Etsuko Abe

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.

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.

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.

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1.

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 - 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.

The specific production of the third component of complement by osteoblastic cells treated with 1α,25‐dihydroxyvitamin D3

A 190 kDa protein was purified from conditioned media of mouse marrow‐derived stromal cell (ST2) cultures treated with 1α 25‐dihydroxyvitamin D3 (1α,25(OH)2D3) and identified as the third component of mouse complement (C3). Northern and Western blot analysis revealed that the production of C3 by ST2 and primary osteoblastic cells was strictly dependent on 1α,25(OH)2D3, but the production by hepatocytes was not. Adding 1α,25(OH)2D3 together with mouse C3 antibody to bone marrow cultures greatly inhibited the formation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like multinucleated cells. Adding C3 alone induced no TRAP‐positive cell formation. These results suggest that, in bone tissues, C3 is specifically produced by osteoblasts in response to 1α,25(OH)2D3 and somehow involved in inducing differentiation of bone marrow cells into osteoclasts in concert with other factors produced by osteoblasts in response to 1α,25(OH)2D3.

Circadian rhythm of 1α,25-dihydroxyvitamin D3 production in egg-laying hens

Abstract The activity of renal 25-hydroxyvitamin D3(25-OH-D3)-1α- and 24-hydroxylase and the plasma concentrations of vitamin D metabolites were investigated in relation to the ovulatory cycle in egg-laying hens. The time after ovulation was estimated from the position of the egg in the oviduct and the dry weight of the egg-shell. The in vitro renal 25-OH-D3-1α-hydroxylase activity was significantly enhanced 14–16 hr after ovulation, whereas 25-OH-D3-24-hydroxylase activity remained unchanged. The plasma level of 1α,25-dihydroxyvitamin D [1α,25-(OH)2-D] was also increased 14–16 hr after ovulation in accord with the enhancement of the renal 1α-hydroxylase activity. The plasma level of 24,25-dihydroxyvitamin D did not change during the ovulatory cycle. These results strongly suggest that 1α,25-(OH)2-D3 production in the kidney varies in a circadian rhythm during the ovulatory cycle in egg-laying hens.

Comparison of the mechanisms of bone resorption induced by 1α,25-dihydroxyvitamin D3 and lipopolysaccharides

SummaryThe mechanisms of increase in bone resorption induced by 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and bacterial lipopolysaccharides (LPS) were compared in anin vitro dead bone assay and a living bone assay. 1α,25(OH)2D3 at concentrations of 0.05–5 ng/ml dose-dependently enhanced the ability of alveolar macrophages to release45Ca from prelabeled dead bone particles (dead bone assay). In addition, the vitamin promoted fusion of the macrophages to form multinucleated cells and also enhanced glucose consumption, a marker of activation of macrophages. LPS at 0.05–5 μg/ml similarly enhanced the release of45Ca from the dead bone particles and glucose consumption by alveolar macrophages, but it did not induce fusion of the cells at any concentration. Both 1α,25(OH)2D3 and LPS dose-dependently stimulated the release of45Ca from fetal mouse calvaria prelabeled with45Ca (living bone assay). Compared to control bone, there were several times as many osteoclasts per given length of trabecular bone surface in calvaria treated for 5 days with either 5 ng/ml of 1α,25(OH)2D3 or 5 μg/ml of LPS. Indomethacin (10−5 M) completely inhibited the LPS-induced increase of osteoclasts, but not the 1α,25(OH)2D3-induced increase. These results suggest that 1α,25(OH)2D3 and LPS similarly stimulate bone resorption by activating macrophages as well as by promoting fusion of precursor cells to form multinucleated cells. 1α,25(OH)2D3 induced formation of multinucleated cells with bone-resorbing activity directly, whereas LPS appeared to induce multinucleated cells through prostaglandin synthesis by some other types of cells present in living bone tissues.

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.

The role of vitamin D in the medullary bone formation in egg-laying japanese quail and in immature male chicks treated with sex hormones

SummaryThe effect of vitamin D3 on medullary bone formation was investigated in egg-laying Japanese quail and in immature male chicks treated with sex hormones. When laying quail were fed a vitamin D-deficient diet for 16 days, their eggshell weights and egg production rate were markedly reduced in a time-dependent manner with a significant decrease in plasma calcium and 25-hydroxyvitamin D3 levels. The calcium content of the medullary bone of femurs decreased markedly with the progress of vitamin D deficiency, whereas that of the cortical bone remained unchanged. Quantitative histological examination also showed that the area of the mineralized portion of medullary bone in quail that were fed the vitamin D-deficient diet markedly decreased compared with that in the control laying quail, whereas the total area of the mineralized and unmineralized portions of medullary bone in the bone marrow cavity increased moderately. Daily administration of vitamin D3 (0.75 µg/day) to the vitamin D-deficient quail increased the mineralization of medullary bone as early as day 4. Daily administration of both estradiol (0.3 mg/day) and testosterone (0.9 mg/day) for 3 weeks to immature male chicks induced an apparent hypercalcemia and matrix formation of medullary bone, regardless of the vitamin D status of the chicks. Mineralization of medullary bone was observed only when vitamin D3 was administered together with the sex hormones. These results suggest that vitamin D3 is directly involved in the mineralization of medullary bone in birds.