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...
The predominant cell responsible for bone resorption, the multinucleated osteoclast, has been difficult to study because of inaccessibility. When feline marrow-derived mononuclear cells are established in long-term culture, multinucleated cells form within 48 h, reaching maximum numbers at 16 d. We have observed that these cultured cells have many of the features of osteoclasts. Morphologically, they are multinucleated, contain large numbers of branched mitochondria, have a peripheral cytoplasm lacking organelles (a clear zone), and have extensive cell-surface processes. In addition to these ultrastructural features, the cells contain a tartrate- resistant acid phosphatase, the activity of which is increased by parathyroid hormone (PTH) and inhibited by calcitonin. PTH, prostaglandin E2, and 1,25(OH)2 vitamin D3 increased multinucleated cell formation, while calcitonin inhibited the stimulatory effects of PTH. Time-lapse cinemicrographic and autoradiographic studies indicated that the multinucleated cells formed by fusion of the mononuclear progenitors. The multinucleated cells were phagocytic and stained with nonspecific esterase, consistent with their being derived from immature monocytes. Further, cell populations enriched for multinucleated cells release 45Ca from devitalized bone. Density-gradient centrifugation on Percoll was used to enrich and characterize the mononuclear progenitors of these multinucleated cells. The progenitor cells were found predominantly in Percoll density layers of 1.065 to 1.08 g/ml and were enriched up to 30-fold as compared to unfractionated cells. The bone marrow mononuclear cells that formed the multinucleated cells were initially nonadherent to plastic, stained heavily with nonspecific esterase, and appeared to be immature monocytes histologically. These data indicate that the multinucleated osteoclast-like cells in our cultures are derived from nonadherent monocytic progenitor cells that are responsive to osteotropic hormones. The ability to grow and characterize these cells in vitro should facilitate studies to elucidate the role these cells play in normal and pathologic states of bone resorption.
Human recombinant transforming growth factor a (TGFa), which binds to the epidermal growth factor (EGF) receptor and causes several biological effects similar to those caused by EGF, was compared with murine EGF for its effects on a number of parameters of bone cell metabolism. TGFa stimulated bone resorption in two organ culture systems, the fetal rat long bone and neonatal mouse calvarial systems. TGFa stimulated bone resorption at concentrations as low as 0.1 ng/ml. TGFa effects on bone resorption in mouse calvariae were inhibited by indomethacin, suggesting that, like EGF, its effects were mediated by prostaglandin synthesis. TGFa had a different time course of action on bone resorption from that of EGF, causing more rapid release of previously incorporated 45Ca from bone cultures, suggesting that TGFa does not function on bone as a simple EGF analogue. TGFa also caused effects on osteoblast function resembling those of EGF. It inhibited alkaline phosphatase activity in cultured rat osteosarcoma cells with the osteoblast phenotype and inhibited collagen synthesis in fetal rat calvaria at concentrations of 1.0 ng/ml. The lowest concentration of TGFa (expressed as nanogram equivalents of EGF per ml) required to produce a response in all of the systems tested was about 1/10th of that needed for EGF to produce a similar effect. These results indicate that TGFa is a potent stimulator of bone resorption and inhibitor of bone formation as assessed by inhibition of collagen synthesis and alkaline phosphatase activity and are consistent with the hypothesis that TGFa may be responsible, at least in part, for the bone resorption associated with some tumors.Transforming growth factor alpha (TGFa) is secreted by a variety of human and rodent tumor cells and binds to the epidermal growth factor (EGF) receptor. TGFa causes many of the known biological effects of EGF (1-3). Multiple peptides of different molecular weights with TGFa activity (as assessed by inhibition of EGF binding and stimulation of anchorage-independent growth) have been detected in culture media and extracts of tumor cells. A small species of 50 amino acids has been purified (4, 5). It is about 40% homologous with EGF (4, 6). Cloning of the cDNA reveals that it is initially synthesized as a larger precursor, which undergoes subsequent posttranslational cleavage. Proper engineering of the sequence coding for the 50 amino acid TGFa has allowed the expression of biologically active human TGFa in Escherichia coli (6).EGF causes osteoclastic bone resorption (7,8) and inhibits bone formation as assessed by collagen synthesis in organ cultures of fetal rat calvariae (7-9) and alkaline phosphatase activity in osteoblast-like cells (10). Since EGF stimulates osteoclastic bone resorption and TGFa competes with EGF for the same receptor, it has been suggested that tumor production of TGFa could be responsible for the bone destruction that is associated with some neoplasms (11,12). Partially purified bone-resorbing activity associated with an animal tumor co...
Evidence is presented that a tumor-derived transforming growth factor is responsible for stimulating bone resorption and causing hypercalcemia in an animal tumor model of the hypercalcemia of malignancy. Both conditioned medium harvested from cultured tumor cells and tumor extracts of the transplantable rat Leydig cell tumor associated with hypercalcemia contained a macromolecular bone resorbing factor with the chemical characteristics of a tumor-derived transforming growth factor.
Amounts of prostaglandin E and prostaglandin F have been measured by radioimmunoassay in extracts of renal cortical carcinoma and benign and malignant breast tumours after solvent extraction and column chromatography. 2. Substantial amounts of prostaglandin E were found in extracts of benign and malignant breast tumours and in renal tumours. Much lower amounts of prostaglandin F were present in all tumour types. 3. Co-cultivation of tumour explants with mouse calvaria led to significant bone resorption in 10 of 13 renal carcinomas, three of eight malignant breast tumours, and two of nine benign breast tumours. Tumours associated with bone resorption had higher concentrations of prostaglandin E in culture media at the end of incubation than did non-resorbers. 4. Indomethacin (14 mumol/1) greatly reduced bone resorption in the presence of the tumour, but this was not always complete, particularly with breast tumours. Indomethacin had no effect on prostaglandin-induced bone resorption. Theophylline (2.2 mmol/1) significantly increased prostaglandin E production and resorption by an effect on the tumour. 5. It is concluded that prostaglandins may be important in mediating the effects of renal cortical carcinoma and possibly breast cancer on bone destruction. A non-prostaglandin mechanism may also contribute to bone destruction by breast carcinoma.
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