PRL-3 mediates the protein maturation of ULBP2 by regulating the tyrosine phosphorylation of HSP60.

Many malignant cells release the NKG2D ligand ULBP2 from their cell surface to evade immunosurveillance by NK cells and CD8 T cells. Although the shedding mechanism remains unclear, various inhibitors of matrix metalloproteinases have been shown to efficiently block the release of soluble ULBP2. The clinical use of these inhibitors, however, is limited because of adverse side effects. Using high-throughput screening technique, we identified a specific inhibitor of phosphatase of regenerating liver 3 (PRL-3) that could reduce the level of soluble ULBP2 in the culture supernatant of various cancer cell lines. Inhibition or gene knockdown of PRL-3 did not reduce ULBP2 shedding, but rather suppressed posttranslational maturation of ULBP2, resulting in intracellular retention of immature ULBP2. We then found that ULBP2 was constitutively associated with heat shock protein HSP60. Complete maturation of ULBP2 required tyrosine phosphorylation of HSP60 which was mediated by PRL-3.

TOX2 regulates human natural killer cell development by controlling T-BET expression.

Thymocyte selection-associated high mobility group box protein family member 2 (TOX2) is a transcription factor belonging to the TOX family that shares a highly conserved high mobility group DNA-binding domain with the other TOX members. Although TOX1 has been shown to be an essential regulator of T-cell and natural killer (NK) cell differentiation in mice, little is known about the roles of the other TOX family members in lymphocyte development, particularly in humans. In this study, we found that TOX2 was preferentially expressed in mature human NK cells (mNK) and was upregulated during in vitro differentiation of NK cells from human umbilical cord blood (UCB)-derived CD34(+) cells. Gene silencing of TOX2 intrinsically hindered the transition between early developmental stages of NK cells, whereas overexpression of TOX2 enhanced the development of mNK cells from UCB CD34(+) cells. We subsequently found that TOX2 was independent of ETS-1 but could directly upregulate the transcription of TBX21 (encoding T-BET). Overexpression of T-BET rescued the TOX2 knockdown phenotypes. Given the essential function of T-BET in NK cell differentiation, TOX2 therefore plays a crucial role in controlling normal NK cell development by acting upstream of TBX21 transcriptional regulation.

CLASPing microtubules at the trans-Golgi network.

Microtubule (MT) arrays can be formed either from centrosomes or from noncentrosomal locations. In this issue of Developmental Cell, Efimov and colleagues report a role of CLASPs, the MT plus end-binding proteins, in MT formation from Golgi, implicating Golgi-originated MT arrays in efficient cell migration (Efimov et al., 2007).

Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of arginine 245.

Killer immunoglobulin-like receptors (KIRs) play an essential role in the regulation of natural killer cell functions. KIR genes are highly polymorphic in nature, showing both haplotypic and allelic variations among people. We demonstrated in both in vitro and in vivo models a significant heterogeneity in function among different KIR2DL1 alleles, including their ability to inhibit YT-Indy cells from degranulation, interferon gamma production, and cytotoxicity against target cells expressing the HLA-Cw6 ligand. Subsequent experiments showed that the molecular determinant was an arginine residue at position 245 (R245) in its transmembrane domain that mechanistically affects both the efficiency of inhibitory signaling and durability of surface expression. Specifically, in comparison with R245-negative alleles, KIR2DL1 that included R245 recruited more Src-homology-2 domain-containing protein tyrosine phosphatase 2 and beta-arrestin 2, showed higher inhibition of lipid raft polarization at immune synapse, and had less down-regulation of cell-surface expression upon interaction with its ligand. Thus, our findings provide novel insights into the molecular determinant of KIR2DL1 and conceivably a fundamental understanding of KIR2DL1 allelic polymorphism in human disease susceptibility, transplant outcome, and donor selection.

A role for borg5 during trophectoderm differentiation.

Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.

Serial analysis of gene expression in adrenocortical hyperplasia caused by a germline PRKAR1A mutation.

CONTEXT: Adrenocortical tumors have been studied at the molecular genetic and cytogenetic levels, but the gene expression profiles of normal and tumor adrenal tissue have not been extensively investigated. OBJECTIVE: The objective of this study was to obtain information about transcriptome differences in hyperplastic adrenal cells. DESIGN AND PATIENTS: We performed serial analysis of gene expression (SAGE) on control adrenal tissue and primary pigmented nodular adrenocortical disease (PPNAD) tissue from two adolescent female patients. MAIN OUTCOME MEASURE: The main outcome measure was to provide quantitative datasets of the vast majority of the transcripts implicated in normal and pathogenic adrenal functioning. RESULTS: The libraries of 28,705 and 31,278 tags represented 14,846 and 16,698 unique mRNAs from the control and PPNAD tissue, respectively. A total of 842 tags from the two libraries did not match any known sequences. We found 127 tags, including 70 no-match tags, to be expressed almost exclusively in control and/or PPNAD adrenals and to be absent or very rare in other human tissues. Examples of well-characterized genes expressed at significantly higher levels in PPNAD included steroidogenic acute regulator, chromogranin A, and those coding for the steroidogenic enzymes P450 cytochromes CYP17A1 and CYP21A2. Pathway analysis revealed Wnt signaling as the most up-regulated in PPNAD. These data were confirmed for selected genes by quantitative RT-PCR and/or immunohistochemistry. CONCLUSIONS: This study was the first of its kind for adrenal tissue and provides important information about the adrenal transcriptome and aberrant signaling in an inherited form of adrenocortical hyperplasia.

Structural characterization and expression studies of Dby and its homologs in the mouse.

In spite of recent evidence showing the importance of DBY (DEAD-box RNA helicase Y) in spermatogenesis in human, the biologic role of its homolog Dby (also known as Ddx3y) in the mouse is less clear. The present study aims at characterizing the molecular structure of Dby and comparing its expression with its X- and autosome-linked homologs in embryonic gonads and developing germ cells in mice. Molecular cloning by rapid amplification of 3'-cDNA ends showed that the Dby gene in the mouse gives rise to 2 transcripts that differ only in the length of the 3'-untranslated region as a consequence of the use of alternative polyadenylation signals. Measurement by quantitative real-time polymerase chain reaction showed that both transcripts were ubiquitously expressed and were present in male germ cells and Sertoli cells. They were more abundant in type A spermatogonia compared with pachytene spermatocytes and round spermatids. Expression of Dby in the embryonic gonad increased from day 10.5 and reached a peak at day 17.5. The expression level of Dby decreased after birth and remained low in adult male gonads. Although the level of expression of Dby was much lower than its X chromosome homolog, Ddx3 (also known as Ddx3x) in all samples examined, the pattern of expression of the 2 genes was comparable. In contrast, their autosomal homolog, D1Pas1(also known as PL10), was predominantly expressed in pachytene spermatocytes and round spermatids. This result is in accord with meiotic sex chromosome inactivation in that Dby and Ddx are replaced in pachytene spermatocytes by their autosomal retroposon. These observations indicate that unlike DBY in humans, the role of Dby in spermatogenesis is less obvious in the mouse and its biologic activity may be replaced by that of Ddx3 and D1Pas1.

PCR-cloning and gene expression studies in common carp (Cyprinus carpio) insulin-like growth factor-II.

Insulin-like growth factor-II (IGF-II) is a member of a growth factor family related to fetal growth in mammals but its physiological role has not been clearly identified in fish. In teleosts, the basic mechanism of the growth hormone (GH)-IGF axis is known to be operative but in a different manner. For instance, IGF-I exhibits GH dependence whereas for IGF-II, its GH dependence varies in different fish species. In this study, we used polymerase chain reaction (PCR) to obtain a common carp IGF-II (ccIGF-II) cDNA fragment and methods of rapid amplification of cDNA ends (RACEs) to obtain a full-length ccIGF-II sequence. The ccIGF-II encodes for a predicted amino acid sequence showing identities of 70.6%, 68.7%, 63.4% and 35% in comparison with salmon, barramundi, tilapia and human IGF-II, respectively. The nucleotide identity between the open reading frame (ORF) of the ccIGF-II and ccIGF-I cDNA sequence is only 36.2%. Distribution of ccIGF-II mRNA levels in common carp tissues was also studied; ccIGF-II expressed in hepatopancreas, heart, and many other tissues in adult carps are similar to the levels of ccIGF-I except in gills and testis. ccIGF-II levels were significantly higher than that of ccIGF-I in most juvenile tissues except in hepatopancreas, where ccIGF-I was higher (threefold) than that of ccIGF-II. The levels of ccIGF-I were also higher than ccIGF-II in carp larvae, from pre-hatched stage to day 30 post-hatching. Injection of porcine GH (pGH) increased the IGF-I and IGF-II mRNA levels in the hepatopancreas and brain of juvenile carps. However, hepatic IGF-I mRNA levels were induced more than IGF-II by pGH, whereas ccIGF-II levels gave a higher response than IGF-I in the brain in response to GH induction.

Genome-wide single-nucleotide polymorphism analysis revealed SUFU suppression of acute graft-versus-host disease through downregulation of HLA-DR expression in recipient dendritic cells.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). To identify recipient risk factors, a genome-wide study was performed including 481,820 single-nucleotide polymorphisms (SNPs). Two GVHD susceptibility loci (rs17114803 and rs17114808) within the SUFU gene were identified in the discovery cohort (p = 2.85 × 10(-5)). The incidence of acute GVHD among patients homozygous for CC at SUFU rs17114808 was 69%, which was significantly higher than the 8% rate observed in CT heterozygous patients (p = 0.0002). In an independent validation cohort of 100 patients, 50% of the patients with the CC genotype developed GVHD compared to 8% of the patients with either CT or TT genotype (p = 0.01). In comparison to CC dendritic cells, those from CT expressed higher levels of SUFU mRNA and protein, had lower levels of surface HLA-DR, and induced less allogeneic mixed leukocyte response (MLR). Ectopic expression of SUFU in THP-1 derived DCs reduced HLA-DR expression and suppressed MLR, whereas silencing of SUFU enhanced HLA-DR expression and increased MLR. Thus our findings provide novel evidence that recipient SUFU germline polymorphism is associated with acute GVHD and is a novel molecular target for GVHD prevention and treatment.

Common carp insulin-like growth factor-I gene: complete nucleotide sequence and functional characterization of the 5'-flanking region.

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of fish. To understand the molecular mechanism which controls the transcription of the IGF-I gene in common carp, we have cloned and completely sequenced the IGF-I gene and the 5'-flanking region from a local tropical fish, the common carp (Cyprinus carpio), and characterized its promoter activity by transfection into human embryonic kidney (293GHR) cells which express the human growth hormone (GH) receptor. The common carp gene is the smallest IGF-I gene known so far, spanning approximately 13 kb, and is consisted of five exons and four introns. The sequence of the gene is consistent with the single type of IGF-I cDNA that we have isolated previously from a common carp liver cDNA library. The expression pattern of IGF-I is similar between juvenile carp and adult carp. While liver was found to be the major site of IGF-I gene expression in common carp, the expression levels in other tissues are relatively low. Like many other IGF-I gene promoters, there are no apparent TATA box and CCAAT box upstream of the transcription initiation site. However, sequence analysis of the common carp IGF-I promoter region identified several consensus liver-enriched transcription factor binding sites, including HNF-1alpha, HNF-3beta, C/EBP, and one STAT5. We have analyzed the promoter activity of the 5'-flanking region of the common carp IGF-I gene by performing luciferase reporter assays in transfected 293GHR cells. Addition of human GH to the transfected cells led to an increased expression of the reporter gene, indicating that the cloned genomic fragment possessed promoter activity. This was confirmed by the lack of promoter activity of a construct in which the putative promoter was cloned in a reverse orientation upstream of the reporter gene. The liver-specific transcription factor, hepatic nuclear factor (HNF)-1alpha, was also found to be involved in the regulation of the common carp IGF-I transcription. Transfection results from a set of deletion mutants helped to map the locations of the responsive elements on the promoter responsible for the GH effect and for the interaction with HNF-1alpha. These observations provide information for delineating the transcriptional regulation of IGF-I gene expression in common carp.

Borg5 is required for angiogenesis by regulating persistent directional migration of the cardiac microvascular endothelial cells.

The microvasculature is important for vertebrate organ development and homeostasis. However, the molecular mechanism of microvascular angiogenesis remains incompletely understood. Through studying Borg5 (Binder of the Rho GTPase 5), which belongs to a family of poorly understood effector proteins of the Cdc42 GTPase, we uncover a role for Borg5 in microvascular angiogenesis. Deletion of Borg5 in mice results in defects in retinal and cardiac microvasculature as well as heart development. Borg5 promotes angiogenesis by regulating persistent directional migration of the endothelial cells (ECs). In primary mouse cardiac ECs (MCECs), Borg5 associates with septins in the perinuclear region and colocalizes with actomyosin fibers. Both Borg5 deletion and septin 7 knockdown lead to a disruption of the perinuclear actomyosin and persistent directional migration. Our findings suggest that Borg5 and septin cytoskeleton spatially control actomyosin activity to ensure persistent directional migration of MCECs and efficient microvascular angiogenesis. Our studies reported here should offer a new avenue to further investigate the functions of Borg5, septin, and actomyosin in the microvasculature in the context of development and disease.

Modulation of NKG2D ligand expression and metastasis in tumors by spironolactone via RXRγ activation.

Tumor metastasis and lack of NKG2D ligand (NKG2DL) expression are associated with poor prognosis in patients with colon cancer. Here, we found that spironolactone (SPIR), an FDA-approved diuretic drug with a long-term safety profile, can up-regulate NKG2DL expression in multiple colon cancer cell lines by activating the ATM-Chk2-mediated checkpoint pathway, which in turn enhances tumor elimination by natural killer cells. SPIR can also up-regulate the expression of metastasis-suppressor genes TIMP2 and TIMP3, thereby reducing tumor cell invasiveness. Although SPIR is an aldosterone antagonist, its antitumor effects are independent of the mineralocorticoid receptor pathway. By screening the human nuclear hormone receptor siRNA library, we identified retinoid X receptor γ (RXRγ) instead as being indispensable for the antitumor functions of SPIR. Collectively, our results strongly support the use of SPIR or other RXRγ agonists with minimal side effects for colon cancer prevention and therapy.

Ex vivo activation of CD56(+) immune cells that eradicate neuroblastoma.

Despite the use of intensive contemporary multimodal therapy, the overall survival of patients with high-risk neuroblastoma is still less than 50%. Therefore, immunotherapy without cross-resistance and overlapping toxicity has been proposed. In this study, we report the development of a novel strategy to specifically activate and expand human CD56(+) (NCAM1) natural killer (NK) immune cells from normal donors and patients with neuroblastoma. Enriched CD56(+) cells from peripheral blood were mixed with CD56(-) fraction at 1:1 ratio and cultured in the presence of OKT3, interleukin (IL)-2, and -15 for five days and then without OKT3 for 16 more days. The final products contained more than 90% CD56(+) cells and could kill neuroblastoma cells effectively that were originally highly resistant to nonprocessed NK cells. Mechanistically, cytolysis of neuroblastoma was mediated through natural cytotoxicity receptor (NCR), DNAX accessory molecule-1 (DNAM-1; CD226), perforin, and granzyme B. Successful clinical scale-up in a good manufacturing practices (GMP)-compliant bioreactor yielded effector cells that in a neuroblastoma xenograft model slowed tumor growth and extended survival without GVHD. Investigation of CD56(+) cells from patients with neuroblastoma revealed a similar postactivation phenotype and lytic activity. Our findings establish a novel and clinically expedient strategy to generate allogeneic or autologous CD56(+) cells that are highly cytotoxic against neuroblastoma with minimal risk of GVHD.

Using ES cells labeled with GFP for analyzing cell behavior during differentiation.

Mouse embryonic stem (ES) cell is a unique yet highly successful system to study stem cell maintenance and differentiation. In this protocol, we describe the generation of engineered mouse ES cells that stably express GFP alone or GFP fused with histone H2B, which allow the cells to be traced live or in fixed samples. This system can be used to study cell behavior changes as ES cells differentiate into different lineages.

Developmental staging of male murine embryonic gonad by SAGE analysis.

Despite the identification of key genes such as Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process is still poorly understood. To better understand the genetic regulation of gonadal development, we performed Serial Analysis of Gene Expression (SAGE) to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Utilizing an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Rather than providing a static descriptive analysis, we developed algorithms to cluster the SAGE data and assign SAGE tags to a corresponding chromosomal position; these data are displayed in chromosome graphic format. A prominent increase in global genomic activity from E10.5 to E17.5 was observed. Important chromosomal regions related to the developmental processes were identified and validated based on established mouse models with developmental disorders. These regions may represent markers for early diagnosis for disorders of male gonad development as well as potential treatment targets.

Chromosome alignment and segregation regulated by ubiquitination of survivin.

Proper chromosome segregation requires the attachment of sister kinetochores to microtubules from opposite spindle poles to form bi-oriented chromosomes on the metaphase spindle. The chromosome passenger complex containing Survivin and the kinase Aurora B regulates this process from the centromeres. We report that a de-ubiquitinating enzyme, hFAM, regulates chromosome alignment and segregation by controlling both the dynamic association of Survivin with centromeres and the proper targeting of Survivin and Aurora B to centromeres. Survivin is ubiquitinated in mitosis through both Lys(48) and Lys(63) ubiquitin linkages. Lys(63) de-ubiquitination mediated by hFAM is required for the dissociation of Survivin from centromeres, whereas Lys(63) ubiquitination mediated by the ubiquitin binding protein Ufd1 is required for the association of Survivin with centromeres. Thus, ubiquitinaton regulates dynamic protein-protein interactions and chromosome segregation independently of protein degradation.