Kazuki Kuroda

Notch–RBP-J signaling is involved in cell fate determination of marginal zone B cells

RBP-J is a key mediator of Notch signaling that regulates cell fate determination in various lineages. To investigate the function of Notch–RBP-J in mature B cell differentiation, we generated mice that selectively lacked B cell RBP-J expression using conditional mutagenesis. Absence of RBP-J led to the loss of marginal zone B (MZB) cells with a concomitant increase in follicular B cells; in contrast, B1 cells in the peritoneal cavity were unaffected. Lack of RBP-J caused no defects in B cells maintenance, survival, plasma cell differentiation or activation. It is therefore likely that Notch–RBP-J signaling regulates the lineage commitment of mature B cells into follicular versus MZB cells. In addition, in mice with RBP-J–deficient B cells, had no obvious changes in immunoglobulin production in response to Ficoll, lipopolysaccharide or chicken gammaglobulin. In contrast, these mice exhibited increased mortality rates after blood-borne bacterial infection, which indicates that MZB cells play pivotal roles in the clearance of these bacteria.

Delta-induced Notch Signaling Mediated by RBP-J Inhibits MyoD Expression and Myogenesis

Signaling induced by interaction between the receptor Notch and its ligand Delta plays an important role in cell fate determination in vertebrates as well as invertebrates. Vertebrate Notch signaling has been investigated using its constitutively active form, i.e. the truncated intracellular region which is believed to mimic Notch-Delta signaling by interaction with a DNA-binding protein RBP-J. However, the molecular mechanism for Notch signaling triggered by ligand binding, which leads to inhibition of differentiation, is not clear. We have established a myeloma cell line expressing mouse Delta1 on its cell surface which can block muscle differentiation by co-culture with C2C12 muscle progenitor cells. We showed that Delta-induced Notch signaling stimulated transcriptional activation of RBP-J binding motif, containing promoters including the HES1 promoter. Furthermore, ligand-induced Notch signaling up-regulated HES1 mRNA expression within 1 h and subsequently reduced expression of MyoD mRNA. Since cycloheximide treatment did not inhibit induction of HES1 mRNA, the HES1 promoter appears to be a primary target of activated Notch. In addition, a transcriptionally active form of RBP-J, i.e. VP16-RBP-J, inhibited muscle differentiation of C2C12 cells by blocking the expression of MyoD protein. These results suggest that HES1 induction by the Delta1/Notch signaling is mediated by RBP-J and blocks myogenic differentiation of C2C12 cells by subsequent inhibition of MyoD expression.

Regulation of marginal zone B cell development by MINT, a suppressor of notch/RBP-J signaling pathway

We found that Msx2-interacting nuclear target protein (MINT) competed with the intracellular region of Notch for binding to a DNA binding protein RBP-J and suppressed the transactivation activity of Notch signaling. Although MINT null mutant mice were embryonic lethal, MINT-deficient splenic B cells differentiated about three times more efficiently into marginal zone B cells with a concomitant reduction of follicular B cells. MINT is expressed in a cell-specific manner: high in follicular B cells and low in marginal zone B cells. Since Notch signaling directs differentiation of marginal zone B lymphocytes and suppresses that of follicular B lymphocytes in mouse spleen, the results indicate that high levels of MINT negatively regulate Notch signaling and block differentiation of precursor B cells into marginal zone B cells. MINT may serve as a functional homolog of Drosophila Hairless.

Stimulation of Osteoblastic Cell Differentiation by Notch

Notch is a transmembrane protein that plays a critical role in the determination of cellular differentiation pathways. Although its importance in the development of mesenchymal tissues has been suggested, its role in skeletal tissues has not been well investigated. Northern blot experiments showed the expression of Notch1 in MC3T3‐E1 osteoblastic cells at early differentiation stages. When a Notch1 cytoplasmic domain (Notch‐IC [NIC]) delivered by an adenovirus vector was expressed in osteoblastic MC3T3‐E1 cells, a significant increase in calcified nodule formation was observed in long‐term cultures. Activation of endogenous Notch in MC3T3‐E1 by coculturing them with Delta‐like‐1 (Dll1)‐expressing myeloma cells also resulted in a stimulation of calcified nodule formation. Not only affecting nodule formation, Notch activation also had effects on osteoblastic differentiation of multipotent mesenchymal cells. Osteoblastic differentiation of C3H10T1/2 cells induced by bone morphogenetic protein 2 (BMP‐2) was significantly stimulated, whereas adipogenic differentiation was suppressed strongly, resulting in a dominant differentiation of osteoblastic cells. NIC expression in primary human bone marrow mesenchymal stem cells (hMSCs) also induced both spontaneous and stimulated osteoblastic cell differentiation. These observations suggest that osteoblastic cell differentiation is regulated positively by Notch and that Notch could be a unique and interesting target molecule for the treatment of osteoporosis.

Suppression of differentiation and proliferation of early chondrogenic cells by Notch.

Notch is a transmembrane protein involved in cell fate determination. In the present study, we observed temporally and spatially restricted expression of Notch1 in developing cartilage. Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immunoreactivity indicating its expression was retained in the perichondrial region. Next, we investigated the expression of Notch1 and related molecules in a chondrogenic cell line, ATDC5 cells. Notch1, Delta-like (Dll)1, Deltex2, and Deltex3 were coexpressed after 6-day insulin treatment. Expression of Hairy and Enhancer of split homologue (HES)-1 followed thereafter. These results suggest that Notch may have a role in the early stage of chondrogenesis. To assess the effect of Notch activation, we cultured ATDC5 cells with a myeloma clone constitutively expressing Dll1, a ligand of Notch. We also used an adenovirus vector to express the constitutively active Notch1 intracellular domain (NIC). Activating either the endogenous or exogenous Notch receptor dramatically inhibited chondrogenic cell differentiation of ATDC5 cells, as assessed by Alcian blue staining of the cells and chondrocyte differentiation markers. Last, we investigated the effect of NIC on the proliferation of the ATDC5 cells. Expression of NIC by the adenovirus strongly suppressed thymidine incorporation. These results indicate that Notch is expressed in the initial stage of chondrogenic cell differentiation and has a strong inhibitory effect on both differentiation and proliferation of the cells when activated. The expression of Notch decreases as chondrogenic differentiation proceeds; however, a population of the cells with sustained expression of Notch1 become perichondrial cells. Considering that the perichondrium acts as a stem cell source of osteoblasts and chondrocytes, Notch1 may have a role in the formation of these cells by suppressing both differentiation and proliferation.

Msx2-interacting nuclear target protein (Mint) deficiency reveals negative regulation of early thymocyte differentiation by Notch/RBP-J signaling

Notch/RBP-J signaling is required for generation of early T progenitors (ETP) and promotion of double-negative (DN) 4 cells from DN3 cells in thymocyte differentiation. However, whether Notch affects other steps during thymocyte differentiation remains unknown. Msx2-interacting nuclear target protein (Mint) is an endogenous inhibitor of Notch regulation. Concordantly, by ex vivo analyses of embryonic thymi and in vitro differentiation studies of fetal liver progenitors, we find that Mint deficiency enhances generation of ETP and DN4 cells. Unexpectedly, however, Mint deficiency impairs differentiation of ETP into DN2 cells, suggesting that Notch/RBP-J signaling negatively regulates DN1–DN2 transition.

Regulation of B cell development by Notch/RBP-J signaling.

RBP-J is an essential signal mediator of all four Notches in nuclei. Loss-of-function analyses clearly show the crucial roles of RBP-J in commitment of T cells versus B cells as well as MZ B cells versus Fo B cells. Such Notch/RBP-J regulation of dichotomic differentiation steps in lymphocyte is reminiscent of the development of sensory organ precursors (SOPs) in Drosophila. Studies on RBP-J conditional knockout mice that have lost MZ B cells without affecting Fo B cell functions have shown that MZ B cells play pivotal roles in immune responses to blood-borne bacteria.

LL5β Directs the Translocation of Filamin A and SHIP2 to Sites of Phosphatidylinositol 3,4,5-Triphosphate (PtdIns(3,4,5)P3) Accumulation, and PtdIns(3,4,5)P3 Localization Is Mutually Modified by Co-recruited SHIP2

Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P3 regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P3 and the reciprocal regulation of PtdIns(3,4,5)P3 itself. LL5β, whose subcellular localization is directed by membrane PtdIns(3,4,5)P3, recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P3 accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5β by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5β or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5β without its PtdIns(3,4,5)P3-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P3 accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P3 at the same location.

Chromosomal mapping of two RBP-J-related genes: Kyo-T and RBP-L.

AbstractWe have recently isolated two genes encoding proteins which have either homology or affinity to RBP-J, a transcription factor involved in Notch signaling. Kyo-T interacts with RBP-J and possibly regulates the function of RBP-J. RBP-L has a highly homologous region with RBP-J but the function of RBP-L is unknown. Fluorescence in situ hybridization analysis of human metaphase chromosomes localized Kyo-T and RBP-L to Xq26 and 20q12–13.1, respectively.