascular endothelial growth factor, VEGF), inflammatory cytokines (e.g. tumor necrosis factor-α, TNF-α), and thrombosis mediator (e.g. thrombin), activate gene transcription in endothelial cells, resulting in changes in hemostatic balance, increased leukocyte adhesion, loss of barrier function, increased permeability, migration, proliferation and successive angiogenesis (Minami and Aird, 2005). The tight control of these processes is essential for homeostasis - endothelial cell activation, if excessive, sustained or spatially and temporally misplaced, may result in vasculopathic disease. Indeed, different extra-cellular mediators engage the endothelium in ways that differ from one signal to the next. A major important point is to survey the temporal and spatial dynamics of endothelial cell activation. Using DNA microarrays, I carried out a global survey of mRNA in human umbilical vein endothelial cells (HUVEC) treated in the VEGF, thrombin, or TNF-α. Clustering analyses of the data revealed a far closer relationship between VEGF and thrombin, than between other pairings (Fig. 1A). Of the various transcripts that were responsive both to VEGF and thrombin, DSCR-1 was the most highly induced at the earliest time point (1 h). Compared with VEGF and thrombin, TNF-α treatment of HUVEC resulted in far less induction of DSCR-1 (3.2-fold at 1 h) (not shown). The rest of the VEGF-mediated induced gene was early growth response (Egr)-3, nerve growth factor inducible (NGFI)-Bβ, cyclooxigenase (COX)-2, and ADAMTS-1 (Fig. 1B).
2.2. Molecular information of the DSCR-1
The DSCR-1 gene consists of 7 exons, of which exons 1-4 can be alternatively spliced, resulting in a number of different mRNA isoforms, each of which exhibit different expression patterns. In adult, there are two major isoforms, DSCR-1 long variant (DSCR-1L) and DSCR-1 short variant (DSCR-1s), expressed in organs (Fuentes et al., 1997). DSCR-1L, encoded by exons 1, 5, 6, and 7, is highly expressed in brain. Exon 1 was originally thought to encode a 29 amino acid region, but later studies revealed a start site further upstream, resulting in a larger 84 amino acid region (Genesca et al., 2003). In contrast, DSCR-1s is encoded by exons 4-7 and is under the control of a different promoter located in intron 3 (intergenic promoter) (Fig. 1C). Each promoter contains different regulatory transcriptional subunits. For example, DSCR-1s is mainly regulated by the calcineurin-NFAT pathway, which is highly induced by angiogenic and inflammatory stimuli in endothelial cells (Minami et al., 2004; Minami et al., 2006).
While, the DSCR-1L isoform is under the control of a Notch and Hes-1-dependent pathway (Mammucari et al., 2005) or TEF-1 dependent pathway (Liu et al., 2008). DSCR-1s inhibits calcineurin phosphatase activity, and the C-terminal 57 residues are sufficient for this activity. DSCR-1s strongly inhibits the calcineurin mediated NFAT signaling via two ways; its ability to disrupt binding of calcineurin to NFAT, and to disrupt calcineurin enzymatic activities (Fig. 1D).
2.3. DSCR-1 expression in cultured cells
VEGF or thrombin induces the DSCR-1s expression in endothelial cells, through the coordinate binding of NFATc and GATA to closely positioned NFAT and GATA motifs in the intergenic promoter (Minami et al., 2004). VEGF/thrombin induces NFATc nuclear localization, and overexpression of the nuclear NFATc1 greatly induces the targeted DSCR-1s expression (Hesser et al., 2004; Minami et al., 2004; Minami et al., 2006). In addition, endothelial cells from the Down syndrome model mice (Ts65Dn) increased DSCR-1 mRNA by 1.7-2.0 fold (Baek et al., 2009). NFATc is an important factor for regulating the vertebrate development (Graef et al., 2001). In endothelial cells, NFATc1, c2, and c3 are expressed (Minami et al., 2009). To survey the NFATc1 binding in genome-widely, we carried out the chromatin immunoprecipitation using the antibody against NFATc1 following the comprehensive sequencing (ChIP-seq) in endothelial cells. We found totally 10,938 regions (
In contrast, erythroid lineage cells isolated from leukemia; K562 indicated the H3K4me3 positive signals within the proximal DSCR-1L promoter region, but not proximal DSCR-1s promoter region (Fig. 2). DSCR-1L reported the proceeding the pathological function in neurons (Cook et al., 2005). Moreover, Down syndrome patients have an increased risk of leukemia (Lott, 1982). Collectively, DSCR-1s and DSCR-1L obtained separate transcriptional machinery. VEGF mediated NFATc activation selectively transactivates the DSCR-1s via the profound binding within the promoter.
2.4. Characterization of the NFAT dependent genes overexpressed in Down syndrome
Besides DSCR-1, other genes encoded in chromosome 21 also reported as a candidate for pathogenesis on the Down syndrome. By using the combination of several NFATc knockout mice, dysfunction of NFAT was shown as a key point for the onset of Down syndrome (Arron et al., 2006). Around 1.5-fold increasing of both DSCR-1 and DYRK1A caused complete NFAT dysfunction. Thus, we test whether many Down syndrome genes obtain the NFATc1 binding on the each proximal promoter, by using the whole-genome NFATc1 ChIP-seq data (Table 1). Interestingly, DYRK1A obtained positive NFATc1 binding. VEGF inducible ADAMTS-1 (see Fig. 1B) also showed the NFATc1 positive binding. Ets family, Ets2, ERG, and GABPα, were highly expressed in endothelial cells, which was shown the regulation for the endothelial cell-specific expression or-essential function. All of them have a possibility as a NFATc1 direct target downstream gene.
|
|
|
|
|
|
||||
ADAMTS1 | A disintegrin and metalloproteinase with thrombospondin motifs, type 1 | + | 5’UTR | |
ERG | v-ets erythroblastosis virus E26 oncogene homolog | + | +5367 & 5th intron | |
Ets2 | v-ets erythroblastosis virus E26 oncogene homolog 2 | + | +55 | |
JAM2 | junction adhesion molecule 2 | - | ||
PTTG1IP | pituitary tumor-transforming 1 interacting protein | + | +883 | |
|
||||
Energy and reactive oxygen species metabolism | ||||
BTG3 | B-cell translocation gene 3 | +, weak | 1st intron | |
MRPL39 | mitochondrial ribosomal protein L39 | +, weak | 1st exon | |
ATP5J | ATP synthase, H+ transporting, subunit F6 | +, weak | 1st exon | |
GABPA | GA binding protein transcription factor, alpha | +, weak | +80 | |
BACH1 | BTB and CNC homology 1, basic leucine zipper transcription factor 1 | +, weak | 1st intron | |
SOD1 | superoxide dismutase 1 | + | 5’UTR | |
CRYZL1 | crystallin, zeta-like 1 | +, weak | 1st Exon & +520 | |
ATP5O | ATP synthase, H+ transporting, O subunit | + | 5’UTR | |
MRPS6 | mitochondrial ribosomal protein S6 | +, weak | 5'-UTR | |
DSCR-1 | Down syndrome critical region gene 1 | + | Indicated in Fig. 2 | |
CBR1 | carbonyl reductase 1 | +, weak | 1st exon | |
CBR3 | carbonyl reductase 3 | + | 1st exon | |
SH3BGR | SH3 domain binding glutamic acid-rich protein | + | 5’UTR | |
NDUFV3 | NADH dehydrogenase flavoprotein 3, 10kDa | + | +139 | |
SNF1LK | salt-inducible kinase 1 | - | ||
C21orf2 | chromosome 21 open reading frame 2 | +, weak | +140 | |
Brain development, neuronal loss, and Alzheimer's type neuropathology | ||||
SIM2 | single-minded homolog 2 | - | ||
DYRK1A | dual-specificity tyrosine-phosphorylation regulated kinase 1A | + | +1680 | |
GART | phosphoribosylglycinamide formyltransferase | + | +513 & 5'-UTR | |
PCP4 | Purkinje cell protein 4 | - | ||
DSCAM | Down syndrome cell adhesion molecule | - | ||
GRIK1 | glutamate receptor, ionotropic, kainate 1 | - | ||
APP | amyloid beta (A4) precursor protein | +, weak | 1st intron | |
S100B | S100 Ca-binding protein B | - | ||
Folate methyl group metabolism | ||||
N6AMT1 | N-6 adenine-specific DNA methyltransferase 1 | +, weak | 1st exon | |
CBS | cystathionine-beta-synthase | - | ||
DNMT3L | DNA methyltransferase 3-like | - | ||
SLC19A1 | Solute carrier family 19 , member 1 | - | ||
FTCD | formiminotransferase cyclodeaminase | - | ||
HRMT1L1 (PRMT2) | Protein arginine methyltransferase 2 | + | 5’UTR |
2.5. DSCR-1 expression in organ
Increased DSCR-1 expression was observed in human fetal Down syndrome kidney versus age-matched control kidney (Fig. 3A). To determine whether the DSCR-1s promoter region directed inducible expression
Subsequently, to determine whether the DSCR-1s promoter confers response to inflammatory or angiogenic stimuli
Real-time PCR analysis was used to quantify changes in transgene expression. Under basal conditions,
2.6. DSCR-1 expression in tumor
Solid tumors produce a variety of pro-angiogenic molecules and inflammatory cytokines, which have important paracrine effects on surrounding endothelial cells. To investigated whether the DSCR-1s transgene is activated in tumor blood vessels, B16-F1 melanoma and Lewis lung carcinoma (LLC) cells were implanted subcutaneously into the flank of DSCR-1s-
3. Biological function of DSCR-1
3.1. DSCR-1 inhibits nuclear localization of NFATc
Adenovirus mediated overexpression of DSCR-1, but not control, inhibited VEGF mediated nuclear localization of NFATc1 and NFATc2 (Minami et al., 2004). DYRK1A is another potential NFAT regulators, which encodes a nuclear serine/threonine kinase that primes substrates for phsphorylation by Glycogen synthase kinase (GSK) 3(Gwack et al., 2006). GSK3 phosphorylates NFATc proteins in the nucleus, resulting in their inactivation and export (Beals et al., 1997). DYRK1A is expressed at elevated levels in some human Down syndrome fetal tissues (Arron et al., 2006). In neuronal cells, DYRK1A inhibits FGF8-mediated induction of NFAT activity. Moreover, it has been shown that a 1.5-fold increase in the dosage of DSCR-1 and DYRK1A, both of which lie within the critical region of human chromosome 21, cooperatively destabilized the calcineurin-NFAT regulatory circuit (Fig. 5), leading to many of the features of Down syndrome (Arron et al., 2006).
3.2. DSCR-1s auto-inhibits the VEGF-mediated vascular activation
VEGF is an endothelial cell specific mitogen, and chemotactic agent, which is involved in wound repair, angiogenesis of ischemic tissue, tumor growth, microvascular permeability, hemostasis and endothelial cell survival (Isner and Losordo, 1999). DSCR-1 overexpression inhibits VEGF-mediated vessel growth, and monocyte cell adhesion (Minami et al., 2006). DSCR-1 overexpression did not lead in increased apoptosis (Minami et al., 2009). Taken together, these findings suggest that DSCR-1 constitutive expression lead the endothelial cells to quiescent status form the VEGF-mediated activated status.
3.3. DSCR-1s attenuates septic inflammation
As shown above, DSCR-1s attenuates VEGF-mediated activation of cultured endothelial cells. These data led us to hypothesize that VEGF- and LPS-inducible expression of DSCR-1s in mice may serve as a negative feedback inhibitor of endothelial activation
To assay for endothelial activation, real-time PCR was performed to measure mRNA expression of E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in tissues from mice 6 h following injection of PBS (control) or LPS. Compared with wild-type littermate controls, LPS-treated DSCR-1-/- mice demonstrated super-induction of E-selectin in the heart and lung, ICAM-1 in heart, and VCAM-1 in lung. In contrast, LPS-mediated induction of cell adhesion molecules was attenuated by overexpression of DSCR-1s (data not shown).
I have recently shown that hyper-activation of the VEGF-calcineurin-NFAT pathway triggers apoptosis in DSCR-1-deficient tumor endothelial cells (Minami et al., 2009). Given that DSCR-1-/- mice have elevated circulating levels of VEGF level (see Fig. 6A), I hypothesized that endotoxemia may result in increased endothelial cell apoptosis in DSCR-1-/- mice. To test this hypothesis, TUNEL assay was carried out in tissue sections from the heart and lung of LPS-treated DSCR-1-null mice and their wild-type littermates. Endotoxemic wild-type mice demonstrated a small number of TUNEL-positive endothelial cells in the heart, and even fewer in the lung. However, in DSCR-1-/- mice, LPS administration resulted in a significant increase in the number of TUNEL-positive cells in both organs (Minami et al., 2009).
Finally in survival studies, LPS-treated DSCR-1-/- mice demonstrated markedly increased mortality compared with endotoxemic wild-type littermates (Fig.6B,
3.4. DSCR-1s attenuates tumor progression
Having established an inhibitory role for DSCR-1 on inflammation
3.5. DSCR-1s attenuates tumor metastasis
During the study for the DSCR-1s promoter activity
3.6. Lacking DSCR-1 results with controversy
While our findings reported here lend further evidence toward DSCR-1s as a negative regulator of NFAT-calcineurin signaling
Interestingly in the endothelial cells, Sandra.et.al., indicated that DSCR-1-/- mice demonstrated reduced blood vessel formation in Matrigel, cornel micropocket, and tumor xenograft assays (Ryeom et al., 2008). DSCR-1-/- endothelial cells displayed hyper-activation of the calcineurin/NFAT pathway and increased sensitivity to VEGF signaling. However, rather than inducing cell proliferation, VEGF-mediated activation of calcineurin/NFAT in DSCR-1-/- endothelial cells ‘re-routed’ downstream signaling, resulting in increased apoptosis, which thus explains the paradoxical reduction in neovascularization. Collectively, considered with the data from DSCR-1 stable expression and null mutation, calcineurin/NFAT activity and DSCR-1s expression level was tightly regulated, resulting the balance would define the endothelial cell growth, viability and tumor angiogenesis (Fig. 8). Future animal studies of DSCR-1 function should be performed by endothelial cell-specific knockout mice targeting either DSCR-1s or DSCR-1L separately.
4. Conclusion
DSCR-1 was identified by the study with vascular activation. DSCR-1 was highest induced by VEGF treatment in primary cultured endothelial cells. Previously, DSCR-1 was simply termed by the localization of the human chromosome 21. However, DSCR-1 indeed highly expressed in Down syndrome individuals, and clearly upregulated with NFAT activation in cells. Moreover, combined with same 21st chromosome encoded protein; ‘DYRK1A’, DSCR-1 strongly feedback attenuated the NFAT activation, resulting the pathogenesis of Down syndrome. I show here that DSCR-1s is highly expressed during embryonic vascular development, and then largely downregulated in adult, yet was highly activated predominantly in endothelium in response to the administration of VEGF or LPS. Stimulated DSCR-1s worked in the auto-inhibition of endothelial cell activation and inflammation. It has still unanswered problems with understanding the phenotypes from DSCR-1 lacking condition, and pathogenesis from DSCR-1L overexpression in neuron. However, based on this knowledge, I believe that DSCR-1s stable expression or the way of DSCR-1s stabilization may lend itself to therapeutic manipulation in vasculopathic disease states, including tumor angiogenesis, metastasis, and inflammation.
Acknowledgments
This study was supported by the Leading-edge Research Promotion fund from Japan Society for the Promotion of Science, and in part supported by a Grant-in-Aid for Scientific Research on Innovative Areas from ministry of Education, Culture, Sports, Science, and Technology in Japan and in part by Mochida Memorial and Sankyo Science Foundation in Japan. I am grateful to Dr. Junichi Suehiro (The University of Tokyo, Japan) for providing the ChIP-seq information with NFATc1.
References
- 1.
Abbasi S. Lee J. D. Su B. Chen X. Alcon J. L. Yang J. Kellems R. E. Xia Y. 2006 Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1. The Journal of biological chemistry281 7717 7726 - 2.
Arron J. R. Winslow M. M. Polleri A. Chang C. P. Wu H. Gao X. Neilson J. R. Chen L. Heit J. J. Kim S. K. et al. 2006 NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21. Nature441 595 600 - 3.
Baek K. H. Zaslavsky A. Lynch R. C. Britt C. Okada Y. Siarey R. J. Lensch M. W. Park I. H. Yoon S. S. Minami T. et al. 2009 Down’s syndrome suppression of tumour growth and the role of the calcineurin inhibitor DSCR1. Nature459 1126 1130 - 4.
Beals C. R. Sheridan C. M. Turck C. W. Gardner P. Crabtree G. R. 1997 Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science275 1930 1934 - 5.
Biancone L. Araki M. Araki K. Vassalli P. Stamenkovic I. 1996 Redirection of tumor metastasis by expression of E-selectin in vivo. The Journal of experimental medicine183 581 587 - 6.
Cook C. N. Hejna M. J. Magnuson D. J. Lee J. M. 2005 Expression of calcipressin1, an inhibitor of the phosphatase calcineurin, is altered with aging and Alzheimer’s disease. J Alzheimers Dis8 63 73 - 7.
Cvetkovic B. Yang B. Williamson R. A. Sigmund C. D. 2000 Appropriate tissue- and cell-specific expression of a single copy human angiotensinogen transgene specifically targeted upstream of the HPRT locus by homologous recombination. The Journal of biological chemistry275 1073 1078 - 8.
Fuentes J. J. Pritchard M. A. Estivill X. 1997 Genomic organization, alternative splicing, and expression patterns of the DSCR1 (Down syndrome candidate region 1) gene. Genomics44 358 361 - 9.
Fukuda M. N. Ohyama C. Lowitz K. Matsuo O. Pasqualini R. Ruoslahti E. Fukuda M. 2000 A peptide mimic of E-selectin ligand inhibits sialyl Lewis X-dependent lung colonization of tumor cells. Cancer research60 450 456 - 10.
Futakuchi M. Ogawa K. Tamano S. Takahashi S. Shirai T. 2004 Suppression of metastasis by nuclear factor kappaB inhibitors in an in vivo lung metastasis model of chemically induced hepatocellular carcinoma. Cancer science95 18 24 - 11.
Genesca L. Aubareda A. Fuentes J. J. Estivill X. De La Luna S. Perez-Riba M. 2003 Phosphorylation of calcipressin 1 increases its ability to inhibit calcineurin and decreases calcipressin half-life. The Biochemical journal374 567 575 - 12.
Graef I. A. Chen F. Crabtree G. R. 2001 NFAT signaling in vertebrate development. Curr Opin Genet Dev11 505 512 - 13.
Gwack Y. Sharma S. Nardone J. Tanasa B. Iuga A. Srikanth S. Okamura H. Bolton D. Feske S. Hogan P. G. et al. 2006 A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. Nature441 646 650 - 14.
Hesser B. A. Liang X. H. Camenisch G. Yang S. Lewin D. A. Scheller R. Ferrara N. Gerber H. P. 2004 Down syndrome critical region protein 1 (DSCR1), a novel VEGF target gene that regulates expression of inflammatory markers on activated endothelial cells. Blood104 149 158 - 15.
Isner J. M. Losordo D. W. 1999 Therapeutic angiogenesis for heart failure. Nat Med5 491 492 - 16.
Liu X. Zhao D. Qin L. Li J. Zeng H. 2008 Transcription enhancer factor 3 (TEF3) mediates the expression of Down syndrome candidate region 1 isoform 1 (DSCR1-1L) in endothelial cells. The Journal of biological chemistry283 34159 34167 - 17.
Lott I. T. 1982 Down’s syndrome, aging, and Alzheimer’s disease: a clinical review. Annals of the New York Academy of Sciences396 15 27 - 18.
Mammucari C. Tommasi di Vignano. A. Sharov A. A. Neilson J. Havrda M. C. Roop D. R. Botchkarev V. A. Crabtree G. R. Dotto G. P. 2005 Integration of Notch 1 and calcineurin/NFAT signaling pathways in keratinocyte growth and differentiation control. Developmental cell8 665 676 - 19.
Minami T. Aird W. C. 2005 Endothelial cell gene regulation. Trends in cardiovascular medicine15 174 184 - 20.
Minami T. Donovan D. J. Tsai J. C. Rosenberg R. D. Aird W. C. 2002 Differential regulation of the von Willebrand factor and Flt-1 promoters in the endothelium of hypoxanthine phosphoribosyltransferase-targeted mice. Blood100 4019 4025 - 21.
Minami T. Horiuchi K. Miura M. Abid M. R. Takabe W. Noguchi N. Kohro T. Ge X. Aburatani H. Hamakubo T. et al. 2004 Vascular endothelial growth factor- and thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis. The Journal of biological chemistry279 50537 50554 - 22.
Minami T. Kuivenhoven J. A. Evans V. Kodama T. Rosenberg R. D. Aird W. C. 2003 Ets motifs are necessary for endothelial cell-specific expression of a 723-bp Tie-2 promoter/enhancer in Hprt targeted transgenic mice. Arteriosclerosis, thrombosis, and vascular biology23 2041 2047 - 23.
Minami T. Miura M. Aird W. C. Kodama T. 2006 Thrombin-induced autoinhibitory factor, Down syndrome critical region-1, attenuates NFAT-dependent vascular cell adhesion molecule-1 expression and inflammation in the endothelium. The Journal of biological chemistry281 20503 20520 - 24.
Minami T. Yano K. Miura M. Kobayashi M. Suehiro J. Reid P. C. Hamakubo T. Ryeom S. Aird W. C. Kodama T. 2009 The Down syndrome critical region gene 1 short variant promoters direct vascular bed-specific gene expression during inflammation in mice. J Clin Invest119 2257 2270 - 25.
Okada Y. Yano K. Jin E. Funahashi N. Kitayama M. Doi T. Spokes K. Beeler D. L. Shih S. C. Okada H. et al. 2007 A three-kilobase fragment of the human Robo4 promoter directs cell type-specific expression in endothelium. Circulation research100 1712 1722 - 26.
Qin L. Zhao D. Liu X. Nagy J. A. Hoang M. V. Brown L. F. Dvorak H. F. Zeng H. 2006 Down syndrome candidate region 1 isoform 1 mediates angiogenesis through the calcineurin-NFAT pathway. Mol Cancer Res4 811 820 - 27.
Reynolds L. E. Watson A. R. Baker M. Jones T. A. D’Amico G. Robinson S. D. Joffre C. Garrido-Urbani S. Rodriguez-Manzaneque J. C. Martino-Echarri E. et al. 465 813 817 - 28.
Roizen N. J. Patterson D. 2003 Down’s syndrome. Lancet361 1281 1289 - 29.
Ryan M. J. Sigmund C. D. 2003 HPRT targeting: "Ets" a powerful tool for investigating endothelial-cell specific gene expression. Arteriosclerosis, thrombosis, and vascular biology23 1960 1962 - 30.
Ryeom S. Baek K. H. Rioth M. J. Lynch R. C. Zaslavsky A. Birsner A. Yoon S. S. Mc Keon F. 2008 Targeted deletion of the calcineurin inhibitor DSCR1 suppresses tumor growth. Cancer Cell13 420 431 - 31.
Salvolini E. Orciani M. Lucarini G. Vignini A. Tranquilli A. L. Di Primio R. V. E. G. F. nitric oxide. synthase immunoexpression. in Down’s. syndrome amniotic. fluid stem. cells 41 23 29 - 32.
Sanna B. Brandt E. B. Kaiser R. A. Pfluger P. Witt S. A. Kimball T. R. van Rooij E. De Windt L. J. Rothenberg M. E. Tschop M. H. et al. 2006 Modulatory calcineurin-interacting proteins 1 and 2 function as calcineurin facilitators in vivo. Proceedings of the National Academy of Sciences of the United States of America103 7327 7332 - 33.
Shin S. Y. Choo S. M. Kim D. Baek S. J. Wolkenhauer O. Cho K. H. 2006 Switching feedback mechanisms realize the dual role of MCIP in the regulation of calcineurin activity. FEBS letters580 5965 5973 - 34.
Vega R. B. Rothermel B. A. Weinheimer C. J. Kovacs A. Naseem R. H. Bassel-Duby R. Williams R. S. Olson E. N. 2003 Dual roles of modulatory calcineurin-interacting protein 1 in cardiac hypertrophy. Proceedings of the National Academy of Sciences of the United States of America100 669 674 - 35.
Yano K. Liaw P. C. Mullington J. M. Shih S. C. Okada H. Bodyak N. Kang P. M. Toltl L. Belikoff B. Buras J. et al. 2006 Vascular endothelial growth factor is an important determinant of sepsis morbidity and mortality. The Journal of experimental medicine203 1447 1458 - 36.
Yano K. Okada Y. Beldi G. Shih S. C. Bodyak N. Okada H. Kang P. M. Luscinskas W. Robson S. C. Carmeliet P. et al. 2008 Elevated levels of placental growth factor represent an adaptive host response in sepsis. The Journal of experimental medicine205 2623 2631