Characteristics of tumor cell bioactivity in oncogenic osteomalacia.

Oncogenic osteomalacia is a condition where renal phosphate wasting occurs causing defective mineralisation, in the presence of a tumor. Cultures of cells were established from a hemangiopericytoma resected from a patient with oncogenic osteomalacia. Conditioned media from the cells inhibited phosphate uptake in opossum kidney cells and stimulated of cAMP in rat osteosarcoma cells, a standard parathyroid hormone (PTH)-like assay. This cAMP stimulation was suppressed by the PTH analogue, 3-34 bPTH and also by heat and trypsin treatment of the media. Tests of conditioned media for PTH and parathyroid hormone related protein (PTHrP) immunoreactivity were negative, however, and no hybridisation to probes for PTH, PTHrP or human stanniocalcin was detected in tumor cell RNA on Northern blot. These data support the hypothesis that tumors responsible for oncogenic osteomalacia produce a humoral substance that reduces renal phosphate reabsorption and provide evidence that the factor may act via PTH/PTHrP receptors.

Involvement of platelets in stimulating osteogenic activity.

Osteoblast-like cells have been shown to be sensitive to the proliferative action of a wide variety of growth factors. Many of these growth factors have been isolated from platelets and are thought to be released at local sites in response to injury. In this study, we tested whether human platelet concentrate, as a supplement to basic medium, would support the proliferative and functional activity of human fetal osteoblast-like cells in both short-term and long-term culture. In short-term studies, uptake of [3H]thymidine was increased in platelet-treated cultures by more than 4-fold compared with 10% serum-supplemented controls. When cultured for prolonged periods on coverslips, the cells formed multilayers, with a collagen-based matrix separating the layers. Long-term cultures that were treated with 1.5% (vol/vol) platelets in serum-supplemented medium showed increases in the depth of the multilayers of as much as 36-fold at 30 days after confluence, compared with the 10% serum-supplemented controls; this difference persisted until day 50. Incorporation of growth factor in the matrix was examined with the use of colloidal gold immunoelectron microscopy. Immunogold labeling intensities for transforming growth factor-beta 1 were significantly lower in the platelet-treated cultures at 20 days and then increased to a maximum level of 2.1-fold more than in the controls at 40 days. Labeling intensities for insulin-like growth factor-I and basic fibroblast growth factor were significantly lower in the platelet-treated cultures than in the controls at all stages of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombospondin co-localises with TGF beta and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17 beta estradiol.

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor beta-I (TGF beta), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF beta and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 beta estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF beta and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10(-10) M-10(-11) M of added estradiol. Treatment with 17 alpha estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF beta and IGF-I, in terms of their distribution and regulation by 17 beta estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.

Modulation of growth factor incorporation into ECM of human osteoblast-like cells in vitro by 17 beta-estradiol.

Human fetal osteoblast-like cells formed a regular multilayered structure in vitro with an extensive collagen-based extracellular matrix. With colloidal gold immunocytochemistry, labels for alkaline phosphatase and osteocalcin were distributed in a relatively diffuse pattern, in contrast to the bone growth factors, insulin-like growth factors I and II (IGF-I and IGF-II), transforming growth factor-beta 1 (TGF-beta 1), and basic fibroblast growth factor, which were colocalized in the collagenous matrix of the multilayer. The inclusion of 17 beta-estradiol (10(-11) to 10(-9) M) in the culture medium increased multilayer depths, increased labeling for IGF-I, IGF-II, and TGF-beta 1, and resulted in earlier detection of TGF-beta 1 label. In contrast, the increase in multilayer depth resulting from treatment with human platelets, an exogenous source of growth factors, was not accompanied by an increase in matrix IGF-I, IGF-II, or TGF-beta 1 label, suggesting a particular effect of estradiol to facilitate this process. Because growth factors in bone matrix may act as coupling agents when released during resorption, reduced growth factor incorporation in the presence of reduced sex steroid concentrations may lead to uncoupling of resorption and subsequent formation.

Role of chondroitin sulfate glycosaminoglycans in mineralizing osteoblast-like cells: effects of hormonal manipulation.

A monoclonal anti-chondroitin sulfate antibody (CS-56) that recognizes native chondroitin sulfate glycosaminoglycans (CSGAG) was used to quantify changes in CSGAG labeling levels in mineralizing human fetal osteoblast-like cell multilayers up to 40 days postconfluence. In control cultures, mean labeling of CSGAG increased in nonmineralized areas from around eight gold probes per micron 2 (gpm) at 20 days to 26 gpm at 40 days. Labeling was markedly increased in the mineralized tissue, to 560 gpm at 30 days and 580 gpm at 40 days. In beta-glycerophosphate-treated cultures, the mineralized areas were increased and appeared earlier (20 days) than in the control cultures. In these cultures, mean CSGASG labeling increased in nonmineralized areas from around 5 gpm at 20 days to 26 gpm at 30 days and was further increased in mineralized areas to 270 gpm at 20 days and 298 gpm at 30 days. Mineralization was not noted in cultures treated with 10(-8) M 1,25-dihydroxyvitamin D, and CSGAG labeling remained low (< 5 gpm) during the study period. These results indicate that an increase in immunoreactive CSGAG is associated with mineralization in this culture system. One possible interpretation of these findings is that proteoglycan molecules or at least their CSGAG side chains may be involved in the mineralization process.