Seiji Miyatani

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.

Notch Signaling Suppresses IgH Gene Expression in Chicken B Cells: Implication in Spatially Restricted Expression of Serrate2/Notch1 in the Bursa of Fabricius

The bursa of Fabricius is a central organ for chicken B cell development and provides an essential microenvironment for expansion of the B cell pool and for generation of a diversified B cell repertoire. We report here that genes encoding the Notch family of transmembrane proteins, key regulators of cell fate determination in development, are differentially expressed in the bursa of Fabricius: Notch1 is expressed in medullary B cells located close to the basement membrane-associated epithelium (BMAE). In contrast, a Notch ligand, Serrate2, is expressed exclusively in the BMAE, which surrounds bursal medulla. A basic helix-loop-helix-type transcription factor, Hairy1, a downstream target of Notch signaling, is expressed in the bursa coordinately with Notch1 and Serrate2 and an immature B cell line, TLT1, which expresses both Notch1 and Serrate2. Furthermore, stable expression of a constitutively active form of chicken Notch1 or Notch2 in a B cell line results in a down-regulation of surface IgM expression, which is accompanied by the reduction of IgH gene transcripts. Transient reporter assay with the human IgH gene intronic enhancer reveals that an active form of Notch1 inhibits the IgH enhancer activity in chicken B cells, suggesting that Notch-mediated signals suppress the IgH gene expression via influencing the IgH intronic enhancer. These findings raise the possibility that the local activation of Notch1 in a subset of B cells by Serrate2 expressed in BMAE may influence the cell fate decision that is involved in B cell differentiation and selection inside the bursa.

Restricting Elements in the Immunological Circuitry: the Role of I Region‐Controlled Determinants

Unlike the network concept that applies to the regulation mediated by the variable region of antigen-recognition molecules regardless of the cell types carrying such variable region structures, the circuit idea is primarily concerned with the sequential activation of different types of immunocompetent cells, regardless of the structure responsible for such selective interactions. Two major restricting elements have been proposed in the circuit type regulation based on the fact that the matching of either immunoglobulin V, or major histocompatibility complex (MHC) between two different cell types was necessary for an effective and meaningful cell interaction. We have reported in the carrier-specific regulation of the antibody response by T cells that MHC products play important roles in the sequential activation of the suppressor or augmenting pathway.’ In the suppressor circuit, the antigen-specific T-suppressor cell factor (TsF) derived from the Lyt-2+ T-suppressor cell (Ts) carries I region determinants controlled by an I-J subregion gene and acts on the responding cells derived from strains sharing the same I-J subregion haplotype.2 In the case of augmentation, the augmenting factor (TAF) derived from the Lyt-I+ T cell (TA) has determinants controlled by an I-A subregion gene and augments the response of I-A subregion compatible strains.’ The treatment of responding cells with conventional anti-I-J and anti-I-A results in the inability of acceptance of TsF and T A F effects. In addition, some but not all T-helper cells (TH2) carry an I-J determinant, which is serologically distinguishable from that expressed on T-suppressor cells.4 These results suggested that the I region controls a series of determinants selectively expressed on T cells and that they are the major elements that restrict the cell interaction to lead to the suppression or augmentation of the immune response. In order to delineate the mechanism of such restrictions, we have attempted to study the immunochemical properties of I region-controlled (Iat) determinants on T cells. Thus, we made a number of functional hybridomas and T-cell clones with Ts and Ta nature expressing different I region determinants distinguishable serologically with I region-specific a l l~ant ibodies .~-~ We also developed several monoclonal antibodies that define such I region determinants with their exquisite specificites.8 This paper deals with the serological and immunochemical analysis of Iat structures on hybridomas and cell lines, and considers the roles of such polymorphic determinants in the restricted interactions among the members in the immunological circuit.

IgVH and MHC-Restricted Regulatory Circuit in the Immune Response

The term “immune circuit” has been used to designate a train process of regulatory cell interactions in which the initial input such as antigenic stimulation activates a consecutive series of lymphoid cells finally producing an output which neutralizes the input signal. Thus, it is supposed that antigenic stimulation induces antibody formation, but at the same time it does activate a series of cells that finally suppress the antibody formation. Such an internal regulatory system is maintained by selective interactions between one cell type and the other which are restricted by mutual recognition of polymorphic structures expressed on partner cells.

Epitope Organizations of I Region Products Associated with Antigen-Specific Augmenting and Suppressor T Cell Factors

We have reported previously two distinct antigen specific T cell factors having the ability to augment (TaF) or to suppress (TsF) the in vitro secondary antibody response (1). Subsequently, several T cell hybridomas producing these factors have been established and maintained in our laboratory (2,3). TaF and TsF produced by these hybridomas had identical functional properties to those from normal antigen-primed T cells. Previous studies revealed a two-chain structure of these soluble products: one chain possesses antigen-binding ability and the other bears determinants encoded by the major histocompatibility complex (MHC); I-A subregion gene product for TaF and I-J subregion gene product for TsF. Both these determinants have been found to be different from conventional class II Ia antigens.