Takao Sudo

NAD-dependent histone deacetylase, SIRT1, plays essential roles in the maintenance of hematopoietic stem cells.

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-Kit(high)Sca-1(+)Lineage(-) (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

Identification of functional domains and novel binding partners of STIM proteins

With a signal trap method, we previously identified stromal interaction molecule (STIM: originally named as SIM) as a protein, which has a signal peptide in 1996. However, recent works have accumulated evidences that STIM1 and STIM2 reside in endoplasmic reticulum (ER) and that both mainly sense ER Ca2+ depletion, which plays an essential role in store operated calcium entry. In the present study, we extensively analyzed the domain functions and associated molecules of STIMs. A STIM1 mutant lacking the coiled‐coil domains was massively expressed on the cell surface while mutants with the coiled‐coil domains localized in ER. In addition, STIM1 mutants with the coiled‐coil domains showed a longer half‐life of proteins than those without them. These results are likely to indicate that the coiled‐coil domains of STIM1 are essential for its ER‐retention and its stability. Furthermore, we tried to comprehensively identify STIM1‐associated molecules with mass spectrometry analysis of co‐immunoprecipitated proteins for STIM1. This screening clarified that both STIM1 and STIM2 have a capacity to bind to a chaperone, calnexin as well as two protein‐transporters, exportin1 and transportin1. Of importance, our result that glycosylation on STIM1 was not required for the association between STIM1 and calnexin seems to indicate that calnexin might function on STIM1 beyond a chaperone protein. Further information concerning regulatory mechanisms for STIM proteins including the data shown here will provide a model of Ca2+ control as well as a useful strategy to develop therapeutic drugs for intracellular Ca2+‐related diseases including inflammation and allergy. J. Cell. Biochem. 112: 147–156, 2011.

The Endothelial Antigen ESAM Monitors Hematopoietic Stem Cell Status between Quiescence and Self-Renewal

Whereas most hematopoietic stem cells (HSC) are quiescent in homeostasis, they actively proliferate in response to bone marrow (BM) injury. Signals from the BM microenvironment are thought to promote entry of HSC into the cell cycle. However, it has been cumbersome to assess cycle status of viable HSC and thus explore unique features associated with division. In this study, we show that expression of endothelial cell-selective adhesion molecule (ESAM) can be a powerful indicator of HSC activation. ESAM levels clearly mirrored the shift of HSC between quiescence and activation, and it was prominent in comparison with other HSC-related Ags. ESAMhi HSC were actively dividing, but had surprisingly high long-term reconstituting capacity. Immunohistochemical analyses showed that most ESAMhi HSC were located near vascular endothelium in the BM after 5-fluorouracil treatment. To determine the importance of ESAM in the process of BM recovery, ESAM knockout mice were treated with 5-fluorouracil and their hematopoietic reconstruction was examined. The ESAM deficiency caused severe and prolonged BM suppression, suggesting that ESAM is functionally indispensable for HSC to re-establish homeostatic hematopoiesis. With respect to intracellular regulators, NF-κB and topoisomerase II levels correlated with the ESAM upregulation. Thus, our data demonstrate that the intensity of ESAM expression is useful to trace activated HSC and to understand molecular events involved in stem cell states.

The efficacy of tocilizumab in a patient with pulmonary arterial hypertension associated with Castleman’s disease

Castleman’s disease is a highly heterogeneous clinical-pathological entity that belongs to the lymphoproliferative disorders and is associated with pulmonary arterial hypertension (PAH) in some patients. It is linked to excessive immune stimulation by interleukin-6 (IL-6), which is also involved in the pathogenesis of PAH. A 31-year-old woman with Castleman’s disease demonstrated PAH characterized by severe right heart failure. Since she was resistant to various conventional therapies including steroids, prostacyclins, bosentan, and sildenafil, tocilizumab (anti-IL-6 receptor antibody) therapy was started. Her clinical course was followed for 6 months, with significant improvement without any adverse effect. This is the first reported case of use of tocilizumab in addition to steroids and conventional PAH therapy in a patient with PAH associated with Castleman’s disease.

Complementary regulation of early B-lymphoid differentiation by genetic and epigenetic mechanisms.

Abstract Although B lymphopoiesis is one of the best-defined paradigms in cell differentiation, our knowledge of the regulatory mechanisms underlying its earliest processes, in which hematopoietic stem cells (HSCs) enter the B lineage, is limited. However, recent methodological advances in sorting progenitor cells and monitoring their epigenetic features have increased our understanding of HSC activities. It is now known that even the highly enriched HSC fraction is heterogeneous in terms of lymphopoietic potential. While surface markers and reporter proteins provide information on the sequential differentiation of B-lineage progenitors, complex interactions between transcription factors have also been shown to play a major role in this process. Epigenetic regulation of histones, nucleosomes, and chromatin appears to play a crucial background role in this elaborate transcription network. In this review, we summarize recent findings on the physiological processes of early B-lineage differentiation, which provides a new paradigm for understanding the harmonious action of genetic and epigenetic mechanisms.

ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias.

Reliable markers are essential to increase our understanding of the biological features of human hematopoietic stem cells and to facilitate the application of hematopoietic stem cells in the field of transplantation and regenerative medicine. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel functional marker of hematopoietic stem cells in mice. Here, we found that ESAM can also be used to purify human hematopoietic stem cells from all the currently available sources (adult bone marrow, mobilized peripheral blood, and cord blood). Multipotent colony-forming units and long-term hematopoietic-reconstituting cells in immunodeficient mice were found exclusively in the ESAM(High) fraction of CD34(+)CD38(-) cells. The CD34(+)CD38(-) fraction of cord blood and collagenase-treated bone marrow contained cells exhibiting extremely high expression of ESAM; these cells are likely to be related to the endothelial lineage. Leukemia cell lines of erythroid and megakaryocyte origin, but not those of myeloid or lymphoid descent, were ESAM positive. However, high ESAM expression was observed in some primary acute myeloid leukemia cells. Furthermore, KG-1a myeloid leukemia cells switched from ESAM negative to ESAM positive with repeated leukemia reconstitution in vivo. Thus, ESAM is a useful marker for studying both human hematopoietic stem cells and leukemia cells.

Estrogen-inducible sFRP5 inhibits early B-lymphopoiesis in vivo, but not during pregnancy.

Mammals have evolved to protect their offspring during early fetal development. Elaborated mechanisms induce tolerance in the maternal immune system for the fetus. Female hormones, mainly estrogen, play a role in suppressing maternal lymphopoiesis. However, the molecular mechanisms involved in the maternal immune tolerance are largely unknown. Here, we show that estrogen‐induced soluble Frizzled‐related proteins (sFRPs), and particularly sFRP5, suppress B‐lymphopoiesis in vivo in transgenic mice. Mice overexpressing sFRP5 had fewer B‐lymphocytes in the peripheral blood and spleen. High levels of sFRP5 inhibited early B‐cell differentiation in the bone marrow (BM), resulting in the accumulation of cells with a common lymphoid progenitor (CLP) phenotype. Conversely, sFRP5 deficiency reduced the number of hematopoietic stem cells (HSCs) and primitive lymphoid progenitors in the BM, particularly when estrogen was administered. Furthermore, a significant reduction in CLPs and B‐lineage‐committed progenitors was observed in the BM of sfrp5‐null pregnant females. We concluded that, although high sFRP5 expression inhibits B‐lymphopoiesis in vivo, physiologically, it contributes to the preservation of very primitive lymphopoietic progenitors, including HSCs, under high estrogen levels. Thus, sFRP5 regulates early lympho‐hematopoiesis in the maternal BM, but the maternal–fetal immune tolerance still involves other molecular mechanisms that remain to be uncovered.

The anti-apoptotic gene Anamorsin is essential for both autonomous and extrinsic regulation of murine fetal liver hematopoiesis

Anamorsin (AM) is an antiapoptotic molecule that confers factor-independent survival on hematopoietic cells. AM-deficient (AM(-/-)) mice are embryonic lethal because of a defect in definitive hematopoiesis; however, the significance of AM in embryonic hematopoiesis remains unknown. This study characterized the hematopoietic defects in AM(-/-) fetal livers. The AM(-/-) fetal liver displayed significantly reduced numbers of c-Kit(+)Sca-1(+)Lin(-) (KSL) cells. An in vitro colony-forming unit assay showed that fetal liver cells isolated from AM(-/-) embryos gave rise to fewer colonies in all cell types. The reconstitution activity in AM(-/-) hematopoietic stem cells (HSCs) was markedly reduced in all lineages. Furthermore, the limiting dilution assay revealed that the number of fetal liver HSCs was reduced because of AM deficiency. Retrovirus-mediated AM expression rescued the defective hematopoietic colony-forming activities of AM(-/-) KSL cells. We also investigated the effects of AM deficiency on fetal liver stromal cells, which support hematopoiesis. Interestingly, primary stromal cell cultures from wild type fetal liver supported the growth of AM(-/-) KSL cells, but stromal cultures from AM(-/-) fetal liver provided little support of wild type KSL cell growth. These results demonstrated that AM was essential for both autonomous and extrinsic regulation of fetal liver hematopoiesis. This study provided new insight into the molecular regulation of hematopoiesis.

Non-labeling multiphoton excitation microscopy as a novel diagnostic tool for discriminating normal tissue and colorectal cancer lesions

Multiphoton excitation microscopy (MPM) is regarded as an effective tool that enables the visualization of deep regions within living tissues and organs, with little damage. Here, we report novel non-labeling MPM (NL-MPM) imaging of fresh human colorectal mucosa, which is useful for discriminating cancer lesions from normal tissues quantitatively without any need for resection, fixation, or staining. Using NL-MPM, we visualized three components in human colorectal mucosa, epithelial cells, immune cells, and basement membranes, based on their characteristic patterns of fluorescence. These patterns are characterized by the different auto-fluorescence properties of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and flavin adenine dinucleotide and from second harmonic generation (SHG). NL-MPM images were at least as informative to pathologists as were ‘conventional’ images of fixed tissue sections stained with hematoxylin and eosin. Additionally, two quantitative parameters extracted from NL-MPM images – the nucleus diameter (index N) and the intensity of SHG in the basement membrane (index S) – rendered it possible to diagnose cancer regions effectively. In conclusion, NL-MPM is a novel, promising method for real-time clinical diagnosis of colorectal cancers, and is associated with minimal invasiveness.

Endothelial Cell-Selective Adhesion Molecule Expression in Hematopoietic Stem/Progenitor Cells Is Essential for Erythropoiesis Recovery after Bone Marrow Injury

Numerous red blood cells are generated every second from proliferative progenitor cells under a homeostatic state. Increased erythropoietic activity is required after myelo-suppression as a result of chemo-radio therapies. Our previous study revealed that the endothelial cell-selective adhesion molecule (ESAM), an authentic hematopoietic stem cell marker, plays essential roles in stress-induced hematopoiesis. To determine the physiological importance of ESAM in erythroid recovery, ESAM-knockout (KO) mice were treated with the anti-cancer drug, 5-fluorouracil (5-FU). ESAM-KO mice experienced severe and prolonged anemia after 5-FU treatment compared to wild-type (WT) mice. Eight days after the 5-FU injection, compared to WT mice, ESAM-KO mice showed reduced numbers of erythroid progenitors in bone marrow (BM) and spleen, and reticulocytes in peripheral blood. Megakaryocyte-erythrocyte progenitors (MEPs) from the BM of 5-FU-treated ESAM-KO mice showed reduced burst forming unit-erythrocyte (BFU-E) capacities than those from WT mice. BM transplantation revealed that hematopoietic stem/progenitor cells from ESAM-KO donors were more sensitive to 5-FU treatment than that from WT donors in the WT host mice. However, hematopoietic cells from WT donors transplanted into ESAM-KO host mice could normally reconstitute the erythroid lineage after a BM injury. These results suggested that ESAM expression in hematopoietic cells, but not environmental cells, is critical for hematopoietic recovery. We also found that 5-FU treatment induces the up-regulation of ESAM in primitive erythroid progenitors and macrophages that do not express ESAM under homeostatic conditions. The phenotypic change seen in macrophages might be functionally involved in the interaction between erythroid progenitors and their niche components during stress-induced acute erythropoiesis. Microarray analyses of primitive erythroid progenitors from 5-FU-treated WT and ESAM-KO mice revealed that various signaling pathways, including the GATA1 system, were impaired in ESAM-KO mice. Thus, our data demonstrate that ESAM expression in hematopoietic progenitors is essential for erythroid recovery after a BM injury.