Chisato Miyaura

1α,25-Dihydroxyvitamin D3 induces differentiation of human myeloid leukemia cells

A human myeloid leukemia cell line [HL-60] could be induced to differentiate into mature myeloid cells by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], the active form of vitamin D3. At 10−10–10−8 M, 1α,25(OH)2D3 suppressed cell growth in a dose-dependent manner and markedly induced phagocytosis and C3 rosette formation. The potency of 1α,25(OH)2D3 in inducing differentiation was nearly equivalent to that of known synthetic inducers such as dimethyl sulfoxide, actinomycin D or a phorbol ester (12-o-tetra-decanoyl-phorbol-13-acetate). These results clearly indicate that 1α,25(OH)2D3, besides its well known biological effect in enhancing intestinal calcium transport and bone mineral mobilization activities, is involved in the cell grwoth and differentiation of HL-60 cells.

Modulation of osteoclast differentiation by local factors.

Bone-resorbing osteoclasts are of hemopoietic cell origin, probably of the CFU-M-derived monocyte-macrophage family. Bone marrow-derived osteoblastic stromal cells play an important role in modulating the differentiation of osteoclast progenitors in two different ways: one is the production of soluble factors, and the other is cell-to-cell recognition between osteoclast progenitors and osteoblastic stromal cells. M-CSF is probably the most important soluble factor, which appears to be necessary for not only proliferation of osteoclast progenitors, but also differentiation into mature osteoclasts and their survival. A number of local factors as well as systemic hormones induce osteoclast differentiation. They are classified into three categories in terms of the signal transduction: vitamin D receptor-mediated signals [1 alpha,25(OH)2D3]; protein kinase A-mediated signals (PTH, PTHrP, PGE2, and IL-1); and gp130-mediated signals (IL-6, IL-11, oncostatin M, and leukemia inhibitory factor). All of these osteoclast-inducing factors appear to act on osteoblastic cells to commonly induce osteoclast differentiation factor (ODF), which recognizes osteoclast progenitors and prepares them to differentiate into mature osteoclasts. This line of approach will undoubtedly produce new ways to treat several metabolic bone diseases caused by abnormal osteoclast recruitment such as osteoporosis, osteopetrosis, Pagets disease, rheumatoid arthritis, and periodontal disease.

Expression of Estrogen Receptor β in Rat Bone

A novel estrogen receptor, estrogen receptor beta (ERbeta), has recently been cloned from a rat prostate cDNA library. In bone, which is an important target tissue of estrogen, ER alpha has been reported to be present preferentially in osteoblasts, but the mechanism of action of estrogen in bone is still not known. In the present study, we examined expression of ERbeta mRNA in bone. Expression of ERbeta mRNA was evident in primary osteoblastic cells isolated from 1-day-old rat calvaria and rat osteosarcoma cells (ROS 17/2.8), and its level was higher than that of ER alpha mRNA. When osteoblastic cells were cultured for 28 days to induce differentiation into mature osteoblasts capable of forming bone nodules, ERbeta mRNA was constantly and highly expressed during the entire culture period. In contrast, the level of ER alpha mRNA was very low at the beginning of culture and it gradually increased during the differentiation of osteoblastic cells. Various tissues including bone were isolated from 8-week-old rats of both sexes, and total RNA was extracted to compare the tissue distribution of expression levels of ERbeta mRNA. In cancellous bone of the distal femoral metaphysis and lumbar vertebra, expression of ERbeta mRNA was obvious, and its level was equivalent to those in the uterus and testis, but lower than those in the ovary and prostate. The level of ERbeta mRNA in femoral cortical bone was lower than that in cancellous bone. There was no appreciable differences between female and male rats in the distribution and expression levels of ERbeta mRNA in bone. These results indicate that ERbeta mRNA is highly expressed in osteoblasts in rat bone, suggesting that there is a distinct mechanism of estrogen action mediated by ERbeta in bone.

Estrogen deficiency stimulates B lymphopoiesis in mouse bone marrow.

We have found that an estrogen deficiency causes a marked increase in bone marrow cells. To examine the effect of estrogen on hemopoiesis, we characterized the increased population of bone marrow cells after ovariectomy (OVX). In OVX mice, the percentage of myeloid cells and granulocytes was decreased, whereas that of B220-positive B lymphocytes was selectively increased 2-4 wk after surgery. The total number of myeloid cells and granulocytes did not change appreciably, but that of B220-positive cells was greatly increased by OVX. When OVX mice were treated with estrogen, the increased B lymphopoiesis returned to normal. B220-positive cells were classified into two subpopulations, B220low and B220high. The majority of the B220low cells were negative for the IgM mu chain, whereas most of the B220high cells were mu-positive. OVX selectively increased the precursors of B lymphocytes identified by B220low. mu-negative phenotype, suggesting that an estrogen deficiency stimulates accumulation of B lymphocyte precursors. When bone marrow-derived stromal cells (ST2) were pretreated with estrogen then co-cultured with bone marrow cells in the presence of estrogen, the stromal cell-dependent B lymphopoiesis was greatly inhibited. The present study suggests that estrogen plays an important role in the regulation of B lymphocyte development in mouse bone marrow.

1α,25-Dihyroxyvitamin D3 induces differentiation of human promyelocytic leukemia cells (HL-60) into monocyte-macrophages, but not into granulocytes

The differentiating action of 1 alpha,25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3] in hematopoietic cells was examined in 3 tumor cell lines. 1 alpha,25-(OH)2D3 induced common differentiation-associated properties in macrophages and granulocytes similarly in mouse myeloblastic leukemia cells (M1), human promyelocytic leukemia cells (HL-60) and human histiocytic monoblast-like lymphoma cells (U937). 1 alpha,25(OH)2D3 markedly induced alpha-naphthyl acetate esterase activity, a typical marker of monocyte-macrophages, in M1 and HL-60 cells. In HL-60 and U937 cells, the vitamin also induced binding of the monoclonal antibody MAS 072, specific for monocyte-macrophages, but not of MAS 067, specific for granulocytes. These results clearly indicate that 1 alpha, 25(OH)2D3 induces differentiation of all cell lines examined preferentially along the monocyte-macrophage pathway.

Recombinant human interleukin 6 (B-cell stimulatory factor 2) is a potent inducer of differentiation of mouse myeloid leukemia cells (M1)

Recombinant human interleukin 6 (IL‐6), a lymphokine involved in the final differentiation of activated B‐cells into antibody‐forming cells, greatly suppressed proliferation and induced differentiation of murine myeloid leukemia cells (M1) into mature macrophage‐like cells. When M1 cells were treated with IL‐6, their growth was completely arrested as early as on day 2, and they were induced to differentiate morphologically into macrophage‐like cells. Differentiation‐associated properties such as phagocytic activity, adherence to the dish surface, Fc and C3 receptors, were also induced within 24 h by IL‐6, and they reached their respective maximal levels on day 2 or 3. The potency of IL‐6 in suppressing proliferation and inducing differentiation was much greater than that of 1α,25‐dihydroxyvitamin D3 one of the most potent inducers of M1 cells. The present report indicates that IL‐6 is involved in the differentiation of not only B‐cells but also myeloid leukemia cells.

Synthetic analogues of vitamin D3 with an oxygen atom in the side chain skeleton A trial of the development of vitamin D compounds which exhibit potent differentiation‐inducing activity without inducing hypercalcemia

Four analogues of vitamin D3 with an oxygen atom in the side chain skeleton were synthesized to determine whether their differentiation‐inducing activity could be separated structurally from their activity to induce hypercalcemia. The order of the in vitro potency to reduce nitroblue tetrazolium in human myeloid leukemia cells (HL‐60) was 22‐oxa‐1α25‐(OH)2D3 > 1α,25‐(OH)2D3 > 20‐oxa‐1α,25‐(OH)2D3≒22‐oxa‐1α‐(OH) D3 > 1α‐(OH)D3 > 20‐oxa‐1α‐(OH)D3. 22‐Oxa‐1α,25‐(OH)2D3, was also about 10‐times more potent than 1α,25‐(OH)2D3 in suppressing proliferation and inducing differentiation of mouse myelomonocytic leukemia cells (WEHI‐3), but the former was much weaker than the latter in inducing the release of 45Ca from prelabeled fetal mouse calvaria. These results suggest that the differentiation‐inducing activity of vitamin D compounds can be separated structurally from their activity to induce hypercalcemia.

Production of interleukin 6 and its relation to the macrophage differentiation of mouse myeloid leukemia cells (M1) treated with differentiation-inducing factor and 1α,25-dihydroxyvitamin D3

We have studied the production of interleukin 6 (IL-6) and its relation to the macrophage differentiation in murine myeloid leukemia cells (M1). As has been reported, differentiation-inducing factor (D-factor), 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3], and recombinant IL-6 similarly induced differentiation of M1 cells into macrophages. The three compounds also induced mRNA expression of IL-6 in M1 cells. M1 cells treated with D-factor or 1 alpha, 25(OH)2D3 produced biologically active IL-6, but the amounts of IL-6 secreted into culture media did not appear to be enough to induce differentiation of M1 cells. Furthermore, simultaneous addition of anti-IL-6 antibody did not suppress the differentiation of M1 cells induced by D-factor or 1 alpha, 25(OH)2D3. These results show that IL-6 production is an essential property associated with the macrophage differentiation of M1 cells, but it may not be responsible for the D-factor- and 1 alpha, 25(OH)2D3-induced differentiation.

Cooperative effect of 1 α,25-dihydroxyvitamin D3 and dexamethasone in inducing differentiation of mouse myeloid leukemia cells

Murine myeloid leukemia cells (MI) are induced to differentiate into macrophages by the metabolically active form of vitamin D3,1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] (E. Abe et al., (1981) Proc. Natl. Acad. Sci. USA 78, 4990-4994). At 0.12-120 nM, 1 alpha,25(OH)2D3 suppressed cell growth in a dose-dependent manner and markedly induced phagocytic activity, lysozyme activity, and C3-receptor formation. The potency of 1 alpha,25(OH)2D3, at 0.12-120 nM, in inducing differentiation was nearly equivalent to that of 10-10,000 nM of dexamethasone, one of the most potent stimulators of Ml cells. Simultaneous treatment with low physiological plasma concentrations of 1 alpha,25(OH)2D3 (0.12 nM) and dexamethasone (10 nM) induced differentiation of Ml cells equivalent to the responses obtained only by using much higher concentrations of the respective steroids when used separately. In addition, two variant clones of Ml cells resistant to either 1 alpha,25(OH)2D3 or dexamethasone were isolated. One was resistant to 120 nM of 1 alpha,25(OH)2D3 but sensitive to 10-1000 nM of dexamethasone. The other was resistant to 1000 nM of dexamethasone but sensitive to 12 nM of 1 alpha,25(OH)2D3. This suggests that the mechanism of action of 1 alpha,25(OH)2D3 in inducing differentiation of Ml cells is different at least in part from that of dexamethasone, and that combination therapy by both steroids may be useful in reducing leukemogenicity of Ml cells in vivo.

1α,25‐Dihydroxyvitamin D3 directly induces fusion of alveolar macrophages by a mechanism involving RNA and protein synthesis, but not DNA synthesis

The results of our present study indicate that 1α,25‐dihydroxyvitamin D3[1α,25(OH)2D3] directly induces fusion of mouse alveolar macrophages without any participation of T‐lymphocytes by a mechanism involving RNA and protein synthesis but not DNA synthesis. We have reported that 1α,25(OH)3D3 induces fusion of alveolar macrophages by a direct mechanism and by a spleen cell‐mediated indirect mechanism [(1983) Proc. Natl. Acad. Sci. USA 80, 5583‐5587]. Alveolar macrophages pretreated with or without anti‐Thy 1.2 antibody and complement fused similarly when they were incubated with 1α,25(OH)2D3. The vitamin suppressed DNA synthesis, but it significantly enhanced RNA and protein synthesis. The 1α,25(OH)2D3‐induced fusion was blocked by adding actinomycin D or cycloheximide, but not by hydroxyurea.