Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions.

Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.

O-fucose modulates Notch-controlled blood lineage commitment.

Notch receptors are cell surface molecules essential for cell fate determination. Notch signaling is subject to tight regulation at multiple levels, including the posttranslational modification of Notch receptors by O-linked fucosylation, a reaction that is catalyzed by protein O-fucosyltransferase-1 (Pofut1). Our previous studies identified a myeloproliferative phenotype in mice conditionally deficient in cellular fucosylation that is attributable to a loss of Notch-dependent suppression of myelopoiesis. Here, we report that hematopoietic stem cells deficient in cellular fucosylation display decreased frequency and defective repopulating ability as well as decreased lymphoid but increased myeloid developmental potential. This phenotype may be attributed to suppressed Notch ligand binding and reduced downstream signaling of Notch activity in hematopoietic stem cells. Consistent with this finding, we further demonstrate that mouse embryonic stem cells deficient in Notch1 (Notch1(-/-)) or Pofut1 (Pofut1(-/-)) fail to generate T lymphocytes but differentiate into myeloid cells while coculturing with Notch ligand-expressing bone marrow stromal cells in vitro. Moreover, in vivo hematopoietic reconstitution of CD34(+) progenitor cells derived from either Notch1(-/-) or Pofut1(-/-) embryonic stem cells show enhanced granulopoiesis with depressed lymphoid lineage development. Together, these results indicate that Notch signaling maintains hematopoietic lineage homeostasis by promoting lymphoid development and suppressing overt myelopoiesis, in part through processes controlled by O-linked fucosylation of Notch receptors.

Notch-dependent control of myelopoiesis is regulated by fucosylation.

Cell-cell contact-dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX(-/-) mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors.

Fucose-deficient hematopoietic stem cells have decreased self-renewal and aberrant marrow niche occupancy.

BACKGROUND: Modification of Notch receptors by O-linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study was to assess the self-renewal and stem cell niche features of fucose-deficient HSCs. STUDY DESIGN AND METHODS: Homeostasis and maintenance of HSCs derived from FX(-/-) mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two-photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose-deficient and wild-type (WT) HSCs. RESULTS: Marrow progenitors from FX(-/-) mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose-deficient HSCs from FX(-/-) mice displayed decreased self-renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by two-photon intravital imaging, the fucose-deficient HSCs were found localized farther from the endosteum of the calvarium marrow than the WT HSCs. CONCLUSIONS: The current reported aberrant niche occupancy by HSCs from FX(-/-) mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O-fucosylation, suggests that fucosylation-modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.

[Effect of ginkgo biloba extract and dipyridamole on transcription and translation of inducible NO synthbase in rabbits after myocardial ischemia-reperfusion injury].

OBJECTIVE: To investigate the effects of Egb761, an extract of ginkgo biloba , and dipyridamole on inducible NO synthase (iNOS) in rabbits after myocardial ischemia-reperfusion injury. METHODS: After being established into ischemia-reperfusion injury model, 35 rabbits were divided randomly into 5 groups: Group A (the sham group), Group B (the model group), Group C (treated with dipyridamole 0.8 mg/kg), Group D (treated with Egb761, 40 mg/kg), and Group E (treated with Egb761 40 mg/kg combined with dipyridamole 0.8 mg/kg), all the medications were administered by intravenous injection 30 min after reperfusion. After administration, myocardial iNOS mRNA expression was detected by RT-PCR and western blot. RESULTS: Myocardial iNOS mRNA transcriptive expression in the 5 groups were A 0, B 157.11 +/- 17.73, C 202.6 +/- 21.84, D 356.13 +/- 24.18 and E 562.34 +/- 35.19 respectively, showing significant difference between the treated groups and group B (P <0.01). The translative expression of myocardial iNOS in the 5 groups were A 34.24 +/- 15.78, B 75.70 +/- 13.71, C 116.89 +/- 22.57, D 143.75 +/- 16.05 and E 195.09 +/- 22.25 respectively, showing significant difference between the treated groups and group B as well (P < 0.05, P < 0.01). CONCLUSION: Both Egb761 and dipyridamole could increase myocardial iNOS expression in transcriptive and translative levels in rabbits after myocardial ischemia-reperfusion injury, and the combined treatment of them shows a more significant effect.

NANOG changes in mouse kidneys with age.

NANOG is essential for mouse and human embryonic stem cell (ESC) pluripotency and selfrenewal. It is also expressed in several adult murine tissues as shown by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. However, human NANOG transcripts have been isolated from adult bone marrow (EST; GenBank accession no. BF893620). Here, we study the NANOG gene expression profile in isolated mouse renal papillary cells by Northern blot and RT-PCR. The whole RNA of mouse renal cells was obtained from fresh renal tissues, renal tissues infused by phosphate-buffered saline (PBS), and isolated renal papillary cells of mouse, respectively, as well as the renal papillary tissue from 18.5 days postcoitum (d.p.c.; fetal), 1-2-week-old (young), 1-8-month-old (adult), and 24-month-old (aging) mice. Our analysis shows that a very low expression level was detected in mouse renal tissues, and the renal papillary cells express more than other tissues as determined with Northern blot and RT-PCR. These data suggest that the kidney has its own cells expressing NANOG, and loss of NANOG expression occurs in an age-dependent manner in the kidney, either due to developmental factors or aging, particularly in renal papillary tissue.

Identification of over-expressed genes in human renal cell carcinoma by combining suppression subtractive hybridization and cDNA library array.

To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis, we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon membrane and the over-expressed genes were then screened by hybridizing the filter with radioactively labeled cDNA from RCC and matched normal kidney tissues. 67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank database. 4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some of them were RCC-related, including vascular endothelial growth factor, vimentin and tissue factor. Most of the known genes were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohistochemical and Western blot analysis for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed genes.

[Expression and function of zinc ribbon gene ZNRD1 in drug-resistant gastric cancer cells].

OBJECTIVE: To study the expression and function of zinc ribbon gene ZNRD1 in drug-resistant cells of gastric cancer. METHODS: Two tumor cell lines were used in this study: gastric cancer SGC7901 and its drug-resistant counterpart SGC7901/VCR stepwise-selected by vincristine. The expression of ZNRD1 in SGC7901 and SGC7901/VCR was detected by northern blot and semiquantitative RT-PCR. ZNRD1 antisense nucleic acid was transfected into SGC7901/VCR cells by lipofectamine. The expression of protein in SGC7901/VCR cells and the transfectants was detected by immunochemical method. Fluorescence activated cell scan (FACS) was applied to observe the cell cycle alteration. Growth curve and drug sensitization of cells for vincristine (VCR) and adriamycin (ADM) were analyzed by MTT assay. RESULTS: The expression of ZNRD1 was higher in SGC7901/VCR than in SGC7901 cells. Immunochemical results showed that the expression level of ZNRD1 protein was lower in anti ZNRD1-SGC7901/VCR cells than in non-transfectants. The anti ZNRD1-SGC7901/VCR cells were gradually accumulated in G(1) phase, with a concomitant decrease of cell population in S phase. MTT assay showed that transfectant cell proliferation was lagged and more sensitive to VCR and ADM than non-transfectants. CONCLUSION: ZNRD1 gene displays high expression in VCR resistant gastric cancer cells. Expression of ZNRD1 protein is effectively blocked in anti ZNRD1-SGC7901/VCR cells by gene transfection. ZNRD1 antisense nucleic acid could reverse, to some degree, the MDR of human drug-resistant gastric cancer cell SGC7901/VCR.

[The porcine alpha1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte negative express GT].

OBJECTIVE: To study whether the porcine alpha1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte negatively expresses GT mRNA and resists to the cytotoxicity of nature antibody in human serum. METHODS: The porcine alpha1, 3 galactosyltransferase gene siRNA targeted vector (pPNTloxPGTsiRNA) were construct with pPNTloxPGT and pMXSV/U6 vector. Positive-negative selection was used to produce a heterozygous pPNTloxPGTsiRNA knockout (+/-) clone. The GT mRNA expressions were detected with northern blot. Complement-mediated NAb cytotoxicity after incubation of hepatocytes with NAbs and complement was determined using 3- (4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS, tetrazolium salt) colorimetric assay. RESULTS: The pPNTloxPGTsiRNA targeted porcine hepatocyte (+/-) negative express GT mRNA. Only 14% to 18% cytotoxicity can be detected at the highest serum concentration. The pPNTloxPGT targeted porcine hepatocyte (+/-) express GT mRNA just as the wild type porcine cells and the cytotoxicity are 77% to 83%. CONCLUSION: The porcine a1, 3 galactosyltransferase gene siRNA targeted heterozygous hepatocyte (+/-) negative express GT and resisted to nature antibody in human serum.