Tohru Ikeda

The non-osteogenic mouse pluripotent cell line, C3H10T1/2, is induced to differentiate into osteoblastic cells by recombinant human bone morphogenetic protein-2.

The possibility that the non-osteogenic mouse pluripotent cell line, C3H10T1/2 (10T1/2), could be induced to differentiate into osteogenic cells by various hormones and cytokines was examined in vitro. Of a number of agents tested, recombinant human bone morphogenetic protein-2 (rhBMP-2) and retinoic acid induced alkaline phosphatase (ALP) activity in 10T1/2 cells. rhBMP-2 also induced mRNA expression of ALP in the cells. Dexamethasone, 1 alpha, 25-dihydroxyvitamin D3, transforming growth factor-beta 1 and insulin-like growth factor-I did not stimulate ALP activity. Treatment with rhBMP-2 greatly induced cAMP production in response to parathyroid hormone in 10T1/2 cells. No ALP activity was induced in NIH3T3 fibroblasts treated with rhBMP-2 or retinoic acid. These results indicate that 10T1/2 cells have a potential to differentiate into osteogenic cells under the control of BMP-2.

Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant‐negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Here we report the cloning of Aiolos, a gene which encodes an Ikaros homologue that heterodimerizes with Ikaros proteins. In contrast to Ikaros_which is expressed from the pluripotent stem cell to the mature lymphocyte_Aiolos is first detected in more committed progenitors with a lymphoid potential and is strongly up‐regulated as these differentiate into pre‐T and pre‐B cell precursors. The expression patterns of Aiolos and Ikaros, the relative transcriptional activity of their homo‐ and heteromeric complexes, and the dominant interfering effect of mutant Ikaros isoforms on Aiolos activity all strongly suggest that Aiolos acts in concert with Ikaros during lymphocyte development. We therefore propose that increasing levels of Ikaros and Aiolos homo‐ and heteromeric complexes in differentiating lymphocytes are essential for normal progression to a mature and immunocompetent state.

Helios, a novel dimerization partner of Ikaros expressed in the earliest hematopoietic progenitors

BACKGROUND Normal hematopoietic development depends on the activity of the Ikaros transcription factor, which contains distinct zinc-finger domains that mediate DNA binding and protein dimerization. Mice homozygous for a transgene encoding a dominant-negative version of Ikaros that lacks the DNA-binding domain but not the dimerization domain have a more severe phenotype than Ikaros null mice. This observation suggests the presence of factor(s) that can dimerize with Ikaros and partially complement its function. One previously identified factor, Aiolos, probably serves this role in the lymphoid system; a related factor involved in hematopoietic progenitors remains unknown, however. RESULTS Here, we describe the cloning of an Ikaros-related gene, Helios. Analysis of the primary sequences of Helios, Ikaros and Aiolos revealed that the DNA-binding, transcriptional activation and dimerization domains are functionally conserved. Helios activated transcription from Ikaros DNA-binding sites and could dimerize with itself, Ikaros or Aiolos. Expression of Helios was detected in the earliest hematopoietic sites of the embryo, in hematopoietic stem cells in the adult and was subsequently restricted to a subset of cells in the T cell lineage. Helios co-localized with Ikaros and Aiolos proteins in macromolecular nuclear structures and formed stable complexes in vivo with the dominant-negative version of Ikaros. CONCLUSIONS Distinct but overlapping expression patterns of members of the Ikaros gene family during hematopoiesis might result in the formation of different multimeric complexes that have specific roles in lineage progression. The preferential expression of Helios in the earliest stages of hematopoiesis suggests that this gene functions predominantly in early progenitors.

Age-related reduction in bone matrix protein mRNA expression in rat bone tissues: application of histomorphometry to in situ hybridization

Age-related changes in the biological activity of osteoblastic cells have been studied extensively using histomorphometry, especially in relation to osteoporosis. Nevertheless, the changes occurring in the biological activity of individual osteoblastic cells are not sufficiently clarified by this technique. In the present study, age-related changes in the expression of bone matrix protein mRNAs in individual osteoblastic cells were analyzed in vivo by in situ hybridization using undecalcified bone sections. In the femurs of 8-week-old male rats, strong expression of type I collagen and osteocalcin mRNAs was detected in cuboidal osteoblasts on the bone formation surface. Osteopontin mRNA was detected in some of the mononuclear cells and osteoclasts on the bone resorption surface, and also in osteocytes. In the femurs of 60-week-old and 100-week-old male rats, expression of these bone matrix protein mRNAs was markedly decreased. Histomorphometrical analysis of 8-week-old and 60-week-old rats indicated that both the activity and number of osteoblasts expressing type I collagen mRNA, as well as the number of osteoclasts, were reduced in these tissues in older animals. These results indicate age-related reductions in both biological activity and numbers of osteoblasts.

Cloning of rat type I receptor cDNA for bone morphogenetic protein-2 and bone morphogenetic protein-4, and the localization compared with that of the ligands

A rat homologue cDNA of mouse (Koenig et al. [1994] Mol. Cell Biol. 14:5961–5974; Suzuki et al. [1994] Proc. Natl. Acad. Sci. USA 91: 10255–10259) and human (ten Dijke et al. [1994] J. Biol. Chem. 269:16985–16988) type I receptors for BMP‐2 and BMP‐4 was cloned. Tissue distribution of the receptor mRNA was studied by in situ hybridization using rats at embryonic days 9, 13, 15, and 18 as well as 1‐ and 5‐day‐old postnatal rats. In the rats at embryonic days 9, 13, and 15, the receptor mRNA was diffusely expressed over the embryonic bodies. At embryonic day 18, the receptor mRNA expression was high in the hair and whisker follicles, tooth bud, cartilage, bone, digestive organs, lung, kidney, heart, and meninges. The receptor mRNA was expressed over a much wider area than those of the ligands in many organs. In the lung and digestive organs, the receptor mRNA was diffusely expressed and most highly expressed in the bronchial epithelium and muscle layer, respectively, in both of which mRNA expression of the ligands was undetectable. The receptor mRNA was highly expressed in the meninges, although neither of the ligands was expressed in or near this region. These results suggest that this receptor participates in both mesoderm formation in early embryogenesis and differentiation of mesodermal cells during maturation of organs, and further suggest the presence of another factor(s) that binds the type I receptor.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos−/−) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos−/− Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos−/− mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4+ Tconvs. Eos−/− mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.