1,25-Dihydroxyvitamin D3 causes formation of multinucleated cells with several osteoclast characteristics in cultures of primate marrow.

Abstract

The osteoclast is thought to be derived from immature marrow cells of the monocyte/macrophage lineage (1-3). However, the osteoclast has been difficult to study directly because ofits relative inaccessibility. Therefore investigators have used other cells of the monocyte/macrophage series, such as peritoneal macrophages (4), alveolar macrophages (5), HL-60 cells (6), and U937 cells (7), as models for osteoclasts or their precursors. Recently, it has been shown that the most potent biologically active metabolite of vitamin D, la,25-dihydroxyvitamin D3, causes fusion of alveolar macrophages (5) and differentiation of mouse leukemic cells and HL-60 and U937 human leukemic cells in culture into macrophages (6)(7)(8). Since the normal osteoclast precursor is believed to be present in the marrow mononuclear cell population, we cultured normal primate marrow mononuclear cells with 1,25-dihydroxyvitamin D3. We utilized the long-term culture system of Testa et al. (9), which they and we have used to examine formation of multinucleated cells with biologic and morphologic characteristics of osteoclasts in cultures of normal feline marrow (9, 10). We found that in the presence of 1,25-dihydroxyvitamin D3, a subpopulation of primate marrow mononuclear cells formed multinucleated cells with some of the characteristics of osteoclasts. MATERIALS AND METHODSCollection and Processing of Baboon Marrow Cells. Bone marrow was aspirated from the sternum or posterior iliac crest of adult baboons anesthetized with ketamine (10 mg/kg). Marrow was collected in syringes containing minimal essential medium alpha (a-MEM, GIBCO) supplemented with 5% (vol/vol) fetal bovine serum (Sterile Systems, Logan, UT) and preservative-free heparin (100 units/ml; Sigma). Marrow mononuclear cells were harvested from the interface of Ficoll/Hypaque density gradients (11) that had been centrifuged at 400 x g for 30 min at 12TC. The marrow mononuclear cells were washed twice with medium and then cultured in 24-well plates (Linbro) at 106 cells per ml, in a-MEM with 20o horse serum. In some experiments marrow mononuclear cells were incubated for 1 hr in a-MEM containing 20% fetal calf serum in 35-mm plastic tissue culture dishes, and the nonadherent cells were collected. The nonadherent cells were cultured in a similar fashion as the unfractionated marrow mononuclear cells. All cultures were maintained in a humidified 4% CO2 atmosphere at 370C. In selected experiments, various concentrations of osteotropic hormones were added at the initiation ofthe cultures and with each feeding. Cultures were fed weekly by removing half of the medium and nonadherent cells and replacing it with an equal volume of fresh medium. No attempt was made to recover the nonadherent cells. After culture, cells were fixed with 5% (vol/vol) glutaraldehyde (Sigma) and then stained with Wright stain. Cells were examined with an inverted phase-contrast microscope; those cells containing more than three nuclei were counted as multinucleated.Assay of Acid Phosphatase Activity. Marrow cells w...

Vitamin D endocrine system and osteoclasts

“…When the bone marrow cells were cultured, multinucleated cells having the characteristics of osteoclasts were formed in response to bone-resorbing factors, including 1a,25(OH) 2 D 3 . [9][10][11] These results established the concept that 1a,25(OH) 2 D 3 was an inducer of osteoclastic bone resorption.…”

The epithelial Ca 2+ channel TRPV5 is essential for proper osteoclastic bone resorption

“…These data indicated that lack of resorption and release of Ca 2ϩ did not cause the observed increased osteoclastogenesis in TRPV5 Ϫ/Ϫ mice. Early studies showed that 1,25(OH) 2 D 3 stimulates the formation of bone-resorbing osteoclasts from precursors or in cocultures of bone marrow cells and osteoblasts (15)(16)(17)(18). Because TRPV5 Ϫ/Ϫ mice have increased 1,25(OH) 2 D 3 levels (7) and increased osteoclastogenesis in vivo, we assessed whether more osteoclast precursors exist in bone marrow of TRPV5 Ϫ/Ϫ mice explaining the observed enhanced osteoclast formation.…”

Sphingosine-1-phosphate-mediated osteoclast precursor monocyte migration is a critical point of control in antibone-resorptive action of active vitamin D

“…Active vitamin D analogs have been used clinically in several countries for treating bone and mineral disorders associated with chronic kidney diseases or osteoporosis, although their direct pharmacologic mechanisms in bone are not fully understood (11,12). Notably, active vitamin D metabolites, such as calcitriol [1α,25(OH) 2 D 3 (1,25-D)], have been shown to increase the expression of RANKL in bone marrow stromal cells, thereby acting as osteoclastogenic, bone-resorbing factors (13,14). These findings have raised an intractable paradox regarding the action on bone of vitamin D (15)(16)(17).…”